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Minglei Ma, Tao Jiang, Dongxue Zhang, Xiaoai Yao, Zhen Wen, Lei Xiu Department of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, People’s Republic of ChinaCorrespondence: Tao Jiang, Email jiangtao@bjsjth.cnObjective: To investigate the association of bedtime with the risk of early-onset diabetes mellitus (DM) and islet beta cell function.Methods: 138 participants with treat-naïve DM were included in this study. All participants underwent a 75g oral glucose tolerance test. Sleep habit was obtained through a standardized questionnaire. Bedtime was categorized as < 22:00, 22:00– 24:00, and ≥ 24:00 in this study. Multivariate logistic regression and multiple linear regression were used to estimate the association between bedtime and risk of early-onset DM and islet beta cell function, respectively.Results: Patients with early-onset DM had a later bedtime than those with late-onset DM. Individuals with bedtime ≥ 24:00 had a higher prevalence of early-onset DM than those with bedtime at 22:00– 24:00 and < 22:00 (51.2% vs 29.3% vs 14.3%, respectively, p = 0.028). The multivariate logistic regression showed that per hour later in bedtime was associated with a 52% increased risk of early-onset DM (p = 0.023). Patients with bedtime after 24:00 had a 146% increased risk of early-onset DM compared to those went to bed between 22:00 to 24:00 (OR = 2.46, 95% CI 1.05 to 5.77, p = 0.039). The multiple linear regression showed that bedtime was independently negatively correlated with late-phase insulin secretion (assessed by disposition index, DI120).Conclusion: Later bedtime was associated with worse late-phase insulin secretion and may be a risk factor for early-onset DM. Proper bedtime to lower risk of early-onset DM deserves further investigation.Keywords: diabetes mellitus, early-onset, sleep, bedtime, islet beta cell function

Minglei Ma, Tao Jiang, Zhen Wen, Dongxue Zhang, Lei Xiu Department of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Tao Jiang, Email jiangtao@bjsjth.cnObjective: To assess the correlation between body components with insulin resistance (IR) and islet beta cell function in patients with newly diagnosed type 2 diabetes mellitus (T2DM) or pre-diabetes mellitus (pre-DM) and to explore whether this correlation differs in males and females.Methods: 111 newly diagnosed diabetic or pre-diabetic patients were recruited in this cross-sectional study. 75g oral glucose tolerance test was used to determine the diagnosis of DM or pre-DM. Homeostasis model assessment of insulin resistance (HOMA-IR) and glucose disposition index (DI30) was calculated to assess IR and islet beta cell function, respectively. Whole-body and regional lean mass (LM) and fat mass (FM) were obtained by dual-energy X-ray absorptiometry (DEXA). Partial correlation and multiple linear regression analyses were used to determine the associations between body composition, IR, and islet beta cell function.Results: More body fat and appendicular fat was observed in female patients than in male, though with similar BMI. Legs fat % was negatively correlated with HOMA-IR, whereas legs lean % was positively associated with HOMA-IR in females (r = − 0.673, p = 0.017; r =0.664, p = 0.018, respectively). The regression analysis showed that legs LM was positively correlated with HOMA-IR in females. However, in male patients, android FM was positively correlated with HOMA-IR (r = 0.462, p = 0.007), and trunk LM was negatively associated with DI30 (r = − 0.458, p = 0.007). Nevertheless, no significant correlation was observed between body composition and islet beta cell function in female patients.Conclusion: Android FM was positively correlated with IR only in male patients but not in females. Besides, relative legs fat and LM were independently associated with IR in female patients but not in males. Further studies are needed to explore the underlying mechanism.Keywords: body composition, diabetes mellitus, insulin resistance, islet beta cell function

