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Abstract Animal translocations provide striking examples of the human footprint on biodiversity. Combining continental-wide genomic and DNA-barcoding analyses, we reconstructed the historical biogeography of the Asian black-spined toad (Duttaphrynus melanostictus), a toxic commensal amphibian that currently threatens two biodiversity hotspots through biological invasions (Wallacea and Madagascar). The results emphasize a complex diversification shaped by speciation and mitochondrial introgression that comprises two distinct species. One species (true D. melanostictus) is distributed in the Indian subcontinent and is invasive in Wallacea. The other species, whose nomenclature remains unsettled, diverged from D. melanostictus in the Miocene era (~7 Mya) and diversified across Southeast Asia, from where it was introduced to Madagascar. Remarkably, the Indonesian population of D. melanostictus was recently established from India, which suggests historical, possibly human-assisted dispersal across the Bay of Bengal, reflecting the centuries-old connection between these regions.

Abstract Background Thyroid hormone (T3) has an inhibitory effect on tissue/organ regeneration. It is still elusive how T3 regulates this process. It is well established that the developmental effects of T3 are primarily mediated through transcriptional regulation by thyroid hormone receptors (TRs). Here we have taken advantage of mutant tadpoles lacking both TRα and TRβ (TRDKO), the only receptor genes in vertebrates, for RNA-seq analyses to investigate the transcriptome changes underlying the initiation of tail regeneration, i.e., wound healing and blastema formation, because this crucial initial step determines the extent of the functional regeneration in the later phase of tissue regrowth. Results We discovered that GO (gene ontology) terms related to inflammatory response, metabolic process, cell apoptosis, and epithelial cell migration were highly enriched among commonly regulated genes during wound healing at either stage 56 or 61 or with either wild type (WT) or TRDKO tadpoles, consistent with the morphological changes associated with wound healing occurring in both regenerative (WT stage 56, TRDKO stage 56, TRDKO stage 61) and nonregenerative (WT stage 61) animals. Interestingly, ECM-receptor interaction and cytokine-cytokine receptor interaction, which are essential for blastema formation and regeneration, were significantly enriched among regulated genes in the 3 regenerative groups but not the non-regenerative group at the blastema formation period. In addition, the regulated genes specific to the nonregenerative group were highly enriched with genes involved in cellular senescence. Finally, T3 treatment at stage 56, while not inducing any measurable tail resorption, inhibited tail regeneration in the wild type but not TRDKO tadpoles. Conclusions Our study suggests that TR-mediated, T3-induced gene regulation changed the permissive environment during the initial period of regeneration and affected the subsequent patterning/outgrowth period of the regeneration process. Specifically, T3 signaling via TRs inhibits the expression of ECM-related genes while promoting the expression of inflammation-related genes during the blastema formation period. Interestingly, our findings indicate that amputation-induced changes in DNA replication-related pathways can occur during this nonregenerative period. Further studies, particularly on the regenerative microenvironment that may depend on ECM-receptor interaction and cytokine-cytokine receptor interaction, should provide important insights on the regulation of regenerative capacity during vertebrate development.

Abstract Dried roots of Zanthoxylum nitidum (2n=2x=70, family Rutaceae) was referred as “Liang-Mian-Zhen” in Chinese, acting as a valuable species due to its notable pharmacological activities. Herein, we combined PacBio HiFi data together with Hi-C mapping technology to construct a chromosome-scale reference genome assembly for Z. nitidum. The assembly reached a length of 2.24 Gb, successfully anchoring 99.31% of sequences onto 35 pseudo-chromosomes. Among these, 26 chromosomes achieved telomere-to-telomere assembly, and 11 chromosomes were gap-free. The contigs N50 and scaffolds N50 reached 57.61 Mb and 78.00 Mb, respectively. Transposable elements comprised 81.44% of the Z. nitidum genome, and over 78% of them were long-terminal repeat retrotransposon elements. Furthermore, 32,737 protein-coding genes were identified and 99.38% of all were functionally annotated. The completeness of the genome assembly and final gene sets reached 97.83% and 96.47% based on Benchmarking Universal Single-Copy Orthologs (BUSCO), respectively. Taken together, our results provided a high-quality chromosome-level assembly of Z. nitidum genome and will be a valuable resource that will facilitate breeding varieties with higher alkaloids content.

