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Back to Journals » Journal of Asthma and Allergy » Volume 14
Short-Term Evaluation of Dupilumab Effects in Patients with Severe Asthma and Nasal Polyposis
Authors Pelaia C , Lombardo N , Busceti MT, Piazzetta G, Crimi C , Calabrese C , Vatrella A , Pelaia G 
Received 12 July 2021
Accepted for publication 19 August 2021
Published 24 September 2021 Volume 2021:14 Pages 1165—1172
DOI https://doi.org/10.2147/JAA.S328988
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 3
Editor who approved publication: Dr Amrita Dosanjh
Corrado Pelaia,1 Nicola Lombardo,2 Maria Teresa Busceti,1 Giovanna Piazzetta,2 Claudia Crimi,3 Cecilia Calabrese,4 Alessandro Vatrella,5 Girolamo Pelaia1

1Department of Health Sciences, Magna Graecia University of Catanzaro, Catanzaro, Italy; 2Department of Medical and Surgical Sciences, Magna Graecia University of Catanzaro, Catanzaro, Italy; 3Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy; 4Department of Translational Medical Sciences, Luigi Vanvitelli University of Campania, Naples, Italy; 5Department of Medicine, Surgery and Dentistry, University of Salerno, Salerno, Italy

Correspondence: Corrado Pelaia
Department of Health Sciences, Magna Graecia University of Catanzaro, Catanzaro, Italy
Tel + 39 0961 364-7007
Email [email protected]

Background: Having been approved for biological treatment of atopic dermatitis, dupilumab has also been recently licensed as add-on therapy for severe asthma and nasal polyposis. With regard to the latter diseases, few real-life clinical investigations have been carried out to date.
Objective: The primary end point of this single-center observational study was to evaluate in a real-life setting the short-term therapeutic effects of dupilumab in patients with severe asthma and nasal polyposis.
Methods: At baseline and after 4 weeks of add-on therapy with dupilumab, several clinical and functional parameters were assessed in 20 patients with severe asthma and nasal polyposis, including both allergic and nonallergic subjects.
Results: After 4 weeks of treatment with dupilumab, all patients experienced remarkable improvement in both severe asthma and nasal polyposis. In particular, asthma-control test and sinonasal outcome test 22 scores had significantly increased (p< 0.0001) and decreased (p< 0.0001), respectively. Oral corticosteroid intake got to zero within 4 weeks (p< 0.0001). Moreover, in week 4, significant increases were detected with regard to both prebronchodilator forced expiratory volume in the first second (p< 0.01) and forced vital capacity (FVC; p< 0.05). At the same time point, dupilumab had significantly reduced residual volume (p< 0.0001) and total lung capacity (p< 0.001), whereas it had enhanced forced midexpiratory flow of 25%– 75% FVC (p< 0.01) and peak expiratory flow (p< 0.01). After 4 weeks of treatment, dupilumab had also lowered levels of fractional exhaled nitric oxide (p< 0.0001).
Conclusion: The results of this real-life study suggest that dupilumab can be utilized in both allergic and nonallergic patients with severe asthma and nasal polyposis as a valuable add-on biological therapy with rapid onset of action.

Keywords: severe asthma, nasal polyposis, interleukin 4 receptor, dupilumab

Severe asthma is characterized by heterogeneous phenotypes underpinned by several inflammatory patterns, which are driven by complex pathobiological mechanisms known as endotypes.1–3 Among the latter, two distinct entities have been identified and defined as type 2 (T2-high) or non–type 2 (T2-low) asthma, respectively.4–9 The most important cellular elements involved in the development, persistence, and amplification of type 2 asthma are T-helper 2 (Th2) lymphocytes and group 2 innate lymphoid cells (ILC2).10–13 Upon activation, Th2 and ILC2 cells release large amounts of IL4, IL13, and IL5.5,14 IL4 induces immunoglobulin class switching, thereby stimulating B lymphocytes to synthesize IgE.15 IL13 cooperates with IL4 in promoting IgE production, and is also responsible for bronchial hyperresponsiveness, goblet-cell metaplasia, and proliferation of airway fibroblasts and smooth-muscle cells.16,17 IL13 also upregulates the expression of the inducible isoform of nitric oxide synthase (iNOS), thus increasing the emission of NO from airway epithelial cells, as well as the levels of fractional NO in exhaled breath (FeNO).18 IL5 plays a pivotal pathogenic role in eosinophilic inflammation by inducing the differentiation, survival, and activation of eosinophils.19
Therefore, high serum concentrations of IgE, minimum FeNO levels of 20–25 parts per billion (ppb), and blood eosinophils counts of at least 150–300 cells/μL are considered reliable biomarkers of T2-high asthma.2 Given the key biological links connecting these biomarkers with the aforementioned cytokines, it can be reasonably inferred that IL4, IL13, IL5, and their receptors represent suitable molecular targets for biological therapies for severe type 2 asthma, refractory to both standard inhaled treatments and oral corticosteroids (OCS).20–22 Within such a therapeutic context, very interesting pharmacologic features characterize dupilumab, a fully human IgG4 monoclonal antibody that binds specifically to the α subunit of the IL4 receptor (IL4Rα).23 Because utilization of IL4Rα is shared by IL4 and IL13, which activate the type I (IL4Rα/γc) and type II (IL4Rα/IL13Rα1) receptor dimers, respectively, dupilumab behaves as a receptor antagonist of both these cytokines.24 Dupilumab has been recently evaluated by two phase III randomized clinical trials (RCTs; Liberty Asthma QUEST and Liberty Asthma VENTURE),25,26 the results of which have led to the approval of this biologic for add-on treatment of type 2 severe asthma. Dupilumab has also been licensed for biological therapy of severe chronic rhinosinusitis with nasal polyps (CRSwNP), effectively treatable with this drug, as shown by two phase III RCTs: Liberty NP SINUS-24 and Liberty NP SINUS-52.27–29 However, because of the recent introduction of dupilumab in clinical practice, few real-life studies have been published to date.30–32
Based on a real-world approach, we decided to investigate the short-term effects of dupilumab in patients with severe type 2 asthma and nasal polyposis. It has been shown that within 2 hours after the first administration, dupilumab effectively neutralized the functions of IL4Rα expressed by CD4+ T cells and CD19+ B cells, and this receptor blockade persisted stably during the following 4 weeks.33 Therefore, the aim of our real-life, single-center study was to verify the rapidity of dupilumab therapeutic effects by evaluating at baseline and after 4 weeks of treatment several outcomes, including symptom control of severe asthma and CRSwNP, lung function, FeNO, and OCS consumption.
We enrolled adult outpatients with severe eosinophilic asthma and nasal polyposis under treatment with dupilumab. Twenty subjects were recruited, eleven women and nine men, with a mean age of 55.8±15.17 years and mean BMI of 26.55±5.68 kg/m2. Mean baseline FEV1 was 67.8%±20.01% of predicted value. Fourteen participants had positive skin-prick tests for perennial and/or seasonal aeroallergens. All 20 enrolled patients were referred to the Respiratory Unit and the Otolaryngology Unit of Magna Graecia University Hospital in Catanzaro, Italy. They complained of persistent asthma symptoms and CRSwNP, and needed high dosages of inhaled CS (ICS)–long-acting β2-adrenergic agonist combinations, along with long-acting muscarinic antagonists (LAMAs). Fourteen patients had not been previously treated with biologics, and the other six underwent a washout period of at least 2 months before switching to dupilumab from previous ineffective biological therapies with omalizumab (three patients), mepolizumab (two patients), or benralizumab (one patient). These patients were consecutively recruited at our University Clinical Center, and the only inclusion criteria regarded fulfilment of the items regulating eligibility for dupilumab treatment. CRSwNP was characterized by an eosinophilic pattern in all 20 patients. Skin-prick testing was performed by placing a drop of each allergen solution on the forearm marked by a skin marker pen, and every drop was pricked by a sterile lancet. Skin sensitivity was then determined by comparing the eventual reactive wheal with that produced by histamine.34 Blood eosinophil and basophil counts were obtained using automated hematology analyzers.35,36
At baseline, all participants had blood eosinophil counts of at least 150 cells/μL and/or FeNO levels >25 ppb, and/or required permanent or nearly continuous OCS treatment. All patients met the European Respiratory Society (ERS)/American Thoracic Society (ATS) criteria that define severe uncontrolled asthma.37 Study participants also reported a history of nasal polyposis, and a diagnosis of CRSwNP was obtained on the basis of clinical symptoms, nasal endoscopy, and computed tomography scans.38,39 Dupilumab was prescribed according to the existing eligibility indications and administered subcutaneously at an initial dose of 600 mg (two injections of 300 mg at different sites), followed by maintenance dosing of 300 mg every 2 weeks.40 Baseline characteristics of patients are summarized in Table 1.

Table 1 Baseline patient features

Table 1 Baseline patient features
The main aim of this real-life investigation was to evaluate the effects of dupilumab on clinical, functional, and laboratory parameters. Asthma control test (ACT) scores, Sino-Nasal Outcome Test (SNOT-22) questionnaire scores, prednisone intake, forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), residual volume (RV), total lung capacity (TLC), forced midexpiratory flow of 25%–75% FVC (FEF25–75), peak expiratory flow (PEF), FeNO levels, and blood eosinophil and basophil counts were assessed at baseline and 4 weeks after the first injection of dupilumab. Spirometry and body plethysmography were performed using the MasterScreen pulmonary function testing system and MasterScreen Body (Jaeger‐Viasys; CareFusion, Höchberg, Germany), according to ATS/ERS guidelines.41 FeNO levels were measured with a Vivatmo Pro FeNO device (Bosch, Waiblingen, Germany) in accordance with ATS/ERS recommendations.42,43 For 2 hours after dupilumab administration, patients were monitored to detect the eventual onset of adverse reactions. Moreover, the possible occurrence of side effects was investigated through a once-weekly telephone call, as well as each time every patient underwent dupilumab injection.
This study met the standards of Good Clinical Practice and the principles of the Declaration of Helsinki. All patients signed a written informed consent. The study was also carried out in agreement with a statement from the local Ethical Committee of Calabria Region (Catanzaro, Italy; document 182–20th May 2021).
All data are expressed as means ± SD if normally distributed and otherwise as medians with IQRs. Normality of data distribution was checked using the Anderson–Darling and Kolmogorov–Smirnov tests. Student’s t test and Mann–Whitney U test were used to compare variables when appropriate. p<0.05 (two-sided) was considered statistically significant. Statistical analysis and figures were created using Prism 9.1.2 (GraphPad Software, San Diego, CA, USA).
After 4 weeks of add-on therapy with dupilumab, ACT scores had significantly increased from a baseline value of 11.85±4.58 to 21.30±3.08 (p<0.0001, Figure 1A). When compared to the baseline measurement of 58.30±21.59, SNOT-22 scores had significantly lowered to 18.90±16.48 after 4 weeks (p<0.0001, Figure 1B). The better control of respiratory symptoms made it possible to quickly and progressively reduce OCS intake, which was eleminated after 4 weeks of treatment with dupilumab. In fact, within 4 weeks, median prednisone consumption fell from a baseline daily dosage of 12.5 (5–25) mg/day to 0 (0–0) mg/day (p<0.0001, Figure 1C).

Figure 1 Clinical effects of dupilumab, with regard to ACT score (A), SNOT-22 score (B), and prednisone intake (C). ACT and SNOT-22 values expressed as means ± SD; prednisone intake expressed as medians (IQR). ****p<0.0001.