Lei Xiu, Tao Jiang, Xiao-ai Yao, Zhen Wen Department of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, People’s Republic of ChinaCorrespondence: Tao JiangDepartment of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, People’s Republic of ChinaEmail jiangtao@bjsjth.cnPurpose: We aimed to analyze the serum vitamin D level in Chinese patients with type 2 diabetes mellitus (T2DM) and discuss its correlation with nonalcoholic fatty liver disease (NAFLD).Patients and Methods: A total of 300 patients with T2DM (92 patients without NAFLD and 208 patients with NAFLD) were enrolled, and 25-hydroxyvitamin D [25-(OH)D] levels were compared between the two groups. Second, the NAFLD fibrosis score (NFS) and fatty liver index (FLI) were used to group patients with T2DM complicated by NAFLD, and the differences in serum 25-(OH)D in patients with different degrees of liver fibrosis were compared. Third, multiple regression analysis was used to analyze the independent predictors of liver fibrosis in patients with T2DM complicated by NAFLD.Results: The level of 25-(OH)D in patients with T2DM complicated by NAFLD was significantly lower than that in patients with T2DM alone. Based on the NFS and FLI, the 25-(OH)D level of the hepatic fibrosis subgroup was significantly lower than that of the subgroup without liver fibrosis. 25-(OH)D was found to be an independent predictor of liver fibrosis in patients with T2DM complicated by NAFLD.Conclusion: The serum 25-(OH)D level in patients with T2DM complicated by NAFLD was significantly reduced, and the 25-(OH)D level showed a gradual downward trend with the degree of liver fibrosis. Low concentrations of 25-(OH)D may be indicative of the degree of liver fibrosis in diabetic patients.Keywords: type 2 diabetes mellitus, nonalcoholic fatty liver disease, 25-hydroxyvitamin D, liver fibrosis

Fowl adenovirus serotype 4 (FAdV-4) is a highly pathogenic virus with a broad host range that causes huge economic losses for the poultry industry worldwide. RNA sequencing has provided valuable and important mechanistic clues regarding FAdV-4–host interactions. However, the pathogenic mechanism and host's responses after FAdV-4 infection remains limited. In this study, we used transcriptome analysis to identify dynamic changes in differentially expressed genes (DEGs) at five characteristic stages (12, 24, 36, 48, and 60 h) post infection (hpi) with FAdV-4. A total of 8,242 DEGs were identified based on comparison of five infection stages: 0 and 12, 12 and 24, 24 and 36, 36 and 48, and 48 and 60 hpi. In addition, at these five important time points, we found 37 common upregulated or downregulated DEGs, suggesting a common role for these genes in host response to viral infection. The predicted function of these DEGs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these DEGs were associated with viral invasion, host metabolic pathways and host immunosuppression. Interestingly, genes involved in viral invasion, probably EGR1, SOCS3, and THBS1, were related to FAdV-4 infection. Validation of nine randomly selected DEGs using quantitative reverse-transcription PCR produced results that were highly consistent with those of RNA sequencing. This transcriptomic profiling provides valuable information for investigating the molecular mechanisms underlying host–FAdV-4 interactions. These data support the current molecular knowledge regarding FAdV-4 infection and chicken defense mechanisms.