Abstract Objective To investigate the clinical application value of microfluidic-based in vitro diagnostic (IVD) technology in pathogenic detection of respiratory tract infections. Methods A total of 300 clinical samples, including blood, bronchoalveolar lavage fluid, and pleural effusion, were collected from patients with respiratory tract infections. The samples were randomly divided into three groups: A, B, and C, with 100 cases in each group. Group A used traditional microbiological detection methods, Group B used metagenomic next-generation sequencing (mNGS) technology, and Group C used both microfluidic-based IVD technology and traditional microbiological detection methods to detect pathogenic microorganisms in the clinical samples. The positive detection rate, detection time, and detection cost were compared among the groups. The diagnostic performance of each group was compared using the Receiver Operating Characteristic (ROC) curve. Results Traditional microbiological detection identified 38 positive samples (38%), including 45 pathogens; mNGS technology identified 95 positive samples (95%), including 210 pathogens; microfluidic-based IVD technology identified 96 positive samples (96%), including 158 pathogens. Microfluidic-based IVD technology had a significantly higher positive detection rate for pathogenic microorganisms compared to traditional culture techniques (96% vs 38%, χ2 = 122.0, P

Abstract The evolution and development of vertebrate lungs have been widely studied due to their significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring their evolutionary importance in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development remains poorly understood. Using single-cell RNA sequencing, we identify 13 cell types in the developing lungs of a land-dwelling frog (Microhyla fissipes). We elucidate the differentiation trajectories and mechanisms of mesenchymal cells, identifying five cell fates and their respective driver genes. Using temporal dynamics analyses, we reveal the gene expression switches of epithelial cells, which facilitate air breathing during metamorphosis. Furthermore, by integrating the published data from another amphibian and two terrestrial mammals, we illuminate both conserved and divergent cellular repertoires during the evolution of tetrapod lungs. These findings uncover the frog lung cell differentiation trajectories and functionalization for breathing in air and provide valuable insights into the cell-type evolution of vertebrate lungs.

Herpetofauna have attracted considerable global attention due to their high risk of extinction. The Mountains of Southwest China are among the 36 global biodiversity hotspots due to their high biodiversity and irreplaceability, and the Shaluli Mountains are the core area of this region. However, the diversity and conservation status of herpetofauna in the Shaluli Mountains remain unclear. In this study, we updated the species richness and composition information of herpetofauna in the Shaluli Mountains based on extensive field expeditions, published literature, and museum specimens. In addition, we identified the local hotspots and key environmental factors limiting species distribution using environmental niche models (ENMs). Furthermore, we evaluated the conservation status of the key species via the Red List Index and gap analysis. Our results revealed a total of 68 herpetofauna species, representing 30 amphibians (21 genera in nine families) and 38 reptiles (19 genera in six families). The amphibian richness showed a hump-shaped pattern along the elevational gradient, while the reptile richness showed a monotonically decreasing pattern. The dry-hot river valley was identified as a local hotspot, and the key environmental factors limiting the richness of herpetofauna species differed. Moreover, the regional Red List indices for amphibians and reptiles were 0.87 and 0.8, respectively, showing that the overall threat level of this region was low. However, there are large conservation gaps in the protection of key species. Our study indicated that new protected areas should be established and the existing protected areas should be upgraded to protect herpetofauna with a large conservation gap.

Abstract Background Anuran metamorphosis, which is driven by thyroid hormone (TH)-mediated processes, orchestrates intricate morphological and functional transformations for the transition from aquatic tadpoles to terrestrial life, providing a valuable model for studying organ functionalization, remodeling, and regression. Larva-specific organ regression is one of the most striking phenomena observed during the anuran metamorphic climax. While previous studies extensively analyzed the regression mechanisms of the tail, the molecular processes governing gill resorption remain elusive. Results We employed Microhyla fissipes as a model, and utilized a comprehensive approach involving histological analysis, transmission electron microscopy, and transcriptomics to unravel gill development and resorption. The pro-metamorphic stages revealed highly developed gill structures, emphasizing their crucial role as the primary respiratory organ for tadpoles. The transcriptomic analysis highlighted the upregulation of genes associated with enhanced respiratory efficiency, such as hemoglobin and mucins. However, as metamorphosis progressed, gill filaments underwent shrinkage, decreases in blood vessel density, and structural changes that signified a decline in respiratory function. The molecular mechanisms driving gill resorption involved the TH pathway—in particular, the upregulation of thyroid hormone receptor (TR) β, genes associated with the tumor necrosis factor pathway and matrix metalloproteinases. Two distinct pathways orchestrate gill resorption, involving apoptosis directly induced by TH and cell death through the degradation of the extracellular matrix. In addition, metabolic reorganization during metamorphosis is a complex process, with tadpoles adapting their feeding behavior and mobilizing energy storage organs. The gills, which were previously overlooked, have been unveiled as potential energy storage organs that undergo metabolic reorganization. The transcriptomic analysis revealed dynamic changes in metabolism-related genes, indicating decreased protein synthesis and energy production and enhanced substrate transport and metabolism during metamorphic climax. Conclusion This study sheds light on the structural, molecular, and metabolic dynamics during gill development and resorption in M. fissipes. The findings deepen our understanding of the intricate mechanisms governing organ regression and underscore the pivotal role of the gills in facilitating the transition from aquatic to terrestrial habitats.