Figure 1 Clinical effects of dupilumab, with regard to ACT score (A), SNOT-22 score (B), and prednisone intake (C). ACT and SNOT-22 values expressed as means ± SD; prednisone intake expressed as medians (IQR). ****p<0.0001.
In association with these relevant clinical outcomes, we detected parallel improvements in lung function. In particular, prebronchodilator FEV1 had improved from a baseline mean of 2.04±0.94 L to 2.30±0.96 L after 4 weeks of add-on treatment with dupilumab (p<0.01, Figure 2A). Furthermore, when compared to baseline, at the week 4, FVC had significantly risen from 2.85±1.23 L to 3.14±1.37 L (p<0.05, Figure 2B). Dupilumab also induced a significant reduction in lung hyperinflation caused by airflow limitation. In comparison to baseline, 4 weeks after the first administration, median RV had significantly diminished from 2.78±0.83 L to 2.09±0.75 L (p<0.0001, Figure 2C). RV reduction was associated with a concomitant decrease in TLC, which had declined from 5.93±1.58 L at baseline to 5.47±1.51 L after 4 weeks (p<0.001, Figure 2D). In addition to such rapid and impressive deflating effects, we also observed that dupilumab had improved small-airway obstruction, with a significant increase in median FEF25–75 from 1.47±0.85 L/sec to 1.80±0.86 L/sec (p<0.01, Figure 2E). In the same period, PEF had increased from 5.69±2.41 L/sec to 6.32±2.25 L/sec (p<0.01, Figure 2F).

Figure 2 Functional effects of dupilumab with regard to FEV1 (A), FVC (B), RV (C), TLC (D), FEF25–75 (E), and PEF (F). All parameters expressed as means ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2 Functional effects of dupilumab with regard to FEV1 (A), FVC (B), RV (C), TLC (D), FEF25–75 (E), and PEF (F). All parameters expressed as means ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
With regard to the effects on some biomarkers of type 2 inflammation, in comparison to baseline, median FeNO levels had sharply decreased from 27.50 (15.25–33) ppb to 2.50 (0–6) ppb (p<0.0001) after 4 weeks of treatment with dupilumab (Figure 3A). Moreover, 4 weeks after the first dupilumab administration, median blood eosinophil and basophil counts did not significantly change with respect to baseline, fluctuating from 310 (192.5–524.3) cells/μL to 325 (215–555) cells/μL (p=0.81, Figure 3B) and from 45 (30–67.5) cells/μL to 50 (30–77.5) cells/μL (p=0.84, Figure 3C), respectively.

Figure 3 Effects of dupilumab on biomarkers with regard to FeNO (A), blood eosinophils (B), and blood basophils (C). All parameters expressed as medians (IQR). ****p<0.0001.

Figure 3 Effects of dupilumab on biomarkers with regard to FeNO (A), blood eosinophils (B), and blood basophils (C). All parameters expressed as medians (IQR). ****p<0.0001.
Add-on treatment with dupilumab was well tolerated, and no serious adverse reactions occurred throughout this study.
Taken together, the results of this single-center observational study carried out in patients with severe asthma and nasal polyposis clearly indicate that dupilumab rapidly induced powerful beneficial effects. At 4 weeks after the first administration, this drug had exerted a positive impact on many clinical and functional parameters, as well as on relevant biomarkers of airway type 2 inflammation. Therefore, such findings further corroborate in a real-world setting and in a short time previous data reported by several RCTs and preliminary real-life investigations.25–27,30–32
After 4 weeks of treatment, mean ACT score significantly increased in comparison to the low baseline measurement, thus rapidly overcoming the critical threshold of 20, which reflects adequate asthma control.44 This result is consistent with similar data reported by the Liberty Asthma QUEST trial, which showed a very quick improvement in the Asthma Control Questionnaire (ACQ-5) scores already detectable at week 2 of add-on biological therapy with dupilumab.25 However, within a real-life context, the ACQ can be usefully substituted by the ACT, which is likely more understandable and easier to be filled in by asthmatic patients routinely referring to clinical centers, who are less strictly monitored than their counterparts enrolled in RCTs.45 In fact, the ACT rather than the ACQ has been used in recent real-world studies with the aim of verifying the therapeutic effectiveness of dupilumab in providing satisfactory control of severe asthma symptoms.30–32 We noticed that the marked improvement in asthma control quickly elicited by dupilumab allowed a fast and progressive tapering of prednisone dosage, which within only 4 weeks culminated in a complete withdrawal of OCS treatment, more quickly than that observed in the Liberty Asthma VENTURE study, thereby confirming that real-life findings can be even better than those of RCTs.26,30 The suspension of OCS intake is very important for patients with severe asthma, a majority of whom are OCS-dependent and exposed to the negative side effects of OCSs, including adrenal insufficiency, respiratory infections, diabetes, hypertension, osteoporosis, glaucoma, and cataracts.46,47 In our patients with severe asthma and CRSwNP, amelioration of clinical symptoms also involved upper airways, as shown by the significant decrease in SNOT-22 scores. This improvement in nasal symptoms confirmed the results of RCTs, thus reiterating the efficacy of dupilumab in the treatment of CRSwNP.27,48 We plan to further corroborate and extend these results by evaluating in future studies the effects of dupilumab on a numeric rating scale, endoscopic nasal polyp score, resistance of nasal cavity, and Lund–Mackay computed tomography score.
With regard to lung function, dupilumab rapidly improved airflow limitation in both large and small airways by significantly enhancing prebronchodilator FEV1, FVC, FEF25–75, and PEF. Consistently with both RCT and preliminary real-life observations,25,26,30–32 FEV1 increase was remarkable, and after just 4 weeks had neared 300 mL. Interestingly, in our severe asthmatic patients, dupilumab also exerted a fast and effective deflating action, demonstrated by the marked reduction in RV and TLC. This therapeutic effect is very important in severe asthma. Differently from mild–moderate disease, severe asthma is frequently characterized by relevant degrees of lung hyperinflation.49
In relation to the effects of dupilumab on biomarkers of type 2 airway inflammation, we found that this monoclonal antibody was able to induce a quick and sharp drop in FeNO levels. Through its peculiar dual-receptor antagonism, dupilumab neutralizes the stimulatory actions of IL4 and especially IL13 on iNOS expression at the level of the bronchial epithelium.50,51 FeNO is a useful biomarker of type 2 asthma, the levels of which correlate with disease severity, lung-function deterioration, and exacerbation risk.52 In addition, FeNO is a reliable tool in choosing tailored biological therapies for severe asthma and monitoring the effects of such treatments.53 With regard to observational investigations like ours, FeNO can thus be considered a valuable predictor of therapeutic response to dupilumab detectable in patients with severe asthma, as well as a good biological indicator associated with the clinical and functional outcomes evaluated in these subjects. Differently from FeNO, we did not detect any change induced by dupilumab on blood counts of eosinophils and basophils. These results can be reasonably explained by the distinct biological actions of the various type 2 cytokines. Dupilumab suppresses the functions of IL4 and IL13, which are the main inducers of NO production. Blood and airway eosinophilia are predominantly promoted by IL5, which is not a target of dupilumab. On the other hand, given the small size of our study population, the lack of dupilumab effects on blood eosinophils is not surprising. Indeed, the results reported by two relevant RCTs showed that dupilumab increased blood-eosinophil numbers in relatively low percentages of treated patients, amounting to 4.1% (versus 0.6% with placebo) and 14% (versus 1% with placebo) in the Liberty Asthma QUEST and Liberty Asthma VENTURE trials, respectively.25,26
Another interesting aspect of our present real-life experience is the mixed nature of the study population, which included atopic and nonatopic patients. In both these subgroups, very relevant clinical and functional effects were rapidly elicited by dupilumab, due to its capacity to interfere with allergic and nonallergic pathways underlying type 2 immunomechanisms implicated in airway inflammation. By impeding stimulation of IL4 and IL13 receptors, dupilumab inhibits the bioactivities of the main cellular sources of these cytokines, namely Th2 and ILC2 cells.5 ILC2 and Th2 cells are the key players in the integrated cross talk between innate and adaptive immunoresponses underpinning atopic and nonatopic asthma.6,12,54 Hence, by dampening such cellular and molecular circuits, dupilumab can extend its therapeutic actions on both upper and lower airways, given the common pathogenic mechanisms shared by type 2 asthma and CRSwNP.54,55
In our opinion, the most relevant finding of this real-life study refers to the fast onset of the therapeutic effects of dupilumab. In relation to such a pharmacodynamic pattern, our patients behaved as a quite homogeneous group, highly responsive to the very quick benefits provided by dupilumab. This therapeutic celerity was particularly valuable for those subjects who did not respond satisfactorily to previous biological treatments. It is likely that the early responsiveness experienced by our patients to dupilumab depends on high expression levels of both IL4 and IL13, whose biological actions can be promptly blocked by this monoclonal antibody on several immune/inflammatory cells.33
In conclusion, we herein show in a real-world setting that within 4 weeks of treatment, dupilumab induced in patients with severe asthma and nasal polyposis several therapeutic benefits, including significant improvements in symptom control and lung function associated with a drastic reduction in a viable biomarker of type 2 airway inflammation, such as FeNO. In our opinion, the main strengths of this observational investigation are demonstration of the valuable rapidity of the therapeutic effects prompted by dupilumab with regard to both bronchial and nasal symptoms, assessment of the inhibitory action exerted by dupilumab on lung hyperinflation, detection of similar therapeutic effects in both allergic and nonallergic subjects, and good short-term profile for the safety and tolerability of dupilumab. On the other hand, the most important limitations are quite common to other single-center real-life studies, and include the relatively low number of enrolled patients, as well the lack of randomization design and placebo control.
All authors contributed to data analysis, drafting, or revising the article, have agreed on the journal to which the article will be submitted, gave final approval to the version to be published, and agree to be accountable for all aspects of the work.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
The authors declare that there are no conflicts of interest in this work.
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40. Wenzel S, Castro M, Corren J, et al. Dupilumab efficacy and safety in adults with uncontrolled persistent asthma despite use of medium-to-high-dose inhaled corticosteroids plus a long-acting β2 agonist: a randomised double-blind placebo-controlled pivotal phase 2b dose-ranging trial. Lancet. 2016;388:31–44. doi:10.1016/S0140-6736(16)30307-5
41. Graham BL, Steenbruggen I, Miller MR, et al. Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European Respiratory Society Technical Statement. Am J Respir Crit Care Med. 2019;200:e70–e88. doi:10.1164/rccm.201908-1590ST
42. American Thoracic Society, European Respiratory Society. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med. 2005;171:912–930. doi:10.1164/rccm.200406-710ST
43. Horváth I, Barnes PJ, Loukides S, et al. A European Respiratory Society technical standard: exhaled biomarkers in lung disease. Eur Respir J. 2017;49:1600965. doi:10.1183/13993003.00965-2016
44. Nathan RA, Sorkness CA, Kosinski M, et al. Development of the asthma control test: a survey for assessing asthma control. J Allergy Clin Immunol. 2004;113:59–65. doi:10.1016/j.jaci.2003.09.008
45. Jia CE, Zhang HP, Lv Y, et al. The asthma control test and the asthma control questionnaire for assessing asthma control: systematic review and meta-analysis. J Allergy Clin Immunol. 2013;131:695–703. doi:10.1016/j.jaci.2012.08.023
46. Heffler E, Blasi F, Latorre M, et al. The severe asthma network in Italy: findings and perspectives. J Allergy Clin Immunol Pract. 2019;7:1462–1468. doi:10.1016/j.jaip.2018.10.016
47. Canonica GW, Colombo GL, Bruno GM, et al. Shadow cost of oral corticosteroids-related adverse events: a pharmacoeconomic evaluation applied to real-life data from the severe asthma network in Italy (SANI) registry. World Allergy Organ J. 2019;12:100007. doi:10.1016/j.waojou.2018.12.001
48. Bachert C, Hellings PW, Mullol J, et al. Dupilumab improves health-related quality of life in patients with chronic rhinosinusitis with nasal polyposis. Allergy. 2020;75:148–157. doi:10.1111/all.13984
49. Jarjour NN, Erzurum SC, Bleecker ER, et al. Severe asthma – lessons learned from the National Heart, Lung and Blood Institute severe asthma research program. Am J Respir Crit Care Med. 2012;185:356–362. doi:10.1164/rccm.201107-1317PP
50. Carr TF, Kraft M. Use of biomarkers to identify phenotypes and endotypes of severe asthma. Ann Allergy Asthma Immunol. 2018;121:414–420. doi:10.1016/j.anai.2018.07.029
51. Busse WW, Kraft M, Rabe KF, et al. Understanding the key issues in the treatment of uncontrolled persistent asthma with type 2 inflammation. Eur Respir J. 2021;4:2003393. doi:10.1183/13993003.03393-2020
52. Ulrik CS, Lange P, Hilberg O. Fractional exhaled nitric oxide as a determinant for the clinical course of asthma: a systematic review. Eur Clin Respir J. 2021;8:1891725. doi:10.1080/20018525.2021.1891725
53. Rolla G, Heffler E, Pizzimenti S. An emerging role for exhaled nitric oxide in guiding biological treatment in severe asthma. Curr Med Chem. 2020;27:7159–7167. doi:10.2174/0929867327666200713184659
54. Scadding GK, Scadding GW. Innate and adaptive immunity: ILC2 and Th2 cells in upper and lower airway allergic diseases. J Allergy Clin Immunol Pract. 2021;9:1851–1857. doi:10.1016/j.jaip.2021.02.013
55. Matucci A, Bormioli S, Nencini F, et al. Asthma and chronic rhinosinusitis: how similar are they in pathogenesis and treatment responses? Int J Mol Sci. 2021;22:3340. doi:10.3390/ijms22073340z
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Game-by-game score predictions for Wisconsin's 2021 football season – Badgers Wire

