Candida, and antioxidant activity of gibberellin A4 (GA4) and A7 (GA7) compounds, and further determined their toxicity in vivo. Methods: GA4 and GA7 in vitro toxicity was determined by MTT method, and nontoxic concentrations were then tested to evaluate the GA4 and GA7 anti-NF-κB activity in LPS-activated RAW-luc macrophage cell culture (luminescence assay). GA4 in silico anti-NF-κB activity was evaluated by molecular docking with the software “AutoDock Vina”, “MGLTools”, “Pymol”, and “LigPlot+”, based on data obtained from “The Uniprot database”, “Protein Data Bank”, and “PubChem database”. The GA4 and GA7 in vitro anti-Candida effects against Candida albicans (MYA 2876) were determined (MIC and MFC). GA7 was also evaluated regarding the viability of C. albicans preformed biofilm (microplate assay). In vitro antioxidant activity of GA4 and GA7 was evaluated against peroxyl radicals, superoxide anions, hypochlorous acid, and reactive nitrogen species. GA4 and GA7 in vivo toxicity was determined on the invertebrate Galleria mellonella larvae model. Results: Our data show that GA4 at 30 µM is nontoxic and capable of reducing 32% of the NF-κB activation on RAW-luc macrophages in vitro. In vitro results were confirmed via molecular docking assay (in silico), since GA4 presented binding affinity to NF-κB p65 and p50 subunits. GA7 did not present anti-NF-κB effects, but exhibited anti-Candida activity with low MIC (94 mM) and MFC (188 mM) values. GA7 also presented antibiofilm properties at 940 mM concentration. GA4 did not present anti-Candida effects. Moreover, GA4 and GA7 showed antioxidant activity against peroxyl radicals, but did not show scavenging activity against the other tested radicals. Both compounds did not affect the survival of G. mellonella larvae, even at extremely high doses (10 g/Kg). Conclusion: Our study provides preclinical evidence indicating that GA4 and GA7 have a favorable low toxicity profile. The study also points to GA4 and GA7 interference with the NF-κB via, anti-Candida activity, and a peroxyl radical scavenger, which we argue are relevant biological effects.Introduction: Gibberellins (GA) are terpenoids that serve as important plant hormones by acting as growth and response modulators against injuries and parasitism. In this study, we investigated the in vitro anti-NF-κB, anti- Gibberellins (GA) are a large group of tetracyclic diterpenoid carboxylic acids that serve as important plant hormones by acting as growth modulators ,2. In seEscherichia coli, Raoultella planticola, Pseudomonas aeruginosa, Salmonella nottinglham, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus), while GA7 seems to be less effective, inhibiting the growth of P. aeruginosa, S. nottinglham, B. cereus, L. monocytogenes, and S. aureus ” and gene: nfkb1 AND organism: “Mus musculus (Mouse) [10090]”. A new search was performed in the Protein Data Bank [To investigate the binding capacity of GA4 to NF-κB subunits, we carried out a molecular docking analysis using the AutoDock Vina program . The Uniata Bank  to obtaiata Bank , in whicCandida activity of GA4 and GA7 was determined against Candida albicans MYA 2876. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the compounds were determined by the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) protocols. Briefly, an inoculum of 103 colony-forming units (CFU) per mL was prepared in RPMI medium supplemented with L-glutamine (2 mM), glucose (2%), and MOPS buffer . GA4 and GA7 were diluted in RPMI medium and tested at concentrations ranging from 2.9 mM to 188 mM. Amphotericin B (AmB) was used as a positive control at concentrations ranging from 0.54 mM to 1.1 mM. The plates with the inocula and treatments were incubated statically for 24 h. The MIC was determined visually as the lowest concentration of the drug that did not produce any visible turbidity in the wells. Aliquots from the wells with no visible cell growth were plated onto agar sabouraud medium (SDA) plates and incubated for 24 h in an aerobic atmosphere. The MFC was defined as the lowest concentration of the subculture at which no visible growth is observed on solid media. The assays were carried out in triplicate via three independent experiments [The anti-Candida albicans MYA 2876 biofilms were grown in 96-well plates by adding 200 μL of inoculum (108 CFU/mL) to YNB media. The plates were incubated statically at 37 °C for 24 h in an aerobic atmosphere for biofilm formation. The supernatant was discarded, and the wells were washed to remove non-adhered cells. GA7 or AmB (positive control) were prepared in YNB at 2×MIC or 10×MIC . GA4 was not tested against the C. albicans biofilm as it showed no antimicrobial activity. Biofilms were treated at 37 °C for 24 h in an aerobic atmosphere. The supernatants were discarded, and the wells were washed to remove non-adhered or killed cells. Biofilms were homogenized, serially diluted, and plated onto SDA for CFU counting. The assays were carried out in triplicate via three independent experiments [® M3, Molecular Devices LLC, Sunnyvale, CA, USA). Trolox was used as a standard (12.5 μM to 400 μM), and the results are expressed as μmol Trolox equivalents per mg of GA4 or GA7 (μmol TE/mg).The peroxyl radical (ROO•) scavenging activity of the gibberellins was determined according to previous authors . Briefly50, which indicates the minimum amount (mg/mL) of GA4 or GA7 required to quench 50% of the superoxide radicals [NADH (166 µM), nitrotetrazolium blue chloride (NBT) (107.5 µM), GA4 or GA7 (23 mM to 727 mM), phenazine methosulfate (PMS) (2.7 µM), and the diluent potassium phosphate buffer  were added to a final volume of 300 µL. The assay was carried out at 25 °C, and absorbance was measured at 560 nm after 5 min. Potassium phosphate buffer was used as a blank. Trolox was used as a standard (12.5 μM to 400 μM), and the results are expressed as ICradicals .2SO4) were added to a final volume of 300 µL into a 96-well microplate. Fluorescence was measured immediately in a microplate reader  with emission at 528 ± 20 nm and excitation at 485 ± 20 nm. The results are expressed as IC50 (μg/mL) of GA4 or GA7 [Briefly, GA4 or GA7 solution (23 μM to 727 mM) homogenized in phosphate buffer , DHR (1.25 µM diluted in 1.15 mM dimethylformamide), and HOCl (5 µM diluted in 1% sodium hypochlorite (NaOCl) solution, adjusted to 6.2 with H4 or GA7 .® M3, Molecular Devices LLC, Sunnyvale, CA, USA) with excitation at 495 nm and emission at 515 nm. Measurements were taken over a 120 min period at 5 min intervals. The results are expressed as IC50 (μg/mL) of GA4 and GA7 [Briefly, 50 μL of GA4 or GA7 solution (23 mM to 727 mM) were added to a 96-well microplate with 50 μL of sodium nitroprusside (SNP) solution 10 mM), 50 μL of phosphate buffer , and 50 μL of diaminofluorescein (DAF) solution (25 μM). Fluorescence was measured in a microplate reader . Larvae were grown at 37 °C, and those with no signs of melanization, and weighing 200 to 300 mg, were randomly selected (n = 10/group). Each larva received 10 μL of GA4 or GA7 at 58.8 mM, 117.5 mM, 235.0 mM, 470.0 mM, and 940.0 mM  or saline solution (control group) in the hemocoel via the last left proleg using a 10 μL Hamilton syringe. All groups were incubated at 37 °C, and survival was monitored at selected intervals for up to 72 h. Larvae showing high melanization and absence of movements upon touch were counted as dead [To determine the in vivo toxicity of GA4 and GA7, we carried out a survival assay on  as dead .G. mellonella larvae was compared using the log-rank Mantel−Cox test. In all assays, the sample size was calculated a priori considering an 80% statistical power and a 5% α error. The data were analyzed in GraphPad Prism, version 6.0 for Windows .Data were checked for homoscedasticity and normality, and showed normal distribution. NF-κB activation and biofilm data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, and cell viability data were analyzed by unpaired Student’s t-test. The percent survival of Candida, and antioxidant properties of commercially available gibberellin A4 (GA4) and gibberellin A7 (GA7). Briefly, our data show that GA4 significantly reduced NF-κB activation, whereas GA7 exhibited anti-Candida activity against Candidaalbicans, with low MIC/MBC values and antibiofilm properties. Moreover, GA4 and GA7 showed peroxyl radical scavenging activity, and did not affect the survival of Galleria mellonella larvae, even at extremely high doses (10 g/Kg).This study reports on the anti-NF-κB, anti-A molecular docking assay was further carried out to confirm the binding capacity of GA4 to the p65 and p50 subunits of NF-κB. The five best results were selected, and are shown in GA4 interacted with the protein 2LWW_model_1 A via hydNF-κB is a mediator of an intracellular signaling pathway that is vital to human life, especially for inflammation and immunity responses . Yet, thSeveral external and internal stimuli can trigger the activation of the NF-κB pathway, such as lipopolysaccharides (LPS) from Gram-negative bacteria, and TNF-α and IL1β, respectively . Once acPeriodontal disease is a condition characterized by an exacerbated inflammatory response, which is highly variable in each individual, as a result of an oral microbial dysbiosis . This coC. albicans. GA4 did not show any inhibitory (MIC) or killing (MFC) effects at the tested concentrations, whereas GA7 showed MIC and MFC values of 94 mM and 188 mM, respectively. As expected, the gold standard AmB showed low MIC and MFC values , which were lower than those of GA7.As shown in Candida albicans infections play an important role in the development of systemic infectious diseases [C. albicans cells colonize most healthy individuals and are often recovered from the dorsum of the tongue. As long as the individual’s immunity is functional, the presence of yeasts tends not to cause great harm; however, immunocompromised patients may suffer from candidiasis. Candida albicans oral infection causes discomfort, pain, loss of taste, difficulty swallowing food and liquid, and poor nutrition. However, depending on the severity of the immunosuppression, systemic infections may develop and progress into severe and fatal conditions in 79% of cases due to lack of effective treatment [diseases . C. albireatment ,22 In threatment . TherefoCandida activity [Candida activity of GA7 observed in our study led us to reason that gibberellins are responsible, at least in part, for the anti-Candida properties of their originating Brazilian organic propolis extract. However, other molecules may act synergistically with GA regarding the antibiofilm properties of the extract.Our study hypothesis was conceived after we detected the occurrence of GA7 in a Brazilian propolis sample that showed anti-activity . This pe2•−), hypochlorous acid (HOCl), or nitrogen species (RNS). The peroxyl radical is generated in biological systems from lipid peroxidation. An example of this are the polyunsaturated fatty acids present on the surface of most mammal cells that work as key factors for oxidative stress [G. mellonella model is a low-cost, ethical, and reliable method that provides preliminary evidence on the toxicological profile of natural compounds before their further experimentation in other animal models, such as mammal models [Candida (GA7) concentrations determined in vitro. Of note, the injected doses of GA4 and GA7 were extremely high and impossible to be consumed by humans; even so, the compounds showed negligible toxicity in this model. Therefore, we can infer that GA4 and GA7 consumption is likely to be nontoxic to mammals, which warrants further investigation.The antioxidant assay showed that both compounds were effective against the ROO● radical, named peroxyl (220 ± 50 μmol TE/g for GA4 and 610 ± 40 μmol TE/g for GA7), but not against superoxide anion , and which, in addition to being associated with changes in the oral bacterial flora, has a strong inflammatory characteristic and notorious participation in the NF-κB pathway . Likewised hosts . In addiCandida activity against planktonic and biofilm Candida albicans cells. Both GA4 and GA7 showed a significant capacity to scavenge the free radical, peroxyl, and were nontoxic to Galleria mellonella larvae, even at extremely high doses. Collectively, the data show that GA4 and GA7 have biological activities of great interest to future application in medical and dental therapy.To conclude, GA4 showed inhibitory effects on NF-κB activation (in vitro) and binding affinity to NF-κB subunits (molecular docking); GA7 exhibited anti-