Abstract Background Captivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision. Results Both captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota. Conclusions Our aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.

Background color matching is essential for camouflage and thermoregulation in ectothermic vertebrates, yet several key cellular-level questions remain unresolved. For instance, it is unclear whether the number of chromatophores or the activity of individual chromatophores plays a more critical role in this process. Using single-cell RNA sequencing (scRNA-seq), we investigated the cellular and molecular mechanisms underlying color change in Rhacophorus dugritei, which adapted to its background by displaying light-green skin on white and black skin on black within two days. We identified two types of chromatophores in their skin, both responsible for the observed color differences. Our findings reveal that morphological color change (MCC) is the dominant process, with the number of chromatophores being more influential in driving color change than the transcriptional activity of melanogenesis in individual cells. Additionally, melanophores from darker individuals exhibited increased activity in energy metabolism pathways, while those from lighter individuals showed stronger immune-related gene expression, suggesting that background adaptation involves more than just morphological changes. Overall, this study successfully applied single-cell sequencing technology to investigate skin pigmentation in a non-model organism. Our results suggest that MCC driven by chromatophore proliferation is a key mechanism of background adaptation, offering new insights into amphibian color adaptation and environmental adaptation in other vertebrates.

MAFLD has become a major global health problem and is the leading cause of liver disease worldwide. The disease progresses from a simple fatty liver to gradual fibrosis, which progresses to cirrhosis and even hepatocellular liver cancer. However, the methods currently used for diagnosis are invasive and do not facilitate clinical assessment of the condition. As a result, research on markers for the diagnosis of MAFLD is increasing. In addition, there are no clinical medications for the treatment of MAFLD, and lifestyle interventions remain effective in the prevention and treatment of MAFLD. In this review, we attempt to make a summary of the emerging diagnostic indicators and effective lifestyle interventions for MAFLD and to provide new insights into the diagnosis and treatment of MAFLD.

Summary: Comprehending the determinants of host-associated microbiota is pivotal in microbial ecology. Yet, the links between climatic factors and variations in host-associated microbiota necessitate further clarification. Mountain-dwelling amphibians, with limited dispersal abilities, serve as valuable models for addressing these questions. Our study, using 126 amphibian-associated microbial samples (64 gut and 62 skin) and 101 environmental microbial samples (51 soil and 50 water) from the eastern Tibetan Plateau, revealed host factors as primary drivers of the variations in host-associated microbiota. However, climatic factors contributed to additional variations in gut microbial beta-diversity and skin microbial function. Water microbiota were identified as a significant contributor to the amphibian-associated microbiomes, with their climate-driven variations mediating an indirect association between the variations in climatic factors and host-associated microbiota. These findings extend our understanding of the assembly of host-associated microbiota in amphibians, emphasizing the significance of microbiota in evaluating the impact of climate change on animals.

Abstract Zanthoxylum nitidum (Roxb.) is a commonly used traditional Chinese medicine. However, the collection and protection of wild germplasm resources of Z. nitidum are still insufficient, and there is limited research on its genetic diversity and fingerprint. In the present study, 15 simple sequence repeat (SSR) markers were developed by genotyping based on multiplexed shotgun sequencing. The genetic diversity of 51 populations (142 individuals) of Z. nitidum was evaluated using these 15 SSRs. A total of 245 alleles (Na) were detected, with an average value of 16.333, and the average polymorphism information content was 0.756. The genetic distance among 51 populations was 0.164~1.000, with an average of 0.659. Analysis of molecular variance showed low genetic differentiation (40%) and high genetic differentiation (60%) between populations and individuals, respectively. The genetic differentiation coefficient (Fst) of the population was 0.338, indicating that 66.2% of the genetic variation occurred within the population, and the gene flow (Nm) was 0.636, demonstrating that the gene exchange between populations was low. Clustering analysis revealed that the genetic similarity coefficient was 0.30, dividing the 51 populations into 4 groups of 2, 17, 3, and 29 populations. There was no specific relationship between geographical location differences and genetic distance. The genetic diversity level of Z. nitidum is relatively high, and our results provide a theoretical basis for the rapid identification of Z. nitidum germplasm resources and variety selection.