Wisconsin stands 10 days away from opening its 2021 football season.
The beginning of their schedule will prove to be the toughest, as the team must face a ranked Penn State team Week 1, travel to Chicago to face Notre Dame Week 3, return home to play Michigan and have a sneaky-tough out-of-conference game against Army soon after.
Overall, though, it’s an extremely manageable schedule. They avoid a crossover game with Ohio State and every tough game before Minnesota is either at home or at a neutral site.
Related: Three Wisconsin Badgers will miss the 2021 season due to injury
Since we’re now 10 days away from kickoff, I’m here with game-by-game score prediction for Wisconsin’s 2021 football season:
Apr 17, 2021; University Park, PA, USA; Penn State Nittany Lions head coach James Franklin looks on during the Penn State spring practice at Beaver Stadium. Mandatory Credit: Mark Alberti-USA TODAY Sports
Wisconsin and Penn State enter Week 1 with a similar profile.
Both teams have quarterbacks that have shown great play, and also struggled at times. Both teams had rough 2020 campaigns—Wisconsin 4-3 and Penn State 4-5. And, most importantly, both enter the year with significant expectations of a bounce-back year.
Big matchups to watch in this one include Penn State wide receivers Jahan Dotson and Parker Washington against the Badger secondary, Wisconsin running backs against P.J. Mustipher and the Nittany Lion defensive line and the overall play of quarterbacks Graham Mertz and Spencer Petras.
Wisconsin will be around 3.5-point favorites in this contest and boast a huge home-field advantage with Camp Randall set to be at full capacity.
It think both offenses find real success in this one and both teams answer a lot of questions about their poor 2020 seasons. But in the end, Wisconsin’s defense comes up with a few huge takeaways and the Badgers pull away with a victory.
 
Final Score: Wisconsin 27, Penn State 23
Sep 14, 2019; Champaign, IL, USA; Eastern Michigan Eagles head coach Chris Creighton reacts during the second half against the Illinois Fighting Illini at Memorial Stadium. Mandatory Credit: Patrick Gorski-USA TODAY Sports
Wisconsin shouldn’t have much trouble with this one after opening their season with a pivotal matchup.
 
Final Score: Wisconsin 34, Eastern Michigan 7
Nov 21, 2015; Madison, WI, USA; A general view of nnow covered seats in the stands of Camp Randall Stadium prior to the game between the Northwestern Wildcats and Wisconsin Badgers. Mandatory Credit: Jeff Hanisch-USA TODAY Sports
May 1, 2021; Notre Dame, Indiana, USA; Notre Dame Fighting Irish quarterback Jack Coan (17) runs the ball in the first half of the Blue-Gold Game at Notre Dame Stadium. Mandatory Credit: Matt Cashore-USA TODAY Sports
The Jack Coan revenge game looks like a pivotal game for Paul Chryst’s team, but it isn’t nearly one of the Badgers’ most important. It’s out-of-conference and will be a great test, but the path to Indy and beyond won’t be affected much by the outcome in Chicago.
That said, I see Coan showing more than people expect in Notre Dame’s offense this season. Pair that with talent everywhere and a team fresh off the playoff, and signs point towards a loss for me in this one.
 
Final Score: Notre Dame 27, Wisconsin 21
Nov 14, 2020; Ann Arbor, Michigan, USA; Michigan Wolverines running back Chris Evans (9) is tackled by Wisconsin Badgers safety Eric Burrell (25) in the first half at Michigan Stadium. Mandatory Credit: Rick Osentoski-USA TODAY Sports
This game doesn’t scare me that much.
Michigan hasn’t won in Madison in two decades, Jim Harbaugh is still in search of a quarterback and the Wolverines have been physically dominated by the Badgers during the last few contests.
 
Final Score: Wisconsin 30, Michigan 21
Oct 20, 2018; Madison, WI, USA; Illinois Fighting Illini wide receiver Ricky Smalling (4) rushes with the football after catching a pass during the second quarter against the Wisconsin Badgers at Camp Randall Stadium. Mandatory Credit: Jeff Hanisch-USA TODAY Sports
It’s lining up to be a rough first year at Illinois for Bret Bielema, though he does have a few things going for him: quarterback Brandon Peters returns and the offensive line is littered with experience.
On defense, though, it should be more of the same for the Fighting Illini.
Wisconsin lost the last matchup in Champaign in heartbreaking fashion. I think that loss still haunts those who were on the 2019 team and they will be set on avoiding a repeat performance.
 
Final Score: Wisconsin 34, Illinois 14
Mandatory Credit: Danny Wild-USA TODAY Sports
While this screams close contest, something tells me Jim Leonhard will have the day of his life stopping Army’s triple-option attack.
If Wisconsin’s offense is healthy, I don’t see Army being able to stop their versatility and multi-dimensional attack. Though we saw Jeff Monken’s team take Michigan to overtime a few years ago, this is a defense that Graham Mertz and company should not have much trouble with.
 
Final Score: Wisconsin 24, Army 13
Oct 31, 2020; Champaign, Illinois, USA; Illinois Fighting Illini defensive lineman Owen Carney Jr. (99) sacks Purdue Boilermakers quarterback Aidan O’Connell (16) during the second half at Memorial Stadium. Mandatory Credit: Patrick Gorski-USA TODAY Sports
Weird stuff happens in West Lafayette in October.
This is probably the biggest trap game for me on the schedule, with a crucial, likely Big Ten West-deciding contest looming against Iowa.
Quarterback Jack Plummer figures to have a solid season, wide receiver David Bell is as dangerous as anybody and defensive end George Karlaftis is one of the conference’s best.
I think Wisconsin wins, but it will test the heart rates of every Badger fan.
 
Final Score: Wisconsin 27, Purdue 24 (OT)
Dec 12, 2020; Iowa City, Iowa, USA; The line of scrimmage between the Iowa Hawkeyes and the Wisconsin Badgers at Kinnick Stadium. Mandatory Credit: Jeffrey Becker-USA TODAY Sports
As mentioned, this game will most likely decide the Big Ten West.
Similar to the Penn State game, Iowa enters the year with much of their success riding on the arm of an up-and-down quarterback in Spencer Petras.
But Iowa again will have one of the best offensive lines in the conference led by Tyler Linderbaum, running back Tyler Goodson is dangerous and the Iowa defense is the Iowa defense.
While Wisconsin–Iowa contests often come down to who wins up front, it’ll be a Graham Mertz vs. Spencer Petras matchup here with each team great in the trenches.
 
Final Score: Wisconsin 24, Iowa 21
Oct 12, 2019; Bloomington, IN, USA; Indiana Hoosiers wide receiver Donavan Hale (6) catches a ball that is ruled out of bounds against Rutgers Scarlet Knights defensive back Avery Young (2) during the second quarter of the game at Memorial Stadium. Mandatory Credit: Marc Lebryk-USA TODAY Sports
While Greg Schiano is poised for a second-year jump at Rutgers, I can’t predict a let-down loss in this spot.
The Big Ten West at this point will be down to the Badgers, Hawkeyes and Minnesota Golden Gophers, with Wisconsin having a shot at controlling their own destiny with two of three wins down the stretch.
They’ll get one here at Rutgers.
 
Final Score: Wisconsin 31, Rutgers 20
Nov 21, 2020; Evanston, Illinois, USA; Northwestern Wildcats wide receiver Riley Lees (19) runs with the ball as Wisconsin Badgers linebacker Leo Chenal (45) tries to tackle him during the first half at Ryan Field. Mandatory Credit: David Banks-USA TODAY Sports
I’m up on Penn State, Rutgers, Iowa and Minnesota. That said, I’m extremely down on Northwestern this season.
Hunter Johnson could finally turn into a solid player at quarterback, but the amount of high-end talent the Wildcats lost after last season will prove significant.
If Graham Mertz can avoid Brandon Joseph in the Wildcat secondary, the Badgers should be able to muscle themselves to victory. Yes, I’m saying that knowing that Wisconsin—Northwestern contests are always way too close for comfort.
 
Final Score: Wisconsin 21, Northwestern 14
Nov 27, 2020; Iowa City, Iowa, USA; Nebraska Cornhuskers quarterback Adrian Martinez (2) and Iowa Hawkeyes defensive tackle Daviyon Nixon (54) in action against the Iowa Hawkeyes at Kinnick Stadium. Mandatory Credit: Jeffrey Becker-USA TODAY Sports
Another year, another spot for Wisconsin’s running game to absolutely dominate the Cornhuskers.
Unless Adrian Martinez takes a massive step, the Nebraska defense finds an answer against the Badgers up front and Scott Frost turns things around quickly, it could be a Big Ten West-clinching win for the Badgers.
 
Final Score: Wisconsin 34, Nebraska 24
Wisconsin players celebrate with the Paul Bunyan Axe after they defeated Minnesota in overtime of an NCAA college football game Saturday, Dec. 19, 2020, in Madison, Wis. (AP Photo/Andy Manis)
While it sounds crazy, this will be the first true road test of Graham Mertz’s career.
I’m pretty high on the Gophers this year, that assuming their defense can’t go anywhere but up. But Tanner Morgan still has Mohamed Ibrahim in the backfield and Chris Autman-Bell on the outside, and P.J. Fleck didn’t suddenly forget how to coach.
I think Wisconsin will have the West locked up by this point thanks to Iowa having one or two losses and the Gophers losing to Ohio State and either Indiana or Iowa.
So, while it sounds crazy, I think Minnesota catches the Badgers looking ahead and takes Paul Bunyan’s Axe.
 