Lei Xiu, Xiao-ai Yao, Tao Jiang Department of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, People’s Republic of ChinaCorrespondence: Tao JiangDepartment of Endocrinology, Beijing Shijitan Hospital, Capital Medical University, No. 10 Tieyilu, Yang Fang Dian, Beijing, 100038, People’s Republic of ChinaTel/Fax +86-10-63926692Email jiangtao@bjsjth.cnPurpose: Chinese adults with early-onset type 2 diabetes mellitus have impaired diastolic function. This study aims to analyse the association between serum vitamin D levels and cardiac diastolic dysfunction in Chinese adults with early-onset type 2 diabetes mellitus.Patients and Methods: We enrolled Chinese adults with early-onset type 2 diabetes mellitus in this study. These patients were divided into two groups: those with diastolic dysfunction and those without diastolic dysfunction. We then compared the levels of serum 25-hydroxyvitamin D [25-(OH)D] between the two groups. The correlation between diastolic function and 25-(OH)D was evaluated by Pearson correlation analysis. Finally, binary logistic regression was used to analyse the relationship between the decrease in diastolic function and 25-(OH)D and other indexes in Chinese adults with early-onset type 2 diabetes mellitus.Results: The level of 25-(OH)D in patients with early-onset type 2 diabetes mellitus complicated with cardiac diastolic dysfunction was significantly lower than that in patients without cardiac diastolic dysfunction (P< 0.01). The degree of liver fibrosis in adult patients with early-onset type 2 diabetes mellitus complicated with diastolic dysfunction was significantly higher than that in adult patients without diastolic dysfunction (P< 0.01). Moreover, decreased 25-(OH)D levels were associated with decreased diastolic function in adults with early-onset type 2 diabetes.Conclusion: 25-(OH)-D was identified as an independent predictor of decreased diastolic function in adults with early-onset type 2 diabetes. The serum 25-(OH)D level in adults with early-onset type 2 diabetes was significantly reduced. 25-(OH)D influences the reduction in diastolic function in adults with early-onset type 2 diabetes and can be used as a predictor of decreased diastolic function in such patients.Keywords: early-onset type 2 diabetes in adults, 25-hydroxyvitamin D, decreased diastolic function, non-alcoholic fatty liver disease

Chi Zhang,1,* Tao Hu,2,* Leshan Xiu,1 Yamei Li,1 Junping Peng1 1NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China; 2Department of Neurosurgery, Shanxi Provincial People’s Hospital, Taiyuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Junping PengNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 6, Rongjing East Street, BDA, Beijing, People’s Republic of ChinaTel/Fax +86 10 67878493Email pengjp@hotmail.comAbstract: Unbiased metagenomic next-generation sequencing (mNGS) has been widely applied in clinical microbiology for infectious disease diagnostics. Some successful applications in central nervous system infections and respiratory tract infections have proved it to be a promising tool for pathogen detection. Here, mNGS was used to confirm the infectious etiology in a case of tuberculous coxitis, which is a common type of extrapulmonary tuberculosis (TB). A 77-year-old Asian male presented with hip pain and fever was diagnosed with smear- and culture-negative tuberculous coxitis based on the evidence of imaging and interferon-γ release assay. At the same time as the anti-TB therapy started, the patient underwent joint clearance surgery. Whole blood and synovial fluid sampled from surgery were kept for bacteriological confirmation using mNGS with high sequencing depth. However, only two paired-end reads from synovial fluid samples were identified as Mycobacterium tuberculosis. Before leaving hospital, the patient was still receiving anti-TB treatment and began to recover. Considering the obvious escalation of the cost and analysis time as the depth of sequencing increases, although we got a positive result here, the scarce number of reads obtained through ultra-deep sequencing indicates its limitations in extrapulmonary tuberculosis.Keywords: extrapulmonary tuberculosis, metagenomics, molecular diagnostic techniques, Mycobacterium tuberculosis

Leshan Xiu,1 Chi Zhang,1 Yamei Li,1 Feng Wang,2 Junping Peng11NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China; 2Shenzhen Center for Chronic Disease Control, Shenzhen, People’s Republic of ChinaCorrespondence: Junping PengNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 6 Rongjing Eastern Street, BDA, Beijing 100176, People’s Republic of ChinaTel/fax +86 106 787 8493Email pengjp@hotmail.comPurpose: Sexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs.Methods: We developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, and Haemophilus ducreyi) and 3 controversial microorganisms as pathogens (Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum).Results: The results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target.Conclusion: STI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI.Keywords: sexually transmitted infections, timely diagnosis, MALDI-TOF mass spectrometry, digital PCR, high-throughput