Dedifferentiated liposarcoma (DDLPS) is a rare and aggressive type of cancer primarily reported in humans, with no documented cases in animals. In this study, we present the first case of DDLPS in a wild amphibian species, Maculopaa medogensis. The tumor was discovered during a routine examination and diagnosed through a combination of advanced diagnostic methods, including micro-CT imaging, gross anatomical inspection, histological analysis, and immunohistochemistry. The tumor exhibited both well-differentiated and dedifferentiated components, characteristic of DDLPS, with evidence of tissue invasion and multiple metastases. Immunohistochemical analysis revealed the strong positive expression of markers such as S100A4, CDK4, MDM2, and CD34, while Leptin expression was negative, further confirming the diagnosis. This is the first reported case of DDLPS in a non-human species, expanding our understanding of cancer in wildlife and underscoring the significance of amphibians as environmental indicators. These findings provide valuable insights for veterinary medicine and wildlife conservation, particularly regarding the role of environmental stressors in cancer development. This study highlights the need for comprehensive diagnostic approaches in wildlife pathology.

Amphibians and reptiles play a critical role in the evolution of Tetrapoda, showcasing significant diversity in terms of their genetics, species, morphology, life history traits, and evolutionary functions [...]

Amphibians serve as reliable indicators of ecosystem health and are the most threatened group of vertebrates. Studies on their spatial distribution pattern and threats are crucial to formulate conservation strategies. Gongga Mountains, with a peak at 7509 m a.s.l. and running latitudinally, are in the center of the Hengduan Mountains Range and at the eastern steep edge of the Qinghai–Xizang Plateau, providing heterogeneous habitats and varied niches for amphibians. In this study, we combined 83 days of field work with information from 3894 museum specimens that were collected over the past 80 years, and identified twenty amphibian species belonging to seven families and twelve genera by morphology. Of these species, seven were listed in the threatened categories of the Red List of China’s Biodiversity and thirteen were endemic to China. Ten species were found on the plateau side (western slope) and eleven species were found on the other side close to the Sichuan Basin (eastern slope). Only one species was found on both sides, indicating different community structures horizontally. The species richness was unimodal vertically and peaking at mid elevation on both sides, with the maximum number (ten vs. nine) of species occurring at 3300–3700 vs. 1700–1900 m a.s.l. and in different types of vegetation. The elevation span and body length of species distributed on both slopes did not show significant differences. These findings help to understand the horizontal and vertical distribution pattern of amphibian diversity, laying a foundation for future biogeographical and conservation research in this area.

Climate change has been considered to pose critical threats for wildlife. During the past decade, species distribution models were widely used to assess the effects of climate change on the distribution of species’ suitable habitats. Among all the vertebrates, amphibians are most vulnerable to climate change. This is especially true for salamanders, which possess some specific traits such as cutaneous respiration and low vagility. The Wushan salamander (Liua shihi) is a threatened and protected salamander in China, with its wild population decreasing continuously. The main objective of this study was to predict the distribution of suitable habitat for L. shihi using the ENMeval parameter-optimized MaxEnt model under current and future climate conditions. Our results showed that precipitation, cloud density, vegetation type, and ultraviolet radiation were the main environmental factors affecting the distribution of L. shihi. Currently, the suitable habitats for L. shihi are mainly concentrated in the Daba Mountains, including northeastern Chongqing and western Hubei Provinces. Under the future climate conditions, the area of suitable habitats increased, which mainly occurred in central Guizhou Province. This study provided important information for the conservation of L. shihi. Future studies can incorporate more species distribution models to better understand the effects of climate change on the distribution of L. shihi.

