Bacteria undergo significant shifts while archaea maintain stability in Pocillopora damicornis under sustained heat stress.

Global warming reportedly poses a critical risk to coral reef ecosystems. Bacteria and archaea are crucial components of the coral holobiont. The response of archaea associated with warming is less well understood than that of the bacterial community in corals. Also, there have been few studies on the dynamics of the microbial community in the coral holobiont under long-term heat stress. In order to track the dynamic alternations in the microbial communities within the heat-stressed coral holobiont, three-week heat-stress monitoring was carried out on the coral Pocillopora damicornis. The findings demonstrate that the corals were stressed at 32 °C, and showed a gradual decrease in Symbiodiniaceae density with increasing duration of heat stress. The archaeal community in the coral holobiont remained relatively unaltered by the increasing temperature, whereas the bacterial community was considerably altered. Sustained heat stress exacerbated the dissimilarities among parallel samples of the bacterial community, confirming the Anna Karenina Principle in animal microbiomes. Heat stress leads to more complex and unstable microbial networks, characterized by an increased average degree and decreased modularity, respectively. With the extension of heat stress duration, the relative abundances of the gene (nifH) and genus (Tistlia) associated with nitrogen fixation increased in coral samples, as well as the potential pathogenic bacteria (Flavobacteriales) and opportunistic bacteria (Bacteroides). Hence, our findings suggest that coral hosts might recruit nitrogen-fixing bacteria during the initial stages of suffering heat stress. An environment that is conducive to the colonization and development of opportunistic and pathogenic bacteria when the coral host becomes more susceptible as heat stress duration increases.

[The number of FOXP3+regulatory T cells (Tregs) decreased and transformed into RORγt+FOXP3+Tregs in lung tissues of mice with bronchopulmonary dysplasia].

Objective To explore the phenotypic conversion of regulatory T cells (Tregs) in the lungs of mice with bronchopulmonary dysplasia (BPD)-affected mice. Methods A total of 20 newborn C57BL/6 mice were divided into air group and hyperoxia group, with 10 mice in each group. The BPD model was established by exposing the newborn mice to hyperoxia. Lung tissues from five mice in each group were collected on postnatal days 7 and 14, respectively. Histopathological changes of the lung tissues was detected by HE staining. The expression level of surfactant protein C (SP-C) in the lung tissues was examined by Western blot analysis. Flow cytometry was performed to assess the proportion of FOXP3+ Tregs and RORγt+FOXP3+ Tregs in CD4+ lymphocytes. The concentrations of interleukin-17A (IL-17A) and IL-6 in lung homogenate were measured by using ELISA. Spearman correlation analysis was used to analyze the correlation between FOXP3+Treg and the expression of SP-C and the correlation between RORγt+FOXP3+ Tregs and the content of IL-17A and IL-6. Results The hyperoxia group exhibited significantly decreased levels of SP-C and radical alveolar counts in comparison to the control group. The proportion of FOXP3+Tregs was reduced and that of RORγt+FOXP3+Tregs was increased. IL-17A and IL-6 concentrations were significantly increased. SP-C was positively correlated with the expression level of RORγt+FOXP3+ Tregs. RORγt+FOXP3+ Tregs and IL-17A and IL-6 concentrations were also positively correlated. Conclusion The number of FOXP3+ Tregs in lung tissue of BPD mice is decreased and converted to RORγt+ FOXP3+ Tregs, which may be involved in hyperoxy-induced lung injury.

Targeted metabolome and transcriptome analyses reveal changes in gibberellin and related cell wall-acting enzyme-encoding genes during stipe elongation in Flammulina filiformis.