ABSTRACT The gut microbiota plays an essential role in the health of bees. To elucidate the effect of feed and Nosema ceranae infection on the gut microbiota of honey bee (Apis cerana), we used 16S rRNA sequencing to survey the gut microbiota of honey bee workers fed with sugar water or beebread and inoculated with or without N. ceranae. The gut microbiota of A. cerana is dominated by Serratia, Snodgrassella, and Lactobacillus genera. The overall gut microbiota diversity was show to be significantly differential by feeding type. N. ceranae infection significantly affects the gut microbiota only in bees fed with sugar water. Higher abundances of Lactobacillus, Gluconacetobacter, and Snodgrassella and lower abundances of Serratia were found in bees fed with beebread than in those fed with sugar water. N. ceranae infection led to a higher abundance of Snodgrassella and a lower abundance of Serratia in sugar-fed bees. Imputed bacterial Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed the significant metagenomics functional differences by feeding and N. ceranae infections. Furthermore, A. cerana workers fed with sugar water showed lower N. ceranae spore loads but higher mortality than those fed with beebread. The cumulative mortality was strongly positive correlated (rho = 0.61) with the changes of overall microbiota dissimilarities by N. ceranae infection. Both feeding types and N. ceranae infection significantly affect the gut microbiota in A. cerana workers. Beebread not only provides better nutrition but also helps establish a more stable gut microbiota and therefore protects bees in response to N. ceranae infection. IMPORTANCE The gut microbiota plays an essential role in the health of bees. Scientific evidence suggests that diet and infection can affect the gut microbiota and modulate the health of the gut; however, the interplay between those two factors and the bee gut microbiota is not well known. In this study, we used a high-throughput sequencing method to monitor the changes of gut microbiota associated with both feeding types and Nosema ceranae infection. Our results showed that the gut microbiota composition and diversity of Asian honey bee were significantly associated with both feeding types and the N. ceranae infection. More interestingly, bees fed with beebread showed higher microbiota stability and lower mortality rates than those fed with sugar water when infected by N. ceranae. Those data suggest that beebread has the potential not only to provide better nutrition but also help to establish a more stable gut microbiota to protect bees against N. ceranae infection.

The freshwater climbing perch, Anabas testudineus, is an euryhaline teleost and an obligate air-breather with the ability to actively excrete ammonia. Members of the Na+/H+ exchanger (NHE) family help maintain intracellular pH homeostasis and ionic balance through the electroneutral exchange of Na+ and H+. This study aimed to obtain, from the gills of A. testudineus, the full cDNA coding sequence of nhe3, and to determine the effects of exposure to seawater or 100 mmol l−1 of NH4Cl in fresh water on its mRNA and protein expression levels. Efforts were also made to elucidate the type of ionocyte that Nhe3 was associated with in the branchial epithelium of A. testudineus. The transcript level and protein abundance of nhe3/Nhe3 were very low in the gills of freshwater A. testudineus, but they increased significantly in the gills of fish acclimated to seawater. In the gills of fish exposed to seawater, Nhe3 was expressed in two distinct types of seawater-inducible Na+/K+-ATPase (Nka)-immunoreactive ionocytes. In Nkaα1b-immunoreactive ionocytes, Nhe3 had an apical localization. As these ionocytes also expressed apical Rhcg1 and basolateral Rhcg2, which are known to transport ammonia, they probably participated in proton-facilitated ammonia excretion in A. testudineus during seawater acclimation. In Nkaα1c-immunoreactive ionocytes, Nhe3 was atypically expressed in the basolateral membrane, and its physiological function is uncertain. For A. testudineus exposed to NH4Cl in fresh water, the transcript and protein expression levels of nhe3/Nhe3 remained low. In conclusion, the branchial Nhe3 of A. testudineus plays a greater physiological role in passive ammonia transport and acid-base balance during seawater acclimation than in active ammonia excretion during environmental ammonia exposure.