Drug-resistant Neisseria gonorrhoeae poses an urgent threat to public health. Recently, sitafloxacin, a new-generation fluoroquinolone, has shown high in vitro activity against drug-resistant N. gonorrhoeae. However, data on its effectiveness in clinical isolates remains limited. In this study, we collected 507 N. gonorrhoeae isolates from 21 hospitals in Shanghai, China, during 2020 and 2021. Antimicrobial susceptibility testing revealed that sitafloxacin minimum inhibitory concentrations (MICs) exhibited a bimodal distribution, ranging from 50 and MIC90 for sitafloxacin were 0.125 mg/L and 0.5 mg/L, respectively, which are 32 and 16 times lower than those for ciprofloxacin (4 mg/L and 8 mg/L, respectively). Sitafloxacin demonstrated high in vitro activity against isolates resistant to either ceftriaxone, azithromycin, or both. Notably, among the isolates with reduced sitafloxacin susceptibility (MIC ≥ MIC90), 83.7% (36/43) were identified as sequence type (ST) 8123. Further phylogenetic analysis showed that ST8123 has evolved into two subclades, designated as subclade-I and subclade-II. A majority of the isolates (80%, 36/45) within subclade-I exhibited reduced susceptibility to sitafloxacin. In contrast, all isolates from subclade-II were found to be susceptible to sitafloxacin. Subsequent genomic investigations revealed that the GyrA-S91F, D95Y, and ParC-S87N mutations, which were exclusively found in ST8123 subclade-I, might be linked to reduced sitafloxacin susceptibility. Our study reveals that sitafloxacin is a promising antibiotic for combating drug-resistant N. gonorrhoeae. However, caution is advised in the clinical application of sitafloxacin for treating N. gonorrhoeae infections due to the emergence of a clone exhibiting reduced susceptibility.

Chemical communication is an important mode of communication in the courtship and breeding processes of amphibians. In caudates, multiple components of sexual pheromones have been identified and functionally verified. One of these pheromone systems is plethodontid modulating factor (PMF). In anurans, the pheromone called amplexin was found in nuptial pads of ranids and was considered a member of the PMF system, yet its bio-function has not been tested. In this study, we obtained 18 amplexin transcript sequences from nuptial pads of Nidirana pleuraden (Amphibia, Ranidae) by transcriptome sequencing and found that the proteins translated by these transcripts are diversified, hydrophilic, and relatively stable. We also acquired a N. pleuraden amplexin isoform with the highest expression level in the transcriptome analysis through the prokaryotic expression system. Using two different animal behavioral experimental settings, we have tested the bio-function of the recombinant PMF protein (rPMF) in N. pleuraden’s reproduction and found that the rPMF does not attract females but shortens the duration of amplexus significantly. This is the first study to verify the function of the PMF pheromone in Anura, indicating the pervasiveness of chemical communication during breeding in amphibians.

Accumulating experimental data have shown that endogenous hormones play important roles in regulating seed dormancy and germination. Zanthoxylum nitidum is a medicinal plant that propagates via seeds, which require a long dormancy period for normal germination, and complex changes in metabolites occur during the germination process. However, the regulatory network of endogenous hormones and metabolites during the germination of Z. nitidum seeds remains unclear. This study investigated the dynamic changes in the levels of metabolites and endogenous hormones during the germination of Z. nitidum seeds. The results revealed an increase in the levels of gibberellin 3 (GA3), 12-oxophytodienoic acid (OPDA), 1-aminocyclopropane-1-carboxylic acid (ACC) and trans-zeatin (TZ) and decrease in the levels of abscisic acid (ABA), jasmonic acid (JA), N-[(-)-jasmonoyl]-(S)-isoleucine (JA-Ile) and trans-zeatin riboside (TZR). Overall, 112 differential metabolites (DAMs) were screened from 3 seed samples (Sa, Sb and Sc), most of which are related to primary metabolism. A total of 16 DAMs (including 3 monosaccharides, 3 phosphate lipids, 3 carboxylic acids, 1 amino acid, 2 pyrimidines, and 4 nucleotides) were identified in the three sample comparison pairs (Sa vs Sb, Sa vs Sc, and Sb vs Sc); these DAMs were significantly enriched in purine metabolism; glycerophospholipid metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism and pyruvate metabolism. OPDA, ACC and GAs were significantly positively correlated with upregulated metabolites, whereas ABA and JA were significantly positively correlated with downregulated metabolites. Finally, a hypothetical metabolic network of endogenous hormones that regulate seed germination was constructed. This study deepens our understanding of the importance of endogenous hormonal profiles that mediate seed germination.