Flammulina filiformis, a typical agaric fungus, is a widely cultivated and consumed edible mushroom. Elongation of its stipe (as the main edible part) is closely related to its yield and commercial traits; however, the endogenous hormones during stipe elongation and their regulatory mechanisms are not well understood. Gibberellin (GA) plays an important role in the regulation of plant growth, but little has been reported in macro fungi. In this study, we first treated F. filiformis stipes in the young stage with PBZ (an inhibitor of GA) and found that PBZ significantly inhibited elongation of the stipe. Then, we performed GA-targeted metabolome and transcriptome analyses of the stipe at both the young and elongation stages. A total of 13 types of GAs were detected in F. filiformis; the contents of ten of them, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, were significantly decreased, and the contents of three (GA5, GA9, and GA29) were significantly increased during stipe elongation. Transcriptome analysis showed that the genes in the terpenoid backbone biosynthesis pathway showed varying expression patterns: HMGS, HMGR, GPS, and FPPS were significantly upregulated, while CPS/KS had no significant difference in transcript level during stipe elongation. In total, 37 P450 genes were annotated to be involved in GA biosynthesis; eight of them were upregulated, twelve were downregulated, and the rest were not differentially expressed. In addition, four types of differentially expressed genes involved in stipe elongation were identified, including six signal transduction genes, five cell cycle-controlling genes, twelve cell wall-related enzymes and six transcription factors. The results identified the types and content of GAs and the expression patterns of their synthesis pathways during elongation in F. filiformis and revealed the molecular mechanisms by which GAs may affect the synthesis of cell wall components and the cell cycle of the stipe through the downstream action of cell wall-related enzymes, transcription factors, signal transduction and cell cycle control, thus regulating stipe elongation. This study is helpful for understanding the roles of GAs in stipe development in mushrooms and lays the foundation for the rational regulation of stipe length in agaric mushrooms during production.

ILC2 influence the differentiation of alveolar type II epithelial cells in bronchopulmonary dysplasia mice.

Bronchopulmonary dysplasia, a common complication of premature infants, is mainly characterized by blocked alveolarization. Proverbially, the injury of alveolar type II epithelial cells is regarded as the pathologic basis of occurrence and development of bronchopulmonary dysplasia. In the case of alveolar epithelial damage, alveolar type II epithelial cells can also differentiate to alveolar type I epithelial cells as progenitor cells. During bronchopulmonary dysplasia, the differentiation of alveolar type II epithelial cells becomes abnormal. Group 2 innate lymphoid cells can produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines IL-25, IL-33, and thymic stromal lymphopoietin. Previous studies have shown that group 2 innate lymphoid cells can inhibit the alveolarization process of bronchopulmonary dysplasia by secreting IL-13. However, whether group 2 innate lymphoid cells can affect the differentiation of alveolar type II epithelial cells in the pathologic process of bronchopulmonary dysplasia remains unclear. In this study, we have shown that IL-13 secreted by group 2 innate lymphoid cells increased during bronchopulmonary dysplasia, which was related to the release of large amounts of IL-33 by impaired alveolar type II epithelial cells. This led to abnormal differentiation of alveolar type II epithelial cells, reduced differentiation to alveolar type I epithelial cells, and increased transdifferentiation to mesenchymal cells through the epithelial-mesenchymal transition. Taken together, our study provides a complementary understanding of the development of bronchopulmonary dysplasia and highlights a novel immune mechanism in the pathogenesis of bronchopulmonary dysplasia.

Type 2 innate lymphoid cell-derived amphiregulin regulates type II alveolar epithelial cell transdifferentiation in a mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common complication in preterm infants characterized by alveolar growth arrest. Interleukin (IL)-33 and type 2 innate lymphoid cell (ILC2) affect type II alveolar epithelial cell (AECII) differentiation in BPD mice and may cause increased lung epithelial-mesenchymal transition (EMT). Amphiregulin (AREG) can be produced by ILC2 and is associated with tissue repair. However, the action mechanism of AREG produced by ILC2 to alveolar development in BPD is unclear. In this study, we aimed to demonstrate the role and mechanism of AREG in influencing AECII transdifferentiation in the lung tissue of BPD mice. The effects of ILC2-derived AREG on AECII transdifferentiation were verified in vivo and in vitro, and the role of IL-33 on ILC2-derived AREG in AECII transdifferentiation in BPD mice and a preliminary investigation of the role of AREG's receptor-epidermal growth factor receptor (EGFR) on AECII transdifferentiation. The results showed that neonatal mice developed severe lung injury after hyperoxia, and IL-33 induced AREG production via ILC2 affected normal AECII differentiation and promoted EMT. In addition, the blockade of EGFR was found to alleviate the impaired AECII differentiation under hyperoxia in an in vitro study. In summary, our study demonstrates that AREG secreted by ILC2 affects AECII transdifferentiation in BPD mice, which provides a new idea for the clinical treatment of BPD.

