However, gene expression networks deciphering [Ca2+]cyt and [Ca2+]nuc signalling pathways remain elusive. Here, using transgenic A. thaliana to selectively impair abscisic acid (ABA)- or methyl jasmonate (MeJA)-induced [Ca2+]cyt and [Ca2+]nuc increases, we identified [Ca2+]cyt- and [Ca2+]nuc-regulated ABA- or MeJA-responsive genes with a genome oligo-array. Gene co-expression network analysis revealed four Ca2+ signal-decoding genes, CAM1, CIPK8, GAD1, and CPN20, as hub genes co-expressed with Ca2+-regulated hormone-responsive genes and hormone signalling genes. Luciferase complementation imaging assays showed interactions among CAM1, CIPK8, and GAD1; they also showed interactions with several proteins encoded by Ca2+-regulated hormone-responsive genes. Furthermore, CAM1 and CIPK8 were required for MeJA-induced stomatal closure; they were associated with ABA-inhibited seed germination. Quantitative reverse transcription polymerase chain reaction analysis showed the unique expression pattern of [Ca2+]-regulated hormone-responsive genes in cam1, cipk8, and gad1. This comprehensive understanding of distinct Ca2+ and hormonal signalling will allow the application of approaches to uncover novel molecular foundations for responses to developmental and stress signals in plants.Calcium acts as a universal secondary messenger that transfers developmental cues and stress signals for gene expression and adaptive growth. A prior study showed that abiotic stresses induce mutually independent cytosolic Ca Krebs et al. [2+]nuc and [Ca2+]cyt in Arabidopsis thaliana root cells and Nod factor-induced spikes in [Ca2+]nuc and [Ca2+]cyt in Lotus japonicus root hair cells. Additionally, osmotic and salt stresses can separately trigger mutually independent increases in [Ca2+]nuc and [Ca2+]cyt in A. thaliana roots; such increases are involved in specific gene expression, root growth, and lateral root development [2+]cyt- and [Ca2+]nuc-regulated molecular networks and their crosstalk with hormone signalling pathways is essential for greater comprehension of Ca2+ signalling in plants.Sessile plants respond to developmental and environmental cues through various secondary messengers and phytohormones. Calcium ion  and calmodulin-like proteins (CMLs) nuc and [Ca2+]cyt modulate gene expression through sensor relays or sensor responders via different mechanisms. CaMs and CMLs interact with DNA-binding proteins and regulate their activities, resulting in activation or repression of targeted gene expression. Furthermore, CPKs, CRKs, CCaMKs, and CBLs-CIPKs phosphorylate specific DNA-binding proteins to regulate gene expression cyt in plant cells [2+]cyt-responsive genes. Furthermore, 269 [Ca2+]cyt-upregulated genes were identified in A. thaliana seedlings, using full genome microarray analysis [2+]nuc-regulated genes have not been identified; the crosstalk between Ca2+ and hormone signalling pathways in response to diverse stresses and developmental cues remains elusive.In plants, Cathaliana ,23. In cpression . In addied genes . CaM antnt cells ,27. Theranalysis . HoweverA. thaliana transcriptome for [Ca2+]nuc- and [Ca2+]cyt-regulated genes in cytosolic-localised parvalbumin (PV-NES) and nucleosolic-localised parvalbumin (PV-NLS) transgenic A. thaliana seedlings in response to ABA and MeJA using full genome microarray analysis. This work revealed 244 [Ca2+]cyt- and [Ca2+]nuc-regulated ABA-responsive genes and 144 [Ca2+]cyt- and [Ca2+]nuc-regulated MeJA-responsive genes. Moreover, using systems biology approaches focusing on genes transcriptionally regulated by Ca2+ and hormones, we defined a highly reliable gene co-expression network among [Ca2+]cyt- and [Ca2+]nuc-regulated genes, Ca2+ signal-decoding proteins, and ABA/JA signalling pathway proteins to uncover dynamic subnetwork structures in response to hormone treatment in A. thaliana.This study represents a comprehensive investigation of the A. thaliana (Col-0 ecotype) plants were grown with a cycle of 16 h light (120 μmol m−2 s−1)/8 h dark at 22 °C and 60% relative humidity. T-DNA insertion mutants cam1 (SALK_202076c), gad1 (SALK_022227), and cipk8 (SALK_139697c) were obtained from The A. thaliana Information Resource (https://www.arabidopsis.org/ (accessed on 6 September 2017)) ) . After gr 2017))  and asser 2017)) , we obtaA. thaliana seeds were surface-sterilised with 75% ethanol and plated on half-strength Murashige and Skoog (MS) salts, 2% sucrose, and 0.8% (w/v) agar at pH 5.8. After stratification at 4 °C in the dark for 3 days, the plates were transferred to a growth chamber under 16 h light (120 μmol m−2 s−1)/8 h dark at 22 °C for another 3 days. Seedlings were then transferred to half-strength MS plates containing 10 μM ABA or 50 μM MeJA, cultured for 5 additional days, and collected for RNA extraction. For the germination assay, seeds were plated on half-strength MS plates containing 0.5 μM ABA or 50 μM MeJA at 4 °C in the dark for 3 days. The plates were then transferred to a growth chamber for 3 days, and the seed germination rate was calculated.2+] measurements were performed in mature root sections of 1-week-old A. thaliana seedlings, as previously described [A. thaliana lines containing cytosolic-localised yellow Cameleon YC 3.6 (NES-YC3.6) or nuclear-localised yellow Cameleon YC 3.6 (NLS-YC3.6) were used to monitor the effects of ABA and MeJA on changes in [Ca2+]cyt and [Ca2+]nuc in root cells. After germination, A. thaliana seedlings were grown vertically on half-strength MS medium for 5–7 days; the roots were immobilised by overlaying 1% (w/v) low-melting-point agarose  in an Attofluor® Cell Chamber . After a small tunnel in the agarose had been made to expose the root, 200 μL of bathing solution buffer  were applied to the chamber. ABA (10 µM) and MeJA (50 µM) in the same bathing solution were separately perfused as the stimulus into the chamber. The mean fluorescence resonance energy transfer values in response to different stimuli represented measurement of 20–30 root cells from at least nine independent seedlings, each of which included three to six root cells. Analyses of statistical significance were performed using the unpaired Student’s t-test with GraphPad Prism 5.0  and the results are presented as means ± standard deviations (SDs).cyt and [Ca2+]nuc in root cells. [Ca2+] measurements were performed in mature root sections of 1-week-old A. thaliana seedlings, as previously described [2+]cyt elevations in A. thaliana roots , calcineurin B-like protein-interacting protein kinase 8 (CIPK8), and glutamate decarboxylase 1 (GAD1)—were hub genes in the main submodules of Ca2+-regulated ABA and JA signalling co-expression networks H-quinone oxidoreductase subunit 1 (NDHA), HYPERSENSITIVE INDUCED REACTION 2 (HIR2), CAPE2, PGSIP2, and PSBI; [Ca2+]cyt- and [Ca2+]nuc-co-regulated ABA-responsive genes such as NTMC2T6.1, RCI2B, and AT4G26190; [Ca2+]cyt-regulated JA-responsive genes such as AT3G45160, AGP14, and HIPP25; [Ca2+]nuc-regulated JA-responsive genes such as PELPK1 and NET2D; [Ca2+]cyt- and [Ca2+]nuc-co-regulated JA-responsive genes such as AT3G19370, AT1G54410, and AT2G21560; and the [Ca2+]cyt-regulated ABA-responsive and [Ca2+]cyt- and [Ca2+]nuc-co-regulated JA-responsive gene AT1G28400  cells. In plants, [Ca2+]nuc oscillations in response to Nod factor treatment are mediated by three nuclear-localised cyclic nucleotide-gated channels (CNGC15a/b/c); such oscillations are required for the establishment of symbiosis by nitrogen-fixing rhizobial bacteria in Medicago truncatula [2+]cyt-activated/repressed, [Ca2+]nuc-activated/repressed, and [Ca2+]cyt- and [Ca2+]nuc-co-regulated genes in response to ABA and MeJA treatment in A. thaliana seedlings. Furthermore, some genes were shared among the [Ca2+]-regulated ABA-responsive and [Ca2+]-regulated MeJA-responsive genes, indicating that both cytosolic and nucleosolic calcium are involved in the transcriptional regulation triggered by ABA or MeJA treatment in A. thaliana. Notably, we found that four calcium signal-decoding genes, CAM1, GAD1, CIPK8, and CPN20, were hub genes in two modules of Ca2+-regulated ABA and JA signalling co-expression networks. Additionally, we found the direct interactions among hub genes and Ca2+-regulated hormone-responsive genes with different combinations in LCI assays. These results provide important clues concerning hub genes CAM1, GAD1, CIPK8, and CPN20 that mediate crosstalk between Ca signalling and hormone pathways in A. thaliana, suggesting the implications for other plants or crops under biotic and abiotic stress cyt-activated ABA-responsive genes, including PSBI, YCF9, PSBJ, RPOA, RPL14, RPS3, RPL22, RPS19, YCF1.1, PSAC, ndhE, and ndhA, as well as one [Ca2+]cyt- and [Ca2+]nuc-activated ABA-responsive gene, PSBT, and one [Ca2+]nuc-repressed MeJA-responsive gene, PSBD. A previous study showed that the chloroplast can function as a sensor for environmental stimuli, such as drought stress, and initiate signals that regulate nuclear gene expression cyt, but not in [Ca2+]nuc, has a vital role in combining and coordinating the expression patterns of nuclear and plastid genes for bi-directional communication between chloroplasts and the nucleus cyt and [Ca2+]nuc. We first showed that both ABA and MeJA induce [Ca2+]cyt and [Ca2+]nuc increases in A. thaliana roots; ABA- and MeJA-induced stomatal closure is impaired in PV-NES, but not PV-NLS, plants. In addition, ABA- and MeJA-induced stomatal closure is impaired in cam1 plants. These results indicated that CAM1 perceives ABA- and MeJA-induced increases in [Ca2+]cyt to regulate stomatal closure in A. thaliana. Hossain et al. [2+]cyt elevation and stomatal closure are disrupted in the ABA-deficient mutant aba2-2 or by treatment with the ABA synthetic inhibitor fluridon in WT plants, consistent with our results in cipk8 plants; thus, CIPK8 presumably regulates ABA biosynthesis in A. thaliana. Furthermore, we found that ABA-inhibited seed germination is more sensitive in PV-NES, PV-NLS, cam1, and cipk8 plants than in WT plants. These results indicated that both CAM1 and CIPK8 participate in cross-talk during ABA- and JA-regulated biological processes in A. thaliana. Using LCI assays, we showed that CIPK8 interacts with CAM1; many Ca2+-regulated ABA- and JA-responsive genes, such as ERF104, PR1, and AGL21, exhibit various expression patterns in cam1 and cipk8 plants. These results suggested that the CAM1–CIPK8 complex could be a connection between the ABA and JA signalling pathways. Further genetic and molecular studies are needed to explore this potential connection.Crosstalk between JA and ABA signalling through interactions of ABA receptor PYRABACTIN RESISTANCE1-Like proteins (PYLs) and JASMONATE ZIM DOMAIN (JAZ) proteins, transcription inhibitors of JA signalling, is known to coordinate the balance between plant growth and defence resistance . Here, wn et al.  showed tPV-NES and PV-NLS separately impair ABA- and MeJA-induced increases in [Ca2+]cyt or [Ca2+]nuc. We also identified 244 [Ca2+]cyt- and [Ca2+]nuc-regulated ABA-responsive and 144 [Ca2+]cyt- and [Ca2+]nuc-regulated MeJA-responsive genes using an A. thaliana genome oligo array. Moreover, gene co-expression network analysis identified four Ca2+ signal-decoding genes, CAM1, CIPK8, GAD1, and CPN20, as hub genes in two [Ca2+]-regulated ABA/JA-responsive gene co-expression modules. Finally, LCI assays, phenotypic observations, and gene expression analyses showed that the CAM1–CIPK8 complex is a key regulator of stomatal movement, seed germination, and expression of [Ca2+]-regulated hormone-responsive genes in response to hormonal signals in A. thaliana.Here, we found that