Acropora muricata corals changed over 9 months following a reciprocal transplant experiment. Using a combination of metabarcoding, genomics, and comparative genomics approaches, we found that coral colonies separated by a small distance harbored different dominant Endozoicomonas-related phylotypes belonging to two different species, including a novel species, “Candidatus Endozoicomonas penghunesis” 4G, whose chromosome-level (complete) genome was also sequenced in this study. Furthermore, the two dominant Endozoicomonas species had different potentials to scavenge reactive oxygen species, suggesting potential differences in responding to the environment. Differential capabilities of dominant members of the microbiome to respond to environmental change can (i) provide distinct advantages or disadvantages to coral hosts when subjected to changing environmental conditions and (ii) have positive or negative implications for future reefs.Bacteria in the coral microbiome play a crucial role in determining coral health and fitness, and the coral host often restructures its microbiome composition in response to external factors. An important but often neglected factor determining this microbiome restructuring is the ability of microbiome members to respond to changes in the environment. To address this issue, we examined how the microbiome structure of IMPORTANCE The coral microbiome has been known to play a crucial role in host health. In recent years, we have known that the coral microbiome changes in response to external stressors and that coral hosts structure their microbiome in a host-specific manner. However, an important internal factor, the ability of microbiome members to respond to change, has been often neglected. In this study, we combine metabarcoding, culturing, and genomics to delineate the differential ability of two dominant Endozoicomonas species, including a novel “Ca. Endozoicomonas penghunesis” 4G, to respond to change in the environment following a reciprocal transplant experiment. Bacteria are among the main microbial partners in the coral holobiont . They ma5–5–9–13–16–Acropora hemprichii and Pocillopora verrucosa in a long-term cross-transplantation experiment and identified that A. hemprichii harbors a highly flexible microbiome, whereas P. verrucosa has a remarkably stable microbiome, even after exposure to different levels of chronic pollution, suggesting that the bacterial communities of different coral species exhibit differential dynamics under environmental change/perturbation.Of the factors involved in restructuring the coral-associated bacterial community, environmental changes and host specificity are two major drivers influencing the composition of the bacterial community in corals. In terms of environmental changes, numerous studies have reported shifts in the bacterial community composition of corals in response to variations in temperature –25, nutr–In most of the studies conducted to date, factors influencing the changes in the coral-associated bacterial community are often external, such as those mentioned above. Only recently have internal factors like host genotype been shown to also influence the coral microbiome –34. Howe–Endozoicomonas , which, along with many other coral-associated bacteria  or, more recently, amplicon sequence variants (ASVs) from metabarcoding surveys. One such group belongs to the genus bacteria , 36, plabacteria . A recenl stress . If EndoEndozoicomonas, as members of this genus show lower relative abundance in degraded or anthropogenically impacted reefs compared to the one that is healthy  in the Penghu Archipelago, Taiwan. These coral species in the Penghu Archipelago have been reported to harbor Endozoicomonas as their dominant bacteria  had 1,815,002 reads  and 1,966 zOTUs, and seawater (n = 12) had 149,638 reads  and 1,521 zOTUs.We collected a total of 110 coral and 12 seawater samples from the experiment, of which 10 coral fragments were removed  during the experiment  different between coral and seawater samples as denoted by different group annotations (a and b). Download FIG S1, PDF file, 1.6 MB.Alpha diversity analysis. (A) Richness. (B) Shannon-Weiner index. (C) Chao1. (D) Inverse Simpson. No significant differences were observed in coral samples from the Inner or Outer Bay . Alpha diversity estimates were significantly   and Bacteroidetes (22.76%) (class Flavobacteriia) were the dominant phyla in seawater samples across all time points, followed by Cyanobacteria (17.75%), which was particularly abundant in Inner Bay samples. There was heavy rainfall during the week in August that samples were collected, and we noticed a higher abundance of Marinimicrobia (class Marinimicrobia SAR406 clade), Planctomycetes, and Verrucomicrobia in those samples, which might explain the different bacterial diversity and evenness results obtained that month. On the contrary, coral samples were dominated by Proteobacteria  (73.08%), Chlamydiae (13.70%), and Tenericutes (8.99%) (class Mollicutes). Dead coral samples had a bacterial community composition similar to that of seawater samples  from the two locations throughout the experiment. We observed shifts in the microbial community of control samples (IC and OC) from April to August and December. The relative abundance of Chlamydiae (family Simkaniaceae), the second abundant phylum in the Outer Bay (OC), with all zOTUs (10 in count) belonging to “Unclassified Simakaniaceae,” decreased from April to August before increasing slightly in December. Tenericutes , the second dominant phylum in the Inner Bay control samples (IC), increased in abundance over time, peaking in December. One zOTU annotated as “Unclassified Entomoplasmatales” had the highest abundance among different members of Tenericutes, including zOTUs belonging to Acholeplasma, Mycoplasma, “Candidatus Bacilloplasma,” and “Candidatus Hepatoplasma.” In July, we observed a sudden spike in Verrucomicrobia abundance in two samples from IC, but soon after in August the community composition became similar to that in June. We also observed patterns of community dynamics in Chlamydiae and Tenericutes in cross-transplant samples (I→O and O→I) over the sampling period. Chlamydiae became the most dominant group in O→I (May) samples, but its abundance decreased sharply thereafter, whereas in I→O samples, Chlamydiae and Tenericutes both remained stable, with Chlamydiae being dominant in May and June and Tenericutes being dominant in July and August  were taxonomically classified as Endozoicomonas. These 48 zOTUs accounted for an average of ~54% relative abundance in coral fragments and 0% in sea water samples. Of these 48 zOTUs, 13 contributed ~90% of the total Endozoicomonas abundance. Interestingly, IC and OC samples harbored different dominant Endozoicomonas zOTUs, with zOTU1 and zOTU2 being dominant in OC and zOTU7 and zOTU9 in IC. Across the sampling time, we also observed shifts in the dominant Endozoicomonas phylotypes. zOTU2 was dominant from April to June, whereas zOTU1 became dominant in July-December OC samples. In IC samples, zOTU7 was dominant from April to May, but after that its relative abundance declined , with zOTU2 being dominant across all sampling times. For I→O transplanted samples, however, instead of Endozoicomonas phylotypes from IC, we observed that zOTU1, another dominant Endozoicomonas phylotype in OC samples, was dominant , whereas samples whose final destination was the Outer Bay (OC and I→O) had no significant difference , indicating that the microbiome had location specificity. Therefore, samples whose final destinations were the Inner Bay and Outer Bay were analyzed independently to test for differences in community composition between the control and transplant groups. Ordination analysis using nonmultidimensional scaling (nMDS), followed by permutational multivariate analysis of variance (PERMANOVA), identified the significant influence of coral sample, sampling month, and their combined effect (interaction term)  were significantly different from each other  . Ellipseon term)  and B. Ton term)  and B.Endozoicomonas, but zOTUs belonging to diverse taxa, including the BD1-7 clade (Gammaproteobacteria), Entomoplasmatales , and Alteromonadaceae (class Gammaproteobacteria), were differentially abundant in I→O samples , Synechococcus (class Cyanobacteria), and others  analysis identified differentially abundant zOTUs of different taxa across all the sampling groups: nine zOTUs were differentially abundant in OC samples, 13 in I→O samples, and 16 in IC and O→I samples  and D. I samples . Similard others .Endozoicomonas-related phylotypes in the coral samples and their differential robustness after transplantation (i) motivated us to determine their phylogenetic position and (ii) provided an opportunity to isolate and culture these phylotypes. A phylogenetic tree based on 16S rRNA gene sequences and the percent identity match between these sequences confirmed that zOTU1 and zOTU2 were 99.02 and 98.05% identical (16S rRNA V6-V8 region), respectively, to Endozoicomonas acroporae Acr-14T (E. acroporae Acr-14T 16S rRNA gene (Endozoicomonas species (Ca. Endozoicomonas penghunesis” 4G) described in this study (described below), and zOTU9 had 98.70% identity (16S rRNA gene V6-V8 region) with “Ca. Endozoicomonas penghunesis” 4G (Ca. Endozoicomonas penghunesis” 4G 16S rRNA gene (Ca. Endozoicomonas penghunesis” 4G identified seven copies of 16S rRNA (described below) based on percent similarity; 16S rRNA gene copy 1  “Ca. Endozoicomonas penghunesis 4G” 16S rRNA (copy1) and zOTUs related to Endozoicomonas. (C) “Ca. Endozoicomonas penghunesis” 4G 16S rRNA copies percent identity to each other. (D) Phylogenetic tree of 16S rRNA copies of “Ca. Endozoicomonas penghunesis 4G” with Endozoicomonas acroporae Acr-14T as an outgroup. zOTUs represented here is the same used in Endozoicomonas. Download FIG S3, PDF file, 0.6 MB.16S rRNA gene percentage identity heatmaps (A) between Copyright © 2022 Tandon et al.2022Tandon 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 Ca. Endozoicomonas penghunesis” 4G is a Gram-negative, facultatively anaerobic, and slightly motile bacterium that forms beige-colored colonies  and is slightly susceptible to the antibiotics streptomycin and ampicillin. No catalase enzymatic activity was reported for this bacterium, but the bacterial culture was trypsin and oxidase positive (Endozoicomonas montiporae CL-33 (GenBank accession no. CP013251) with 96.17% identity; based on a species identity cutoff of 97%, this suggests that the bacterium is a novel Endozoicomonas species. Further, average nucleotide identity (ANI) analysis on genome sequences confirmed our assertion that “Ca. Endozoicomonas penghunesis” 4G is a novel species (“positive . This nepositive . The pH positive  surroundpositive  and withpositive . A 16S r species E.10.1128/msystems.00359-22.4FIG S4Candidatus Endozoicomonas penghunesis 4G”. (A) Negative stain by 2% phosphotungstate, (B) thin section, and (C) surface of a bacterial colony. Micrographs in panels A and B were imaged under transmission electron microscope; panel C was observed under cyro-scanning electron microscopy. Download FIG S4, PDF file, 12.4 MB.Electron micrographs of “Copyright © 2022 Tandon et al.2022Tandon 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 10.1128/msystems.00359-22.9TABLE S2Candidatus Endozoicomonas penghunesis 4G” and E. acroporae Acr-14T. Download Table S2, DOCX file, 0.02 MB.Differential phenotypic characteristics of “Copyright © 2022 Tandon et al.2022Tandon 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 10.1128/msystems.00359-22.10TABLE S3Candidatus Endozoicomonas penghunesis 4G” and related Endozoicomonas species strain growth conditions. Download Table S3, DOCX file, 0.02 MB.