Short-time mortality and severe complications of very premature infants-a multicenter retrospective cohort study from Jiangsu Province during 2019-2021.

Background: The short-term and long-term severe complications of preterm infants have brought serious psychological and economic burdens to the society and family. Therefore, our study aimed to investigate the risk factors for the mortality and serious complications in very premature infants less than 32 weeks of gestational age (GA), so as to guide the antenatal and postnatal care of very premature. Methods: The very premature infants from 1 January 2019 to 31 December 2021 from 15 member hospitals of the Neonatal Intensive Care Unit (NICU) Multi-center Clinical Research Collaboration Group in Jiangsu Province were recruited. In accordance with the plan of the intensive care unit for unified management, recruitment of premature infants is carried out on the day of admission, and discharge or death is the outcome indicator in 1-2 months by telephone follow-up. The research content mainly includes three aspects: clinical information of mother and infant, outcomes and complications. According to the final outcomes, very premature infants were divided into two categories: survival without severe complications, survival with severe complications and death. Then, univariate and multivariate logistic regression model and receiver operating characteristic (ROC) analyses were used to analyze the independent risk factors. Results: A total of 3,200 very premature infants with GA less than 32 weeks were recruited. The median GA is 30.00 (28.57, 31.14) weeks, the average birth weight is 1,350 (1,110, 1,590) g, among whom 375 premature infants survived with severe complications, and 2,391 premature infants survived without severe complications. Then, it was found that GA at birth was a protective factor for death and severe complications, whereas severe neonatal asphyxia and persistent pulmonary hypertension of the newborn (PPHN) were independent risk factors for death and severe complications in very premature infants born at less than 32 weeks of gestation. Conclusions: The prognosis of very premature infants in NICU treatment depends not only on GA, but also on various perinatal factors and their clinical management, such as preterm asphyxia and PPHN occurrence, so the next step is necessary for multicenter continuous quality improvement to improve outcomes in very preterm infants.

ILC2 regulates hyperoxia-induced lung injury via an enhanced Th17 cell response in the BPD mouse model.

BACKGROUD: Recent research has focused on the role of immune cells and immune responses in the pathogenesis of bronchopulmonary dysplasia (BPD), but the exact mechanisms have not yet been elucidated. Previously, the key roles of type 2 innate lymphoid cells (ILC2) in the lung immune network of BPD were explored. Here, we investigated the role Th17 cell response in hyperoxia-induced lung injury of BPD, as well as the relationship between ILC2 and Th17 cell response. METHODS: A hyperoxia-induced BPD mouse model was constructed and the pathologic changes of lung tissues were evaluated by Hematoxylin-Eosin staining. Flow cytometry analysis was conducted to determine the levels of Th17 cell, ILC2 and IL-6+ILC2. The expression levels of IL-6, IL-17 A, IL-17 F, and IL-22 in the blood serum and lung tissues of BPD mice were measured by ELISA. To further confirm the relationship between ILC2 and Th17 cell differentiation, ILC2 depletion was performed in BPD mice. Furthermore, we used immunomagnetic beads to enrich ILC2 and then flow-sorted mouse lung CD45+Lin-CD90.2+Sca-1+ILC2. The sorted ILC2s were injected into BPD mice via tail vein. Following ILC2 adoptive transfusion, the changes of Th17 cell response and lung injury were detected in BPD mice. RESULTS: The expression levels of Th17 cells and Th17 cell-related cytokines, including IL-17 A, IL-17 F, and IL-22, were significantly increased in BPD mice. Concurrently, there was a significant increase in the amount of ILC2 and IL-6+ILC2 during hyperoxia-induced lung injury, which was consistent with the trend for Th17 cell response. Compared to the control BPD group, ILC2 depletion was found to partially abolish the Th17 cell response and had protective effects against lung injury after hyperoxia. Furthermore, the adoptive transfer of ILC2 enhanced the Th17 cell response and aggravated lung injury in BPD mice. CONCLUSIONS: This study found that ILC2 regulates hyperoxia-induced lung injury by targeting the Th17 cell response in BPD, which shows a novel strategy for BPD immunotherapy.