Final Score: Minnesota 28, Wisconsin 20
Final Record: 10-2 (8-1 Big Ten)
 
 
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PM Modi And Japanese PM Suga Agree To Work For Timely Implementation Of Mumbai – Ahmedabad Bullet Train Project – Swarajya

Prime Minister Narendra Modi met Suga Yoshihide, Prime Minister of Japan on the sidelines of the Quad Leaders’ Summit.
The two Prime Ministers reaffirmed their commitment to advance efforts to facilitate the smooth and timely implementation of the Mumbai-Ahmedabad High-Speed Rail (MAHSR) project.
The MAHSR project is being executed with technical and financial assistance from the Government of Japan.
Prime Minister Narendra Modi met Suga Yoshihide, Prime Minister of Japan, in Washington DC on 23 September 2021 on the sidelines of the Quad Leaders’ Summit.
The two Prime Ministers welcomed the launch of the Supply Chain Resilience Initiative (SCRI) between India, Japan and Australia earlier this year as a collaborative mechanism to enable resilient, diversified and trustworthy supply chains.
Discussions also took place on climate change issues and green energy transition, and the potential for Japanese collaboration with India’s National Hydrogen Energy Mission.
The two Prime Ministers reaffirmed their commitment to advance efforts to facilitate the smooth and timely implementation of the Mumbai-Ahmedabad High-Speed Rail (MAHSR) project.
The MAHSR project is the first High-Speed Rail corridor to be implemented in India. It is being executed with technical and financial assistance from the Government of Japan.
From Mumbai To Ahmedabad Within Two Hours
With a total of twelve stations in the States of Maharashtra, Gujarat and Union Territory of Dadra and Nagar Haveli, the MAHSR corridor will have a length of 508.17 Km.
While a limited-stop (in Surat and Vadodara) service will cover this distance in one hour and 58 minutes, all stops service will take two hours 57 minutes to cover this distance.
High-speed rail will be operating at a speed of 320 Kmph at an elevated (10 to 15 m) track above the ground on a viaduct all along except 26 km in Mumbai, which will be underground. All stations will be elevated except Bandra Kurla Complex station (Mumbai), which will be underground.
National High-Speed Rail Corporation Limited (NHSRCL) is the executing agency of the MAHSR project.
According to NHSRCL, there will be 35 trains per day in one direction, where there will be one train every 20 minutes in peak hours and one train every 30 minutes in non-peak hours. Train frequency will be further increased to one train every eight minutes in future.
Initially, Mumbai-Ahmedabad High-Speed rail will be equipped to handle 17,900 passengers one way daily, which will be increased up to 92,900 passengers in future.
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Postnatal development in a marsupial model, the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuromorphia: Dasyuridae) | Communications Biology – Nature.com