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens, but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription may precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1/Aqp1 and aqp3/Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens. These results provide insights into how P. annectens regulates branchial Aqp expression to cope with desiccation and rehydration during different phases of aestivation.

Himantura signifer is a freshwater stingray which inhabits rivers in Southeast Asia. It can survive in brackish water but not seawater. In brackish water, it becomes partially ureosmotic, but how it maintains its plasma hypoionic to the external medium is enigmatic because of the lack of a rectal gland. Here, we report for the first time the expression of Na+:K+:2Cl− cotransporter 1 (nkcc1) in the gills of freshwater H. signifer, and its moderate up-regulation (~2-fold) in response to brackish water (salinity 20) acclimation. The absence of the Ste20-related proline-alanine-rich kinase and oxidation stress response kinase 1 interaction site from the N-terminus of H. signifer Nkcc1 suggested that it might not be effectively activated by stress kinases in response to salinity changes as in more euryhaline teleosts. The increased activity of Nkcc1 during salt excretion in brackish water would lead to an influx of Na+ into ionocytes, and the maintenance of intracellular Na+ homeostasis would need the cooperation of Na+/K+-ATPase (Nka). We demonstrated for the first time the expression of nkaα1, nkaα2 and nkaα3 in the gills of H. signifer, and the up-regulation of the mRNA expression of nkaα3 and the overall protein abundance of Nkaα in response to acclimation to brackish water. Immunofluorescence microscopy revealed the presence of a sub-type of ionocyte, co-expressing Nkcc1 and Nkaα, near the base of the secondary lamellae in the gills of H. signifer acclimated to brackish water, but this type of ionocyte was absent from the gills of fish kept in fresh water. Hence, there could be a change in the function of the gills of H. signifer from salt absorption to salt excretion during brackish water acclimation in the absence of a functioning rectal gland.

This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na+/K+-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na+:K+:2Cl- cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl- channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

Na(+):K(+):2Cl(-) cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g(-1) and 2.7 µmol g(-1) after 6 days of exposure to 50 mmol l(-1) NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance.

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l(-1)) and accumulate ammonia to high concentrations in its brain (4.5 µmol g(-1)). Na(+)/K(+)-ATPase (Nka) is an essential transporter in brain cells, and since NH4(+) can substitute for K(+) to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K(+) specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4(+) to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na(+)/K(+)-ATPase and Na(+)/NH4(+)-ATPase activities over a range of K(+)/NH4(+) concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l(-1) NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na(+)/NH4(+)-ATPase activities were significantly lower than the Na(+)/K(+)-ATPase activities assayed at various NH4(+)/K(+) concentrations. Furthermore, the effectiveness of NH4(+) to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K(+) specificity of K(+) binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.

Abstract Background Currently, there is a lack of research on multi-drug resistant Pseudomonas aeruginosa (MDR-PA) isolation in bronchiectasis-related hemoptysis. The aim of this study to analyze the risk factors for recurrent hemoptysis following bronchial artery embolization (BAE) and compare the recurrent hemoptysis-free rates between MDR-PA, non-MDR-PA, and non-PA isolation. Methods A retrospective study was performed of patients diagnosed with idiopathic bronchiectasis-related recurrent hemoptysis who underwent BAE at an university-affiliated hospital. Patients were categorized based on PA susceptibility tests into non-PA, non-MDR-PA, and MDR-PA groups. Univariate and multivariate Cox regression were conducted to identify independent risk factors for recurrent hemoptysis. The Kaplan-Meier curves was conducted to compare recurrent hemoptysis-free rates after BAE for non-PA, non-MDR-PA, and MDR-PA. Results A total of 432 patients were included. 181 (41.90%) patients experienced recurrent hemoptysis during a median follow-up period of 25 months. MDR-PA isolation (adjusted hazard ratio (aHR) 2.120; 95% confidence interval (CI) [1.249, 3.597], p = 0.005) was identified as an independent risk factor for recurrent hemoptysis. Antibiotic treatment (aHR 0.666; 95% CI [0.476, 0.932], p = 0.018) reduced the risk of recurrent hemoptysis. The cumulative recurrent hemoptysis-free rates for non-PA, non-MDR-PA, and MDR-PA were as follows: at 3 months, 88.96%, 88.24%, and 75.86%, respectively; at 1 year, 73.13%, 69.10%, and 51.72%; and at 3 years, 61.91%, 51.69%, and 41.10% (p = 0.034). Conclusion MDR-PA isolation was an independent risk factor of recurrent hemoptysis post-BAE. Reducing the occurrence of MDR-PA may effectively decrease the recurrence rates of hemoptysis.