Comparison of gut viral communities in children under 5 years old and newborns.

OBJECTIVES: The gut virome of humans is mainly composed of bacteriophages and their role in shaping the gut microbiome and influencing human health is increasingly recognized. However, little is known about the dynamic changes of the gut virome in children and its role in growth and development. In this study, we collected fecal samples from newborns and children under 5 years old from the same area during the same time period to investigate the gut viral community using viral metagenomic technique. METHODS: We used viral metagenomics to compare the gut bacteriophage composition between newborns and children under 5 years of age. We collected fecal samples from 45 newborns who were born at the Affiliated Hospital of Jiangsu University and 45 healthy children who were examined at the same hospital. The two groups were classified as the newborn group and the children group. RESULTS: Our sequencing analysis showed that the number of seqeunce reads of the children group were more than that of the newborn group. The results of alpha diversity and beta diversity both indicated that the diversity of the children group was significantly higher than that of the newborn group and the children group is different from the newborn group. The abundance of gut virome in the children group was also higher than that in the newborn group. The analysis of the genetic characteristics of the viruses showed that the phage genome was scattered and clustered with specificity. CONCLUSION: Our findings indicate that the gut bacteriophage communities undergo changes over time, presenting diversity and dynamic characteristics. We characterized the composition of gut virome in children and newborns in this region. However, further research is needed to investigate the function of bacteriophages in the ecology of the gastrointestinal tract.

The function role of ubiquitin proteasome pathway in the ER stress-induced AECII apoptosis during hyperoxia exposure.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar dysplasia and pulmonary microvascular remodeling. In the present study, we have investigated the functional roles of ubiquitin proteasome pathway (UPP) in BPD, and its relationship with endoplasmic reticulum stress (ERS) mediated type II alveolar epithelial cell (AECII) apoptosis. METHODS: A hyperoxia-induced BPD rat model was constructed and the pathologic changes of lung tissues were evaluated by hematoxylin-eosin staining. Cell apoptosis and protein expression were determined by TUNEL assay and Western blotting, respectively. Further reagent kit with specific fluorescent substrate was utilized to measure the activity of 20 s proteasome. Meanwhile, AECII were cultured in vitro and exposed to hyperoxia. AECII apoptosis were measured by flow cytometry. In contrast, MG132 treatment was induced to explore UPP during hyperoxia exposure on AECII apoptosis and ERS sensors expression. RESULTS: A significant increase in apoptosis and total ubiquitinated proteins expression were observed in BPD rats and AECII culture, and the change of UPP was associated with ERS. In order to confirm the role of UPP in AECII apoptosis of BPD, AECII cells were treated by MG132 with the concentration of 10 µmol/L under hyperoxia exposure. We found that the proteins expression of glucose-regulated protein 78 (GRP-78), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), as well as AECII apoptosis were increased following MG132 treatment. Furthermore, the relatively up-regulated in the levels of total ubiquitinated proteins expression and 20 s proteasome activity were correlated with increased ERS sensors expression. CONCLUSIONS: Our findings indicate that UPP may participate in the ERS-induced AECII apoptosis under hyperoxia condition.