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Communications Biology volume 4, Article number: 1028 (2021)
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Marsupials exhibit unique biological features that provide fascinating insights into many aspects of mammalian development. These include their distinctive mode of reproduction, altricial stage at birth, and the associated heterochrony that is required for their crawl to the pouch and teat attachment. Marsupials are also an invaluable resource for mammalian comparative biology, forming a distinct lineage from the extant placental and egg-laying monotreme mammals. Despite their unique biology, marsupial resources are lagging behind those available for placentals. The fat-tailed dunnart (Sminthopsis crassicaudata) is a laboratory based marsupial model, with simple and robust husbandry requirements and a short reproductive cycle making it amenable to experimental manipulations. Here we present a detailed staging series for the fat-tailed dunnart, focusing on their accelerated development of the forelimbs and jaws. This study provides the first skeletal developmental series on S. crassicaudata and provides a fundamental resource for future studies exploring mammalian diversification, development and evolution.
Marsupials (Metatheria) represent a distinct viviparous lineage within extant Mammalia that have been evolving independently from their sister group placental mammals (Eutheria) for over 160 million years1,2. This evolutionary distance has made them exceptionally powerful in comparative biology for understanding mammalian evolution on both a genetic and developmental level. In addition to their use for comparative studies, marsupials also exhibit unique biological features, chief among them is their unusual mode of reproduction. Eutherian mammals (hereafter referred to as placentals) typically give birth to well-developed young after a prolonged period of gestation with a large maternal contribution to the development of the offspring in utero. In contrast, marsupials give birth to highly altricial young (Fig. 1b) after a short gestation with only a minimal maternal investment during pregnancy via a short-lived, simple placenta3. Instead, marsupials have a large maternal contribution post-birth where the young are dependent on maternal milk through an extended lactation period3,4. Newborn marsupials crawl to the mother’s teat, typically located within her pouch, where they undergo the majority of their development, ex utero4,5,6. To facilitate the crawl to the teat, marsupials have well-developed forelimbs and shoulder girdles, but comparatively delayed development of the hindlimbs7,8,9,10,11,12. Similarly, craniofacial structures such as the nasal cavity, tongue, oral bones and musculature are accelerated relative to the development of the posterior end of the body13,14,15. Chondrification and ossification of the facial skeleton, forelimbs and shoulder girdle are significantly accelerated in marsupials compared to placentals13,14,16. This heterochrony is suggested to underpin developmental constraints in the marsupial clade that have restricted their overall morphological diversity within the limb and facial skeleton compared with placental mammals17,18, though this remains controversial19,20. Defining the mechanisms that control these unique developmental events can therefore provide insights into the processes underlying limb and craniofacial patterning across mammals.
a Adult fat-tailed dunnart (S. crassicaudata). Image: Alan Henderson—Minibeast Wildlife. b Schematic comparing extent of development of the fat-tailed dunnart and mouse (M. musculus) neonates on the day of birth.
Traditionally, the marsupial models, Macropus eugenii (tammar wallaby) and Monodelphis domestica (opossum), have been studied in regard to diverse biological phenomena, including sex determination21,22,23,24,25,26,27,28, reproduction29,30,31,32,33,34, genomic imprinting35,36,37,38,39 and other aspects of development40,41,42,43,44,45,46. Given their ease of captive breeding and experimental manipulation, M. domestica has been used in research across North America, for example to model craniofacial phenotypes observed in children treated with thalidomide47, and to reconstruct the evolution of the mammalian middle ear48. However, the 80-million-year divergence between Australian and North American marsupials49 means that an Australian laboratory-based marsupial model with similarly easy husbandry, year-round breeding and experimental manipulation is still needed for a more complete understanding of mammalian biology. The fat-tailed dunnart (Sminthopsis crassicaudata; hereafter referred to as the dunnart) is an established marsupial model that has been used successfully for studies of brain development, fertilisation, reproduction, respiration, nutrition, thermoregulation, vision and immunology50,51,52,53,54,55,56. In this context, expansion of the developmental and genetics resources available for the dunnart would further its value as a model for investigating mammalian biology and evolution.
Fat-tailed dunnarts are small, carnivorous Australian marsupials from the family Dasyuridae that contains about 70 other living species (Fig. 1a). Dunnarts have a short gestation of 13.5 days and represent one of the most altricial mammals known8 (Figs. 1b and 2). They typically give birth to supernumerary young57 and can suckle up to 10 young (equal to the number of teats). Juvenile dunnarts are weaned after ~65–70 days postpartum (D)50,58, with males reaching sexual maturity at approximately D200 and females entering their first oestrus at approximately D8558. The dunnart can breed all year round, which allows for efficient expansion and maintenance of colony numbers and derivation of staged foetuses and pouch young for experimental studies. As such, the dunnart provides an excellent opportunity to study and manipulate early mammalian development, which occurs almost entirely ex utero.
Numbers below pouch young refer to the day postpartum.
Detailed staging series are imperative resources for facilitating the study of any model species, but are especially interesting in marsupials owing to their unique developmental biology. There are currently no published data on the development of the skeleton of the fat-tailed dunnart, and studies on other related marsupials are often from whole-mount bone and cartilage staining on limited stages59,60,61,62, or X-ray computed tomography (CT) scanning of museum specimens where absolute age is typically unknown63,64,65,66. Previous histological studies of ossification patterns in marsupials have suggested that in highly altricial species, ossification occurs after birth60,61,62. If ossification of the dunnart skeleton also occurs post-birth, observations and manipulations of early skeletal patterning and development can be made postnatally in the pouch, further supporting its use as a model organism. However, a detailed description and staging of these events in the dunnart has yet to be established.
Here, we establish a complete postnatal ontogenetic series of the dunnart, emphasizing critical stages underlying the onset and early ossification of the cranial and postcranial skeleton. We provide quantitative developmental comparisons of craniofacial and limb heterochrony and development compared to the established placental mouse model, Mus musculus. This developmental series lays the foundation for further studies into the molecular and genetic control of the many unique aspects of marsupial development including mechanisms underlying extreme skeletal heterochrony. Our findings complement the ongoing development of additional S. crassicaudata resources as a marsupial laboratory model, including gold standard genetic resources, such as a chromosome level genome, inbred laboratory strains, and induced pluripotent cell lines.
Newborn dunnarts (D0) had an average head length of 2 mm and average weight of 12 mg (Supplementary Table 2). They were bright pink at birth and had highly vascularised, translucent skin with the developing lungs, heart and bladder clearly visible through the skin. Newborns lacked a definitive neck and had a pronounced cervical swelling under the head between the forelimbs, which disappeared by D4. At birth there were no ear primordia, and very faint retina pigmentation was visible indicating the location of the future eyes (Fig. 2, Supplementary Fig. 1a). At birth the dorsal side of the neurocranium was flat and did not begin to round until 24 h after birth. The mandibular and maxillary swellings were visible along with the hyoid/secondary arch. The medial nasal swelling was the dominant feature of the face and contributed to a large prominent nostril (Supplementary Fig. 2). This stage in the development of the facial prominences is comparable to the mouse on embryonic day (E) 11.5–1267 (Supplementary Fig. 2). On the day of birth, the oral region was completely fused except for a small round opening for attachment to the teat. By D15 the mouth began to delaminate and was fully separated by D30, coinciding with tooth eruption (Supplementary Fig. 3a). Dunnart limb heterochrony was distinct, where on D0 forelimbs were well developed (including cartilaginous skeletal elements and musculature) with interdigital separation and claws present, while the hindlimbs were rudimentary and paddle-like with faint digital grooves. The proximal region of the hindlimb remained fused with the base of the tail until approximately D9. There were no cartilaginous condensations in the dunnart hindlimb at D0 or D1 (Fig. 3c, d), comparable to the E11.5 mouse hindlimb68. The newborn fat-tailed dunnart forelimbs appear similar to those of the E14–E15 mouse with regards to digit development68. However, in the mouse forelimb this coincides with the presence of forelimb ossification centres69, which are not present in dunnart newborns. Unlike diprotodontian marsupials (marsupials with forward facing pouches, order Diprotodontia) which display strong climbing motion of the forelimbs at birth4,6,70,71,72, on D0 the dunnart appeared to have a weaker climbing motion after being removed from the pouch (Supplementary Movie 1).
a D0 head sections stained with haematoxylin and eosin Y (purple = nuclei, pink = cytoplasm), alcian blue (cartilage), alizarin red (mineralised bone) and Masson’s trichrome (blue = collagen, red = muscles, cytoplasm, and keratin). Representative images of D0 at higher magnification (×60) show ossification had begun with collagen present in the maxillary and mandible prominences. b D1 head sections stained with haematoxylin and eosin Y (purple = nuclei, pink = cytoplasm), alcian blue (cartilage), and alizarin red (mineralised bone). Wholemount, c D0 and d D1 fat-tailed dunnart specimens cleared and stained with alcian blue (cartilage) and alizarin red (bone). bv = blood vessel, cc = cartilago cupularis, ma = mandible, mc = Merkel’s cartilage, mx = maxillary, ob = osteoblast, oc = oral cavity, pl = palatine process, ton = tongue.
At birth marsupials display sexual dimorphisms which precede gonad development and are under the control of the X chromosome. The appearance of the pouch and scrotum are determined by the number of X chromosomes present, with one X chromosome determining the development of a scrotum and two X chromosomes leading to the development of a pouch73. Female dunnart pouch young display visible mammary primordia from D2 while the scrotal bulges in males are not easily distinguished until D4. Figure 2 shows the gross morphology from D0 to D30. A table of features for staging young is presented in Table 1.
Iodine-stained pouch young allow the visualisation of soft tissues using microCT scanning. For the early stages (<D10) the pouch young tissue is very lean, so the internal resolution at these stages is less clear than older specimens. Nevertheless, details of the developing organs can be observed. The major phase of organogenesis in the dunnart occurs almost entirely in the postnatal period, captured here in high resolution (Supplementary Movies 23456 and 7). These movies span the major differentiation events for all major organs. The lungs of the neonates were characteristic of primitive airways with few tubular-like structures and limited branching (Supplementary Fig. 1b). Air bubbles under the skin were visible (Supplementary Fig. 1a), consistent with gas exchange occurring via the skin at this early stage. The lung increased in complexity between D30 and D35, with the saccules becoming smaller and increasing the surface area for gas exchange (Supplementary Fig. 3, Supplementary Movies 3 and 4). A large mesonephros was present in the peritoneal cavity at birth (Supplementary Fig. 1b, Supplementary Movie 2). The mesonephros is the functional kidney in most neonatal marsupials74,75. By D3 the metanephros (definitive kidney) was visible and by D10, displayed a distinguished renal cortex and medulla. By D20 the mesonephros appeared to have regressed.
Marsupials are one of the few mammals to undergo testicular descent and inguinal closure31,76. In our developmental series we were able to capture the process of testicular descent through the inguinal canal and into the scrotum. On D40 the testes had migrated to the base of the abdomen and were ready to begin the inguinoscrotal phase of testicular descent (Supplementary Fig. 4a, Supplementary Movie 5). At D50 the testes were visible in the body wall (Supplementary Fig. 4b, Supplementary Movie 6), transitioning the inguinal canal and by D60 they were situated in the scrotum (Supplementary Fig. 4c, Supplementary Movie 7).
At birth, pouch young lack an ossified skeleton, showing a cartilaginous postcranial skeleton and chondrocranium (Fig. 3a). Positive alizarin red staining, which stains mineralised bone, was observed in the maxillary and dentary in D1 frontal sections but not at D0 (Fig. 3a, c). However, when stained with Masson’s trichrome stain, collagen deposits (blue) typical of osteoid matrix and bone were present in the maxillary and dentary tissue (Fig. 3a) suggesting that ossification had started in the newborn pouch young. Within 24 h of birth, ossified bones of the facial and postcranial skeleton were present as observed with wholemount alizarin red staining (Fig. 3d) and microCT scanning (Fig. 4a).
The right lateral side of the skeleton is shown in (a), (d) and (g), the dorsal view of the skull in (b), (e) and (h), and the ventral view of the skull in (c), (f) and (i). Pouch young one day after birth (D1) are shown in (a), (b) and (c). Pouch young six days (D6) after birth are shown in (d), (e) and (f). Pouch young 15 days after birth (D15) are shown in (g), (h) and (i). as = alisphenoid, at = atlas, bo = basioccipital, bs = basisphenoid, cl = clavicle, de = dentary, eo = exooccipital, et = ectotympanic, fd = forelimb digits, fe = femur, fi = fibula, fr = frontal, hu = humerus, hy = hyoid, in = incus, il = ilium, ip = interparietal, is = ischium, ju = jugal, la = acrimal, mc = metacarpals, ml = malleus, mt = metatarsals, mx = maxilla, na = nasal, pa = parietal, pl = palatine process, pm = premaxilla, pt = pterygoid, ra = radius, ri = ribs, sc = scapula, so = supraoccipital, sq = squamosal, st = sternum, ti = tibia, ul = ulna, ve = vertebrae.
On D1, ossification centres were evident in the premaxilla, maxilla, palatine process and exoccipital bones (Fig. 4a–c). The pterygoid and basioccipital bones were observed on D3. The first ossification centre of the zygomatic arch began in the jugal on D4, along with the basisphenoid and frontal bones of the skull. By D5, ossification centres in the supraoccipital, squamosal, nasal, lacrimal, ectotympanic, goniale and parietal were present. Ossification of the alisphenoid was evident on D6 and the malleus on D7 (Fig. 4d–f). Ossification centres of the incus and interparietal were present on D15 (Fig. 4g–i). The petrosal bone of the inner ear was the last cranial bone to begin ossification and was clearly observable on D20.
At birth, the lower jaw consists entirely of a thin rod of cartilage, known as Meckel’s cartilage (Fig. 3a, c). By D1 ossification of the dentary bone (mandible) surrounding the Meckel’s cartilage had begun. The coronoid, condylar and angular processes that form as part of the dentary were visible on D4. The oral bones were the first to ossify in both the dunnart and the mouse77,78,79 (Fig. 5). However, the onset of cranial ossification was more uniform in the mouse with bones of the cranium ossifying alongside the oral bones (Fig. 5). In contrast, ossification in the bones of the fat-tailed dunnart cranium occur long after ossification of the oral bones was initiated (Fig. 5).
We also compared the relative timing of the onset of ossification in the fat-tailed dunnart to two other Dasyuridae species, Dasyurus viverrinus (Eastern quoll)13,80 and Sminthopsis macroura (the stripe-faced dunnart), as well as the reconstructed Marsupialia ancestor64. S. crassicaudata (this study) similarly had early ossification of the maxilla, premaxilla and dentary but greater variation in onset of ossification for other bones of the skull with the exception of the petrosal bone (Supplementary Fig. 5b). Aside from the petrosal bone, there was no overlap in the timing of the onset of ossification in the other cranial bones with another dunnart species, the striped-faced dunnart (S. macroura).
The bones surrounding the oral cavity connected early relative to the bones of the cranium, with the maxilla-palatine bone contact event on D8, lacrimal-maxilla and premaxilla-maxilla on D10, premaxilla-nasal on D15, and maxilla-nasal on D25. On D20 the secondary jaw joint (temporomandibular joint) is present with a mortar-shaped condylar head and a concave-shaped glenoid fossa (Fig. 6a, b). Other early connecting bones include the goniale-ectotympanoid (D5) and goniale-malleus (D10) (Supplementary Figs. 6 and 7). On D30, bone contacts in the cranium had begun with the supraoccipital-interparietal, parietal-frontal, frontal-nasal, frontal-lacrimal and alisphenoid-frontal (Fig. 7a–c; Supplementary Fig. 6). The latest bone contacts observed in this cranial series were the supraoccipital-petrosal, supraoccipital-squamosal, and basioccipital-basisphenoid which had just begun to make contact at weaning (D70; Fig. 7g–i, Supplementary Fig. 6). The onset of bone-contacts observed in the fat-tailed dunnart showed a similar pattern to both the Eastern quoll13,80 and the reconstructed Marsupialia ancestor64 with the exception of the supraoccipital-squamosal bone contact (relative timing Marsupialia = 0.17 and S. crassicaudata = 1.0), maxilla-jugal (Marsupialia = 0.20, D. viverrinus = 0.14, dunnart = 0.67), supraoccipital-interparietal (Marsupialia = 0.29, D. viverrinus = 0.14, S. crassicaudata = 0.67), jugal-squamosal (Marsupialia = 0.39, D. viverrinus = 0.50, S. crassicaudata = 0.78; Supplementary Fig. 5a).
Specimens were ranked in order of bone onset or bone contact timing and relative ranks were normalised for comparison between species. M. musculus is shown with pink circles and S. crassicaudata is shown with green triangles, highlighting the early onset of ossification of the oral region in both species.
a Ventral view of the secondary jaw joint with inset white box showing entire skull. b Posterior to anterior view of the secondary jaw joint. c = condyle (orange), et = ectotympanic ring (purple), gf = glenoid fossa (light green), in = incus (yellow), ml = malleus (blue) and sq = squamosal (dark green). Scale bar = 1 mm.
The right lateral side of the skeleton is shown in (a), (d) and (g), the dorsal view of the skull in (b), (e) and (h), and the ventral view of the skull in (c), (f) and (i). Pouch young on D35 are shown in (a), (b) and (c). Pouch young on D50 are shown in (d), (e) and (f). Pouch young on D70 are shown in (g), (h) and (i).
On D1, ossification centres of thoracic and cervical vertebrae were present either side of the neural arch (Fig. 4a). The atlas had begun to ossify by D2 and ossification centres of the lumbar vertebrae were present on D3. The caudal vertebrae did not begin ossification until two days later on D5, coinciding with the onset of ossification of the first bone of the pelvic girdle, the ilium (Fig. 4d). The ischium ossifies later and was evident on D15 (Fig. 4g). The ossification centre of the distal section of the epipubic bone was present on D20. Ossification centres were present in the scapula, clavicle and the body of the first six rib pairs on D1 (Fig. 4a). By D4 the body of the true, false and floating ribs were all undergoing ossification. The capitulum of each rib had an ossification centre on D7. The first ossification centres of the sternum were evident on D15 in the xiphoid process, manubrium and the first three anterior sternebrae of the body (Fig. 4g). The fourth sternebrae began ossification later and was evident on D25.
Cartilage was present in the forelimb but there was no cartilage present in the hindlimb on the day of birth or D1 (Fig. 3c, d). One day after birth, ossification centres in the humerus, radius and ulna were evident (Fig. 4a; Table 2). On D5 the bones of the hindlimbs began to ossify, with the femur, tibia and fibula present (Fig. 4a). Ossification centres were present on D6 in the forelimb phalanges but not until D15 in the hindlimb phalanges (Fig. 4d; Table 2). The metacarpals of the forelimbs had begun ossification on D10 and the metatarsals of the hindlimbs by D15 (Fig. 4g; Table 2). Ossification centres of the carpals and tarsals were the last to ossify and were evident on D40 (Fig. 7d; Table 2).
Pouch young display heterochrony of the limb bones from day one to approximately day 50 (Fig. 8). Individual bones of the dunnart limbs also displayed pronounced differences in allometric scaling during growth (Fig. 8). Following birth, pouch young possess well-developed forelimbs, showing ossification of the humerus, radius and tibia, but possess underdeveloped, paddle-like hindlimbs lacking any bone. By D5, the hindlimbs show their first signs of extension and development, emphasizing the strong developmental heterochrony. The autopod of the forelimb (carpus) and hindlimb (tarsus) are roughly the same length from D15 to D30 (Fig. 8). After D30 the autopod of the hindlimb overtakes the carpus and this difference in length continues to expand until the weaned juvenile stage.
Relative lengths of forelimb and hindlimb elements throughout development, shown as shaded grey bars (n = 1). Limbs are divided into proximal and distal segments: the stylopod (humerus and femur), zeugopod (ulna/radius and tibia/fibula) and autopod (carpals, metacarpals, tarsal, metatarsals and digits).
Here, we present a high-resolution, digital reconstruction of the complete postnatal ontogeny of S. crassicaudata. Our study focused on characterizing early skeletal development in the fat-tailed dunnart to establish it as a comparative laboratory-based model for mammalian skeletal osteogenesis. Our description of postnatal gross morphology including head length, weight and crown-to-rump length provides a staging tool for future studies using this marsupial model. This expands upon a previous staging series54 to include detailed skeletal descriptions and a general overview of some interesting aspects of organ development.