The impacts of lead/Pb2+ on ecosystems have received widespread attention. Growth suppression is a major toxic effect of Pb compounds on aquatic animals, however, some studies have also reported their growth-promoting effects. These complex outcomes may be explained by anions that accompany Pb2+ or by the multiple toxic mechanisms/pathways of Pb2+. To examine these hypotheses, we tested how Bufo gargarizans tadpoles responded to Pb(NO3)2 (100 and 200 μg/L Pb2+) using transcriptomics and microbiomics, with NaNO3 and blank groups as controls. Tadpoles exposed to Pb(NO3)2 showed delayed development while increased somatic growth in a dose-dependent manner, which can be attributed to the effects of NO3- and Pb2+, respectively. Tadpole transcriptomics revealed that exposure to NO3- downregulated the MAPK pathway at transcriptional level, explaining the development-suppressing effect of NO3-; while Pb2+ upregulated the transcription of detoxification pathways (e.g., xenobiotics metabolism by cytochrome P450 and glutathione metabolism), indicating cellular stress and thus contradicting the growth advantage of Pb2+-exposed tadpoles. Pb2+ exposure changed the tadpole gut microbiota drastically, characterized by increased polysaccharides and carbohydrate utilization while decreased fatty acid and amino acid consumption according to microbial functional analysis. Similar gut microbial variations were observed in field-collected tadpoles from different Pb2+ environments. This metabolic shift in gut microbiota likely improved the overall food utilization efficiency and increased the allocation of fatty acids and amino acids to the host, explaining the growth advantage of Pb2+-exposed tadpoles. In summary, our results suggest multiple toxic pathways of Pb2+, and the gut microbiota may affect the pollution outcomes on animals.

Abstract Background The efficacy of reduced-port laparoscopic surgery (RLS) for total gastrectomy remains unclear. This study focused on evaluating the short-term outcomes of RLS compared with conventional laparoscopic surgery (CLS) for total gastrectomy. Methods One hundred and ten patients who underwent completed laparoscopic total gastrectomy for gastric cancer between September 2018 and June 2022 were retrospectively collected and classified into two groups (65 CLS and 45 RLS) according to different operation approach. Twenty-four RLS cases underwent single-incision plus two ports laparoscopic surgery (SILS + 2) and twenty-one underwent single-incision plus one port laparoscopic surgery (SILS + 1). Surgical outcomes, pain intensity, cosmetic and postoperative morbidity, and mortality were compared between groups. Results The overall incidence of postoperative complications was similar between the CLS group and the RLS group (16.9% vs. 8.9%, P = 0.270). It was also comparable in the Clavien-Dindo classification (P = 0.774). However, compared with the CLS group, the RLS group had a significantly shorter total length of incision (5.6 ± 1.0 cm vs. 7.1 ± 0.7 cm, P = 0.000); shorter time to first ambulation (24.9 ± 5.9 h vs. 27.6 ± 5.0 h, P = 0.009), flatus (3.0 ± 0.8 d vs. 3.5 ± 1.0 d, P = 0.022) and oral intake (4.0 ± 1.6 d vs. 6.1 ± 5.1 d, P = 0.011); lower white blood cell count on the third day after the operation (9.8 ± 4.0*109/L vs. 11.6 ± 4.7*109/L, P = 0.037); and lower visual analogue scale score on postoperative days 1 and 3(3.0 ± 0.7 vs. 3.3 ± 0.7, P = 0.044 and 0.6 ± 0.7 vs. 1.6 ± 0.6, P = 0.000 respectively). On the other hand, it didn’t find any difference in short-term outcomes between the SILS + 2 group and the SILS + 1 group (P > 0.05). But the proximal resection margin was longer in the SILS + 2 group than in the SILS + 1 group (2.6 ± 0.7 cm vs. 1.5 ± 0.9 cm, P = 0.046) in patients with adenocarcinoma of the esophagogastric junction (AEG). Conclusions RLS for total gastrectomy is a feasible and safe technique when performed by an experienced laparoscopic surgeon. Moreover, compared with SILS + 1, SILS + 2 might have some advantages in AEG patients.