Abstract Histamine is a biogenic amine that is critical in various physiological and pathophysiological processes, including but not limited to allergic reactions, wakefulness, gastric acid secretion and neurotransmission. Here, we determine 9 cryo-electron microscopy (cryo-EM) structures of the 4 histamine receptors in complex with four different G protein subtypes, with endogenous or synthetic agonists bound. Inside the ligand pocket, we identify key motifs for the recognition of histamine, the distinct binding orientations of histamine and three subpockets that facilitate the design of specific ligands. In addition, we also identify key residues responsible for the selectivity of immethridine. Moreover, we reveal distinct structural features as determinants of Gq vs. Gs or Gs vs. Gi coupling differences among the histamine receptors. Our study provides a structural framework for understanding the ligand recognition and G protein coupling of all 4 histamine receptors, which may facilitate the rational design of ligands targeting these receptors.

Abstract Background The microbiota and metabolites in the gastrointestinal tracts of female animals at different reproductive periods are very important to the growth, development, and health of themselves and their offspring. However, the changes in the gastrointestinal microbiota and metabolites throughout reproductive period of different sheep breeds and their effects on the growth and development of offspring lambs are still unclear. Hence, this study presents an assessment of the reproductive hormone levels, immune levels, rumen microbiota, and metabolites in Hu sheep and Suffolk ewes at different reproductive periods and their effects on the growth and development of offspring lambs. Results Hu sheep and Suffolk during non-pregnancy, pregnancy, and lactation were used as the research objects to determine reproductive and immune indexes of ewes at different periods, analyze rumen microbiome and metabolome, and track the growth performance and development of offspring lambs. The results showed that the reproductive hormone and immune levels of Hu sheep and Suffolk underwent adaptive changes across different reproductive periods. Compared with non-pregnancy, the microbial energy metabolism and lipid metabolism function decreased during Hu sheep pregnancy, and energy metabolism function decreased during lactation. In Suffolk, energy metabolism, glycan biosynthesis, and metabolism function were enhanced during pregnancy, and the metabolism of cofactors and vitamins was enhanced during lactation. Prevotella increased in Suffolk during pregnancy and lactation (P < 0.05) and was positively correlated with the birth weight and body size of the lambs (P < 0.05). Moreover, the abundances of Butyrivibrio and Rikenellaceae_RC9_gut_group during pregnancy were positively correlated with the intestinal immunity of the offspring lambs (P < 0.05), thereby regulating the intestinal immunity level of the lambs. Metabolomic analysis revealed that the protein digestion, absorption, and amino acid metabolism of Hu sheep were enhanced during pregnancy, which provided amino acids for the growth and development of pregnant ewes and fetuses and was significantly correlated with the birth weight, body size, and intestinal immunity of lambs (P < 0.05). Simultaneously, there was an increase in acetate and propionate during the pregnancy and lactation period of both Hu sheep and Suffolk, providing energy for ewes during reproductive period. Moreover, the microbiota during the lactation period was significantly correlated with the milk quality and lambs daily gain (P < 0.05). Conclusions This study revealed the characteristic succession changes in the rumen microbiota and its metabolites at different reproductive periods in sheep breeds and their regulation of reproductive hormone and immune levels and identified their potential effects on the growth and development of offspring lambs. The findings provide valuable insights into the health and feeding management of different sheep breeds during the reproductive stage. Video Abstract