BCDnet: Parallel heterogeneous eight-class classification model of breast pathology.

Breast cancer is the cancer with the highest incidence of malignant tumors in women, which seriously endangers women's health. With the help of computer vision technology, it has important application value to automatically classify pathological tissue images to assist doctors in rapid and accurate diagnosis. Breast pathological tissue images have complex and diverse characteristics, and the medical data set of breast pathological tissue images is small, which makes it difficult to automatically classify breast pathological tissues. In recent years, most of the researches have focused on the simple binary classification of benign and malignant, which cannot meet the actual needs for classification of pathological tissues. Therefore, based on deep convolutional neural network, model ensembleing, transfer learning, feature fusion technology, this paper designs an eight-class classification breast pathology diagnosis model BCDnet. A user inputs the patient's breast pathological tissue image, and the model can automatically determine what the disease is (Adenosis, Fibroadenoma, Tubular Adenoma, Phyllodes Tumor, Ductal Carcinoma, Lobular Carcinoma, Mucinous Carcinoma or Papillary Carcinoma). The model uses the VGG16 convolution base and Resnet50 convolution base as the parallel convolution base of the model. Two convolutional bases (VGG16 convolutional base and Resnet50 convolutional base) obtain breast tissue image features from different fields of view. After the information output by the fully connected layer of the two convolutional bases is fused, it is classified and output by the SoftMax function. The model experiment uses the publicly available BreaKHis data set. The number of samples of each class in the data set is extremely unevenly distributed. Compared with the binary classification, the number of samples in each class of the eight-class classification is also smaller. Therefore, the image segmentation method is used to expand the data set and the non-repeated random cropping method is used to balance the data set. Based on the balanced data set and the unbalanced data set, the BCDnet model, the pre-trained model Resnet50+ fine-tuning, and the pre-trained model VGG16+ fine-tuning are used for multiple comparison experiments. In the comparison experiment, the BCDnet model performed outstandingly, and the correct recognition rate of the eight-class classification model is higher than 98%. The results show that the model proposed in this paper and the method of improving the data set are reasonable and effective.

Sumoylation of CCAAT-enhancer-binding protein α inhibits lung differentiation in Bronchopulmonary Dysplasia model rats.

Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar simplification, surfactant deficiency, and respiratory distress. In the present study, we have investigated the functional roles of sumoylated CCAAT/enhancer binding protein alpha (C/EBPα) in the BPD rat model. A significant increase in small ubiquitin-like modifier 1 (SUMO1) and sumoylated C/EBPα protein levels were observed in BPD rats, and the levels of the sumoylated C/EBPα were associated with the pulmonary surfactant proteins (SPs). In order to confirm the role of sumoylated C/EBPα in BPD rats, SUMO1 was knocked down by lentiviral transfection of neonatal rat lungs with SUMO1-RNAi-LV. We found that the expression of C/EBPα and surfactant proteins increased following SUMO1 knockdown. Furthermore, the relatively low decrease in the levels of C/EBPα sumoylation was correlated with reduced glycogen consumption. Besides, co-immunoprecipitation assays revealed that sumoylation is involved in the regulation of the interaction between C/EBPα and TGFß2 in the lung. In conclusion, our findings indicate that sumoylation may act as a negative regulator of the C/EBPα-mediated transactivation in BPD rats.

SENP1 has an important role in lung development and influences the differentiation of alveolar type 2 cells.