“Copyright © 2022 Tandon et al.2022Tandon 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 Ca. Endozoicomonas penghunesis” 4G was first assembled using Nanopore reads and later polished with quality filtered Illumina reads, resulting in a single contig of 6,004,453 bp and N50 of 6,004,453. Genome completeness, contamination, and strain heterogeneity were estimated to be 97.52, 0.98, and 0%, respectively. Out of 573 single-copy marker genes (c_Gammaproteobacteria) from the checkM database  and identified 43 WD40 domain proteins (WD40), four ankyrin repeat proteins (ARPs), and 12 tetratricopeptide repeat proteins (TRPs). Almost all the WD40 domain-containing proteins were arranged consecutively  were predicted from the genome. We annotated seven copies of 16S, nine of 23S, eight of 5S rRNA genes, and 80 tRNAs. A sequence similarity analysis of 16S rRNA gene copies revealed that all copies were at least 98.76% identical, with four copies >99.28% identical and two 100% identical to each other . Copy-1 is study . There wcutively  and flanl-proline glycine betaine ABC transport system permease (ProV and OusW)  of the total genes into subsystems. Subsystems (i) carbohydrates and (ii) cofactors, vitamins, prosthetic groups, and pigments had the highest number of annotated genes, 270 and 238, respectively . In the nd OusW) . Multipl10.1128/msystems.00359-22.5FIG S5Ca. Endozoicomonas penghunesis” and E. acroporae Acr-14T using the Rapid Annotation Server Tool. Download FIG S5, PDF file, 0.5 MB.RAST annotation. Subsystem level annotation of open reading frames (as percentages) from “Copyright © 2022 Tandon et al.2022Tandon 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 Ca. Endozoicomonas penghunesis” 4G had genes encoding essential amino acids and pathways including glycolysis and tricarboxylic acid cycle; genes for converting nitrate to nitrite  and ammonia to l-glutamate were identified, but none were related to the conversion of nitrite to ammonia. Assimilatory and dissimilatory sulfate reduction and oxidation pathways were also completely absent. Furthermore, no genes related to the uptake of extracellular taurine or its metabolism to sulfite were identified. Interestingly, siroheme biosynthesis and siroheme-dependent anaerobic sulfite reduction operons were present in “Ca. Endozoicomonas penghunesis” 4G  observed in the calmer waters of the Inner Bay. Comparing the metabolic repertoire of the two species, we found that genes for dimethylsulfoniopropionate (DMSP) metabolism and dimethyl sulfoxide (DMSO) reduction were absent from “Ca. Endozoicomonas penghunesis,” but E. acroporae had a complete operon for DMSP metabolism, as reported in our previous study (Ca. Endozoicomonas penghunesis” in the control (IC) and transplant samples (I→O) in summer (June-August). Furthermore, the robustness of E. acroporae in the control (OC) and transplant samples (O→I) throughout the year could be due to the species’ ability to remove oxidative stress (which increased in summer) more efficiently via DMSP metabolism and the presence of catalase activity. This was further confirmed by using the 2,2 diphenyl-1-picrylhydrazyl (DPPH) assay as a proxy for measuring the ROS scavenging ability, where E. acroporae was identified as a strong free radical scavenger compared to “Ca. Endozoicomonas penghunesis” in ambient (25°C) as well as higher temperature conditions (30°C)  .10.1128/msystems.00359-22.6FIG S6Endozoicomonas species at 25°C and 30°C. The assay was performed after 24 h of incubation. All experiments were performed in triplicate, and data are expressed as means ± standard deviations. Differences between the mean from each two groups were calculated by two-tailed paired Student t test P value indicate statistical significance . Download FIG S6, PDF file, 0.1 MB.Determination of DPPH radical scavenging activity of two Copyright © 2022 Tandon et al.2022Tandon 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 Drupella cornus was also observed in the samples from the Inner Bay during the sampling in May and continued until December  Coral Acropora sp. strain Colony attacked by Drupella cornus (red arrows). (B) Zoomed-in photo of panel A. (C) Another colony being attacked. (D) Zoom-in photo of panel C. Coral colonies in the Inner Bay only suffered from Drupella cornus, which nibbled coral, potentially causing bleaching and death. Download FIG S7, PDF file, 0.9 MB.Copyright © 2022 Tandon et al.2022Tandon 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 Endozoicomonas, one of the most dominant bacterial groups in the coral microbiome toward environmental change. We analyzed the microbiome dynamics of the common Indo-Pacific coral A. muricata over 9 months following a reciprocal transplant experiment at the finest resolution of zOTUs. We identified that different Endozoicomonas phylotypes in the A. muricata coral colonies belong to two dominant species, including a novel species, and have differential adaptation capabilities, with one species more resilient to environmental change than the other. Our results shed light on an often-neglected factor when determining variations in community composition: bacterial species/strains adapt differently when coral hosts are subjected to biotic and abiotic stressors. Furthermore, we also isolated, cultured, and sequenced a single chromosome-level genome of one of the dominant phylotypes belonging to a novel Endozoicomonas species, “Ca. Endozoicomonas penghunesis” 4G, to ascertain the ecological and functional role of this bacterium and add to the growing knowledge and genome data sets of this key microbe in coral reefs.This study aimed to test the differential capability of Acropora muricata microbiome was dominated by members of class Gammaproteobacteria (phylum Proteobacteria) (Silva v132 grouped Gammaproteobacteria into Betaproteobacteria), particularly by Endozoicomonas-related phylotypes , was described as an obligate intracellular bacterium, but its function has remained enigmatic  and the transplanted samples (O→I), with only a change in the dominant zOTU from zOTU1 to zOTU2, whereas those related to “Ca. Endozoicomonas penghunesis” 4G were only dominant in April and May months in the control samples of the Inner Bay (IC)  could play an important role in host survival, since a bacterium is likely to be not as well adapted to a new niche as resident strains, unless it has the genetic capability to mitigate the new stressors , 54. DurBay (IC) . There aEndozoicomonas phylotypes is potentially analogous to the abundance of genotypes of different endosymbiotic algae Symbiodinium, often found in coral colonies  became esis” 4G ) were thcroporae . Furtherhunesis” , so shufEndozoicomonas phylotype identified in the metabarcoding data analysis. Phylogenetic analysis identified that the dominant zOTUs  from the Outer Bay were closer to a previously characterized species, E. acroporae , many coding genes , and complete pathways for essential amino acids, suggesting a free-living life stage. Furthermore, a comparative genomics study identified that Endozoicomonas species are capable of differential functional specificity, and different genotypes may play disparate metabolic roles in their hosts (E. acroporae is the only known Endozoicomonas species capable of metabolizing dimethylsulfoniopropionate (DMSP) to dimethylsulfide (DMS)  for glycine-betaine, another osmolyte, were identified in the genome of “Ca. Endozoicomonas penghunesis.” Other Endozoicomonas species have also been identified to have the ability to scavenge glycine-betaine through transporters (12-dependent anaerobic growth) by oxidizing 1,2-propanediol with tetrathionate as an electron acceptor  it provides to the bacterium and coral host that maintains it.Understanding the functional and ecological roles of coral-associated microbes in reefs has become critical to developing an intervention for coral reef protection, such as developing a coral probiotic , 59. Thecroporae , whose gcroporae , 61. On ir hosts . This isde (DMS) . Genes fsporters , potentiacceptor . PhysiolCa. Endozoicomonas penghunesis” 4G, E. acroporae Acr-14T showed stronger free radical scavenging ability at higher temperatures, no matter in cell lysate or supernatant  were collected at a depth of 3 m from the Outer Bay (O) (N23° 33.097' E119° 38.335′) and Inner Bay (I) (N23° 31.853′ E119° 33.629') along the reef adjacent to the coast of the Penghu Archipelago, Taiwan  at each time point and location. Two- by 2-cm fragments were taken from each colony, rinsed with filtered seawater, and stored in 99% ethanol at −20°C before DNA extraction.2) with ~15 ml 1× TE buffer  and then placed into sterile Ziploc bags. Total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method  method . Coral t) method .AAC GCG AAG AAC CTT AC-3′) (ACG GGC GGT GWG TRC-3′) , 1× TaKaRa Ex Taq buffer, 0.2 mM deoxynucleotide triphosphate mixture (dNTPs), 0.2 mM forward and reverse primers, and template DNA. The PCR conditions consisted of an initial denaturing step at 95°C for 5 min, followed by 30 cycles at 94°C for 30 s, 52°C for 20 s, and 72°C for 45 s and a final extension at 72 °C for 10 min. The amplified product was visually confirmed using 1.5% agarose gel with a 5-μl PCR product. Target bands (~420 bp) were cut and eluted using a QIAEX II gel extraction kit .For DNA library construction, 968F (5′-T AC-3′)  and 1391 TRC-3′)  universaEach bacterial V6-V8 amplicon was tagged with a unique barcode sequence by designing tag primers with 4-base overhangs at 5′ ends. The sample-specific tagging reaction was performed with a 5-cycle PCR, with a reaction program of initial denaturation at 94°C for 3 min, followed by denaturation at 94°C for 30 s, annealing at 52°C for 20 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. The amplified product was purified using the QIAquick PCR purification kit . Purified products were pooled into four independent libraries and sequenced with Illumina MiSeq paired-end sequencing (2 × 300 bp) at Yourgene Biosciences, Taiwan. No kit and PCR negative controls were sequenced in this study.Paired-end raw reads obtained from Illumina sequencing were merged using USEARCH v11  with thepost hoc tests using vegan package P value correction for multiple testing. Multivariate analysis was performed after square root transforming the zOTU count data. Betadisper function was used to calculate the multivariate dispersion of samples (Bray-Curtis distance) between sample groups. Homogeneity of multivariate dispersion was tested with ANOVA. Nonmultidimensional scaling (nMDS) was performed to compare community compositions using the Bray-Curtis distance metric between sample groups. Permutational multivariate analysis of variance (PERMANOVA) with the “adonis” function  was used to statistically test for differences in community compositions between the back and cross transplant samples for each location as dispersion was significantly different between groups. Linear discriminant analysis effect size (LEfSe) implemented in the microbiomeMarker package in R was used to identify shifts in zOTUs between back- and cross-transplant samples for each location with a log Linear discriminant analysis (LDA) cutoff of 3 . z score-transformed abundance profiles of marker zOTUs identified from LEfSe were visualized with a heatmap via pheatmap.All statistical analyses and graphs were generated in R . Stacked bar plots were obtained by converting absolute abundance profiles into relative abundances. Abundance profiles were processed with the R packages phyloseq , vegan , ggplot23, NO3, and PO4, were measured with a LaMotte 1910 SMART 3 colorimeter; pH was measured with a HORIBA LAQUA act water quality meter, and salinity was measured with an ATAGO master refractometer.The water temperatures of the Outer (O) and Inner (I) Bays of the Penghu Archipelago, Taiwan, were obtained from May 2018 through December 2018 using temperature data loggers  located ~3 m deep, close to target colonies, and recording temperatures every 30 min. Abiotic factors, including NHCa. Endozoicomonas penghunesis” 4G was isolated from the coral Acropora muricata off the coast of the Inner Bay, Penghu Archipelago, Taiwan . Coral tissue and mucus were sprayed with TE buffer  and serially diluted to 10−4. All dilutions were plated on modified marine broth version 4 (MMBv4 agar) (AGA GTT TGA TCM TGG CTC AG-3′) and Endozoicomonas-specific reverse En771R (5′-TCA GTG TCA RRC CTG AGT GT-3′)  and 1541R (5′-AAG GAG GTG ATC CAG CC-3′). The full-length 16S rRNA PCR was amplified using 30 cycles with a denaturing step at 95°C for 30 s, annealing at 55°C for 30 s, and a final extension at 72°C for 90 s. Amplified products with target bands  were cut and later sequenced by Sanger sequencing  from Genomics, Taipei, Taiwan. Chromatograms obtained were manually checked and sequences were trimmed. The final length of the high-quality trimmed sequence was ~600 bp. Sequences with ≤98% identity to 16S rRNA genes of type strains from genus Endozoicomonas were deemed new candidates for novel Endozoicomonas species.