Abstract Background Breast cancer is a heterogeneous group of diseases. The polarization of CD4+ T helper (Th) lymphocytes (mainly Th1 and Th2) may differ in breast cancers with different outcomes, but this has not been fully validated. Methods This study is a bioinformatic analysis, in which differentially expressed genes (DEGs) were identified in patients with low and high Th1/Th2 ratios. And then, DEG functions, hub genes and independent predictors were determined. Results Low Th1/Th2 ratio was associated with poor outcome in Luminal A and basal-like breast cancer (p

ABSTRACT Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. This opportunistic pathogen, known as classical K. pneumoniae (cKp), typically causes infections in individuals with comorbidities. cKp is often multidrug resistant, and treatment options are limited. By comparison, hypervirulent K. pneumoniae (hvKp) can cause infections in healthy individuals outside of the healthcare setting. Notably, there has been emergence of strains containing both multidrug resistance and hypervirulence genotypes (MDR hvKp). Whether these strains can circumvent killing by components of the innate immune system remains incompletely determined. Here, we compared the ability of selected hvKp (ST23 and ST86) and MDR hvKp (ST11 and ST147) clinical isolates to survive in human blood and serum and tested phagocytic killing of the microbes by human neutrophils. On average, the hvKp isolates tested had greater survival in blood and serum compared with MDR hvKp isolates. Compared with MDR hvKp isolates, the hvKp isolates had less surface-bound serum complement following culture in normal serum. Consistent with these findings, the percentage of neutrophils with phagocytosed hvKp isolates was limited (67% of neutrophils contained ingested MDR hvKp. Phagocytosis of the MDR hvKp isolates was accompanied by significant bacterial killing (P < 0.05). The inability of neutrophils to ingest and kill these hvKp isolates was, in part, overcome by addition of rabbit antiserum specific for these clinical isolates. The results provide insight into host defense against emerging MDR hvKp and are an initial step toward assessing the potential of a vaccine or immunotherapy approach for treatment of infections. IMPORTANCE Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro, whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

Objectives: Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a huge health challenge worldwide. The aim of this study was to evaluate the incidence of polymyxin resistance in clinical CRKP isolates in China and to characterize the molecular mechanisms underlying these polymyxin-resistant CRKP (PR-CRKP) isolates. Methods: A total of 493 CRKP clinical isolates from patients were collected from six tertiary-care hospitals in China during 2017–2018. Minimum inhibitory concentrations of polymyxin B and colistin were determined using the broth microdilution method. PR-CRKP isolates were identified and subjected to whole-genome sequencing. Quantitative real-time PCR and structural modelling analysis were also performed. Results: We observed a 2.2% (11/493) polymyxin resistance rate in this multicentre cohort. Polymyxin B MICs ranged from 4 to 64 μg/mL and colistin MICs ranged from 8 to 128 μg/mL in 11 PR-CRKP isolates. Key genetic variations identified in PR-CRKP isolates involved eight disruptions (seven insertional inactivation by an insertion sequence [IS] element, one frameshift deletion) in mgrB, and three missense mutations in pmrA, pmrB, and phoP. ISKpn26 was the predominant IS (4/7), and three of these occurred in nucleotide position 74 in the mgrB gene. In addition, we reported a novel mutation S62R in pmrB that may confer polymyxin resistance in K. pneumoniae. Conclusions: Our findings highlight the multifaceted molecular mechanisms of polymyxin resistance in CRKP.

Biological diversity is a concept that contains multiple facets, of which functional diversity is considered to be more integrative to describe the relationship between biodiversity and ecosystem functioning. In recent decades, functional diversity patterns have been assessed along elevational gradients in plants and birds. However, few empirical studies have been conducted to reveal amphibian functional diversity patterns between elevational zonations on mountains. In the present study, we investigated amphibian ecomorphologically functional diversity patterns in different elevational zonations of Nepal Himalaya. Our results indicated that functional space occupied by amphibian assemblages differed in size between elevational zonations, with assemblages in zonation-2 (500–1300 m) occupying the highest proportion. In terms of the position in functional space, assemblages in low elevational zonations (zonation 1 & 2) differed significantly from those in high elevational zonations (zonation 3–5). Distribution of individuals in the five assemblages along the six axes of the functional space was significantly different between most pairs of comparisons, while the difference varied between assemblages along axes. Interestingly, despite morphologically extreme amphibians accounted for small proportions of the total number of amphibians, they filled a large proportion of the six dimensional functional space. More importantly, most of the extreme amphibians were also the most functional uniqueness individuals, and they were widely distributed in all the zonations. Therefore, functional vulnerability of amphibian assemblages exists throughout all the elevational zonations. Accordingly, extreme amphibians with most functional uniqueness should be in particular well protected to maintain amphibian functional diversity. Further research can explore the mechanism underlying the unique amphibian functional diversity patterns in Nepal Himalaya.