Allogeneic stem cell transplant (SCT) recipients are at an increased risk of poor outcomes from COVID-19. While the mRNA-1273 (Moderna) and BNT162b2 (Pfizer) COVID-19 mRNA vaccines are highly immunogenic in the general population, the immune response in SCT recipients is poorly understood. We characterized the immunogenicity and reactogenicity of COVID-19 mRNA vaccines in a cohort of SCT patients.We performed a prospective cohort study of 16 allogeneic SCT patients and 23 healthy controls. Blood samples for both cohorts were collected prior to first vaccination (baseline), at the time of second vaccination, and approximately 28 days post-second vaccination. Anti-Spike (S), anti-S1, anti-receptor binding domain (RBD), and anti-Nucleocapsid (N) IgG levels were measured quantitatively from plasma using a multiplexed single molecule array (Simoa) immunoassay. Reactogenicity was captured for the SCT cohort via a self-reported post-vaccination diary for 7 days after each dose.Demographics and SCT recipients’ characteristics are shown in Table 1. In the SCT cohort, we observed a significantly lower anti-S (p< 0.0001), S1 (p< 0.0001), and RBD (p< 0.0001) IgG responses as compared to healthy controls, both at the time of dose 2 and 28 days post-vaccine series . Overall, 62.5% of SCT recipients were responders after vaccine series completion, as compared to 100% of healthy controls . While no patients had a reported history of COVID-19 diagnosis, 2 patients in the SCT cohort had elevated anti-S IgG levels and 1 showed elevated anti-N at baseline. 10/16 participants in the SCT cohort completed at least one post-vaccination diary. Local and systemic reactions were reported by 67% and 22% of participants, respectively, after dose 1, and 63% and 50% after dose 2 . All reported events were mild. Table 1: DemographicsFigure 1: Plasma IgG TitersAnti-Spike (A), anti-S1 (B), anti-RBD (C), and anti-nucleocapsid (D) IgG titers were measured at baseline, time of second dose, and approximately 28 days after second vaccination. IgG levels were measured quantitatively using multiplexed single molecule array (Simoa) immunoassays, and are reported as Normalized Average Enzymes per Bead (AEB). Allogeneic stem cell transplant recipients (mauve) showed significantly lower anti-S, S1, and RBD IgG responses as compared to healthy controls (mint). Low titers of anti-N IgG demonstrates no history of COVID-19 natural infection during the course of the study.Figure 3. Solicited Local and Systemic Adverse Events10 allogeneic stem cell transplant recipients completed at least one diary for 7 days after vaccination. Reactions after dose 1 are shown in light blue, and reactions after dose 2 are shown in dark blue. Local reactions (A) were reported by 67% (6/9) of participants after dose 1, and 63% (5/8) after dose 2. Systemic reactions (B) were reported by 22% (2/9) of participants after dose 1, and 50% (4/8) after dose 2. All reported events were mild (Grade 1).Among SCT recipients, mRNA COVID-19 vaccines were well-tolerated but less immunogenic than in healthy controls. Further study is warranted to better understand heterogeneous characteristics that may affect the immune response in order to optimize COVID-19 vaccination strategies for SCT recipients.Figure 2: Response Rate to COVID-19 VaccinationAn internally validated threshold for responders was established using pre-pandemic sera from healthy adults. A positive antibody response was was defined as individuals with anti-Spike IgG levels above the 1.07 Normalized AEB threshold.Amy Joyce, NP, Kadmon (Advisor or Review Panel member) Lewis A. Novack, MS, Lumicell Inc. Precision Healing, Inc.  David Walt, PhD, Quanterix Corporation  Robert Soiffer, MD, alexion (Consultant)gilead (Advisor or Review Panel member)jazz (Advisor or Review Panel member)juno/bms (Advisor or Review Panel member)kiadis (Board Member)precision bioscience (Consultant)Rheos (Consultant)takeda (Consultant) Nicolas C. Issa, MD, AiCuris (Scientific Research Study Investigator)Astellas (Scientific Research Study Investigator)GSK (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator)


We detected significantly elevated estradiol levels in critically ill male COVID-19 patients compared to all control cohorts. Testosterone levels were significantly reduced in critically ill male COVID-19 patients compared to control cohorts. No statistically significant differences in sex hormone levels were detected in critically ill female COVID-19 patients, albeit similar trends towards elevated estradiol levels were observed. Linear regression analysis revealed that among a broad range of cytokines and chemokines analysed, IFN-γ levels are positively associated with estradiol levels in male and female COVID-19 patients. Furthermore, male COVID-19 patients with elevated estradiol levels were more likely to receive ECMO treatment. Thus, we herein identified that disturbance of sex hormone metabolism might present a hallmark in critically ill male COVID-19 patients.Male sex was repeatedly identified as a risk factor for death and intensive care admission. However, it is yet unclear whether sex hormones are associated with disease severity in COVID-19 patients. In this study, we analysed sex hormone levels (estradiol and testosterone) of male and female COVID-19 patients ( The current SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus Type 2) pandemic continues taking its toll on human health resulting in 4.1 million lives lost worldwide (as of 20 July 2021). The clinical spectrum of SARS-CoV-2 infection is broad, ranging from mild upper respiratory illnesses to severe primary pneumonia with respiratory failure, multi-organ failure and death .Thus, there is increasing evidence that being male constitutes a major risk factor associated with SARS-CoV-2 fatality. However, the underlying factors of sex disparity observed in COVID-19 remain unclear yet. We have recently shown using the golden hamster model, that SARS-CoV-2 infection attacks the reproductive organs and causes massive dysregulation of sex hormones in infected male and female animals . Thus, wWe herein compared sex hormone levels and cytokine responses in critically ill male and female COVID-19 patients to critically ill non-COVID-19 patients as well as to further control cohorts . Analysis within the critically ill cohort was stratified by sex, age and Sequential Organ Failure Assessment (SOFA) scores.COVID-19). As a COVID-19 negative control cohort, we included 42 patients  with laboratory-confirmed negative SARS-CoV-2 PCR who were admitted to the Department of Intensive Care Medicine at the University Medical Center Hamburg-Eppendorf as a control group (herein referred as ICUnon-COVID-19).This retrospective cohort study includes the first 50 laboratory-confirmed (qRT-PCR) SARS-CoV-2 patients  who were admitted to the Department of Intensive Care Medicine at the University Medical Center Hamburg-Eppendorf from 9 March to 29 April 2020 . We also recruited healthy donors .A detailed description of the individual cohorts as well as respective inclusion and exclusion criteria are summarized in Supplementary Figure 1.The University Medical Center Hamburg-Eppendorf is a tertiary care hospital with 1738 hospital beds. The Department of Intensive Care Medicine includes regularly 12 multidisciplinary ICUs with a total of 140 ICU beds. Since the first wave of SARS-CoV-2 pandemic in March 2020, a minimum of two and a maximum of four intensive care units treated simultaneously up to 40 patients suffering from COVID-19 or suspected COVID-19.The following demographic and clinical variables were collected retrospectively for the COVID-19 and non-COVID-19 cohort from the electronic patient data management system (PDMS) : age, sex, body mass index, comorbidities, admission type and diagnosis, Acute Physiology and Chronic Health Score (APACHE II) and Simplified Acute Physiology Score II (SAPS II) on admission, Sequential Organ Failure Assessment Score (SOFA) within the first 24 h, classification of Acute Respiratory Distress Syndrome (ARDS) using the Berlin definition  and the A panel of 13 hormones was measured in plasma samples by an external laboratory accredited for measurements of human samples . The panel included total testosterone, free testosterone, dihydrotestosterone, androstenedione, 17-β-estradiol (E2), estrone, sex hormone-binding globulin, thyroid-stimulating hormone, free triiodothyronine (T3), free thyroxine (T4), luteinizing hormone, follicle-stimulating hormone and cortisol. In detail, cortisol, TSH, T4, LH, FSH, TT, E2 and SHBG were analyzed by electro-chemiluminescence immunoassay (ECLIA). Free TT was analysed by enzyme-linked immunosorbent assay and DHY-TT was measured by liquid chromatography–mass spectrometry (LC-MS/MS). Estrone levels were measured using a radioimmunoassay (RIA).TM multiplex assay  according to the manufacturer’s instructions in a Bio-Plex200 System with high-throughput fluidics .A panel of 27 cytokines and chemokines , granulocyte-colony stimulating factor (G-CSF), interferon-γ (IFN-γ), interferon γ-induced protein (IP-10), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-1β (IL-1β), interleukin 1 receptor antagonist (IL-1RA), monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation, normal T-cell expressed and presumably secreted chemokine (RANTES), tumour necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF)) was measured in plasma samples of COVID-19 patients. Cytokine and chemokine levels were measured using a Bio-Plex ProU test, Student-T test and the Fisher’s exact test as appropriate. All given p-values in the tables are of descriptive nature and not adjusted for multiple testing.Continuous variables are expressed as median with first to third quartile. Categorical variables are given as absolute and relative numbers. The distribution of data was visually interpreted using histograms. Variables were compared between groups (COVID-19 and non-COVID-19) with the Wilcoxon–Mann–Whitney p < 0.05 .Statistical evaluation for quantitative data was performed with two-way-ANOVA using Holm-Šidák correction for multiple comparisons. For non-normal data unpaired Mann–Whitney or Kruskal–Wallis test ignoring any multiple comparisons were used. Statistical significance was defined as All statistical evaluations mentioned above were performed with SAS®, version 9.4 TS level 1M5  and IBM® SPSS® Statistics 27. Graphical representation of the data was performed via GraphPad Prism 8 v. 8.4.2 .n = 292 SARS-CoV-2 PCR-positive patients were admitted to the Department of Intensive Care Medicine, at the University Medical Center Hamburg-Eppendorf from 9 March to 31 May 2021. Of these, n = 157 (54%) were patients secondarily admitted from referring intensive care units from all over northern Germany. In total, n = 194 (66%) of all COVID-19 patients were male and n = 98 (34%) of all COVID-19 patients were female. Thereof, n = 104 (36%) patients died; of which n = 71 (68%) were male and n = 33 (32%) were female. These data are in line with large epidemiological data analysis identifying being male as a high-risk factor for COVID-19 related death [A total of p = 0.6) (Table S1). A total of 39 (78%) patients met the ARDS criteria [p = 0.856). In the ICU non-COVID-19 group, 14 (33.3%) patients had a suspected bacterial infection on admission to the ICU. CRP was significantly higher in COVID-19 patients (p < 0.001). In total, mortality within the COVID-19 cohort was 30% (n = 15) compared to 11.9% (n = 5) in the non-COVID-19 control group.Critically ill COVID-19 patients were compared to the respective control group of critically ill non-COVID-19 patients with negative SARS-CoV-2 PCR . All COVcriteria  in the Cn = 39). As an additional healthy control group, we recruited age-and sex-matched SARS-CoV-2 negative male blood donors (HC) (n = 50). Furthermore, we recruited SARS-CoV-2 PCR-negative ICU patients (n = 42) as an additional control for critical illness independently of COVID-19 (herein referred to as ICU non-COVID-19). Critically ill male COVID-19 patients