“v4 agar)  and incuT GT-3′) . EndozoiCa. Endozoicomonas penghunesis” 4G was cultivated on MMBv4 medium . Download Copyright © 2022 Tandon et al.2022Tandon 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 Ca. Endozoicomonas penghunesis” 4G on MMB agar in the 2.5-liter Oxoid AnaroGen system (Thermo) and cultured at 25°C for 7 days. Oxidase and catalase activity was tested independently by adding 35% H2O2 and 0.1% tetramethyl–phenylenediamine dihydrochloride, respectively. An antibiotic sensitivity test was performed after spreading bacteria on an MMB plate, with each disc containing different antibiotics (10 μg streptomycin and 10 μg ampicillin). The results were observed after 5 days of incubation at 25°C, and sensitivity was measured based on the distance from the discs to the edge of the clear zone. Bacteria were scored as sensitive if the diameter was greater than 2 mm, slightly sensitive if the diameter was 1 to 2 mm, and resistant otherwise.Three physiological tests  were measured based on the turbidity  of cultures grown at different pH values, NaCl concentrations, and temperatures, respectively. Commercial API 20NE kits  were used to test the ability to metabolize different carbon substrates per the manufacturer’s protocol. Additional carbon utilization was evaluated in the modified marine medium . General cell structure and cell inner structure were studied by transmission electron microscopy (TEM). The bacterial shape on a single colony was observed by scanning electron microscopy (SEM). TEM and SEM observations were made after bacteria were cultured in MMB for 1 day and MMB agar (1.5%) for 3 days, respectively. Colonies were incubated at 25 °C.The morphology of “Ca. Endozoicomonas penghunesis” 4G thin section, bacteria were first centrifuged at 2,500 × g for 5 min, and bacterial pellets were collected and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in a 0.1 M sodium phosphate buffer (pH 7.0) at room temperature for 1 h. After three 20-min buffer rinses, the samples were postfixed in 1% OsO4 in the same buffer for 1 h at room temperature and then rinsed again as described above. Samples were dehydrated in an alcohol series, embedded in Spurr’s resin (EMS), and sectioned with a Leica EM UC6 ultramicrotome . The ultrathin sections (70 to 90 nm) were stained with 5% uranyl acetate in 50% methanol and 0.4% lead citrate in 0.1 N sodium hydroxide. An FEI G2 Tecnai Spirit Twin TEM was used at 80 kV for viewing, and images were captured with a Gatan Orius charge-coupled device (CCD) camera.For the “Ca. Endozoicomonas penghunesis” 4G was observed using cryo-SEM (FEI Quanta 200 SEM/Quorum Cryo System PP2000TR). The MMBv4 agar plate containing a single colony of “Ca. Endozoicomonas penghunesis” 4G was sectioned into 1 mm by 1 mm, loaded onto the medium-containing stub, and then frozen with liquid nitrogen slush. The frozen sample was transferred to the sample preparation chamber at −160°C. After 5 min, the temperature was raised to −85°C, and the samples were etched for 20 min. After coating at −130°C, the samples were transferred to the SEM chamber and observed at −160°C and 20 kV.The colony of “2O twice, and the bacteria were mounted onto grow-discharge carbon-Formvar grids. Bacteria were stained by 2% phosphotungstate for 1 s, and finally the sample was rinsed with sterilized H2O twice and viewed under an FEI G2 Tecnai Spirit Twin TEM at 80 KV. The images were then captured with a Gatan Orius CCD camera.The general cell morphology was studied by negative staining and observed under TEM. Bacteria were enriched in MMB for 1 day before adding a fixative solution  at 37°C for 10 min. To reduce the background signal of TEM observation, MMB was replaced first by PBS and then by sterilized HEndozoicomonas species were incubated in MMbv4 medium with 0.1% glucose at 25°C and 30°C for 24 h, and the bacterial supernatant was harvested by centrifugation at 13,000 rpm for 5 min at 4°C. To collect cell lysate, cell pellets were resuspended by 0.8 ml 50 mM Tris-HCl lysis buffer  and loaded into a 2-mL O-ring-capped plastic microcentrifuge tube. The cell pellets with lysis buffer were vortexed at maximum intensity for 5 min and incubated on ice for 1 min. The step was repeated twice. After that, the cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C to pellet down the cell debris and beads. The hydrogen atom donating ability of bacterial cultural supernatant and bacterial cell lysates was determined by decolorization of methanol solution of DPPH from violet color to yellow color in the presence of antioxidants. The 0.2 mM DPPH solution in 100% methanol was prepared, and 100 μl of the sample (supernatant or cell lysates) was mixed with 100 μl DPPH in 96-well plates in triplicate. Blanks were 100 μl sample mixed with 100 μl DPPH solvent (100% methanol), and control was 100 μl sample solvent (MMbv4 as the supernatant solvent and Tris-HCl lysis buffer as cell lysate sample) mixed with 100 μl DPPH solution. The reaction mixtures were incubated in the dark at 25°C for 30 min. The absorbance of the mixture was measured by a microplate reader  at 517 nm. The DPPH scavenging activity was defined assample is the absorbance of the mixture of DPPH solution with samples, ABSblank is the absorbance of the mixture of samples and DPPH solvent, and ABScontrol is the absorbance of the mixture of sample solvent and DPPH solution.The ability of free radical scavenging of two bacterial species was tested by a 2,2 diphenyl-1-picrylhydrazyl (DPPH) assay. The two Endozoicomonas zOTUs identified and the 16S rRNA gene of “Ca. Endozoicomonas penghunesis” 4G and to identify their closest neighbor within genus Endozoicomonas and its cultured isolates, representative 16S rRNA sequences from type strains  and one outgroup Halospina denitrificans HGD were downloaded from the NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/taxonomy). Sequences were aligned using the RNA homology search tool cmalign , and then the adapters were removed and the reads trimmed with AdapterRemoval v2  using the ANI calculator from the enveomics collection (http://enve-omics.ce.gatech.edu/ani/)  .PRJNA758232, and the “Ca. Endozoicomonas penghunesis” genome was made available under GenBank accession no. SAMN21016876.All sequencing data generated in the manuscript was submitted under the BioProject no.


THRβ) gene mutation. Most individuals with RTH are considered clinical euthyroid. We report a family with a rare heterozygous point mutation, c.959G>T, (p.R320L) of the THRβ gene. The proband developed atrial fibrillation and life-threatening heart failure with pulmonary edema, which was quite different from previously reported THRβ gene mutations. Considering the rareness of RTH and the heterogeneity of its phenotypes, our report allows for a better understanding of the manifestation and management of patients with RTH and THRβ gene mutation.Resistance to thyroid hormone (RTH) is a rare disease typically associated with elevated levels of thyroid hormones and non-suppressed thyroid stimulating hormones. The most common cause of RTH is thyroid hormone receptor β ( THRβ) gene and thyroid hormone receptor α (THRα) gene. There are three receptor isoforms  which are mainly expressed in different tissues  [THRβ gene mutation (termed RTHβ), and the others are related to the mutations in the THRα gene, TH transport, and metabolism genes [THRβ gene mutant has a dominant negative effect. That is, the mutation gene product adversely affects the normal gene product. Thus, cases with heterozygous THRβ gene mutation present more severe disease symptoms as compared with those with homozygous mutation [THRβ gene. Our proband developed atrial fibrillation with decompensated heart failure because he missed a follow-up.Resistance to thyroid hormone (RTH) is a rare disease with impaired tissue responsiveness to thyroid hormones (TH). The prevalence rate is about 1/40,000 with limited studies [sm genes . The climutation . In thisThis study was approved by the Institutional Review Board of Far Eastern Memorial Hospital (111178-C). All participants provided written informed consent.® Predesigned Gene Capture Pools to target the coding regions of THRα and THRβ genes. An NGS was performed using Illumina MiSeq Reagent Kit v3 on a MiSeq platform . The Genome Aggregation Database (gnomAD)  and ClinVar  were used to check for variants. The American College of Medical Genetics and Genomics (ACMG) guidelines were followed for variants interpretation. The variants that met the criteria for pathogenic were confirmed with traditional Sanger sequencing.Genomic DNA was extracted from the subjects’ peripheral blood using a QIAGEN Gentra Puregene Blood kit. The DNA samples were prepared using Illumina TruSeq DNA PCR-Free Sample Preparation Kit. Probes were designed from XGenTHRβ gene (NM_001252634) was noted.A 49-year-old man visited our out-patient-department (OPD) about an enlargement of the neck mass. He denied any symptoms, even having had goiter for years. He has a sister who also has a goiter and has been diagnosed to have thyroid dysfunction. Thyroid sonography revealed bilateral muti-nodular goiter. Laboratory data showed mildly elevated free T4, TSH, anti-TPO, and normal TSH receptor Ab. Specifically, data indicated 2.99 ng/dl (0.2–2) of free T4, 4.22 µIU/mL (0.4–4) of TSH, 11.49% (<15) of TSH receptor Ab, and 101 IU/mL (<35) of anti-TPO. According to the data, TSH-secreting pituitary tumor was suspected. The pituitary MRI showed suspicious microadenoma, but the TRH stimulation test was normal. Other axes of pituitary functions were normal. Under the impression of thyroid hormone resistance syndrome, a genetic study was advised, but the patient refused. The patient did not come for a follow-up until 5 years later. An episode of shortness of breath brought him to our ER. The electrocardiogram (EKG) revealed atrial fibrillation E, and thTHRβ gene (NM_001252634) . Laboratory data showed 2.57 ng/dL (0.2–2) of free T4, 1.52 µIU/mL (0.4–4) of TSH, and 1.91% (<15) of TSHR Ab. Carbimazole 10 mg bid and propranolol 10 mg bid were given, and symptoms improved. Further studies were conducted at our OPD, including thyroid ultrasound, pituitary function tests, pituitary MRI, electrocardiogram (EKG), 24-h Holter EKG, and echocardiogram. Only bilateral multinodular goiter was noted in the thyroid ultrasonography. The pituitary function tests and MRI imaging were normal. The heart examinations were normal in both rhythm and function. The genetic study