Abstract Background Cassava is one of three major potato crops and the sixth most important food crop globally. Improving yield remains a primary aim in cassava breeding. Notably, plant height significantly impacts the yield and quality of crops; however, the mechanisms underlying cassava plant height development are yet to be elucidated. Results In this study, we investigated the mechanisms responsible for cassava plant height development using phenotypic, anatomical, and transcriptomic analyses. Phenotypic and anatomical analysis revealed that compared to the high-stem cassava cultivar, the dwarf-stem cassava cultivar exhibited a significant reduction in plant height and a notable increase in internode tissue xylem area. Meanwhile, physiological analysis demonstrated that the lignin content of dwarf cassava was significantly higher than that of high cassava. Notably, transcriptome analysis of internode tissues identified several differentially expressed genes involved in cell wall synthesis and expansion, plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis between the two cassava cultivars. Conclusions Our findings suggest that internode tissue cell division, secondary wall lignification, and hormone-related gene expression play important roles in cassava plant height development. Ultimately, this study provides new insights into the mechanisms of plant height morphogenesis in cassava and identifies candidate regulatory genes associated with plant height that can serve as valuable genetic resources for future crop dwarfing breeding.

Abstract Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.

Abstract Laser-scanning confocal microscopy serves as a critical instrument for microscopic research in biology. However, it suffers from low imaging speed and high phototoxicity. Here we build a novel deep compressive confocal microscope, which employs a digital micromirror device as a coding mask for single-pixel imaging and a pinhole for confocal microscopic imaging respectively. Combined with a deep learning reconstruction algorithm, our system is able to achieve high-quality confocal microscopic imaging with low phototoxicity. Our imaging experiments with fluorescent microspheres demonstrate its capability of achieving single-pixel confocal imaging with a sampling ratio of only approximately 0.03% in specific sparse scenarios. Moreover, the deep compressive confocal microscope allows single-pixel imaging at the single-photon level, thus reducing the excitation light power requirement for confocal imaging and suppressing the phototoxicity. We believe that our system has great potential for long-duration and high-speed microscopic imaging of living cells.

Nuclear energy plays a crucial role in energy supply in the 21st century, and more and more Nuclear Power Plants (NPPs) will be in operation to contribute to the development of human society. However, as a typical complex system engineering, the operation and development of NPPs require efficient and stable control methods to ensure the safety and efficiency of nuclear power generation. Reinforcement learning (RL) aims at learning optimal control policies via maximizing discounted long-term rewards. The reward-oriented learning paradigm has witnessed remarkable success in many complex systems, such as wind power systems, electric power systems, coal fire power plants, robotics, etc. In this work, we try to present a systematic review of the applications of RL on these complex systems, from which we believe NPPs can borrow experience and insights. We then conduct a block-by-block investigation on the application scenarios of specific tasks in NPPs and carried out algorithmic research for different situations such as power startup, collaborative control, and emergency handling. Moreover, we discuss the possibilities of further application of RL methods on NPPs and detail the challenges when applying RL methods on NPPs. We hope this work can boost the realization of intelligent NPPs, and contribute to more and more research on how to better integrate RL algorithms into NPPs.