Newborn dunnarts have large heads (almost 50% of the total body length) and well-developed forelimbs compared to hindlimbs that are required to facilitate movement to the pouch and teat attachment. They lack an ossified skeleton, showing a cartilaginous postcranial skeleton and chondrocranium. The chondrocranium is a transitory, embryonic structure critical in the developing mammalian head81,82. In fat-tailed dunnarts, the newborn young display large medial nasal swellings. This is consistent with descriptions of the chondrocranium in Sminthopsis virginiae (the red-cheeked dunnart), which has the most extensive cartilago cupularis (the rostral part of the nasal cartilage) of any marsupial examined83. Previous studies have shown that variation in the cupula nasi anterior (the anterior portion of the cartilaginous nasal capsule) reflects the animal’s life history83. The nasal capsule morphology in marsupials is more uniform across the group than observed in placentals and this is thought to be related to the extended fixation of pouch young to the mother’s teat during lactation83. It has also been proposed that there is a relationship between the innervation of the area proximal to the cupla nasi anterior and the sensory requirements of marsupials at birth84,85,86. An in vitro study in M. domestica newborn young found that when pressure was applied to the innervated snout this induced electromyographic responses from the triceps muscle in both forelimbs87. The forelimb responses were absent when the facial skin was removed, suggesting that the journey to the pouch may be aided by the influence of facial mechanosensation on forelimb movement87. Dunnart neonates lack a distinct neck and any skeletal elements to support the head and the journey to the pouch is instead aided by a large cervical swelling which supports the head until D4. The cervical swelling is also thought to assist with teat attachment88. Cervical swellings have been reported in two other Dasyuridae species: the Tasmanian devil, Sarcophilus harrisii and Eastern quoll, Dasyurus viverrinus8,88 and are correlated with the altricial nature of the pouch young. Marsupials with more precocious newborns, such as macropods (e.g., kangaroos, wallabies), lack cervical swellings and have full head movement at birth8,33.
The microCT scans provide an excellent resource for studying the development of internal organs, particularly in the older stages where the scans are clearer. We have briefly described aspects of lung, gonad and kidney development but make these data publicly available for additional studies of organ development in more detail. The lungs of dunnart newborns were characteristic of primitive airways with large tubular-like structures. On the day of birth, dunnart lungs are similar to that of eutherian lungs during the canicular stage of embryonic development89. At birth, in the fat-tailed dunnart, the skin is responsible for almost all gas exchange with pulmonary ventilation unable to satisfy the demand for oxygen until between D23 and D3589. Interestingly, this is when we observe the rapid increase in lung complexity between D30 and D35. It has been suggested that the short gestation time of dunnarts provides insufficient time for development of a complex respiratory system and therefore relies on transcutaneous gas exchange89.
Remarkably, we were able to capture the process of testicular descent through the inguinal canal into the scrotum. On D40, the testes had migrated through the abdomen and were ready to begin the inguinoscrotal phase of testicular descent. At D50, the testes were visible at the neck of the scrotum and by D60 were situated in the base of the scrotum. It has previously been suggested that the time taken for completion of testicular descent in marsupials may be associated with postnatal growth rate90 given the shorter period (3–4 weeks) for testicular descent in the bandicoot (Perameles gunni)91 compared to kangaroos, wallabies and possums (10–11 weeks)74,92,93,94. However, in our study we find that although dunnarts represent one of the most altricial neonates (Grade 18), the process of testicular descent occurs at almost exactly the same postnatal stage as in other more precocial marsupials74,92,93,94. Our results are also supported by a study in Antechinus stuartii (family Dasyuridae), which found that after one month the testes were situated in the inguinal canal and after two months were present at the base on the scrotum95.
Using a combination of microCT scanning and histological techniques we confirmed that on the day of birth (<24-h old), no ossified bones were present in the dunnart. Mineralized bone was observed one day after birth and was limited to bones in the oral region, forelimbs and vertebrae. We observed osteoid matrix in the maxillary and dentary of D0 pouch young head sections showing that ossification had begun. This suggests that microCT is useful for assessing when ossified bone arises, however, histology is still required for investigating the onset of ossification prior to mineralization.
Previous studies of skeletal development in marsupials have similarly shown that the maxilla, premaxilla and dentary are the first bones to ossify59,61,62,63,64. However, there appears to be some differences in the timing of onset of ossification in different marsupials. In Monodelphis domestica and Macropus eugenii these bones have already begun ossification prior to birth59. In contrast, in Isoodon macrourus62, Trichosurus vulpecula62, Didelphis albiventris60 and Sminthopsis macroura61 ossification begins after birth. Although marsupials show inter- and intraspecies variation in the timing of the onset of ossification in bones of the skull, the relative timing of bone contacts in the oral region, middle ear and late occipital region are highly conserved64. Similar to Spiekman and Werneburg64, we observed the first bone contacts occurred in bones surrounding the mouth cavity (maxilla-palatine) and middle ear bones (ectotympanic-goniale and goniale-malleus). In mammals the middle ear bones form as part of the mandible and separate after the secondary jaw joint has formed48,96. However, due to the altriciality of newborns, marsupials and monotremes are born before the jaw joint forms and use their middle ear bones to articulate the lower jaw with the head to allow for feeding82,97,98,99,100. In the dunnart the secondary jaw joint is formed by D20, which is comparable with the opossum (D14–D2082,96,101). The last bones to make contact were the those that form the back of the cranium, particularly in those that connect the occipital bones. As previously observed in marsupials64, the connections within the occipital region occurred around the time of weaning, suggesting that the region is not required for cervical support during suckling64.
Developmental comparisons to the well-known placental model, the laboratory mouse (Mus musculus), are useful for studying the evolution of skeletal elements, particularly given the heterochrony with in marsupials. Most ossification centres in placentals are present at birth, with ossification in mice beginning on embryonic days 12–13102,103,104, while in the dunnart all ossification occurs postnatally. Similar to the dunnart, the bones of the oral region (premaxilla, maxilla, dentary, palatine and pterygoid) are the first to ossify in the mouse, presumably in preparation for feeding, as in marsupials. However, unlike in the dunnart and other marsupials, the mouse cranium (basioccipital, frontal, parietal and sphenoid) ossifies at a similar stage of development as the oral bones. The developmental timing of the facial morphology appears to be less disparate across marsupials than placental mammals, but displays equal variation in the neurocranial morphology105. This difference in cranial developmental timing between marsupials and placentals is thought to be driven by the early functional requirements of the oral apparatus for continuous suckling in marsupials105. However, the bones of the oral regions (premaxilla, maxilla, dentary and palatine) are also the first to ossify in the reconstructed ancestral ossification sequence of Mammalia63 and often in non-mammalian amniotes suggesting prioritized development of the oral apparatus is the ancestral state in amniotes63,64,106,107,108,109. The short gestation time and altricial stage of the marsupial neonate at birth is likely to have driven a more extreme shift in the developmental timing of this event, relative to the rest of the body. This is supported by the extremely high integration of the oral bones in early marsupial postnatal ontogeny in comparison to other cranial regions where levels of integration are similar to placentals17. In particular, cranial bones arising from the migratory neural crest cell population of the first pharyngeal arch have been shown to be constrained between marsupials to facilitate early functional demands66.
Early ossification of the craniofacial bones in Monodelphis domestica (grey short-tailed opossum) is thought to occur through accelerated migration, proliferation and differentiation of the cranial neural crest cells that pattern the facial prominences and skeletal elements110,111. A marsupial-specific region within a Sox9 enhancer was found to drive early and broad expression of Sox9 in pre-migratory neural crest cell domains contributing to early migration of cranial neural crest cells relative to the mouse112,113. The spatio-temporal expression of downstream key ossification genes such as Runx2 and Osx have not been studied and in comparison to placentals, little is known of the molecular and genetic control of craniofacial development in marsupials96,97,114. Recently, Smith (2020) presented previously unpublished work of J.P. Hill and Katherine Watson on the development of neural crest in other marsupial taxa115. They confirmed that early migration of the neural crest occurs in multiple marsupial taxa, particularly for the neural crest cells that end up in the facial and first arch regions115. Interestingly, dasyurids differed from other marsupials with considerable accumulation of neural crest before somitogenesis had begun, along with far earlier migration of the ectomesenchyme into the craniofacial region115. Smith (2020) suggested that the shorter the period of gestation in marsupials the earlier the accumulation and migration of neural crest will be. Being one of the most altricial marsupials in existence, the dunnart provides an ideal model in which to study the molecular drivers of heterochrony in craniofacial ossification in marsupials.
Based on the facial processes present in the newborn dunnart and the lack of ossification centres, we propose that at D0 dunnart craniofacial development corresponds to that of the E11.5–E12 mouse embryo. This suggests that at birth in the dunnart, neural crest cells are still proliferating and differentiating. Given this new developmental information, the dunnart therefore presents a new and exciting mammalian model to expand craniofacial research as facial primordia can be manipulated ex utero and the role of key genes underlying osteogenesis can be directly observed.
The ossification sequence of the dunnart postcranial skeleton follows a similar pattern to that previously described in marsupials59,61,62,65. Like all marsupials, dunnart pouch young display heterochrony in the development of the limb bones with the forelimbs being longer than the hindlimbs from D1 to ~D50. After D50, the limbs undergo a heterochronic shift in their length and the hindlimbs overtake the length of the forelimbs. This corresponds with the pouch young no longer being permanently attached to the teat in the lead up to weaning58. The bones of the carpals and tarsals were the last to ossify in the limbs. The ossification of the metacarpals, metatarsals and digits before the carpals and tarsals is consistent with ossification patterns in S. macroura61, Trichosurus vulpecula62, Thylacinus cynocephalus65 and Didelphis alviventris60. Embryological and molecular studies suggest that marsupial limb heterochrony is driven by both an acceleration in the development of the forelimb buds and a delay in the development of the hindlimb buds7,9,10,116,117,118. In addition to the heterochrony between the fore- and hindlimbs in marsupials, heterochrony is also observed in the timing of the onset of marsupial forelimb and hindlimb development relative to placentals. Dunnart forelimb development is accelerated compared to the hindlimb, and compared to that in the mouse119, where both limb buds begin to differentiate and grow at a similar stage of development119,120. On the day of birth, the dunnart forelimb has well-developed musculature, and digits with claws to facilitate its crawl to pouch. However, the D0 dunnart lacks ossified forelimb bones, suggesting that it does not require fully formed skeletal elements to complete the crawl to the mother’s pouch. Conversely, the hindlimb has no cartilage or bone and is a paddle-like bud with digital condensations beginning to form. Generally, mammalian forelimbs develop ahead of hindlimbs, with the timing relatively close in placentals, while in marsupials the difference in the rate of forelimb and hindlimb development is extreme6,121. The dunnart represents one of the most altricial neonates8, and so is an extreme example of this heterochronic shift in forelimb and hindlimb development. The heterochrony in marsupial limb development involves the early specification and initiation of patterning in the forelimb bud relative to foetal development122. In the opossum (M. domestica), the early initiation of the forelimb field is initiated during the early stages of somitogenesis through accelerated expression of the necessary forelimb transcription factor TBX5, and limb outgrowth and patterning genes FGF10, FGF8 and SHH, and greater myocyte (forming the later limb musculature) allocation42,122. SHH is also expressed early in the forelimbs of tammar wallaby (Macropus eugenii) embryos relative to hindlimb bud expression123.
Research into marsupial limb development is currently limited by the inability to perform in vivo transgenic experiments42,44,122,123,124,125,126,127. Although limb organ cultures have been successfully used in the opossum to investigate the molecular control of limb heterochrony124, limb development has predominantly been studied in the chick and mouse given the ease of observing and manipulating limb patterning in these species. Electroporation technologies previously used in chick128, xenopus129 and mice130 have recently been successfully applied to manipulating dunnart brain development55. This technology provides a tangible system for the manipulation of gene expression in the fat-tailed dunnart in vivo.
The public availability of precisely staged microCT scans for the fat-tailed dunnart as presented here provides a valuable resource for future studies in mammalian development. The highly altricial state of the newborn dunnart allows for precise, tissue-specific manipulation of skeletal development ex utero, prior to the onset of ossification and over an extended period of time. Such manipulations are challenging to perform in placental mammals where ossification is established in utero. The dunnart provides access to key ossification stages in an easily manipulable mammalian model. A staging series of the fat-tailed dunnart therefore presents a fundamental resource that will underpin future work into marsupial development, in particular defining the molecular control of craniofacial and limb heterochrony.
All animal procedures, including laboratory breeding, were conducted in accordance with the current Australian Code for the Care and Use of Animals for Scientific Purposes131 and were approved by The University of Melbourne Animal Ethics Committee (AEC: 1513686.2) and with the appropriate Wildlife Permit (number 10008652) from the Department of Environment, Land, Water and Planning. Animals were housed in a breeding colony in the School of BioSciences, The University of Melbourne. Animals are bred using the Poiley outbreeding system to limit inbreeding132 and breeding boxes were set up with a male:female ratio of 1:2 or 1:3. Animals were kept in cages with water and vitamin supplements (Pentavite; 1 mL into 100 mL water) changed three times a week. All cages had nest boxes with shredded newspaper, empty toilet rolls, drinking bottle, food bowl and native tree branches. Animals were fed each day a diet consisting of live food (2 crickets and 3 mealworms) and wet mince mixture of 51% beef mince (Whiskas), 36% beef and lamb flavoured biscuits (Whiskas), 12.7% wombaroo and 0.3% calcium carbonate. Dunnart were kept on a 16:8-h light:dark cycle and temperature between 21 and 25 C. Females were monitored to track oestrus cycle133. Body weight fluctuations in female animals and daily examination of urine samples under light microscopy were used to determine whether ovulation had occurred. A decrease in body weight and the presence of cornified epithelial cells in the urine is associated with the day of ovulation133. The presence of sperm in the urine of females confirmed that insemination had occurred. Pregnancy is timed with the detection of ovulation or the appearance of sperm set as day 0 of pregnancy133. Female pouches were checked every day for births by gently holding the animal with one hand and opening the pouch with the thumb and index finger of the other hand. On the day of birth, the mothers often have a small amount of blood in the urine and the pouch young will be pink and highly vascularised. Pouch young were removed by gently holding the female on her back with one hand and using the thumb and index finger of the other hand to gently but quickly slide the young from the teat. Pouch young under D15 were placed on ice for 15 min as an anaesthetic followed by immersion-fixation in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Pouch young from D15 to D45 were anaesthetised on ice for 15 min, followed by intraperitoneal (IP) injections of 4% PFA in PBS. Pouch young D50 and older were anaesthetised via an intramuscular injection of 10 mg kg−1 Zoletil (1:1 tiletamine HCl, zolazepam HCl), then killed with an 0.05–0.1 mL volume intraperitoneal injection of 150 mg kg−1 pentobarbital sodium (made up to 60 mg ml−1 in sterile saline) After fixation specimens were washed twice with PBS for 1 h and then dehydrated in increasing ethanol solutions before storing in 70% ethanol. Pouch young were photographed using a Nikon Digital Sight DS-U3 (Nikon, Tokyo, Japan) and all images were processed through NIS Elements Analysis D v.4.300.00 64-bit software (Nikon).
Specimens were collected every day from D0 to D10, then every 5 days from D10 to D50, then every 10 days from D50 to D70. Specimens were scanned using X-ray micro-computed tomography (microCT) at the TrACEES platform, School of Earth Sciences, University of Melbourne. One specimen from each stage was stained with 1% iodine in 100% ethanol in order to visualise soft tissue in the microCT images. Specimens under D40 were stained for 24 h and specimens over D40 were stained for 72 h. Specimens were rinsed in 100% EtOH once before scanning. Specimens were either mounted in 200–1000 μL pipette tips (Axygen) suspended in ethanol or suspended dry with cotton wool spacers between specimens. Larger specimens were wrapped in standard bubble wrap and scanned in either a 15 or 50 mL Falcon tube (Sigma). MicroCT scanning was performed in a Phoenix Nanotom m (GE Sensing & Inspection Technologies GmbH) operated using xs control and Phoenix datos—x acquisition and reconstruction software (GE Sensing & Inspection Technologies). Samples were scanned for 10 minutes at varying resolutions ranging from 4.75 to 26.18 μm pixel size (Supplementary Data 1). The X-ray energy varied from 40 to 55 kV and 300 to 250 μA depending upon the size of the specimen and whether it was stained with iodine (Supplementary Data 1). A Diamond target was used and 1199 2D X-ray projections were collected over a 360° rotation of the specimens. Skeletal reconstructions were performed in VGStudio MAX 3.0 (Volume Graphics, Heidelberg, Germany). Left and right-side limb bones were measured for one individual per sampled age and averaged using polyline length tool in VGStudio MAX 3.0 (Volume Graphics, Heidelberg, Germany). First appearance of skeletal elements and cranial bone contacts were observed in VGStudio MAX 3.0 for one specimen from each precisely staged age and recorded (Volume Graphics, Heidelberg, Germany). To compare to previously published datasets from other mammalian species, specimens were ranked in order of bone onset timing or bone contact timing, and relative ranks were distributed between 0 and 1 as described in Koyabu et al.63. Original and relative ranks are listed in Supplementary Table 1 and Supplementary Data 2 and 3.
Pouch young were collected (see the ‘Collection of pouch young’ section) on D0 and D1 either fixed in 4% paraformaldehyde for serial sectioning or fixed in 95% ethanol for whole-mount staining. Samples for serial sectioning were processed through a series of ethanol and xylene washes (15-min steps; Tissue-Tek VIP, School of BioSciences), embedded into paraffin wax (Leica) and 7-μm sections cut with a microtome (Zeiss, Sydney, Australia) and transferred to superfrost slides (Platinum Pro, Grale). For D0 (n = 1) and D1 (n = 1), sections were either stained with Harris’ Haematoxylin (Australian Biostain) and Eosin Y (Australian Biostain), Alizarin Red (ProSciTech, Australia) or Alician Blue (ProSciTech, Australia) according to standard methods134, or a modified Masson’s trichrome stain135,136. Sections were imaged on an Olympus BX51 Microscope with an Olympus DP70 Camera (Olympus, Sydney, Australia). Bone and cartilage wholemount staining was performed according to the described protocol for mouse embryos137. Briefly, pouch young skin (D0, n = 2; D1, n = 2) was removed with forceps, stained in 0.05% alcian blue stain solution overnight then washed for 8 h in 70% ethanol. Specimens were cleared with 1% potassium hydroxide for 2 h and counterstained with alizarin red stain solution (0.005% (w/v) alizarin red in 1% potassium hydroxide) overnight. Specimens were cleared in 1% potassium hydroxide/20% glycerol for 2 days and stored in glycerol:ethanol (1:1). Stained pouch young were photographed using a Nikon Digital Sight DS-U3 (Nikon, Tokyo, Japan) and all images were processed through NIS Elements Analysis D v.4.300.00 64-bit software (Nikon).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
All X-ray and reconstructed microCT data shown are publicly available through a MorphoSource repository (www.morphosource.org, Project P1150)138. Histology slide images are available through a Figshare repository (www.figshare.com, Project 111930)139. For comparison of ossification patterns between species, timing and ranks were obtained from two previous publication datasets by Koyabu et al.63 and Spiekman et al.64.
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The authors thanks Eva Suric, Tania Long and Darren Cipolla for their technical contributions and help with animal husbandry. We would like to acknowledge the technical support by Dr. Jay Black and the Melbourne TrACEES Platform (Trace Analysis for Chemical, Earth and Environmental Sciences) for access to the GE Phoenix Nanotom m micro-CT scanner. This work was supported by Discovery Project funding (DP160103683) from the Australian Research Council to A.J.P. and a Discovery Early Career Award (DE180100629) to C.A.H.
School of Biosciences, University of Melbourne, Parkville, VIC, Australia
Laura E. Cook, Christy A. Hipsley & Andrew J. Pask
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
Axel H. Newton
Department of Biology, University of Copenhagen, Copenhagen, Denmark
Christy A. Hipsley
Department of Sciences, Museums Victoria, Carlton, VIC, Australia
Christy A. Hipsley & Andrew J. Pask
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A.J.P., L.E.C., C.A.H. and A.H.N. designed and conceived the study. A.J.P. and C.A.H. supervised the study. L.E.C. collected the specimens, described the stages, performed the histological experiments, analysed the data and wrote the manuscript. C.A.H. generated the scan videos. A.H.N. reconstructed the microCT scans and measured the limb stages. All authors reviewed and revised the manuscript and gave final approval for publication.
Correspondence to Andrew J. Pask.
The authors declare no competing interests.
Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Luke R. Grinham. Peer reviewer reports are available.
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Cook, L.E., Newton, A.H., Hipsley, C.A. et al. Postnatal development in a marsupial model, the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuromorphia: Dasyuridae). Commun Biol 4, 1028 (2021). https://doi.org/10.1038/s42003-021-02506-2
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Published: 02 September 2021
DOI: https://doi.org/10.1038/s42003-021-02506-2
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Tesla's Latest Project Is Unlike Anything Else – CarBuzz