revealed a heterozygous mutation c.959G>T, (p.R320L) of the 1252634) . During For patients with persistent elevation of circulating free T3 and free T4 levels, the differential diagnosis, in addition to RTH, was TSH-secreting pituitary adenoma. In our proband, the first brain MRI showed suspicious pituitary microadenoma. In order to rule out TSH-secreting pituitary adenomas, the TRH stimulation test was done. The response was normal, with TSH increasing more than 50% after TRH stimulation, and the diagnosis of TSH-secreting pituitary adenomas was excluded .THRβ gene with heterogeneous mutation c.959 G>T (p.R320L) is exceedingly rare. There had been only two cases with the same mutation reported in one study, and mutation in this region decreased the affinity between thyroid hormone receptor β and triiodothyronine, T3 [THRβ gene, one was classified in GRTH, and the other was in PRTH [THRβ gene with different nucleotides have also been reported, such as R320H, R320C, and R320P [THRα is predominant. Thus, patients with THRβ mutation are likely to have cardiac symptoms. It was observed that cardiovascular features were not corelated with the genotype and progressed with aging [THRβ were given L-T3 and/or propranolol or were not given any medication. In our cases, the cardiac symptoms could only be relieved by the combination of carbimazole and propranolol. Electrocardiogram and echocardiography every 2–3 years were recommended in symptomatic patients [The nine, T3 . In the  in PRTH . In factnd R320P ,8,9,10. th aging ,12. Bothpatients . HoweverTHRβ gene. The proband developed atrial fibrillation and life-threatening heart failure with pulmonary edema, which was quite different from previously reported THRβ gene mutations. Considering the rareness of RTH and the heterogeneity of its phenotypes, our report allows for a better understanding of the manifestation and management of patients with RTH and THRβ gene mutation.RTH is a rare disease with impaired tissue responsiveness to TH. We report a family with a rare heterozygous point mutation, c.959G>T, (p.R320L) of the


In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest. With the improvement of biomedical research and clinical care, the survival of patients with childhood cancer has increased. The quality of life after cancer has therefore become a major issue in the management of these patients. Oncological treatments are mainly based on chemo- and radiotherapy, the gonadotoxicity of which can lead to gonadal dysfunction and infertility . To presSpermatogenesis is an essential process by which haploid spermatozoa develop from SSC in the seminiferous tubules of the testis. Meiosis is the step during which genetic recombinations occur in diploid spermatocytes and, at the end of which, haploid spermatids are generated. A single round of DNA replication is followed by two consecutive rounds of nuclear meiotic divisions. The first meiotic division (meiosis I) is characterized by a prolonged prophase during which chromosome synapsis, crossovers, and homologous recombination take place , and by Meiosis is therefore a complex process involving many genes, and the deregulation of its expression can be at the origin of meiotic arrest, as demonstrated in transgenic mice ,25. NumeIgf-1 [Our previous findings concerning in vitro maturation of prepubertal rat testicular tissue evidenced a blockage during meiosis I , as is aIgf-1 , or upreIgf-1  in cultuIgf-1  or an inIgf-1  led to aIgf-1 .The aim of the present study was therefore to understand the underlying mechanisms of meiotic arrest in organotypic cultures of prepubertal rat testicular tissue, using histological analyses and a bulk transcriptomic approach.As previously described , rat preWe next determined whether germ cell depletion in cultured tissues was the result of a decrease in proliferation or an increase in DNA damage. Ki67 immunostaining revealed that the proliferative activity of intratubular cells was maintained at the end of the culture process . The proThe formation of the XY body was then assessed by immunolabeling γH2AX, a protein involved in DSBs and transcriptional silencing of the XY body, on tissue sections j. When pIn order to identify the molecular mechanisms involved in the spermatogenic arrest during organotypic culture, bulk RNA sequencing analyses were conducted on in vitro- and in vivo-matured testicular tissues. For each time point, three replicates were analysed, with three different testes for in vivo controls and twelve pooled cultured explants per replicate for the in vitro conditions. One 14.5 dpp testicular tissue, which displayed a different transcriptomic profile compared to the other two replicates, was excluded from the downstream analyses. A PCA  revealed the biological relevance of the resulting transcriptomic dataset: indeed, the first component (or dimension), containing 84.8% of variances, appeared strongly correlated with the tissue maturity of samples a. IndeedBased on the above PCA and on histological observations, we compared the transcriptomes of cultured tissues to those of 14.5 dpp tissues to highlight the genes involved in the initiation of germ cell maturation which could be deregulated during cultures, thereby leading to premature spermatogenic arrest c. By appp value < 4.58 × 106), 56 in the cell cycle (4.13 × 1025), and 58 in cell differentiation (0.037)  and cell cycle checkpoints , as well as in genes involved in metaphase/anaphase transition  (7), cell development , cell death , stress , or inflammation  (5) (7), as well as growth factors  (5), but also of the reproductive  and immune system  signaling pathways  enrichment, on the seven resulting expression patterns. This functional analysis highlighted 1056 enriched GO terms  signific (0.037) . The P1– 0.0002) , reflect 0.0002)  appeared, 0.007) . Interes007) (5) . Additio 0.0008) . A KEGG pathways .Cyp17a1, Insl3, Tesk1, Cdc34, Kif11, Ube2c, Incenp, Plk1, Ccnf, Cks2, Alox15, Bub1b, Ccna2, Cbx2, and Aspm for the pattern P1–4  related to the synaptonemal complex. Most of them were down-regulated in cultured tissues, and may be related to the synapsis defect observed in vitro  were down-regulated , reflecting the beginning of testicular maturation , which are essential for spermatogenic differentiation [Cyp17a1, Cyp11a1, and Hsd3b1) in cultured tissues  were up-regulated , spindle organization and chromosomal segregation , cell cycle checkpoints , or meiotic entry   .Cbx2, Cks2, Top2a, and Ube2c mRNA levels during the culture period  software was used. IPA analyses clearly demonstrated that zinc finger and BTB domains containing 17 (ZBTB17), tumor necrosis factor (TNF), and CCAAT/enhancer binding protein beta (CEBPB) were the top three predicted upstream regulators of the 594 deregulated genes between 14.5 dpp and D28 cultured tissues, among the 600 candidate genes . Most ofThe current study explored the in vitro maturation of rat prepubertal testicular tissues at the molecular level in order to unravel the mechanisms underlying meiotic arrest. Indeed, although organotypic culture at a gas–liquid interphase has proven to be a promising approach for generating spermatozoa from immature testicular tissues in the mouse model, a complete in vitro spermatogenesis has not yet been achieved in other species, including rats and humans, thereby delaying its application in clinics for fertility restoration of survivors of childhood cancer.Consistently with our previous findings , no germTwo distinct types of meiotic arrest have been described in humans : (i) arrMeiotic arrest can have a genetic ,157 or aCyp11a1, Cyp17a1, Hsd3b1), and a depletion in CYP17A1 and 3βHSD were evidenced in organotypic cultures in the current study. We therefore assessed the effect of three molecules with anti-inflammatory properties  on spermatogenic progression in organotypic cultures. Among them, TAK 242, a TLR4-NF-κB signaling pathway inhibitor, has previously been used to successfully decrease the number of testicular macrophages in cultures of mouse prepubertal testicular tissues [Then, IPA analyses performed on our dataset led to the identification of predictive upstream regulators. We found that the top 10 upstream regulators were involved in inflammation and in regulation of spermatogenesis. Among them, TNFα is partly produced by macrophages to promote the inflammatory process, and its expression is accompanied by the production of reactive oxygen species (ROS) or chemokines . In addi tissues . Interes tissues ,178,179.In conclusion, through a combination of histological and molecular analyses, we uncovered for the first time several underlying molecular mechanisms that could explain the meiotic arrest observed in cultures of rat prepubertal testicular tissues. Our data converge towards an alteration in BTB integrity, in the steroidogenic pathway, and possibly an inflammatory response. The utilization of the transcriptomic data obtained in this study will be useful to further optimize in vitro tissue maturation protocol, particularly the crucial meiotic step, before considering a clinical application2 asphyxiation, whereas pups aged 8.5 dpp were decapitated. In this study, 106 rats were used.Rats were housed in a temperature- and humidity-controlled environment, under a 12h light/dark cycle. Food and water were provided ad libitum. Pubertal and adult rats were euthanized by CORattus norvegicus) were purchased from Charles River Laboratories  and bred in our animal facility. After euthanasia, testes were biopsied and rinsed in αMEM  at 4 °C. The tunica albuginea was removed with two needles under a binocular magnifier . Wistar rats  or 4% paraformaldehyde . They were then dehydrated in successive ethanol/xylene baths, embedded in paraffin and cut into thin sections (3 µm thick). After two xylene baths, tissue sections were rehydrated in successive descending ethanol baths and plunged into PBS buffer. For immunocytological analyses, cells from in vivo controls or cultured explants were spread onto glass slides. For molecular analyses, the central necrotic area in cultured explants was removed with two thin needles, before being stored in liquid nitrogen.2–95% air at 34 °C for 6, 16, 22, or 28 days. The culture medium was composed of αMEM supplemented with 10% Knock-out serum replacement , 10−6 M retinol  and 3.4 mM vitamin E . To assess the effect of anti-inflammatory molecules, organotypic cultures were supplemented for 28 days with either TAK 242 , Infliximab  or genistein . Cultures supplemented with TAK 242 or genistein were compared to cultures supplemented with DMSO . The media were prepared and replaced every 3–4 days. Juvenile male rats at 14.5, 24.5, 30.5, and 36.5 dpp were used as in vivo controls.Testicular explants were cultured using the gas–liquid interphase method as described before . BrieflyAll the antibodies used in this study for immunohistochemical analyses are presented in Hemalun–Eosin–Saffron staining was performed to identify the different meiotic germ cells and division figures . ® 488 for 1 h at RT. Sections were rinsed, dehydrated with ethanol, and mounted in Vectashield  with Hoechst 33342 .Ki67 and DDX4: To assess cell proliferation (Ki67) and germ cell (DDX4)/Sertoli cell ratio, tissues sections were subjected to a standard immunofluorescence protocol as described below. Tissue sections fixed in PFA were incubated in citrate buffer  at 96 °C for 40 min and cooled for 20 min at RT. After rinsing in distilled water, sections were permeabilized in PBS-Triton X-100 0.01% for 15 min at RT (only for Ki67). To avoid nonspecific staining, sections were blocked with 5% BSA + 5% horse serum (Ki67) or with 20% goat serum + 5% BSA (DDX4). Slides were then incubated overnight with primary antibodies at 4 °C. Negative controls were performed with pre-immune IgGs. After 3 washes in PBST, sections were incubated with secondary antibodies coupled to Alexa Fluor® 594. Positive controls were performed with DNase I before TUNEL assays and negative