presented the highest estradiol levels compared to critically ill male non-COVID-19 patients (p = 0.0293) or male patients with CHD (p = 0.0004) or healthy males (p = 0.001) (p = 0.0250) or male patients with CHD (p = 0.0050) or healthy males (p < 0.0001) , we recruited an age – and sex-matched SARS-CoV-2 negative CHD control cohort  . In line, a substantial proportion of plasma dihydrotestosterone levels in COVID-19 males was even below the lowest reference range, confirming dihydrotestosterone deficiency in men. In contrast, dihydrotestosterone levels were comparable and within clinical references in female COVID-19 and HC cohorts (p = 0.9568) . To assess whether the increase in estradiol levels can be attributed to a general increase in estrogens, we next measured estrone concentrations. Estrone levels in the plasma of COVID-19 males were higher than those measured in HC males (p < 0.0001) . Similarly, estrone levels were elevated in the plasma of female COVID-19 patients unlike HC females (p = 0.0009) .Testosterone is further metabolized to dihydrotestosterone by 5-α reductase. Dihydrotestosterone (DHT) also acts as an androgen and plays a key role in activating the transcription of various genes and activation of various immune cells similar to testosterone . Thus, wCritically ill male COVID-19 patients present additionally elevated estrone levels accompanied by severely reduced DHT levels. Critically ill female COVID-19 patients present elevated estrone levels, while dihydrotestosterone levels remain unchanged.p = 0.0245 and p = 0.0273). In female COVID-19 patients, a trend towards higher estradiol levels with increasing SOFA scores was also observed (p = 0.0307)  scores or the requirement for extracorporeal membrane oxygenation (ECMO) later during the patients’ ICU stay. In critically ill male COVID-19 patients, estradiol levels increased with disease severity  0.0307) . Testost . TestosThese data show that critically ill male COVID-19 patients with elevated estradiol levels are more likely to require ECMO treatment.p = 0.0301), IL-1RA (p = 0.0160), IL-6 (p = 0.0145), MCP-1 (p = 0.0052) and MIP-1α (p = 0.0134) levels were elevated in those with higher SOFA scores (8-11) compared to those with lower SOFA scores (2-3) (p = 0.0476) .We then analyse whether changes in cytokine and chemokine responses in critically ill COVID-19 patients correlate with their respective sex hormone levels given that most immune cells possess androgen and estrogen receptors, using multivariable regression . Among aThese findings suggest an association of estradiol levels with IFN-γ levels, which is in line with previous studies reporting on the estradiol-controlled transcription of IFN-γ due to the presence of an estrogen-responsive element (ERE) in its promoter region .In this study, we show that disturbance of sex hormone metabolism is associated with critical COVID-19 in men but not in women.In critically ill men with COVID-19, we detected significantly elevated estradiol levels compared to control cohorts . Furthermore, testosterone levels were significantly reduced in males with COVID-19 compared to all control cohorts. Our findings were recently confirmed by two independent single-centre studies ,15. A stReduced testosterone levels were likely of testicular origin in 25% (primary hypogonadism) of all male COVID-19 cases in our study, suggesting that 75% can likely be attributed to hypothalamic-hypopituitary origin (secondary hypogonadism). These findings are further confirmed by the Italian single-centre study reporting that low testosterone levels in men with COVID-19 are associated with secondary hypogonadism . The vasIn women with COVID-19, we did not detect any significant alterations in testosterone or estradiol levels. However, some female COVID-19 patients had elevated testosterone levels albeit statistically not significant. It is important to note that all but one female COVID-19 patients were postmenopausal. However, the small female COVID-19 cohort size in our study is a potential limitation with respect to conclusions on female COVID-19 outcome. This variance in cohort size was due to that more men than women were admitted to the ICU, highlighting the importance of sex on critical COVID-19 outcome. Despite these limitations in the female COVID-19 cohort, others postulated an elevated COVID-19 risk for women with polycystic ovary syndrome, a condition characterized by increased androgen levels . This hiComparative analysis of 27 different cytokines/chemokines in the male and female COVID-19 patients and subsequent regression analysis revealed that interferon-γ (IFN-γ) positively correlates with estradiol but not testosterone levels. This correlation is particularly interesting given that IFN-γ possesses an estrogen-responsive element (ERE) in its promoter region . IFN-γ iCollectively, our findings herein highlight that disturbance in circulating sex hormone levels is a hallmark of critical COVID-19 in males. Longitudinal hormone surveillance studies may be warranted during acute and recovery phases in COVID-19 patients to predict short- and potentially long-term risks .APACHE IIAcute Physiology and Chronic Health ScoreARDSAcute Respiratory Distress SyndromeCHDCoronary heart diseasesCOVID-19Coronavirus disease 2019DZIFGerman Center for Infection ResearchE217-β-estradiolECLIAElectro-chemiluminescence immunoassayECMOExtracorporeal membrane oxygenationEREEstrogen responsive elementHCHealthy ControlICUIntensive Care UnitIFN-γInterferon-γIQRInterquartile rangeLHLuteinizing hormonePCOSPolycystic ovary syndromePDMSPatient data management systemRIARadioimmunoassaySAPS IISimplified Acute Physiology Score IISARS-CoV-2Severe Acute Respiratory Syndrome CoronavirusType 2SHBGSex hormone-binding globulinSOFASequential Organ Failure Assessment ScoreT3Free triiodothyronineT4Free thyroxineUKEUniversity Medical Center Hamburg-EppendorfWHOWorld Health OrganizationThe study with laboratory-confirmed COVID-19 and non-COVID-19 ICU patients was reviewed and approved by the ethics committee at the Hamburg State Chamber of Physicians (registration no.: WF-053/20 and WF-073/21). The need for an informed consent for the ICU cohort, healthy blood donors (HC) and patients with coronary heart diseases (CHD) was waived by the ethics committee because data were retrieved anonymously and retrospectively from electronic health records. The study is registered under the number NCT04979091 (HAM-SEX-C19) on ClinicalTrials.gov.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Here we report that strain RB2126, in addition to flp-1(ok2811), also contains two additional background mutations. One of the mutations gives the animals a constitutive dauer phenotype (Daf-c), and complementation testing confirms that it is in the Daf-c gene daf-11. The second mutation is isolated based on the fact that it partially suppresses the elevated body curvature of flp-1(ok2811) mutants, but it remains uncloned. The updated genotype of RB2126 strain is flp-1(ok2811); daf-11(yak155); yak156.The FLP-1 neuropeptide is involved in locomotion, mechanosensation, and reproduction. Strain RB2126 is reported to contain the  C. elegans . Some of the flp genes were found to play roles in locomotion, egg laying, and sleep, but most flp genes have unknown function . flp-1 is one of the few flp genes whose mutations are reported to cause overt behavioral defects . The initial flp-1 deletion alleles that were analyzed behaviorally  included a deletion of part of daf-10 . This resulted in some of the initial behaviors that were attributed to FLP-1 peptides being caused by loss of daf-10 activity . Subsequent analysis of newer flp-1 deletion alleles suggests that FLP-1 peptides are involved in nose touch sensitivity, locomotion, and reproduction .The FLP family of neuropeptides contains at least 30 genes in flp-1(ok2811) is a prospective null allele . We obtained strain RB2126  in order to analyze flp-1(ok2811) null mutant locomotion behavior. When the strain was grown at room temperature (23°C), some animals formed noticeable dauers similar to strains containing mutations in constitutive dauer (Daf-c) genes  . To examine whether the Daf-c phenotype was because of a background mutation, strain RB2126 was outcrossed three times. This outcross resulted in the segregation of two different mutants with readily apparent phenotypes: one resulting in deep body bends and a second in the Daf-c phenotype. PCR genotyping showed that the deep body bends co-segregated with the flp-1(ok2811) deletion. The mutation causing the Daf-c phenotype was mapped to chromosome V close to the daf-11 gene and a complementation test using daf-11(sa195)  showed that it is in daf-11 . These results suggest that flp-1(ok2811) mutants exhibit elevated body curvature, and that strain RB2126 has a background mutation in daf-11. This new daf-11 allele in strain RB2126was named yak155.flp-1(ok2811) mutants are known to exhibit elevated body curvature . Interestingly, strain RB2126 (flp-1(ok2811); daf-11(yak155)) had significantly reduced deep body bends in comparison to strain XZ2314 (flp-1(ok2811); three times outcrossed from strain RB2126) . This suggests that either the daf-11(yak155) mutation partially suppresses the phenotype of flp-1(ok2811) or that the RB2126 strain has other background mutation(s) that affect the body curvature. To test the possibility that the reduced deep body bends of flp-1(ok2811) in RB2126 strain are due to daf-11, double mutant strain flp-1(ok2811); daf-11(sa195) was constructed, and its body curvature was examined. flp-1(ok2811); daf-11(sa195) mutants (strain XZ2380) had similar body curvature as flp-1(ok2811) mutants (strain XZ2314) . In addition, larvae (L2 to L3 stage) of flp-1(ok2811) and flp-1(ok2811); daf-11(sa195) often exhibit a characteristic body posture resembling the number ”8”, but this posture was not observed in RB2126 larvae . This result shows that daf-11(sa195) does not affect the elevated body curvature of flp-1(ok2811) mutants, suggesting that the body curvature of the RB2126 strain may be modified by another background mutation(s) other than daf-11(yak155).flp-1(ok2811); daf-11(yak155) double mutants (strain XZ2406) and flp-1(ok2811) mutants (strain XZ2314) were compared. Body curvature in these strains was found to be similar, suggesting that strain RB2126 has at least one additional background mutation in an unknown gene that affects the body curvature of flp-1(ok2811) . In support of this, strain RB2126 was outcrossed using strain EG7765 that contains the genetic marker oxTi76  and animals of the F2 generation that were not green, and did not exhibit Daf-c or elevated body curvature were picked. This outcross resulted in the isolation of strain XZ2394 that contains the flp-1(ok2811) mutation but does not show the same extent of elevated body curvature as XZ2314(flp-1(ok2811)) strain . This suggests that strain RB2126, besides the mutation in daf-11, contains an additional mutation that partially but significantly suppresses the flp-1(ok2811) elevated body posture. This new allelewas named yak156 and the updated genotype of strain RB2126 is flp-1(ok2811); daf-11(yak155); yak156.To verify the presence of additional background mutation(s), the body curvature of Strain maintenanceE. coli strain OP50 as a food source .Strains were maintained at 15˚C or room temperature (23˚C) on NGM plates with Waveform quantificationFirst-day adult animals were placed on an OP50 plate and allowed to move forward until they had completed at least five tracks. Each animal’s tracks were imaged at 50X magnification using a Nikon SMZ745 stereoscope and an iLabCam. Worm track period and 2X amplitude were measured using the line tool in Image J. For each worm, three to five periods and amplitudes were measured. Each experiment was performed on 5-10 worms.StatisticsP values were determined using GraphPad Prism 9.0 (GraphPad Software). Normally distributed data sets requiring multiple comparisons were analyzed by a one-way ANOVA followed by a Bonferroni test. Normally distributed pairwise data comparisons were analyzed by two-tailed unpaired t tests.StrainsBristol N2: wild typeoxTi76 IV; unc-18(md299) XEG7765: daf-11(sa195) VJT195: unc-42(e270) V daf-11(sa195) VJT6709: flp-1(ok2811) IV; daf-11(yak155) V; yak156RB2126: flp-1(ok2811)IV [3X outcrossed from RB2126]XZ2314: oxTi76 IV; daf-11(yak155) VΧΖ2370: flp-1(ok2811) IV; daf-11(sa195) VXZ2380: flp-1(ok2811) IV; yak156XZ2394: flp-1(ok2811) IV; daf-11(yak155) VXZ2406:


Old miR-29a transgenic mice showed fewer osteoporosis signs, less 5mC, and less 8-OHdG formation than age-matched wild-type mice. miR-29a overexpression reversed age-induced senescence and osteogenesis loss in bone-marrow stromal cells. miR-29a promoted transcriptomic landscapes of redox reaction and forkhead box O (FoxO) pathways, preserving oxidation resistance protein-1 (Oxr1) and FoxO3 in old mice. In vitro, miR-29a interrupted DNA methyltransferase 3b (Dnmt3b)-mediated FoxO3 promoter methylation and senescence-associated β-galactosidase activity in aged osteoblasts. Dnmt3b inhibitor 5′-azacytosine, antioxidant N-acetylcysteine, or Oxr1 recombinant protein attenuated loss in miR-29a and FoxO3 to mitigate oxidative stress, senescence, and mineralization matrix underproduction. Taken together, miR-29a promotes Oxr1, compromising oxidative stress and FoxO3 loss to delay osteoblast aging and bone loss. This study sheds light on a new antioxidation mechanism by which miR-29a protects against osteoblast aging and highlights the remedial effects of miR-29a on osteoporosis.Senescent osteoblast overburden accelerates bone mass loss. Little is understood about microRNA control of oxidative stress and osteoblast senescence in osteoporosis. We revealed an association between microRNA-29a ( Ink4a, p21Cip1 signaling, β-galactosidase activity, DNA damage, and senescence-associated secreted phenotypes [Osteoporosis is a chronic skeletal disease with low bone mass and fragile microstructure , becominenotypes . inhibitenotypes .KLF5 hypermethylation, inhibiting osteogenic differentiation [Epigenetic alternation is one of the hallmarks of cellular aging, whereas a chronic dysregulation of the epigenetic program accelerates the development of age-related diseases . Of epigntiation .miR-219-5p targets retinoic acid receptor-related orphan receptor beta, ameliorating osteogenesis loss [miR-195 function increases angiogenesis, progressively improving bone mass in aged mice [miR-31-5p knockdown slows osteoclastic resorption and age-mediated bone loss [MicroRNAs (miR) bind to the 3′-untranslated region of mRNA targets, which interrupt protein translation and modulate plenty of biological activities . These mged mice . miR-31-one loss  and angione loss  of mesenmiR-29a correlated with oxidative stress and osteoblast aging in human osteoporosis and examined if miR-29a knockout or overexpression affected osteoblast senescence and bone loss in old mice and characterized the epigenetic mechanism by which miR-29a controlled oxidative stress and senescence program in osteoblasts of osteoporotic skeleton.This study aimed to investigate whether Studies on human bone biopsies were approved by the Chang Gung Medical Foundation Institutional Review Board (Affidavit #202000823B0). Informed consent was obtained from patients preoperatively. Thirteen patients with osteoporosis  and 13 patients without osteoporosis  who required lumbar spine decompression, fixation, discectomy, or vertebroplasty were enrolled in the osteoporosis group and control group, respectively. The BMD of the hip was quantified using dual-energy X-ray absorptiometry before surgery. Leftover bone biopsies and 10 mL peripheral blood were harvested.miR-29a gene  (RP23-173G8) of Bacterial Artificial Chromosome (BAC) library  were cloned using PCR protocols. The loxP site and the neomycin (Neo) resistance cassette (PL452) were inserted into PL253 plasmid . The Neo gene driven by T7 promoter in mammalian cells was removed by Cre recombinase. The linearized BAC construct was transferred into embryonic stem cells of C57BL/6 mice to produce miR-29a chimera, which further crossed with Sox2-Cre mice  to breed miR-29aKO mice. The genotype of miR-29aKO mice was confirmed using customized primers . The homologous arms of C57BL/B6 mouse  primers  and PCR miR-29a transgenic mice , which carried osteocalcin (Bgp) promoter-driven human miR-29a precursor construct, were generated. In brief, linear constructs with osteocalcin (Bgp) promoter–human miR-29a precursor (506 bp) –bovine growth hormone (BGH) polyadenylation (polyA) were transfected into fertilized eggs from C57L/B6 mice through microinjection protocols. The micro-injected eggs were grafted into foster mother mice, as previously described [miR-29a precursor were considered miR-29aTg mice, whereas mice that did not carry the gene of interest were considered wild-type (WT) mice [Osteoblast-specific escribed . GenotypWT) mice .Three-month-old and 9-month-old male miR-29aTg or WT mice were divided into young and old groups, respectively. To label bone mineralization, 10 mg/kg calcein was injected 9 days and 3 days before euthanasia. Peripheral blood was drawn and centrifuged to harvest serum. Femurs and tibiae specimens were dissected for subsequent experiments.3), trabecular volume , trabecular thickness , and trabecular number (Tb.N/mm) of proximal tibiae, were measured using SKYSCAN® CT-Analysis software, according to the maker’s instructions. In a subset experiment, μCT radiography of marrow fat in tibiae was performed upon soaking in OsO4, as previously described [4-soaked specimens were reconstructed and merged using the software. Marrow fat volume (mm3) and fat surface (mm2) were calculated automatically.The microstructure of femurs and tibiae was visualized using SkyScan 1176 μCT  and captured 200 radiographs (9-μm voxel size), as previously described . BMD (g/escribed . The ima2) and energy (N.mm), of the middle region of femurs upon 3-point bending were quantified using SHIMADZU EZ-SX Material Test System  with TRAPEZIVMX software, according to the maker’s instructions.Upon measuring the cross-section area of the middle region of femurs using μCT scanning protocols, the biomechanical properties, including breaking force , 5-methylcytosine (5mC) , and 8-hydroxydeoxyguanosine (8-OHdG) . Immunostained osteoblasts in each high-power field (×400 magnification) were counted. Three random fields in each section and 3 sections in each tibia specimen were selected to quantify immunostained cells.The calcein labeling in methyl acrylate -embedded tibia sections was evaluated using a fluorescence microscope . Trabecular bone histology was evaluated using von Kossa staining kits  and hematoxylin–eosin stain kits . Osteoclasts were probed using a tartrate-resistant acid phosphatase (TRAP) staining kit . Trabecular volume , mineral acquisition rate , and osteoclast number (Oc.N/mm) were quantified using Axio Image Analysis System . Six random fields in each section and 3 sections in each animal were selected for histomorphometry. Immunohistochemical staining for paraffine-embedded sections was performed using β-galactosidase , p165 cells/well, 24-well plates) were seeded in an osteogenic condition using StemProTM Osteogenesis Differentiation Kits  and in an adipogenic condition using StemProTM Adipogenic Differentiation Kits (A1007001) for 21 days and 15 days, respectively. Mineralized matrices and adipocytes were stained using von Kossa stain kits and Nile Red stain kits . Bone-marrow macrophage precursor cells  were incubated in αMEM with 15 ng/mL M-CSF and 40 ng/mL RANKL  for 1 week. Osteoclasts were probed using TRAP stain kits. Von-Koss-stained mineralized matrices in each low-power field (x125 magnification) and Nile-red-stained adipocytes and TRAP-stained osteoclasts in each high-power field were measured using the Zeiss Image Analysis System.Mesenchymal cells and macrophage precursor cells in bone marrow were isolated, as previously described . Bone-ma5/well, 6-well plates) were incubated in medium for 3 days. After 10 passages, osteoblasts were designated to the aged group (P10). In some experiments, 105 cells/well (24-well plates) were incubated in 100 nM 5-Aza-2′-deoxycystidine  or 10 μM N-acetylcysteine  for 4 h and incubated in an osteogenic medium.Osteoblasts were multiple-passaged as an in vitro model of age-induced bone loss, as previously described ,27,28. U4 cells/48-well plate) were incubated in DMEM and 10% FBS with or without 300 µm H2O2  [Murine MC3T3-E1 preosteoblasts   or 50 µMmiR-29a mimetic, antisense oligonucleotide, and scramble control  were added to LipofectamineTM 3000  and transferred into P0 or P10 osteoblasts .A total of 10 nM 3 cells/slide) was probed using Senescence β-Galactosidase Staining Kits . SA-β-gal-stained cells in each field, 3 fields in each slide, and 3 slides for each experiment were counted.SA-β-gal activity in osteoblasts , and the threshold value for amplification reaction was probed. Equation 2−ΔΔCt was used to calculate the fold change of mRNA expression.Total RNA was extracted from WT and miR-29aTg osteoblasts using TRI Reagent™ Solution . High-Capacity cDNA Reverse Transcription Kits  were utilized to reversely transcribe 1 μg of total RNA. PCR was investigated using primers , 2× TaqM®. Fold change of the gene expression log2 > 1 was considered differentially expressed. Gene ontology and KEGG pathway were clustered using Kobas bioinformatics engine , as previously described [A total of 10 μg of RNA was amplified using Amino Allyl MessageAmp II aRNA amplification kits  and coupled with CyDye using CyDye Post-Labeling Reactive Dye Packs , according to the makers’ instructions. Upon purification, the labeled aRNA was hybridized onto Mouse MOA2.0 whole-genome arrays . Data and clustering were processed using Rosetta Resolver Biosoftwareescribed .Dnmt3b was constructed into luciferase reporter pCRII-TOPO II vector . A total of 10 ng of luciferase vector and 10 ng Renilla luciferase reporter vector were mixed with LipofectamineTM 3000 Transfection Reagent  and transfected into osteoblasts . The reporter cells were transfected into 30 nM scramble control, miR-29a mimetic, or antisense oligonucleotide. Fluorescent luciferase activity was probed using Dual Luciferase Detection Kits and normalized with Renilla luciferase activity.Wild-type  or 5-bp mutant (5′-UUUUACCUAAUUACAGTAAACAU-3′) of the 3′-UTR of 6 osteoblasts was harvested using MagMAX™-96 DNA Multi-Sample Kits . Methylated DNA was probed using the EZ DNA Methylation™ Kit , according to the maker’s manuals. A total of 10 ng genomic DNA was mixed with 100 μL CT Conversion Reagent with bisulfite at 50 ℃ in the dark for 16 h and incubated at 4 ℃ for 10 min. Upon eluting through Zymo-Spin™ IC Column, the elute was washed with an M-Wash Buffer and mixed with a M-Desulphonation Buffer and an Elution Buffer. PCR analysis of the eluted DNA was performed using the ABI 7900 Detection System together with specific primers, methylated and unmethylated FoxO3 promoter, and miR-29a promoter. PCR analysis of GADPH gene was performed to confirm the equal loading of elute DNA . Immunoblotting of 5mC, 5hmC, Foxo3, Dnmt3b, and actin levels in the lysates was performed using specific antibodies and SuperSignal™ Western Blotting Kits .Lysates of 5 × 10miR-29a, and immunostaining in the non-osteoporosis and osteoporosis groups were analyzed using a Wilcoxon test. The patients’ gender of both groups was analyzed using a Chi-square test. The investigations of WT and miR-29aKO mice were analyzed using a Student’s t-test. The investigations of young and old WT and miR-29aTg mice and in vitro models were analyzed using an ANOVA