It's also very hush-hush.
The recently revealed Tesla Model S Plaid and its weird and possibly illegal yoke steering wheel has dominated the news lately. But at the same time, the California-based automaker has been very quietly embarking on a new, secret project in Texas. Bloomberg reports that Tesla subsidiary Gambit Energy Storage has begun building a more-than-100-megawatt energy storage project in the Lone Star state, specifically in the town of Angleton, about 40 miles south of Houston.
What's so special about a battery like this? It's capable of providing power to around 20,000 homes, assuming it's a hot summer day. The construction of this project is so secret that workers on site are also tasked with hiding equipment from locals and the media.
However, a Tesla logo spotted on a worker’s hardhat provided a dead giveaway of who’s behind the project. Public records confirmed the rest. The timing of this project getting underway could not be better for Tesla. Texas recently suffered mass power outages due to a severe winter storm last month, proving the state’s power grid and general infrastructure is not reliable.
Tesla already offers the PowerPack and larger MegaPack for utility customers and, since 2015, sells the Powerwall home battery system to private customers. The following year, Tesla acquired SolarCity, a solar panel producer and installer. The so-called “solar roof” can now be installed on private homes.
Tesla CEO Elon Musk recently moved from California to Texas, yet another sign that the 49-year-old billionaire wants to keep a close eye on the latest projects, which also includes the Austin Gigafactory that'll soon begin Cybertruck production. This massive new 100-megawatt battery, upon completion, will be one of the world's largest battery projects. Tesla already has a 20-megawatt system online supporting the Los Angeles area. A 182.5-megawatt system is also currently under construction in the San Francisco Bay Area.
What does this all mean? Tesla intends to be an energy provider leader, along with continued plans to dominate the electric vehicle market. Texas has long been a dominant force for US energy production with natural gas, oil, wind, and solar resources. Musk and Tesla Energy intend to add renewables to that list.