controls were carried out by omitting the enzyme solution (TUNEL) or with pre-immune IgGs (SYCP3 immunostaining).To detect DNA fragmentation and spermatocytes in the same samples, tissue sections were first subjected to TUNEL assays , following the manufacturer’s instructions. Then, sections were post-fixed with 4% PFA for 15 min at RT and finally underwent the standard immunofluorescence protocol described above (without the permeabilization step). Rabbit anti-SYCP3 antibodies were used, and revealed with secondary antibodies coupled to Alexa FluorTissue sections fixed with PFA were submitted to a standard immunofluorescence protocol, as previously described. CD68-immunolabeled testicular tissue images were acquired on a Thunder upright microscope . The surface area occupied by CD68+ macrophages was measured both in the peripheral  and central (necrotic) regions of the cultured explants.Tissue sections fixed with Bouin’s solution were incubated in citrate buffer at 96 °C for 10 min for antigen retrieval, and cooled down for 5 min at RT. After rinsing with distilled water, endogenous peroxidases were blocked with HP block  for 15 min at RT. After 3 washes in PBST, nonspecific binding sites were then blocked for 30 min at RT with 20% goat serum, 5% BSA in PBS-Tween 0.05%. Tissue sections were incubated for 2 h at RT with anti-γH2AX antibodies diluted in blocking buffer. Subsequently, primary antibodies were detected with polyvalent secondary antibodies for 5 min at RT. Staining was achieved after incubation with a streptavidin-horseradish peroxidase solution  for 15 min at RT followed by 3,3′-diaminobenzidine substrate for 30 s at RT. After nuclear counterstaining with hematoxylin , sections were dehydrated in successive ethanol baths of increasing concentrations, followed by two xylene baths and mounting with Eukitt .Each in vivo replicate originated from different rats. For quantitative histological analyses, at least 2 levels of the testis/explant (separated by at least 30 µm) and 30 seminiferous tubules were analysed for each replicate. For each in vitro replicate, 2–3 testicular explants were analysed. In total, approximately 360 tubules were counted for in vivo controls and at least 720 tubules for the in vitro condition, per analysis. HES and γH2AX immunostaining were analysed with a DM4000B light microscope  equipped with Leica Application Suite software . Immunofluorescence analyses (except for CD68) were performed on an epifluorescence microscope  equipped with a monochrome CCD IEEE1394 FireWire video camera .Preparations of meiotic chromosome spreads from rat spermatocytes were adapted from previously published protocols ,182 with® 594 coupled to streptavidin for 30 min at RT. Preparations were rinsed, dehydrated with ethanol, and mounted in Vectashield . The slides were sealed with polish for confocal microscopy. For the analysis of chromosome synapsis, spermatocytes were isolated from at least three 36.5 dpp control rat testes and D28 in vitro cultured tissues.After 2 min at 37 °C, chromosome spreads were rehydrated in PBST for SYCP3/SYCP1 immunostaining or in successive Triton X-100 baths  for SYCP3/γH2AX immunostaining. Spreads were blocked for 30 min at RT with 5% BSA + 5% horse serum in PBS (SYCP3/SYCP1) or with 2% BSA + 0.05% Triton X-100 in PBS (SYCP3/γH2AX). They were then incubated overnight with primary antibodies at 4 °C (SYCP3/SYCP1) or RT (SYCP3/γH2AX) in a humidified chamber. Negative controls were performed with pre-immune antibodies. After 3 washes, preparations were incubated with appropriate secondary antibodies. Biotin was revealed with Alexa FluorThe classification was performed as previously described . Briefly® 488 and at 552 nm to excite Alexa Fluor® 594 and a conventional scanner at 400 Hz. Using a 63× , fluorescence emission was sequentially detected through a hybrid detector  in a photon counting mode with a specific band from 500 to 540 nm for Alexa Fluor® 488 and 600 to 650 nm for Alexa Fluor® 594. Z-stack images were acquired using an adapted step-size of 250 nm. 3D images (xyz) reconstruction and analysis were performed using Imaris software . ImageJ  was used to adjust brightness and contrast for representative image treatment.Confocal images (1024 × 1024 pixels) of meiotic chromosome spreads were acquired using an upright fixed-stage Leica TCS SP8 CFS confocal microscope  equipped with diode lasers  at 488 nm to excite Alexa FluorTotal RNA was extracted according to the manufacturer’s instructions using an RNeasy Micro Kit , and stored at −20 °C until use. Genomic DNA was removed by incubation with Turbo DNase  and RNA quality control was performed before RNA sequencing and RT-qPCR on Agilent Bioanalyzer . All the samples chosen for analysis displayed an RNA integrity number (RIN) value greater than or equal to 8.5.RNA-Seq libraries were generated from 400 ng of total RNA using a TruSeq Stranded mRNA LT Sample Preparation Kit , according to the manufacturer’s instructions. Briefly, following purification with poly(T)-oligo attached magnetic beads, mRNA was fragmented using divalent cations at 94 °C for 2 min. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during the second strand cDNA synthesis using DNA polymerase I and RNase H. Following the addition of a single ‘A’ base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR  × 12 cycles; 5 min at 72 °C) to create the cDNA library. The excess of PCR primers was further removed by purification using AMPure XP beads , and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Libraries were then single-read sequenced with a length of 50 bp on an Illumina Hiseq4000 sequencer. Image analysis and base calling were carried out using RTA v.2.7.7  and bcl2fastq v.2.17.1.14 . For each time point, three independent sets of total RNA were isolated and used as a template for RNA-seq library preparation. In addition, for each cultured sample, testicular tissue fragments originated from a minimum of 3 juvenile males. Thus, a minimum of 9 animals per time point was used to minimize/normalize the effect of genetic variability.Reads from each individual sample were aligned to the rn6 release of the rat genome with STAR (version 2.5.2a),  using prRefSeq transcripts were quantified with StringTie (version 1.3.3), with default settings applied . TranscrPrincipal component analysis (PCA) was performed based on the gene abundance values with the FactoMineR package to graphically evaluate the distribution of sequenced samples .p ≤ 0.05) . Finally, we intersected the three sets of DEGs to select 600 genes that showed significant differential abundances in all three comparisons. The resulting 600 genes were next clustered into seven expression patterns (P1-P7) with the unsupervised HCPC algorithm, respectively [The statistical filtration of the genes showing a differential expression (DE) across experimental samples was performed using AMEN . Out of ectively . The resp value was <0.01 and the number of genes in this cluster showing this annotation was >5. Upstream regulator analysis was performed using the Ingenuity Pathway Analysis software .AMEN  was usedTo check the accuracy of the transcriptome data, DEGs identified as important for the process of spermatogenesis were selected for RT-qPCR. Each reaction was performed using three technical replicates and three biological replicates for the robustness of our analysis. RNA extraction and integrity analysis were performed as previously described.cDNA synthesis was performed using qScript™ cDNA SuperMix  according to the manufacturer’s instructions using the following program: 5 min at 25 °C, 30 min at 42 °C, and 5 min at 85 °C.Gapdh and actin β (Actb), which were identified and validated as the most stable and suitable genes for RT-qPCR analysis in rodent testis development [−ΔΔCt method [cDNA amplifications were carried out in a total volume of 13 μL containing 6 ng of cDNA templates, 6.5 μL of SYBR Green , and 300 nM of each primer. Specific primers are listed in elopment . Data wet method . The Kru2 for 30 min at room temperature. Incubation of testicular fragments in 1 mM CaCl2 in PBS was used as a negative control. Tissues were fixed with PFA, dehydrated, and prepared as previously described. DDX4 immunofluorescence was then performed as described above, with slight modifications. After antigenic sites revelation with citrate buffer, nonspecific binding sites were then blocked for 30 min at RT with 5% horse serum and 5% BSA in PBS-Tween 0.05%. Then, sections were incubated with a primary antibody against DDX4, revealed with a secondary goat anti-rabbit antibody coupled with Alexa Fluor® 488. Alexa Fluor® 594-conjugated streptavidin was added at the same time to reveal biotin localization. Sections were rinsed, dehydrated with ethanol, and mounted in Vectashield with Hoechst 33342.The assay was performed as previously described . Brieflyg, 4 °C. The supernatants were collected and stored at −20 °C until use. Tissues were flattened out in a PBS + protease inhibitors  buffer. Samples were centrifuged for 10 min at 12,000× The standards for testosterone and stable labelled isotopes were obtained from Merck Millipore. Working solutions were prepared in methanol. Serial dilutions from working solutions were used to prepare seven-point calibration curves and 3 quality control levels for all analytes. For the final calibration and quality control solutions, PBS was used for testicular fragments and αMEM for culture media. The linearity ranges were from 0.05 to 10 ng/mL.A simple deproteinization was carried out by an automated sample preparation system, the CLAM-2030  coupled with a 2D-UHPLC-MS/MS system. Once the sample was on board, 30 µL was automatically pipetted in a pre-conditioned tube containing a filter, in which reagents were added, then mixed and filtered. Briefly, the PTFE (polytetrafluoroethylene) filter vial (0.45 μm pore size) was previously conditioned with 20 μL of methanol . Successively, 30 μL of sample and 60 μL of a mixture of isotopically labelled internal standards in acetonitrile were added. The mixture was agitated for 120 s (1900 rpm), then filtered, by application of vacuum pressure (−60 to −65 kPa) for 120 s, into a collection vial. Finally, 30 µL of the extract was injected in the 2D-UHPLC-MS/MS system.® modules (Shimadzu Corporation): an isocratic pump LC20AD SP, for pre-treatment mode; a binary pump consisting of coupling two isocratic pumps Nexera LC30AD for the analytical mode; an automated sampler SIL-30AC; a column oven CTO-20AC; and a triple-quadrupole mass spectrometer LCMS-8060.Analysis was performed on a two dimensional ultra-performance liquid chromatograph-tandem mass spectrometer (2D-UHPLC–MS/MS) consisting of the following ShimadzuThe assay was broken down into two stages. The first was the pre-treatment, where the sample was loaded on the perfusion column. The second step was the elution of the compounds of interest to the analytical column.The deproteinized extract performed by the CLAM-2030 was automatically transferred to the automated sampler, where 30 µL was directly analysed in the chromatographic system.