Post­translational modification via small ubiquitin­like modifier (SUMO) is involved in the regulation of various important cellular processes. SUMO modification can be regulated at the level of conjugation, and can also be reversed by the SUMO­specific proteases (SENPs). However, current studies of the regulation and function of SENP in lung development remain limited. In this study, the expression levels of SENP1 and SUMO1 were assessed during lung development in rats. SUMO1 modification occurred during lung development and changes in SENP1 expression were consistent with the changes in the presence of free SUMO1. In order to investigate the function of SENP1, alveolar type (AT) 2 cells were transfected with SENP1­targeting small interfering RNA, and the proliferation, apoptosis and differentiation function of AT2 cells was subsequently evaluated. Marked upregulation of conjugated SUMO1 was observed following SENP1 inhibition. Furthermore, depletion of SENP1 resulted in increased apoptosis, decreased proliferation and impaired differentiation status of AT2 cells. Thus, the results support that SENP1 is an essential regulator of the balance between SUMOylation and deSUMOylation during lung development, specifically affecting the proliferation and differentiation status of AT2 cells.

[Hyperoxia induces AECII apoptosis of premature rats by reducing the activity of 20S proteasome].

Objective To establish a hyperoxia-exposed cell damage model of primary cultured premature rat alveolar type II epithelial cells (AECIIs) and investigate the effect of hyperoxia on ubiquitinated protein degradation and apoptosis of AECIIs. Methods Primary cultured premature rat AECIIs in vitro were divided into air group and hyperoxia group, which were exposed to air and 950 mL/L O2, respectively. After 24-, 48- and 72-hour exposure, isothiocyanate-labeled annexinV/propidium iodide (annexinV-FITC/PI) double staining combined with flow cytometry was utilized to detect the apoptosis of AECIIs. Real-time quantitative PCR and Western blotting were performed to observe the mRNA expression of ubiquitin and the protein levels of total ubiquitinated proteins, Bax/Bcl-2 ratio and caspase-3. Specific fluorescence substrate was used to measure the activity of 20S proteasome. Results Compared with the air group, the apoptosis of AEC II s in the hyperoxia group increased at each time point. The activity of 20S proteasome in AECIIs in the hyperoxia group decreased dramatically, while the levels of ubiquitin mRNA and total ubiquitinated protein increased. At the same time, the expression of caspase-3 and the Bax/Bcl-2 ratio were significantly raised. Conclusion Exposure to hyperoxia decreases the activity of 20S proteasome in AECIIs and the degradation of ubiquitinated proteins, which may induce the apoptosis of AECIIs by upregulating the expression of caspase-3 and the Bax/Bcl-2 ratio.

[Association between endoplasmic reticulum stress pathway mediated by inositol-requiring kinase 1 and AECII apoptosis in preterm rats induced by hyperoxia].

OBJECTIVE: To study the association between endoplasmic reticulum stress (ERS) pathway mediated by inositol-requiring kinase 1 (IRE1) and the apoptosis of type II alveolar epithelial cells (AECIIs) exposed to hyperoxia. METHODS: The primarily cultured AECIIs from preterm rats were devided into an air group and a hyperoxia group. The model of hyperoxia-induced cell injury was established. The cells were harvested at 24, 48, and 72 hours after hyperoxia exposure. An inverted phase-contrast microscope was used to observe morphological changes of the cells. Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis. RT-PCR and Western blot were used to measure the mRNA and protein expression of glucose-regulated protein 78 (GRP78), IRE1, X-box binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP). An immunofluorescence assay was performed to measure the expression of CHOP. RESULTS: Over the time of hyperoxia exposure, the hyperoxia group showed irregular spreading and vacuolization of AECIIs. Compared with the air group, the hyperoxia group showed a significantly increased apoptosis rate of AECIIs and significantly increased mRNA and protein expression of GRP78, IRE1, XBP1, and CHOP compared at all time points (P<0.05). The hyperoxia group had significantly greater fluorescence intensity of CHOP than the air group at all time points. In the hyperoxia group, the protein expression of CHOP was positively correlated with the apoptosis rate of AECIIs and the protein expression of IRE1 and XBP1 (r=0.97, 0.85, and 0.88 respectively; P<0.05). CONCLUSIONS: Hyperoxia induces apoptosis of AECIIs possibly through activating the IRE1-XBP1-CHOP pathway.