test and a Bonferroni post hoc test. p value < 0.05 resembled significant difference.Differences of age, hip BMD, serum miR-29a expression correlated with human osteoporosis. We collected bone biopsies and serum from 13 patients with osteoporosis  and 13 patients without osteoporosis  who required lumbar spine decompression, fixation, discectomy, or vertebroplasty. Serum miR-29a levels  were generated by crossing loxPmiR-29a mice with CreSox2 mice. RT-PCR confirmed miR-29a loss in the bone tissue of 3-month-old miR-29a KO mice  expression and mineralized extracellular matrix underproduction  [miR-29a expression in bone tissue   , glutaredoxin (Glrx), glutathione disulfide reductase (Gsr), cytochrome 1 (cyc1), and peroxiredoxin 5 (Prdx5)  d. RT-PCReoblasts e. Oxr1 aaTg mice f.FoxO3 loss in an old miR-29aTg bone microenvironment was used to verify if the microRNA affected FoxO3 methylation in aged osteoblasts. Multiple passaged osteoblasts   a were inpression b and Foxpression c. Forcedeoblasts c. The mepromoter d was sigeoblasts d, as is miR-29a repressed FoxO3 methylation in aged osteoblasts. A bioinformatics prediction  shows that DNA methyltransferase 3b (Dnmt3b) catalyzes DNA methylation and is a putative target of miR-29a. Dnmt3b expression was increased in multiple-passaged osteoblasts. This effect was downregulated in miR-29a mimetic-treated osteoblasts  of Dnmt3b  repressed Dnmt3b mRNA expression (miR-29a promoter methylation (FoxO3 promoter methylation (miR-29a (FoxO3 mRNA expression (Of interest, forced rescence a and incpression b. We invpression c and propression d in multhylation e and Foxhylation f to reve(miR-29a g and Foxpression h and propression d and imppression i.miR-29a and Orx1 loss were present in senescent osteoblasts, we investigated what role antioxidant protein Oxr1 may play in miR-29a control of osteoblast senescence. Osteoblasts were incubated in H2O2 as an in vitro model of oxidative stress2-induced cellular senescence [2O2 or Oxr1 recombinant protein for 15 days. H2O2 significantly reduced miR-29a (FoxO3 (Bgp expression (p21 expression (2O2-induced loss in miR-29a, FoxO3, and Bgp expression and attenuated p21 expression. Plenty of osteoblasts showed strong SA-β-gal activity upon incubating in H2O2. Oxr1-treated cells showed weak SA-β-gal staining (Given that  miR-29a a, FoxO3 a (FoxO3 c, and Bgpression c, whereapression d as compstaining e.miR-29a slowed osteoblast senescence, preserving extracellular mineralized matrix production to protect from bone loss in old mice. miR-29a attenuated FoxO3 loss and oxidative stress through repressing Dnmt3b-mediated antioxidant protein Oxr1 loss and DNA hypermethylation in aged osteoblasts. Collective evidence conveys a new insight into the antioxidation and anti-aging potential of miR-29a against bone loss.Osteogenic cell senescence involves postnatal bone homeostasis, microstructure integrity, or osteoporosis development . ProgrammiR-29a loss, DNA hypermethylation, osteoblast senescence, and oxidative stress in human osteoporosis. Expanding studies show increased p16 expression [miR-29a may play in DNA methylation and oxidative stress in osteoporosis.Investigations of clinical specimens uncovered a plethora of reactions, such as pression  or DNA mpression  in bone miR-29a knockout mice, suggesting that this molecule was required to maintain skeletal tissue integrity. The adverse effect of miR-29a loss on bone homeostasis is also pointed out by a study manifesting that transgenic overexpression of miR-29-3p tough decoy in mice inhibits bone mineral accretion and trabecular bone volume [miR-29a deletion induced DNA hypermethylation and osteoblast senescence. The impact of miR-29a loss on osteoblast fate and bone integrity in this murine model further explained the histological features of senescent osteoblast overburden in human osteoporotic bone.We, for the first time, have revealed phenotypes of low bone mass in e volume . Nine-moe volume ,27,28,29miR-29a appeared to attenuate excessive bone turnover by preserving the osteogenic differentiation capacity of mesenchymal progenitor cells and reducing RANKL expression to downregulate osteoclastic activity. The senescence-associated secretory phenotype, like IL-6 expression [miR-29a in delaying age-induced skeletal deterioration.Striking findings were that old miR-29Tg mice showed fewer osteoporosis signs, including mild MAR loss and osteoclast formation, than age-matched WT mice. pression , was alsmiR-29a, at least in part, disrupted the Dnmt3b catalysis of FoxO3 promoter hypermethylation to compromise FoxO3 loss, reversing senescence and mineralized matrix underproduction in aged osteoblasts. Transcription factor Foxo3 is important to slow cellular aging, promoting tissue homeostasis and function [FoxO3 interference induces reactive oxygen radical overproduction, inhibiting the osteogenic differentiation capacity of mesenchymal stem cells [FoxO3 gene correlates with human tissue aging [miR-29a and Dnmt3b. Dnmt3b inhibition mitigated miR-29a promoter methylation, in turn reversing miR-29a and FoxO3 functions to drive osteoblasts away from senescence. The inhibition of DNA methyltransferase in bone-forming cells promotes osteogenic activity. DNMT3b knockdown or 5-aza-induced DNA hypomethylation promotes the osteogenic differentiation of human [We revealed that function . FoxO3 iem cells . The metue aging . In addiof human  and muriof human . The arrmiR-29a repressed 8-OHdG formation in aged osteoblasts. A transcriptomic landscape was that miR-29a affected redox reactions by promoting a plethora of antioxidant proteins in osteoblasts. Expanding studies reveal the biological roles of Oxr1, Glrx, and Gsr in cellular senescence. For example, forced Gsr expression downregulates Klotho loss-mediated renal cell aging and dysfunction [Oxr1 knockdown aggravates oxidative stress and apoptosis [2O2-treated osteoblasts and aged skeleton. Oxidative stress dysregulates epigenetic signatures, affecting the differentiation capacity of mesenchymal progenitor cells [miR-29 involves the oxidative stress and viability of human mesenchymal stem cells upon DGCR8 interference [miR-29a and Oxr1 in repressing oxidative stress to downregulate DNA hypermethylation-induced osteoblast dysfunction. In this study, patients in the osteoporosis group were significantly older than the control group. However, the limitation of the experiment for human bone biopsies is the absence of osteoporosis indications in age-matched young patients who required spinal surgery. The possibility cannot be ruled out that ubiquitous miR-29a knockout may change other tissues’ integrity or function, which may influence osteoblast function and bone homeostasis. miR-29a signaling may affect other DNA methylation enzymes or redox regulators to maintain anabolic activity in aged osteoblasts. The investigations related to the interactions of miR-29a, oxidative stress, and epigenetic pathways reveal a new molecular mechanism underlying bone mass loss.Oxidative stress is a prominent deleterious reaction, dysregulating protein stability or epigenetic pathways to hinder survival or biological activity in senescent cells . Of notefunction . Antioxipoptosis . In thisor cells . miR-29 rference . The in miR-29a loss, oxidative stress, and DNA hypermethylation correlated with osteoblast aging in human osteoporosis. miR-29a knockout accelerated osteoblast senescence and bone loss. miR-29a reversed FoxO3 loss to improve senescence program through targeting Dnmt3b-mediated FoxO3 methylation and increased antioxidant proteins, including Oxr1, to downregulate oxidative stress and DNA methylation, compromising age-induced osteoblast loss and osteoporosis (miR-29a, DNA methylation inhibitor, and Oxr1 on age-induced osteoblast dysfunction and bone loss.Taken together, profound evidence revealed that oporosis f. This s


Antimicrobial stewardship programs (ASP) aim to improve appropriate antimicrobial use. Post-operative antibiotics are generally not necessary, especially those without surgical site infections risk factors (e.g. obesity). Few studies have described the impact of ASP interventions on patient outcomes especially in unique populations such as obstetrics. This study aims to evaluate the impact of ASP interventions on post-elective caesarean (eLSCS) oral antibiotic prophylaxis use and patient outcomes including SSI rates.This pre-post quasi-experimental study was conducted over 9 months (2 months pre- and 7 months post-intervention) in all women admitted for eLSCS in our institution. Interventions included eLSCS surgical prophylaxis guideline dissemination, where a single antibiotic dose within 60 minutes before skin incision was recommended. Post-eLSCS oral antibiotics was actively discouraged in those without SSI risk factors. This was followed by ASP intervention notes (phase 1) for 3 months, and an additional phone call to the ward team for the next 7 months (phase 2). Phase 3 (next 6 months) constituted speaking to the operating consultant. The primary outcome was post-operative oral antibiotics prescription rates. Secondary outcomes included rates of 30-day post-operative SSI.A total of 1751 women was reviewed. Appropriateness of pre-operative antibiotic prophylaxis was 98% in our institution. There were 244 women pre-intervention, 274 in post-intervention phase 1, 658 in phase 2 and 575 in phase 3. Pre-intervention post-eLSCS antibiotic prescribing rates was 82% (200), which reduced significantly post-intervention to 54% (148) in phase 1, 50% (331) in phase 2 and 39% (226) in phase 3 (p< 0.001). There was no significant difference in patients who developed post-operative SSI pre-post intervention  and among who received post-operative oral antibiotics compared to those without .ASP interventions can reduce post-eLSCS antibiotic prophylaxis rates without adversely impacting patient safety.All Authors: No reported disclosures


SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical  Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected tens of millions, leading to over 2 million deaths worldwide , and othIn addition to providing new insights into coronavirus receptor interactions and host range, characterizing determinants of ACE2 species restriction also has implications for SARS-CoV-2 animal model development. Mouse models have been essential for understanding the pathogenesis of coronaviruses and have been key resources for the preclinical development of vaccines and antiviral therapies –7. Howev–9–14–Based on previous SARS-CoV research and published structures , 18, we In order to evaluate the impact of mACE2 humanization at each of these interaction hot spots, we first modeled the interaction between SARS-CoV-2 RBD and mACE2 . From thTo experimentally test these models, a panel of hmACE2 receptors was generated to directly evaluate whether these predicted contact residues are essential for SARS-CoV-2 infection. The receptors were introduced into mouse delayed brain tumor astrocytoma (DBT-9) cells , 20, norGiven the importance of the three ACE2 interaction hot spots for SARS-CoV-2 infection, we asked whether these same determinants were important for infection by other preepidemic group 2B coronaviruses. We modeled interactions between hmACE2.3 and two related group 2B CoVs, preepidemic SARS-like bat CoV WIV-1  and pang10.1128/mBio.03149-20.3FIG S3FIG S3, TIF file, 1.6 MB.Structure-based multiple sequence alignment of the RBD from SARS-CoV, SARS-CoV-2, WIV-1, and P5L. Download Copyright © 2021 Adams et al.2021Adams et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro  as well as those from species reported to support little to no infection    25, 27), 27in vichicken) , providi, ferrets (Mustela putorius), mink (Mustela lutreola), domestic cats (Felis catus), pigs (Sus scrofa), guinea pigs (Cavia porcellus), dogs (Canis