® MAYI-ODS (5 mm L × 2 mm I.D.). The first step consisted of loading the extract on the perfusion column with a mobile phase composed of 10 mM ammonium formate in water  at flow rate of 0.5 mL/minute over 2 min. Then, the system switched to the analytical step to elute the analytes from the perfusion column to the analytical column, to achieve chromatographic separation. During this step, the loading line was washed with propan-2-ol  over 3 min. Chromatographic separation was achieved on a Restek® Raptor Biphenyl  maintained at 40 °C and a gradient of (A) 1mM ammonium fluoride buffer in water  and (B) methanol  at flow rate of 0.675 mL/minute as follows: 0.0–2.10 min, 5% (B); 2.1–3.0 min, 5 to 65% (B); 3.0–4.75 min, 65% (B); 4.75–5.0 min, 65 to 70% (B); 5.0–6.6 min, 70% (B); 6.6–8.0 min, 70 to 75% (B); 8.0–8.5 min, 75 to 100% (B); 8.50–9.5 min, 100% (B); 9.5–9.6 min, 100% to 5% (B); 9.6–12.0 min, 5% (B).The LC-integrated online sample clean-up was performed using a perfusion column ShimadzuDetection and quantification were performed by scheduled-MRM (multiple reaction monitoring) using a 1 millisecond pause time and a 50 millisecond dwell time to achieve sufficient points per peak.The interface parameters and common settings were as follows: interface voltage: 1 kV; nebulizing gas flow: 3 L/minute; heating gas flow: 10 L/minute; drying gas flow: 10 L/minute; interface temperature: 400 °C; DL (desolvation line) temperature: 150 °C; heat block temperature: 500 °C; collision gas pressure 300 kPa. Compound-specific MRM parameters are shown in g. The supernatants were collected and stored at −20 °C until used. Protein concentration in lysates was determined using the Bradford assay . Loads were adjusted to 20 µg in sterile water with 4× Laemmli buffer, incubated at 95 °C for 5 min, and separated on Midi 4–15 % precast gels . Proteins were transferred onto nitrocellulose (Androgen receptor and CYP17A1) or PVDF (3βHSD) membranes, using the Transblot Turbo Transfer System . Transfer quality was confirmed by Ponceau S staining. Membranes were washed in TBS and then blocked in 5% non-fat milk (Androgen receptor and CYP17A1) or 5% BSA (3βHSD and β-actin) in TBS + 0.2% Tween-20 (TBST) for 30 min at 37 °C with a gentle rocking action. Membranes were incubated with primary antibodies . Β-actin was used to normalize proteins of interest.Tissues were lysed in an RIPA buffer containing 150 mM NaCl, 0.5% cholic acid, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100 and protease inhibitors. Tissue homogenates were agitated for 30 min at 4 °C and centrifuged for 10 min at 12,000× tibodies  overnightibodies . MembranAntibody dehybridization: Membranes were incubated for 30 min in a pre-warmed dehybridization solution containing 0.5 M Tris base (pH 6.8), 10% SDS, and 0.7% β-mercaptoethanol, in a 50 °C water bath. Then, membranes were rinsed abundantly with distilled water and rinsed twice with TBST. The efficiency of dehybridization was confirmed by the absence of labelling on the membrane after incubation with the secondary antibody.2 tests and Kruskall–Wallis tests, followed by Dunn’s multiple comparison, were performed as specified in each figure. A value of p < 0.05 was considered statistically significant.Statistical analyses were carried out with the GraphPad Prism software . Non-parametric Mann–Whitney tests, Chi


MYOC) gene, within its olfactomedin (OLF) domain, constitute the strongest genetic link to primary open-angle glaucoma via a toxic gain of function, and thus MYOC is an attractive precision-medicine target. However, not all mutations in MYOC cause glaucoma, and common variants are expected to be neutral polymorphisms. The Genome Aggregation Database (gnomAD) lists ∼100 missense variants documented within OLF, all of which are relatively rare  and nearly all are of unknown pathogenicity. To distinguish disease-causing OLF variants from benign OLF variants, we first characterized the most prevalent population-based variants using a suite of cellular and biophysical assays, and identified two variants with features of aggregation-prone familial disease variants. Next, we considered all available biochemical and clinical data to demonstrate that pathogenic and benign variants can be differentiated statistically based on a single metric: the thermal stability of OLF. Our results motivate genotyping MYOC in patients for clinical monitoring of this widespread, painless and irreversible ocular disease.Accurate predictions of the pathogenicity of mutations associated with genetic diseases are key to the success of precision medicine. Inherited missense mutations in the myocilin ( Summary: Differentiating benign from misfolding, glaucoma-associated mutations in the myocilin olfactomedin domain has been a challenge. We show that the pathogenicity of myocilin variants can be predicted by a thermal stability cutoff of 47°C. MYOC, GenBank accession NM_000261.2), predominantly within its olfactomedin (OLF) domain  domain , cause e) domain , and theout mice  or in inout mice  or hemizce   , raisingte.org/) , currentte.org/)  and, forte.org/) , clinicaMYOC do not confer glaucoma susceptibility  were considered likely to be benign A. V449I,patients , includipatients . Finallypatients . To our To assess the toxic gain of function of myocilin misfolding, we compared the extent of myocilin secretion  to its iOverall, only E300K, S313F and N420Y consistently exhibited defects suggestive of disease-like behavior: intracellular insolubility levels >50% of WT levels, decreased cellular secretion <50% of WT levels and/or intracellular solubility levels >50% of WT levels A,B. DespTo complement western blot analysis and further characterize insoluble aggregates produced by cells expressing E300K, S313F and N420Y myocilin, next we stained cells with Proteostat, a dye used to visualize amyloid aggregates . PreviouEscherichia coli expression of the monomeric fusion protein  to soluble aggregates, which we fractionated by size-exclusion chromatography (SEC) (m) within ∼4°C of WT, including two variants with Tm values higher than for WT  . Consisthy (SEC) A; A427T hy (SEC) A. After n for WT .m values had CD spectra that most closely resembled those of WT and differed from the moderately destabilized disease mutant D380A and the severely destabilized disease mutant I499F  CD spectra, respectively . The 13 nt I499F B,C. No dnt I499F . For addnt I499F D; at hignt I499F D.Structural integrity was further confirmed by high-resolution crystal structures, which were solved for 11 OLF variants. No gross changes from WT OLF were detected for the structures of T293K, R296H, L303I, V329M, S331L, E352K, T353I, K398R, A445V, V449I or K500R , indicatIn addition to the five variants that we predicted initially to be benign , our study reveals seven more  that are indistinguishable from WT OLF across multiple assays. Thus, our data indicate these should also be considered benign. Surprisingly, V329M does not appear to harbor any pathogenic signatures despite its location in an internal strand associated with amyloid formation  and its E. coli was notably diminished, suggestive of misfolding, and that of S313F was lower than that of WT, near our designated cutoff  and N420Y (47.3°C) were lower than that of WT, whereas that of S313F (49.5°C) was close to that of WT OLF, similar to that of Mus musculus OLF  and pathogenic  variants are statistically significant (P=0.01). Second, and more impressively, the average thermal stability of benign  and disease variants  segregates with P<0.0001 (P=1.2×10−40). All data can be captured between two standard deviations of the mean for benign and pathogenic variants, with a visual cutoff of 47°C.We next considered alternative relationships between age at diagnosis and thermal stability . Using aMYOC are not generally causal for glaucoma, pathogenicity is currently considered only with prioritized clinical metrics, and where available, laboratory data, such as that curated by ClinGen  and related projects (https://myocilin.com)  . Unlike lin.com) .m ∼15°C higher than OLF  is within the lower bound of the thermal stability of a WT-like variant but outside the range of that of a disease variant. Previously, A427T was considered mildly pathogenic because it was documented in a family with glaucoma, albeit at an age of diagnosis >40 and incomplete penetrance among family members . The onlopensity  and deleEarly monitoring and diagnosis based on genotype, combined with early intervention, could be transformative for precision clinical care of glaucoma . GlaucomThe plasmid for full-length myocilin was custom cloned by GenScript in the pcDNA 3.1 vector and includes a C-terminal FLAG tag, as reported previously . Mutatio2. Cells were plated at 70-80% confluency in six-well plates 24 h prior to transfection. Plasmid transfections were carried out with Lipofectamine 2000 (Invitrogen) at a ratio of 1 µg plasmid to 2.5 µl Lipofectamine 2000 mixed in serum-free Opti-MEM (Invitrogen). Cells were transfected for 48 h prior to collection. The medium was changed to serum-free DMEM with 1% penicillin-streptomycin-glutamine 24 h post transfection.HEK293T cells (American Type Culture Collection) were grown and maintained in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum (Hyclone), and 1% penicillin-streptomycin-glutamine (Gibco) at 37°C with 5% COg for 5 mins to remove cell debris, and then concentrated 10× with an Amicon filtration device (30 kDa molecular mass cutoff) for immunoblotting. Adherent cells were scraped and lysed in Triton X-100 lysis buffer  containing cOmplete protease inhibitor (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), by overnight freeze-thaw at −20°C. Cell lysates were then fractionated into soluble (supernatant) and insoluble (pellet) fractions by centrifugation at 17,000 g for 10 mins. Total protein concentrations of the medium and soluble fraction were measured by BCA assay (Pierce). Samples of media and soluble fractions for immunoblotting were then prepared with a final concentration of 1× Laemmli sample buffer containing 10% β-mercaptoethanol (v/v). Insoluble fractions were washed three times with ice-cold PBS (Gibco), resuspended in 300 μl of 2× Laemmli buffer with β-mercaptoethanol and then sonicated with a rod sonicator (Qsonica Q125) for 5 mins with 10 s on/off pulses at 50% amplitude). Insoluble samples were boiled for 30 mins at 95°C, and the media and soluble samples were boiled for 5 mins at 95°C prior to immunoblotting. Two independent experiments were conducted in analytical duplicates.The medium collected 48 h after transfection was treated with cOmplete protease inhibitor (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), centrifuged at 1000 Samples were loaded into 12% stain-free Tris-glycine SDS-polyacrylamide gels prepared in house. Gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked at room temperature for 1 h in 5% milk. The following primary antibodies  were dilGlass microscope coverslips  were soaked for 30 min in 1% gelatin (Sigma-Aldrich) diluted in PBS. The solution was aspirated and allowed to air dry for 1 h in 24-well tissue-culture plates. HEK293T cells were plated at 70-80% confluency onto treated coverslips and grown overnight as described above. Cells were transfected with plasmids in serum-containing media as described above. Cells were washed 48 h post transfection with PBS and fixed in 10% formalin  for 30 min at room temperature and washed twice with PBS. Cells were then permeabilized with 1× assay buffer containing 0.5% Triton X-100 and 3 mM EDTA, supplied in the Proteostat Aggresome detection kit  and blocked overnight with 2% bovine serum albumin (BSA) in PBS. Primary antibodies [rabbit calnexin polyclonal antibody  and mouse monoclonal anti-FLAG ] for myocilin detection, each diluted 1:200, were prepared in 0.1% BSA in PBS and incubated for 3 h at room temperature. After primary antibody incubation, the coverslips were washed three times with PBS. Secondary antibodies [anti-rabbit Alexa Fluor 488  and anti-mouse Cy5 ] were diluted 1:1000 in 0.1% BSA in PBS and incubated for 45 min at room temperature. After secondary antibody incubation, the coverslips were washed again three times with PBS. Lastly, Proteostat and Hoechst 33342  were diluted 1:2000 and 1:1000, respectively, in 1× assay buffer and incubated for 30 min at room temperature. The coverslips were washed and mounted using Antifade Gold Mounting Reagent (Invitrogen) and allowed to cure for 24 h. Fluorescence images were captured using a laser scanning confocal microscope (Zeiss LSM 700 with AxioObserver and 63×1.4 oil immersion objective).Colocalization measurements were calculated using Pearson's coefficient within ImageJ  using the JACoP plugin . Statist2, and cells were allowed to grow for 16 h. The cells were pelleted, flash frozen in liquid nitrogen