Pathogenic genes in most patients with cleidocranial dysplasia have been confirmed to be runt-related transcription factor 2 (RUNX2), which controls mutations in specific osteoblast transcription factors and affects skull ossification and suture adhesion. This study aimed to explore the role of RUNX2 mutations. Here, we report a rare case of a splice site mutation in a Chinese population with typical cleidocranial dysplasia symptoms, cranial suture insufficiency, clavicle dysplasia, and dental anomalies. Peripheral blood samples from the proband and her mother were subjected to Sanger sequencing. The expression levels of RUNX2 before and after mutation were verified using digital PCR (dPCR). The results revealed a classic mutation at the fifth base of the intron 5 initiation splicing sequence (NM001024630.4: C.685+5G > A). The mutation rate in the proband was 53 %, while the mother did not have any mutations. The secondary RNA structure of the RUNX2 gene in the progenitor was predicted to change, and the structural free energy was low in the wild-type, with the stem folded first and the structure being relatively stable. After the mutation, the free energy increased. This finding enriches the RUNX2 mutation library of CD-related genes in Chinese individuals.

ABSTRACT Ceracris nigricornis Walker is an insect of the Acrididae, which can harm bamboo, rice, corn, sorghum and other crops, and can cause serious economic losses. In this study, based on 234 occurrence sites of C. nigricornis obtained from the Global Biodiversity Information Facility and literature, and data of three future climate scenarios presented by CMIP6, two niche models (GARP, MaxEnt) were used to predict the suitable area of C. nigricornis in China. The result shows that the main environmental factors affecting the distribution of C. nigricornis are min temperature of coldest month (bio6), mean temperature of coldest quarter (bio11), precipitation of driest month (bio14) and precipitation of wettest quarter (bio16). From the result of MaxEnt model, it can be seen that the suitable area of C. nigricornis in China is 128.91 × 104 km2 under current scenario. It will decrease by 3.19% in the 2050s and then increase by 12.04% in the 2090s under the SSP1‐2.6 scenario, increase by 5.79% in the 2050s and then decrease by 7.53% in the 2090s under the SSP2‐4.5 scenario, and increase by 33.03% in 2050s and then decrease by 23.31% in the 2090s under SSP5‐8.5 scenario. From the result of GARP model, it can be seen that the suitable area of C. nigricornis in China is 166.09 × 104 km2 under current scenario. It will increase by 8.41% in 2050s and then continue to increase by 6.11% in 2090s under SSP1‐2.6 scenario, increase by 23.84% in the 2050s and then decrease by 0.88% in the 2090s under the SSP2‐4.5 scenario, and increase by 34.37% in 2050s and then decrease by 1.75% in 2090s under SSP5‐8.5 scenario. The boundaries of suitable areas will expand to the north and southwest of China under future climate change scenarios, specially in Sichuan, Chongqing and Yunnan. Local forestry authorities should strengthen the monitoring of bamboo forests to prevent the damage caused by the introduction of C. nigricornis.

ObjectiveTo develop and validate a prognostic model for patients with BRAF V600E-mutated colorectal cancer.MethodsThe clinical and pathological information of 206 patients with BRAF V600E-mutated colorectal cancer diagnosed in Cancer Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College from 2014 to 2021 was retrospectively collected. Least absolute shrinkage and selection operator (LASSO) regression, Cox regression, and nomograms were used to develop clinical prognostic models. The differentiation was measured using C-statistic, and the predicted variability was evaluated using the calibration curve. The prognostic model was externally validated using validation set data from 164 patients pooled from five studies.ResultsOur clinical prognostic model included three variables: pathological stage, microsatellite status, and primary tumor site. In internal validation, the model had a concordant index of 0.785 (95% CI [0.732–0.839]) and a concordant index of 0.754 (95% CI [0.698–0.810]) using pathological staging. External validation confirmed the robustness of the model with a consistency index of 0.670 (95% CI [0.617–0.724]) and a consistency index of 0.584 (95% CI [0.546–0.622]) using pathological staging. Likelihood ratio test results show that our model is better (internal validation, p = 5.141e−03; external validation, p = 2.728e−05). The calibration graph drawn based on the prediction and the actual situation is close to the 45° diagonal.ConclusionBy adding microsatellite status and primary tumor site on the basis of pathological stage, we improved the discriminability and prediction accuracy of the model and successfully established a prognosis model for patients with BRAF V600E mutation of colorectal cancer.