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Dyne Therapeutics Presents New In Vivo Data for its Myotonic Dystrophy Type 1 Candidate (DYNE-101) Demonstrating Robust Splicing Correction During World Muscle Society 2021 Virtual Congress – Stockhouse

Dyne Therapeutics Presents New In Vivo Data for its Myotonic Dystrophy Type 1 Candidate (DYNE-101) Demonstrating Robust Splicing Correction During World Muscle Society 2021 Virtual Congress  Stockhouse
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Project F-Word: Our F-100 Gets Some Pane Management – Ford Muscle

© 2017 Power Automedia.
All rights reserved.
Back before the OEMs learned about gluing our cars and trucks together, they assembled them using some basic items such as bolts but also used non-conventional items of assembly, such as string. Common, household string was a necessary tool when installing items such as front windshields or rear glass for decades and that holds true for many F-100 pickup trucks, including our own Project F-Word.
Patina is cool, but not when it blurs your vision. Our F-100 has seen many miles and the resultant chips, cracks, and other blemishes needed fixing. Note, our truck has the trim installed, you will need to decide if you’re going to be using this trim again when you order your new weatherstrip.
Swapping glass is one of those chores that ride a fine line between having helping hands on-call, and “too many hands in the pot.” Also, window glass is, well – glass! As such, it can be fragile, if you try and move it in ways differing from its strength axis, but it’s not like trying to install eggshells either. Taking your time is the most important tool when working with glass. F-Word’s front and rear glass had seen better days, and the weatherstripping hadn’t aged gracefully.
Many F-100 windshields exhibit this separation near the edges. It’s unsightly, and needs to go! Also, before you remove all the glass from your ride, check the new pane(s) to ensure they’ve not been damaged in shipping.
Classic Industries offers replacement glass for various 1967 through 1979 Ford F-100 trucks (as well as other vehicles) and they also offer weatherstripping to ensure a tight seal around those vintage body seams. We are re-using the rear glass on F-Word but stepped up with new weatherstripping on both front and rear panes. Check out the pictures to see how the team at Fast Auto Glass in Southaven, Mississippi improved our view both front and rear on Project F-Word. We’ll really appreciate having that new glass and weatherstripping when we drive our Coyote-swapped, hopped-up pick ’em-up truck to Holley’s Intergalactic Ford Festival in about a month.

Since the old weatherstrip isn’t going to be reused, you can simplify removal by cutting it. Some folks prefer to cut the small lip off of the weatherstrip on the inside, while others stick the blade up under the weatherstrip and trim from the outside. If you have trim, it will come off as the weatherstrip falls away. Be careful with sharp, pointy objects!
You will have residue and old glue/weatherstrip to remove. Now is a good time to check and repair any rust that may have occurred over the past few decades.
We were swapping weatherstrip on both the front and rear, so we removed both panes of glass and cleaned both sides of the cab up at the same time.
We started with the back glass. This tool from Classic Industries helps wrap the string around the outside of the weatherstrip once it’s installed. This is where it pays to have friends help hold the glass on the outside while you work the inner part of the weatherstrip into position.
Begin by pulling the string, which brings the inside edge of the weatherstrip with it. Work your way across the bottom on each side, and then up to the top. You should end in the middle at the top.
We then went to the front glass.
After installing the weatherstrip, the trim is next. A little WD40 helps slide it in place. After that, the string is coiled around the weatherstrip channel and the windshield is ready to go in.
Some folks begin at the bottom, while others prefer to start at the top. The guys at Fast Glass started seating the top first.
Either way, you need to make sure the glass is centered in the opening and that the window is seated all the way on the starting edge. This will help you greatly as you’re trying to squeeze the opposing side’s weatherstrip over the lip.

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Legends of Le Detroit storytelling event slated – Grosse Pointe News (subscription)

16980 Kercheval Ave • McCourt Building • Grosse Pointe, MI 48230 • 313.882.6900 • Office Hours: Monday-Friday 9am-5pm
Sunday, September 26, 2021
16980 Kercheval Pl. • Grosse Pointe, Michigan 48236 • 313.882.6900 • Monday-Friday 9am-4pm

Courtesy photos
Trois Bouffons will perform during “Legends of le Detroit” on Oct. 9.
The Legends of Le Detroit storytelling event will take place from 1 to 4 p.m. Saturday, Oct. 9, at Detroit Abloom, 248 Manistique, Detroit. The event will feature music, dancing and storytelling for all ages.
Guests will learn about local lore of Detroit legends from the “Legends of le Détroit,” published in Detroit in 1883, a collection of folklore, genealogy and family narratives related to the founding and early history of the city. Compiled by Marie Caroline Watson Hamlin, a little-known local folklorist, it consists of more than 30 folk stories rooted in Detroit’s early history, as well as Native American and French folklore.
Renowned Michigan storyteller Genot Picor will be the featured storyteller and emcee the event. His interactive program centers on the traditional stories, songs and dances of the Great Lakes region, whereby the audience becomes part of the journey. Talented storytellers from metro Detroit and Grosse Pointe Theatre will share tales such as the Nun of St. Clair, Le Lutin, The Legend of the Windmill Pointe and several more.
Trois Bouffons will perform in the outdoor garden pavilion. A traditional musical ensemble based in southeast Michigan, Trois Bouffons specializes in traditional French Canadian, Great Lakes Maritime, Appalachian and other historic musical selections. The ensemble is comprised of violinist Trae McMaken, upright bass player Mark Szabo and guitarist Picor.
This event is free and open to the public. The event will be held outdoors, rain or shine, under the large pavilion in the garden.
The storytelling program takes place under the pavilion at Detroit Abloom.
Storytelling in the garden offers health benefits in addition to its historical aspect.
“Being in nature, or even viewing scenes of nature, reduces anger, fear and stress and increases pleasant feelings,” said Suzy Berschback, healthy communities manager for Beaumont Health. “Exposure to nature not only makes you feel better emotionally, but it also contributes to your physical well-being, reducing blood pressure, heart rate, muscle tension and the production of stress hormones and Detroit Abloom is a beautiful garden to see.”
Detroit Abloom is a demonstration land-use model project, based on cut flower farming and the creation of sanctuary gardens to repurpose vacant blighted land. It is a nonprofit that offers three levels of activities: horticultural, wellness and community. Learn more about it at detroit
abloom.com.

Street parking is available.
September 22, 2021
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Bam Adebayo details strong connection he's building with Damian Lillard on Team USA – Heat Nation

Miami Heat center Bam Adebayo indicated that he’s in the midst of establishing chemistry with his Team USA teammate Damian Lillard.
Adebayo offered his response to a question asking him to name the one player who’s been in his ear the most since the American team arrived in Japan.
Bam Adebayo asked if there's been a Team USA teammate who has been in his ear: Getting to talk to and getting a chemistry with is Dame. I play center, he plays point guard so we got to build that connection. pic.twitter.com/Mv6GMmTQR6
— Brendan Tobin (@Brendan_Tobin) July 29, 2021

“I wouldn’t say been in my ear, but getting to talk with him and getting a chemistry with him is Dame,” Adebayo said. “Just because he’s had, I play center, he plays point guard, so we got to build a connection in the pick-and-roll.”
Those chemistry issues are vital to Team USA again winning the gold medal, with some early struggles thus far making its path to that goal harder.
Prior to the start of the Olympics, Team USA was upset by both Nigeria and Australia. It was then embarrassed in its first Olympic contest by losing to France.
Lillard’s uncertain future with the Portland Trail Blazers has led to some rumors surfacing about the veteran guard being acquired by the Heat. Of course, to fit Lillard within the team’s salary cap would mean making some major roster changes.
Whether or not that happens, Adebayo and Lillard are hoping that their burgeoning chemistry lasts at least long enough to achieve their goal in Tokyo.
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Brad Sullivan is a freelance writer for HeatNation.com, having been an avid fan of NBA basketball for more than four decades. During that time, he’s watched the Heat evolve from gestation period to expansion team all the way to three-time NBA champions. He’ll follow their quest toward again reaching those lofty heights, and do so by offering some perspective along the way.
The Miami Heat reportedly believe that forward KZ Okpala can become a rotation player if he is able to…
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James "Flex" Lewis Says He Won't Compete at the 2021 Mr. Olympia – BarBend

 
Seven-time Olympia 212 Champion bodybuilder James “Flex” Lewis has formally announced that he will not compete in the 2021 Mr. Olympia. The “Welsh Dragon” announced the news in a video on his YouTube channel that was posted late in the evening on July 8, 2021.
This marks the second year in a row that Lewis has dropped out of the Mr. Olympia. After a dominant reign in the 212 division, Lewis announced his intention to compete in the Open division — a move lauded by fans. He has yet to step on stage since 2018. The announcement begins at the 4:10 mark in the video below.
[Related: How Bodybuilders Cut Weight While Still Holding Onto Muscle]
“I feel really good. That time off last year from competing, did it mentally f*** me up? Yes, it did, because I knew what I look like, and I know I could be doing damage up on that stage,” Lewis says in the video above. “So now this year has come, and there are a wide variety of things that have happened this year.”
Lewis talks about his family’s move to Las Vegas, NV, the opening of his new gym, the Dragon’s Lair Gym, and he reveals a more intimate reason for why he’s choosing to opt-out of this year’s O.
“The main reason, which I didn’t really want to get into, is that myself and my wife are trying for another kid. Bodybuilding has always been a part of my core being. As I’m getting older, my family has replaced what was once first in line, and that was bodybuilding.”
 
 
A post shared by Flex Lewis™ (@flex_lewis)

[Related: What You Need to Know About How to Build Muscle]
Lewis won the 212 Olympia title every year from 2012 to 2018 and is one of the greatest non-heavyweight bodybuilders of all time. He retired from the 212 division after his 2018 victory and took off 2019 focus on preparing to compete in the Open division. Then, Olympia President Dan Solomon sent him an invite to compete at the 2020 Mr. O. Lewis accepted the invite but withdrew from the contest to receive stem cell treatment for a shoulder injury. His invite was extended to Mamdouh “Big Ramy” Elssbiay, who won the title from then-champion Brandon Curry.
Lewis had not formally received a special invite to compete at this year’s contest, currently scheduled to occur October 7-10 in Orlando, FL. As a 212 champion, Flex can enter that contest any year he chooses. However, he needs to qualify or accept an invitation to compete in the Open division (or any other division). Lewis didn’t share specific plans for 2022, but he did emphasize that fans would see him compete again.
“I appreciate everybody’s support. I’m not retiring. There’s still so much burning desire and ambition for me to fulfill my bodybuilding dreams, and, God willing, injury-free, I will be on that bodybuilding stage next year.”
Featured Image: @flex_lewis on Instagram

BarBend is an independent website. The views expressed on this site may come from individual contributors and do not necessarily reflect the view of BarBend or any other organization. BarBend is the Official Media Partner of USA Weightlifting.
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