and stored at −80°C. WT, T256M, R296H, L303I, S313F, S331L, E352K, V449I and K500R variants were purified by amylose affinity chromatography and size-exclusion chromatography as reported previously for WT OLF (OLF variants were recombinantly expressed and purified as described previously  with minr WT OLF . E300K aE300K and MBP-OLFS313F), 50 mM maltose was added to stabilize MBP outside the range of OLF . SYPRO Orange fluorescence was monitored as a function of temperature in an Applied Biosystems Step One Plus RT-PCR system with fixed excitation at 480 nm and a carboxy-X-rhodamine 610 nm emission filter. Thermal melts were conducted in triplicate from 25 to 95°C with a 1°C/min gradient. Fluorescence data were blank subtracted and fitted with a Boltzmann sigmoid in GraphPad Prism v9 to determine the midway point of unfolding, the melting temperature (Tm).The thermal stability of OLF variants was measured by differential scanning fluorimetry . Brieflye of OLF . For ass2HPO4, 10 mM KH2PO4, 200 mM NaCl, pH 7.2) were used. Spectra were acquired at 4°C with ten averaged scans from 300 to 200 nm at a 500 nm/min scan rate, using a 0.1 cm cuvette. Data were blank subtracted ad converted to mean residue ellipticity, θ=(Mres×θobs)/(10×d×c), where Mres= 112.9 is the mean residue mass calculated for OLF, θobs is the observed ellipticity (degrees) at a given wavelength, d is the path length (cm) and c is the protein concentration (g/ml). Near-UV CD experiments were conducted with OLF variants in gel filtration buffer, pH 7.2, at a final concentration of 30-50 µM. For urea experiments, OLF was unfolded in the corresponding urea concentration for at least 1 h at 4°C prior to CD spectrum acquisition. Scans were taken at 4°C and measured from 320 to 250 nm with a scan rate of 50 nm/min in a 0.1 cm cuvette. Each measurement was the average of ten scans and converted to mean residue ellipticity.Far-UV CD and near-UV CD were performed as reported previously  to asses2. Crystals were cryo-cooled in a solution containing their respective reservoir solution supplemented with 30% glycerol. X-ray diffraction datasets were collected at the Advanced Photon Source, Argonne National Labs beamlines, Southeast Regional Collaborative Access Team (SER-CAT) 22-ID  or 22-BM (for K500R), or at the Advanced Light Source, Lawrence Berkeley National Labs beamlines, Berkeley Center for Structural Biology (BCSB) BL-5.0.2 (for R296H). Data were processed using HKL-2000 (4WXQ) as the search model. The models were iteratively refined using Coot  were concentrated to 5-10 mg/ml. Crystals were grown by the hanging-drop method by equilibration against a reservoir containing 8-10% PEG 8000 and 0.05-0.1 M MgClHKL-2000 . StructuHKL-2000 , using Wing Coot  and Phening Coot .m±s.d., the raw values of which were obtained from measurements reported by us in the literature (m±s.d. WT-like variants and disease variants segregated into two clusters. Significance was evaluated by a Student's unpaired two-tailed t-test. Linear regression and statistical analyses were calculated in GraphPad Prism v9.Age at diagnosis was curated from the literature . Individterature , 2015 or10.1242/dmm.049816_sup1Supplementary informationClick here for additional data file.


Previous studies have reported that high fasting plasma glucose (FPG), even that within the normal range, is associated with the risk of type 2 diabetes (T2D). Nevertheless, these findings are limited to specific populations. Thus, studies in the general population are imperative.This study included two cohorts comprising 204 640 individuals who underwent physical examinations at the Rich Healthcare Group present at 32 locations in 11 cities of China from 2010 to 2016 and 15 464 individuals who underwent physical tests at the Murakami Memorial Hospital in Japan. Cox regression, restricted cubic spline (RCS), Kaplan–Meier (KM) curves, and subgroup analysis were used to determine the relationship between FPG and T2D. Receiver operating characteristic (ROC) curves were used to evaluate the predictive power of FPG for T2D.The mean age of the 220 104 participants (204 640 Chinese and 15 464 Japanese participants) was 41.8 years (41.7 years for the Chinese and 43.7 years for the Japanese participants). During follow-up, 2611 individuals developed T2D (2238 Chinese and 373 Japanese participants). The RCS demonstrated a J-shaped relationship between FPG and T2D risk, with inflexion points of 4.5 and 5.2 for the Chinese and Japanese populations, respectively. Multivariate-adjusted hazard ratio (HR) was 7.75 for FPG and T2D risk after the inflexion point (7.3 for Chinese and 21.13 for Japanese participants).In general Chinese and Japanese populations, the normal baseline FPG range showed a J-shaped relationship with the risk of T2D. Baseline FPG levels help identify individuals at high risk of T2D and may enable early primary prevention to improve their outcomes.The online version contains supplementary material available at 10.1186/s12967-023-04006-9. The prevalence of T2D has rapidly increased over the past few decades. The harmful effects and increased healthcare costs associated with it are a growing public concern , 2. AccoFasting plasma glucose (FPG) is closely related to diabetes, not only as an indicator of pancreatic function and insulin resistance  but alsoTherefore, we analysed the relationship between normal FPG and T2D in a general adult population. Considering East Asians have a common ethnicity, they have similar diabetes characteristics and diagnostic criteria . TherefoThis study included the following two cohorts: 1) 685 277 individuals who were examined by the Rich Healthcare Group examined at 32 locations in 11 cities of China from 2010 to 2016  and (2)  685 277 FPG, serum triglyceride (TG), total cholesterol (TC), and alanine aminotransferase (ALT) levels were measured using an automated analyser after > 8 h of fasting. Questionnaires were used to record age, sex, height, weight, smoking, and alcohol history. Physicians measured blood pressure of the participants after 20 min of quiet rest.Hypertension was defined as systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg. The diagnostic criteria for T2D were FPG ≥ 7.0 mmol/L, HbA1c of at least 6.5%, or self-reported T2D. Annual health checks were regarded as follow-up visits.http://www.r-project.org, R Foundation for Statistical Computing, Vienna, Austria) and EmpowerStats packages . Statistical significance was determined by P (two-sided) values below 0.05.First, all continuous data were tested for normality using Kolmogorov–Smirnov tests. For normally distributed data, analysis of variance (ANOVA) tests were used; for others, Kruskal–Wallis nonparametric tests were used to determine differences between the groups. Chi-square tests were used for comparing count data. Second, we conducted receiver operating characteristic (ROC) analysis to assess the prediction accuracy of FPG for T2D onset. Third, we performed Cox regression analysis to examine the linear relationship between FPG and new-onset T2D as well as restricted cubic spline (RCS) analysis to explore the potential nonlinear relationship. Fourth, the relationship between FPG and cumulative T2D incidence diabetes was analysed using Kaplan‒Meier (KM) curves. Finally, subgroup analysis was used to test the sensitivity analysis. All statistical analyses were conducted using R packages (P < 0.001). The Q5 group demonstrated the highest age, body mass index (BMI), SBP, DBP, TC, TG, and ALT, as well as the most significant number of women, smokers and drinkers, and persons with hypertension than the Q1–Q4 group . The Chinese population demonstrated a consistent relationship, i.e, all parameters except drinking (P = 0.304) were positively correlated with T2D incidence. In the Japanese population, all parameters except smoking (P = 0.126) were positively correlated with the T2D incidence. Additional file To compare the role of FPG and other baseline parameters in predicting T2D, univariate Cox proportional hazards analysis was performed. In all populations, every baseline parameter , except drinking were positively associated with the risk of developing T2D .Higher FPG levels were associated with an increased risk of developing T2D in the fully model (Table P for nonlinearity < 0.05). The inflexion points for the relationship between FPG and new-onset T2D in the Chinese and Japanese populations were 4.5 mmol/L and 5.2 mmol/L, respectively (Table P < 0.001).We adjusted the variables of the fully model to further construct the RCS to depict the risk for FPG and T2D. Multivariate-adjusted RCS Fig.  revealedly Table . The risThe areas under the ROC curves (AUCs) were used to evaluate the effectiveness of FPG in predicting T2D risk. The AUCs of the FPG and T2D relationships were 0.772, 0.762, and 0.805 for all populations, Chinese, and Japanese, respectively, indicating good predictive performance . In the Chinese and Japanese populations, a stronger correlation was observed between FPG and T2D in the following groups of people: age < 45 years, BMI (18.5–24 kg/m2), no hypertension, no smoking, and no drinking  are presented as a forest plot  and risk of future T2D occurrence in an East Asian population. The risk of T2D increased significantly when FPG exceeded 4.5 mmol/L and 5.2 mmol/L in the Chinese and Japanese populations, respectively. Overall, the rise in T2D risk in these the East Asian populations was insignificant or sluggish until FPG level reached 4.5 mmol/L.Previous studies involving various populations exhibited similar results –21. Tiro2), no hypertension, no smoking, and no drinking. Previous studies showed that age [Previous epidemiological studies , 27 demothat age , obesitythat age , hypertethat age , and smothat age  are riskthat age . Thus, tCurrently, the pathogenesis of T2D is not fully understood. Studies in European and American populations have shown that insulin resistance triggers T2D and ultimately leads to islet β-cell failure . HoweverThe strength of this study is the large sample size; this is the largest study population  for any study concerning normal FPG ranges and T2D risk. Moreover, our study covers a broader age range (18–97 years). However, our study has certain limitations. For the diagnosis of T2D, we relied solely on FPG, glycosylated haemoglobin, and patient self-reporting; therefore, oral glucose tolerance test (OGTT) might have been overlooked. Furthermore, there might have been confusion related to type 1 diabetes (T1D), which requires insulin antibody tests that time-consuming and laborious for diagnosis. We also did not have access to the medical history records of our study population; therefore, the possibility that certain medications caused T2D cannot be excluded.This study comprising over 200 000 general East Asians showed that a J-shaped relationship exists between normal FPG range and T2D risk, with inflexion points of 4.5 mmol/L and 5.2 mmol/L for the Chinese and Japanese populations, respectively. These results are expected to draw clinicians' attention to the risk of diabetes in a population exhibiting healthy glycaemic index currently. FPG levels below 4.5 mmol/L significantly reduce the risk of progression to T2D. Clinical trial interventions are still required to further validate our results. Future research, regarding early primary prevention and management of populations with healthy glucose levels currently is necessary.Additional file 1: Fig. S1. ROC curve analysis of the relationship between FPG and T2D in different populations. Table S1. Baseline characteristics of the Chinese population according to FPG quintiles. Table S2. Baseline characteristics of the Japanese population according to FPG quintiles. Table S3. Univariate cox regression analysis of T2D. Table S4. Efficacy of FPG in predicting T2D in different populations. Table S5. Subgroup analysis of FPG and T2D risk in the Chinese and Japanese populations.Additional file 2: STROBE-cohort checklist.