lupus), mice (Mus musculus), and chickens . (b to h) Predicted hotspot contacts between SARS-CoV-2 RBD and the ACE2 molecule from humans (b), macaques (c), ferrets (d), mink (e), cats (f), pig (g), guinea pig (h), dog (i), mouse (j), and chicken (k). (l and m) Predicted hotspot contacts between SARS-CoV-2 variant Y453F and mink (l) and human (m) ACE2. (n) Predicted hotspot contacts between SARS-CoV-2 variant N501Y and human ACE2. Download FIG S1, TIF file, 1.2 MB.ACE2 hotspot contacts are predictive of SARS-CoV-2 host range. (a) Aligned hotspot residues of the ACE2 molecule from humans, macaques , pig (b), guinea pig (c), dog (d), chicken (e), and mouse (f). Download Copyright © 2021 Adams et al.2021Adams et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo by gene editing  and SARS-CoV RBD hACE2 complex (PDB ID 2AJF). WIV-1 RBD was modeled using the reference sequence, GenPept accession no. AGZ48828.1, and P5L RBD was modeled using GenBank accession no. MT040335.1. Additional receptor interactions were modeled from the following translated reference sequences: chicken, GenBank accession no. XM_416822.5; guinea pig, GenBank accession no. XM_023562040.1; ferret, GenBank accession no. XM_004758886.2; pig, GenBank accession no. EU518378.1; dog, GenBank accession no. NM_001165260.1; macaque, GenBank accession no. NM_001135696.1; and mink, GenBank accession no. MT560518.1. Contacts were identified and visualized in the PyMOL molecular graphics system (Schrödinger LLC). Structure-based multiple sequence alignment was generated with UCSF Chimera.The ACE2 panel was aligned using Geneious Prime (version 2020.0.5). The hACE2 sequence is found under GenBank accession no. MT461669), icSARS-CoV-2-nLuc (GenBank accession no. MT461671) (KF367457.1). Mouse delayed brain tumor astrocytoma (DBT-9)  , and icW (DBT-9) , 20 cellEach mouse, human, and chimeric ACE2 was expressed in pCDNA3.1 vector that conjugates a C-terminal 6×-His tag to the end of the expressed protein. The hmACE2 panel was generated with a combination of site-directed mutagenesis and custom double-stranded gBlocks gene fragments (IDT). DBT-9 cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen). Expression of each receptor was verified via staining against a 6×-His epitope  and Western blotting.CT) method and fold change over viral RNA in empty vector-transfected cells. SARS-CoV-2 genome equivalents were calculated against a standard curve with a diagnostic genomic assay primer set against Nsp4  of 1 for 1 h. Inoculum was removed, cells were washed with Dulbecco's phosphate-buffered saline (DPBS), and medium was replaced. Receptor usage was analyzed 24 h postinfection. RNA was collected via TRIzol (Invitrogen) and extracted using Direct-zol RNA MiniPrep kit (Zymo Research). Viral RNA was quantified in duplicate via reverse transcriptase quantitative PCR (qRT-PCR) using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) on a QuantStudio 3 (Applied Biosystems). Viral RNA was quantified with a subgenomic diagnostic assay primer set . Host 18nst Nsp4 . ReceptoAll relevant data sets generated or analyzed in this study have been included in the article. Additional information, including replicates, is available upon request.


Moreover, melatonin effectively reduced NLRP3 inflammasome triggering in RLE-6TN cells. Meanwhile, this study demonstrated melatonin thwarting over NLRP3 inflammasome triggering was depending on ROS. In addition, this study found that melatonin activated Nrf2/Heme Oxygenase-1 (HO-1) pathway, with pyroptotic-inhibiting function of melatonin was reverted through a bespoke Nrf2-inhibitor and siNrf2. In summary, this study concluded that melatonin prevents RLE-6TN cellular pyroptosis through Nrf2-triggered ROS downregulation.Pyroptosis has pivotal parts within disease development, rendering this attractive mechanism for novel therapeutics. This investigation aimed at analyzing melatonin roles within pyroptosis together with related mechanistics. RLE-6TN cultures were exposed to varying LPS doses for 4.5 h followed by concomitant culturing in the presence of ATP (5 mM) for 0.5 h to induce injury, and the roles of melatonin, N-Acety-L-cysteine (NAC – a ROS scavenger), ML385 (specific Nrf2 inhibitor) were examined. Apoptosis analysis was performed through lactate dehydrogenase (LDH) activity assays, together with propidium iodide (PI) stain-assay. Intracellular ROS were quantified through 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). Pyrolysis-associated proteins, such as nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific protease-1 P20 (Caspase-1 P20), gasdermin D-N (GSDMD-N), and mature interleukin-1β (IL-1β), were identified through Western blotting. Dataset outcomes demonstrated LPS/ATP induce RLE-6TN cell pyroptosis, while melatonin alleviated this phenomenon, visualized through increased cell survival rate, reduction of LDH discharge and PI Pyroptosis consists of a recently identified pro-inflammation-based type of apoptosis , charactMelatonin (MT) is well known for its role in regulating the circadian rhythm. Furthermore, melatonin also carries anti-oxidant, anti-apoptotic, neuroprotecting, and immune-modulation roles . MelatonThe NLRP3 inflammasome is well studied and plays critical roles in pyroptosis . The NLRROS can be deemed as a major triggering factor for NLRP3 inflammasome . OverwheThis investigation hypothesized that LPS/ATP co-treatment induces pyroptosis in RLE-6TN cells and that melatonin exerts its anti-pyroptotic effect by activating Nrf2 to reduce ROS production and inhibit NLRP3 inflammasome. The study goals were to analyze melatonin roles within pyroptosis, together within related molecular mechanisms.2.2.1All in vivo studies were conducted in line with the National Institutes of Health Guidelines for the Use of Laboratory Animals. Male C57BL/6 murines (Seven-weeks-old) were procured through Chongqing Medical University Animal Center. Animals had ad libitum water/chow-food and were kept in a regulated environment , and they were randomized within three separate groups (n = 6/group): control-group; LPS-group; LPS + melatonin-group. Control-murines were treated with sterilized saline. ALI was induced as previously described . Briefly2.2Lung tissues were fixated with 4% paraformaldehyde, followed by dehydration, paraffin-embedding, and slicing. Slices were stained with hematoxylin and eosin (H&E) and evaluated under a light microscope.2.3TM, USA), and cultured within RPMI-1640 augmented with 10% fetal bovine serum (FBS) using a 5% carbon-dioxide incubator.RLE-6TN cultures were procured through American Type Culture Collection /melatonin  were placed into solution containing sterilized/de-ionized water followed by serial dilution preparations as necessary. ATP, NAC, and ML385 were procured through Sigma-AldrichTM . The classic two-step approach of NLRP3 inflammasome activation in alveolar macrophages (AMs) [LPS  was added each well at 37°C in dark conditions, for 15 minutes. Eventually, DAPI  was employed for staining cells (10 minutes in dark conditions). Post-staining, images of the cells were immediately acquired by fluorescence microscopy. The fluorescence intensity was assessed using ImageJ software.Apoptosis was assessed through LDH discharge/PI stain-testing. LDH present within cellular culture supernatants was evaluated through LDH assay kit®  in line with kit protocols. Regarding PI staining, the methods were followed according to protocols by Su et al. . In brie2.6The half-maximal inhibitory concentration (IC50) for LPS/ATP and optimal study-doses for melatonin were ascertained through CCK-8 assay as previously described . CCK-8 a2.7TM, Tokyo, Japan). Mean fluorescence intensity was evaluated through ImageJ software.ROS was detected as previously described . Photos Malondialdehyde (MDA), superoxidedismutase (SOD) levels within lung tissue homogenates were quantified in line with corresponding kit protocols .2.8TM . RLE-6TN cells were transfected with siNrf2 and negative control (NC) siRNA using the Lipofectamine 3000 reagent , according to the manufacturer’s protocols. Western blotting was applied to assess transfection efficiency.Both siNrf2 and negative control (NC) siRNA were purchased from RiboBio2.9Cytoplasmic and nuclear proteins were extracted as previously described . Nuclear2.10TM, China) containing phenylmethylsulfonyl fluoride  was employed for total cellular proteomic content extraction, with all homogenate samples subjected to centrifuging . SDS-PAGE (8%-10%) gels were employed for separating denatured proteins and consequently transferred onto PVDF membranes. BSA (5%) was employed for membrane-blocking (90 minutes/room temperature). This step was followed by overnight incubating with primary antibodies for NLRP3 (ab214185), ASC (WL02462), Caspase-1 P20 (AF4005), GSDMD-N (ab215203), IL-1β (A11369), Nrf2 (ab62352), HO-1 (ADI-SPA-895), NQO1 (WL04860), GAPDH (Lot 0094782) and Histone-H3 . The final step involved incubation with horseradish peroxidase-conjugated secondary antibodies (90 minutes/room temperature) prior to analysis.Yan et al.  protocol2.11TM, Wuhan, China) and interleukin-18 (IL-18)  pulmonary levels were evaluated through ELISA kits, in line with kit protocols.IL-1β (Boster2.12.TM) for 20 minutes, blocked with 10% goat serum for 60 minutes, and consequently incubated with Nrf2 primary antibodies (1:200) overnight, in a humid chamber at 4°C. Consequently, cells were incubated with Cy3-conjugated secondary antibody (1:40) for 60 minutes, and subsequently stained using DAPI  in dark conditions, for 5 minutes. Images were taken by fluorescence microscopy . ImageJ software was used to evaluate fluorescence intensity.Immunofluorescence staining was performed according to the previously described protocols . RLE-6TN2.13TM version 8.0). The significance of the difference between negative control siRNA and siNrf2 was determined using Student’s t-tests. P values <0.05 were deemed to confer statistical significance.Datasets were presented as mean ± standard-deviation (SD) of three separate replicates. Group variations were assessed through one-way ANOVA/Bonferroni’s multiple comparisons test  represents a highly acute inflammation status together with diffused alveolar/epithelial injuries . ApoptosImmune-system dysregulated function was always deemed to be a major influencer for inflammation-related diseases. Inflammasomes can be intimately linked to inflammatory diseases. The term ‘inflammasome’ was originally coined by Martinon et al. in 2002 . PresentROS are established to have pivotal parts for activating the NLRP3 inflammasome  togetherNrf2/HO-1 pathway serves as a central regulator of antioxidant activity . PreviouMelatonin has been used for treating multiple inflammatory conditions due to its powerful anti-inflammatory and antioxidant effects. This compound is able to scavenge free radicals directly or indirectly by stimulating antioxidant enzyme activity, as well as through inhibition of pro-oxidative enzyme activity . AnotherHowever, some studies have found that melatonin even promotes the production of ROS in several tumor and non-tumor cell lines . These c5.We only discussed the role of a single cell. Alveolar macrophages were confirmed to have a pivotal part within ALI. Therefore, it is necessary to further investigate the associations for alveolar macrophages/epithelial cells and corresponding mechanisms.6.In essence, this investigation revealed that melatonin lowers the generation of ROS, driven by LPS/ATP, through targeting Nrf2 activity, thereby thwarting NLRP3 inflammasome to ease pyroptosis – in vitro and vivo. Moreover, these findings provided novel views into possible cellular and molecular mechanisms implicated within ALI/ARDS. At the same time, it also paves the way for future research on the interaction between alveolar macrophages and epithelial cells.


Hox genes are not expressed in frontonasal and first pharyngeal arch (PA1) CNCCs, whereas a single Hox gene, Hoxa2, is necessary to provide patterning information to second pharyngeal arch (PA2) CNCCs. In frog, chick and mouse embryos, ectopic expression of Hoxa2 in Hox-negative CNCCs induced hypoplastic phenotypes of CNCC derivatives of variable severity, associated or not with homeotic transformation of a subset of PA1 structures into a PA2-like identity. Whether these different morphological outcomes are directly related to distinct Hoxa2 overexpression levels is unknown. To address this issue, we selectively induced Hoxa2 overexpression in mouse CNCCs, using a panel of mouse lines expressing different Hoxa2 ectopic expression levels, including a newly generated Hoxa2 knocked-in mouse line. While ectopic Hoxa2 expression at only 60% of its physiological levels was sufficient for pinna duplication, ectopic Hoxa2 expression at 100% of its normal level was required for complete homeotic repatterning of a subset of PA1 skeletal elements into a duplicated set of PA2-like elements. On the other hand, ectopic Hoxa2 overexpression at non-physiological levels  led to an almost complete loss of craniofacial skeletal structures. Moreover, ectopic Hoxa5 overexpression in CNCCs, while also resulting in severe craniofacial defects, did not induce homeotic changes of PA1-derived CNCCs, indicating Hoxa2 specificity in repatterning a subset of Hox-negative CNCCs. These results reconcile some discrepancies in previously published experiments and indicate that distinct subpopulations of CNCCs are differentially sensitive to ectopic levels of Hox expression.Providing appropriate positional identity and patterning information to distinct rostrocaudal subpopulations of cranial neural crest cells (CNCCs) is central to vertebrate craniofacial morphogenesis.  The multipotent neural crest cells delaminate dorsally along the rostrocaudal axis of the forming neural tube and migrate towards various regions of the developing embryo, where they locally differentiate into a broad range of cell lineages . In the Hox gene expression. CNCCs contributing to the FNP and first pharyngeal arch (PA1) do not express Hox genes, whereas CNCCs contributing to the second and more posterior arches display nested combinatorial Hox expression patterns, providing each PA with distinct regional identities along the rostrocaudal axis [Hox genes in establishing rostrocaudal identity of CNCC derivatives first became evident with the targeted inactivation of Hoxa2 in the mouse, which resulted in homeotic transformation of PA2 into a subset of PA1-like derivatives [Hoxa2 knockout mice, the PA2-derived stapes, styloid process and lesser horns of hyoid bone were absent and replaced by proximal PA1-like structures including the malleus, incus, gonial bone and tympanic ring in a mirror image pattern of their normal counterparts [Hoxa2 downregulation in Xenopus [Hoxa2 for PA2 CNCC identity specification.Rostrocaudally, a fundamental molecular difference among CNCC subpopulations concerns their patterns of dal axis ,4,6,7,8.ivatives ,10. Nameterparts ,10. Furtterparts , was absterparts ,12. Hoxa Xenopus  and zebr Xenopus  also indHoxa2 ectopic expression in Hox-negative CNCCs led to different morphological phenotypes. Several studies reported a repatterning of proximal PA1 into PA2-like derivatives [Hoxa2 in chick Hox-negative CNCCs abolishes the development of the entire facial skeleton, including frontonasal structures and jaws [Hoxa2 in the chondrogenic regions [Hoxa2, both in the CNCCs and surrounding tissues. When only CNCCs were targeted in chick embryos, the loss of facial structures was observed but not homeotic repatterning of PA1 elements [In contrast, distinct experiments analyzing ivatives ,15,16,17ivatives . Rather,and jaws . These d regions . To explelements .Wnt1-Cre-dependent conditional ectopic expression of Hoxa2 in CNCCs is alone sufficient to induce a repatterning of proximal PA1 into PA2-like skeletal elements, including mirror image duplication of the pinna [Hoxa2 to repattern Hox-negative CNCC subpopulations [However, recent studies in the mouse have shown that he pinna ,16. Notahe pinna ,16. Nameulations ,16.Hox genes in the repatterning of Hox-negative CNCC derivatives. In the chick, Creuzet et al., 2002 [Hoxa2, Hoxa3 or Hoxb4 in Hox-negative CNCCs had negative effects on facial skeleton formation, albeit at different extents. While ectopic Hoxa2 expression caused the absence of the entire Hox-negative CNCC derivatives, the effects of Hoxa3 and Hoxb4 were more restricted [Hox-negative CNCCs following Hoxa2 overexpression, it is still unclear whether ectopic expression of Hox genes other than Hoxa2 in the mouse might also induce repatterning into PA2-like derivatives.Lastly, little is known about the specificity of l., 2002 , showed stricted . Howeverstricted , did notHoxa2 expression may differentially affect the morphogenesis of Hox-negative CNCC subpopulations. Here, we evaluated how distinct ectopic Hoxa2 expression levels differentially affect Hox-negative CNCC subpopulation morphogenesis. To this aim, we induced Wnt1::Cre-dependent conditional Hoxa2 overexpression in CNCCs using mouse lines that generate different Hoxa2 ectopic expression levels. Namely, we used our previously reported mouse allele [Hoxa2 ectopic expression at only 60% of endogenous levels in PA2, and a newly generated allele, which expresses high, non-physiological, levels of ectopic Hoxa2, namely 200% of endogenous levels. We further compared the obtained phenotypes with those described in Kitazawa et al., 2015 [Wnt1::Cre-dependent conditional ectopic Hoxa2 expression in Hox-negative CNCCs comparable to physiological endogenous levels (100%) in PA2. In this study, a repatterning of proximal PA1 into a full set of PA2-like derivatives  was observed alongside with a severe impairment of more anterior skeletal structures [Hoxa2 overexpression at 60% of its physiological level induced only a partial repatterning of proximal PA1 into PA2-like skeletal elements, including full duplication of the pinna, and relatively modest hypoplasia of more anterior skeletal structures. In contrast, high, non-physiological, ectopic levels (200%) of Hoxa2 overexpression caused dramatic hypoplasia and/or aplasia of craniofacial elements including PA2 and proximal PA1 derivatives, without clear homeotic phenotype. Lastly, we assessed Hox gene specificity in repatterning of craniofacial elements by analyzing mice displaying Wnt1::Cre-dependent Hoxa5 conditional overexpression in CNCCs. We found that overexpression of Hoxa5 in CNCCs induced a loss of proximal PA1 and PA2–PA4 structures without homeotic transformation, indicating specificity of Hoxa2 in the repatterning of a subset of PA1 into PA2-like CNCC derivatives.One possibility to reconcile the distinct morphological phenotypes reported by the different studies is that different threshold levels of ectopic e allele  and showl., 2015 , which aructures . Here, wAll animal experiments were performed in accordance with Guide for Care and Use of Laboratory Animals and were approved by the Veterinary Department of the Canton of Basel-Stadt.CAG promoter and WPRE sequence to induce high levels of Hoxa2 overexpression (ROSA26CAG::(lox-stop-lox)-Hoxa2-IRES-EGFP-WPRE) in a Cre-dependent manner, with a similar strategy as for the CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREROSA26 mouse line [ROSA26 locus the targeting vector pR26-CAG-lsl-3xFLAG-Hoxa2-IRES-GFP, consisting of a CAG promoter, a lox-stop-lox cassette, a Hoxa2 tagged with a 3xFLAG, an IRES-GFP, a WPRE element, a bGH poly(A) and a PGK-Neo cassette. To generate this plasmid, we used pR26-CAG-lsl-Kir -Hoxa2-IRES-EGFP-WPREROSA26 allele was obtained. Heterozygous and homozygous mice were viable and fertile.We generated a mouse line that utilizes use line . By homoBendito; ), in whiBendito;  and clon(lox-stop-lox)-Hoxa2-IRES-EGFPROSA26 [CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREROSA26 [CAG::(lox-stop-lox)-Hoxa2-IRES-EGFP-WPREROSA26 alleles were initially generated in the background of E14 ESCs. Germline-transmitted alleles were crossed with the CD1 mouse line and maintained as homozygous mice. Homozygous (lox-stop-lox)-Hoxa2-IRES-EGFPROSA26, CAG::(lox-stop-lox)-Hoxa2-IRES-EGFP-WPREROSA26 or CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREROSA26 females were crossed with the Wnt1::Cre transgenic mouse line [(lox-stop-lox)-Hoxa2-IRES-EGFPWnt1::Cre;ROSA26 (Wnt1-Hoxa2-low), CAG::(lox-stop-lox)-Hoxa2-IRES-EGFP-WPREWnt1::Cre; ROSA26 (Wnt1-Hoxa2-high) or CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREWnt1::Cre;ROSA26 (Wnt1-Hoxa5) mice, respectively.RES-EGFP , ROSA26CGFP-WPRE  and newluse line  on C57B/Wnt1-Hoxa2-low (n = 4), Wnt1-Hoxa2-high (n = 6) and Wnt1-Hoxa5 (n = 9) fetuses was performed according to a previously described protocol with minor modifications [(lox-stop-lox)-Hoxa2-IRES-EGFPROSA26  mice with a distinct Wnt1::Cre line [n = 6), showing undistinguishable phenotype from the Wnt1-Hoxa2-low mice reported here. Samples were fixed in 95% ethanol for 5–7 days. Subsequently, embryos were incubated with 0.015% alcian blue 8GS, 0.005% alizarin red S and 5% acetic acid for 3 days with agitation at 37 °C. Samples were cleared in 1% KOH for several days and in 1% KOH/glycerol series until surround tissues turned transparent. The samples were stored in glycerol for a longer term.Skeletal staining of mouse E18.5 ications ,24. Addiications  generateCre line  (n = 6),Wnt1-Hoxa2-low and Wnt1-Hoxa2-high mice in a comparable manner with previous Wnt1-Hoxa2-medium mice analysis [Hoxa2 and Gapdh was performed by ΔΔCt using Gapdh as an internal control. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) post hoc tests.We carried out RNA extraction from PA1 and PA2 in E10.5 wild-type, analysis . The PA1Wnt1::Cre [Hoxa2 overexpression was achieved in mouse Hox-negative premigratory CNCC progenitors by knock-in of a CAG promoter-(lox-stop-lox)-Hoxa2 cassette into ROSA26 to generate the CAG::(lox-stop-lox)-Hoxa2ROSA mouse line. RT-qPCR analysis in E10.5 embryos showed that ectopic Hoxa2 expression in PA1 was at a level comparable to endogenous Hoxa2 expression in wild-type PA2 (100%) [Wnt1-Hoxa2-medium.In a previous study , Wnt1::Cnt1::Cre -dependen2 (100%) . We refeWnt1-Hoxa2-medium fetuses, ectopic Hoxa2 expression resulted in two categories of phenotypes -Hoxa2-IRES-EGFPROSA mouse line [lox-stop-lox)-Hoxa2-IRES-EGFP cassette was knocked-in to the ROSA26 locus. To conditionally overexpress Hoxa2 in CNCCs, this line was crossed with the Wnt1::Cre transgenic mouse line [Hoxa2 expression driven by the ROSA26 promoter was significantly lower, about 60%, as compared to the endogenous Hoxa2 expression in wild-type PA2 -Hoxa2-IRES-EGFPWnt1::Cre; ROSA  line as Wnt1-Hoxa2-low. We also re-analyzed (lox-stop-lox)-Hoxa2-IRES-EGFPROSA26 mice from our previous study [Wnt1::Cre line [Wnt1-Hoxa2-low specimen (see below).To assess how differential ectopic use line ,22, wheruse line . RT-qPCRtype PA2 A, likelyl., 2015 . We refeus study  crossed Cre line , showingWnt1-Hoxa2-low E18.5 fetuses displayed a fully duplicated pinna, as previously reported [Hoxa2 expression can induce pinna duplication, while not sufficient for the complete homeotic transformation of skeletal elements. Moreover, in E18.5 Wnt1-Hoxa2-low fetuses, structures derived from the FNP and distal PA1, e.g., facial, nasal, premaxillary, nasal septum, vomer, palatine, pterygoid, alisphenoid, dentary, maxillary, jugal and squamous, were reduced or absent. This suggests that the severity of hypoplasia and malformation or aplasia of individual elements derived from rostral Hox-negative CNCC subpopulations is differentially dependent on local levels of ectopic Hoxa2 expression.reported  -Hoxa2-IRES-EGFP-woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) cassette into the mouse ROSA26 locus to generate CAG::(lox-stop-lox)-Hoxa2-IRES-EGFP-WPREROSA mice. The CAG promoter is shared with the CAG::(lox-stop-lox)-Hoxa2ROSA mouse line [WPRE sequence further enhances the expression of the transgene by increasing nuclear and cytoplasmic mRNA transcript levels [Hoxa2 in CNCCs, we crossed this line with the Wnt1::Cre transgenic mouse line [IRES sequence -Hoxa2-IRES-EGFP-WPREWnt1::Cre; ROSA line as Wnt1-Hoxa2-high.To assess how high, non-physiological, levels of use line , but thet levels . To overuse line . In E10.sequence C. In addpression B. We refWnt1-Hoxa2-high fetuses showed extensive hypoplasia and/or aplasia of craniofacial structures derived from FNP and distal PA1 -Hoxa5-IRES-EGFP-WPRE cassette, driving high levels of Hoxa5 expression, was knocked-in to the ROSA26 locus to generate the CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREROSA mouse line [Wnt1::Cre transgenic mouse line [Wnt1::Cre; CAG::(lox-stop-lox)-Hoxa5-IRES-EGFP-WPREROSA line as Wnt1-Hoxa5.To assess the specificity of ectopically-expressed use line . This liuse line . We refeHoxa5 overexpression from the Hoxa5-IRES-EGFP-WPRE allele, we detected EGFP expression at E18.5  were sufficient to induce the formation of an ectopic duplicated pinna in Hox-negative CNCCs  allowed for the repatterning of proximal PA1 CNCCs into a full set of PA2-like skeletal elements  generated the most severe phenotype, with almost complete aplasia of craniofacial cartilage and bone elements, including the proximal PA1 and PA2 derivatives. This analysis indicates that the severity of hypoplasia/malformation, or aplasia, of craniofacial structures correlates with Hoxa2 overexpression levels. Altogether, these results reconcile the discrepancies in previously published experiments and indicate that distinct subpopulations of Hox-negative CNCCs are differentially sensitive to ectopic levels of Hox expression.These contradictory results are likely due to the different experimental approaches in distinct model organisms. Here, we investigated whether different Hoxa2 to specify CNCC patterning information is position- and context-dependent. In Hoxa2 knockout mouse fetuses, only derivatives of the proximal part of PA1, including the middle ear ossicles incus and malleus-homologous to the quadrate and articular cartilage forming the primary upper and lower jaw joint articulation in non-mammalian jawed vertebrates (the ‘hinge’ region of PA1 [Hoxa2 specifies PA2 CNCCs’ identity by modifying an underlying Hox-negative ground (default) patterning program, shared by the proximal PA1 and PA2 CNCCs [Hoxa2 was close to its physiological expression levels , only the proximal PA1 CNCC derivatives  could acquire a PA2-like identity. In contrast, the other Hox-negative CNCCs’ subpopulations, including those contributing to the distal PA1 and FNP, could not be repatterned. Their development was instead impaired, with varying degrees of severity, depending on the ectopic expression levels of Hoxa2. The two distinct categories of phenotypes observed by Hoxa2 ectopic expression in Hox-negative CNCCs  indicate that among Hox-negative CNCCs, only those sharing the ground (default) patterning program with PA2 , were dn of PA1 ,10,12. IA2 CNCCs ,17. In oHox-negative CNCCs is not yet established at the premigratory stage. Indeed, Hox-negative premigratory CNCC progenitors can replace each other in building a whole craniofacial skeleton [Hox-negative CNCCs’ subpopulations , driving the morphogenetic program to make the right structure in the right place [Hox-negative ground (default) patterning program shared by proximal PA1 and PA2 CNCCs is established in post-migratory CNCCs.The positional identity of the skeleton . Becauseskeleton . Recentlskeleton . In respht place . This fuHoxa2 to repattern Hox-negative CNCCs would then absolutely depend on the presence of permissive local environmental signal(s). The mirror image homeotic transformation of PA2 into a subset of PA1 derivatives in Hoxa2 knockout mutants strongly implies that (at least part of) PA1 and PA2 derived CNCCs are bi-directionally instructed by the same set of ectodermal and/or endodermal signals at the pharyngeal cleft and/or pouch between PA1 and PA2, and that their distinct morphological readouts solely depend on whether Hoxa2 is expressed  or not  [Hox-negative CNCCs, only those able to respond to the same signal(s) that pattern PA2 CNCCs, i.e., the proximal PA1 CNCCs, would acquire a PA2-like identity upon ectopic expression of Hoxa2. In contrast, overexpression of Hoxa2 in the other Hox-negative CNCCs’ subpopulations, including distal PA1 and FNP, would make them incompatible to appropriately respond to the local environmental signals that normally instruct them, causing a dramatic impairment of the development of the corresponding skeletal structures [Wnt1-Hoxa2-low fetuses occasionally display ectopic structures around the eye, morphologically resembling small ectopic pinnae, suggesting that signals compatible with pinna formation are also present there [As a corollary, the ability of mal PA1) ,12. Hencructures . In thisnt there .Wnt1-Hoxa2-low, Wnt1-Hoxa2-medium and Wnt1-Hoxa2-high mouse lines display hypoplasia of craniofacial structures. However, while hypoplasia was relatively modest in Wnt1-Hoxa2-low and Wnt1-Hoxa2-medium fetuses  in Hox-negative CNCCs resulted in negative, albeit not identical, effects on the development of the craniofacial skeleton, with no homeotic phenotypes [Hoxa3 and Hoxb4 ectopic expression were more restricted, selectively affecting the chondrogenesis of distinct CNCC derivatives [Hox genes other than Hoxa2 might also induce repatterning of PA1 into PA2-like derivatives.In the chick, ectopic expression of enotypes . In partivatives . Given tWnt1-Hoxa2-high and Wnt1-Hoxa5 fetuses, in which Hoxa2 and Hoxa5 were strongly expressed in CNCCs using the same induction systems . We found that overexpression of Hoxa5, a Hox gene normally not expressed in CNCCs giving rise to craniofacial structures, surprisingly induced milder hypoplasia of FNP and distal PA1 derivatives, as compared to Hoxa2 overexpression. In contrast, the pharyngeal arch CNCC derivatives, including proximal PA1 and PA2–PA4 structures  were notably absent in Wnt1-Hoxa5 fetuses, thus marking a remarkable difference as compared with Hoxa2 overexpression. Moreover, in Wnt1-Hoxa5 fetuses there was no homeotic transformation of PA1 into PA2-like elements (summary schemes in Hoxa2 and Hoxa5 overexpression levels in CNCCs were likely similar, they resulted in relatively distinct and specific phenotypes, consistent with the observations in chick embryos by Creuzet et al., 2002 [Here, we compared the craniofacial phenotypes of l., 2002 .IRES-EGFP in Wnt1-Hoxa2-high and Wnt1-Hoxa5 embryos it is possible to isolate them by FACS, as we previously did with Wnt1-Hoxa2-low embryos [Hox gene-mediated cell identity specification during development.Finally, it is still relatively unclear how Hox proteins select and bind to their target sequences in vivo to regulate specific downstream target genes during morphogenesis. In this respect, even though beyond the scope of this study, our experimental design might be useful as it would allow to compare the direct binding sites of different Hox transcription factors, expressed at similar levels, in mouse embryonic tissues and cell types in vivo. For instance, since CNCCs are labelled by  embryos . Moreove


PAX8 defects underlie only 1% of these cases and NKX2-1 does not seem to be involved. Here, we analyzed other TD-related genes in 128 non-related Mexican patients  with non-syndromic CH-TD diagnosis established by clinical evaluation, thyroid hormone serum profiling, and scintigraphy (74%) or ultrasonography (26%). We performed Sanger sequencing of FOXE1, NKX2-5, and TSHR and evaluated copy number variations (CNVs) in TSHR, FOXE1, PAX8, and NKX2-1 by multiplex ligation-dependent probe amplification. Odds ratios for TD risk were explored for FOXE1 polyalanine stretches [polyAla-rs71369530] in cases and controls (N = 116). Five rare missense changes cataloged as benign (NKX2-5:p.(Ala119Ser)-rs137852684), of unknown significance (FOXE1:p.(Ala335Gly)-rs543372757; TSHR:p.(Asp118Asn)-rs1414102266), and likely pathogenic (FOXE1:p.(Gly124Arg)-rs774035532; TSHR:p.(Trp422Arg)-rs746029360) accounted for 1.5% (N = 2/128) of clinically relevant genotypes (supported in part by protein modeling) in CH-TD. No CNVs were identified, nor did polyAla > 14 alanines in FOXE1 significantly protect against TD. The present and previously published data collectively show that small clinically relevant germline variants in PAX8, FOXE1, and TSHR are found in only a very small proportion (2.5%) of isolated CH-TD Mexican patients.Mexico shows a high birth prevalence of congenital hypothyroidism (CH) due to thyroid dysgenesis (TD).  PAX8, FOXE1, NKX2-1, TSHR, etc.,) whose mono- or biallelic loss-of-function genotypes, even interacting in a polygenic model, may explain the etiology of 5–10% of CH-TD patients ;[Gly124Arg] and compound heterozygous TSHR p.[Asp118Asn];[Trp422Arg], were found to account for 1.5% (N = 2/128) of the CH-TD patients, but further functional and segregation analyses are warranted.Two previously unreported and clinically relevant genotypes, homozygous PAX8, NKX2-1, FOXE1, or TSHR.Unlike Polish CH patients , MexicanNKX2-5, our data support the idea that it has negligible etiological participation in TD along with a benign character for its p.(Ala119Ser) allele [Although we did not evaluate CNVs in ) allele .FOXE1 polyAla alleles larger than 16 alanines or their genotypes in French Caucasians [The protective effect on TD risk previously described for ucasians  was not