Zanthoxylum nitidum (Roxb.) DC. (Z. nitidum) is a type of Chinese Dao-di herb, also called Liangmianzhen, which is widely used to treat arthralgia, rheumatic arthralgia, and stomach pain. However, genomic resources for Z. nitidum are still scarce. This study provides transcriptomic resources for Z. nitidum by applying single-molecule real-time (SMRT) sequencing technology. In total, 456,109 circular consensus sequencing (CCS) reads were generated with a mean length of 2,216 bp from Z. nitidum roots, old stems, young branches, leaves, flowers, and fruits. Of these total reads, 353,932 were full-length nonchimeric (FLNC) reads with an average length of 1,996 bp. A total of 16,163 transcripts with a mean length of 1,171 bp were acquired. Of these transcripts, 14,231 (88%) were successfully annotated using public databases. Across all the 16,163 transcripts, we identified 6,255 long non-coding RNAs (lncRNAs) and 22,780 simple sequence repeats (SSRs). Furthermore, 3,482 transcription factors were identified. Among the SSR loci, 1–3 nucleotide repeats were dominant, occupying 99.36% of the total SSR loci, with mono-, di-, and tri-nucleotide repeats accounting for 61.80%, 19.89%, and 5.02% of the total SSR loci, respectively. A total of 36 out of 100 randomly selected primer pairs were verified to be positive, 20 of which showed polymorphism. These findings enrich the genetic resources available for facilitating future studies and research on relevant topics such as population genetics in Z. nitidum.

Jiuzhaigou National Nature Reserve (JNNR) is a renowned World Biosphere Reserve and UNESCO-designated World Nature Heritage Site. The age structure and body size of a population are crucial for assessing the quality of habitats in which a population lives and are essential for the vertebrate conservation and management, especially for amphibians. Unfortunately, information about the life history traits of amphibians is currently unavailable in JNNR. Herein, we first estimated the age structure and body size of Rana kukunoris, which is endemic to the Eastern Qinghai-Xizang Plateau. Then, we compared our data with 28 reported populations along an elevation gradient (1797–3450 m) and investigated how life history traits respond to climatic variations. Our results indicated the following: (1) For individuals from JNNR, the maximum longevity is 8 years, age at sexual maturity (ASM) is 2 years, suggesting a favorable ecological environment in JNNR. Notably, females are significantly larger than males due to the age factor. (2) The average age and ASM show a positive correlation with elevation. However, when the elevation exceeds 3000 m, the average SVL initially increases and then decreases due to the harsh environmental conditions at higher elevation. (3) Temperature and/or UV-B have a significant impact on the average age, ASM, and average SVL variations of R. kukunoris, suggesting adaptive potential of this species via life history variations in light of environmental changes. These accounts provide antecedent information about the life history traits of amphibians in JNNR, and provide insights into the driving factors of the life history variations of the plateau brown frog.

The CRISPR-Cas system in Klebsiella pneumoniae can prevent the entry of blaKPC-IncF plasmids. However, some clinical isolates bear the KPC-2 plasmids despite carrying the CRISPR-Cas system. The purpose of this study was to characterize the molecular features of these isolates. A total of 697 clinical K. pneumoniae isolates were collected from 11 hospitals in China, and tested for the presence of CRISPR-Cas systems using polymerase chain reaction. Overall, 164 (23.5%) of 697 K. pneumoniae isolates had type I-E* (15.9%) or type I-E (7.7%) CRISPR-Cas systems. The most prevalent sequence type among isolates carrying type I-E* CRISPR was ST23 (45.9%), followed by ST15 (18.9%). Isolates with CRISPR-Cas system were more susceptible to ten antimicrobials tested, including carbapenems, compared with the CRISPR-negative isolates. However, there were still 21 CRISPR-Cas-carrying isolates that showed resistance to carbapenems, and these isolates were subjected to whole-genome sequencing. Thirteen of these 21 isolates carried blaKPC-2-bearing plasmids, of which nine had a new plasmid type, IncFIIK34, and two had IncFII(PHN7A8) plasmids. In addition, 12 of these 13 isolates belonged to ST15, while only eight (5.6%, 8/143) isolates belonged to ST15 in carbapenem-susceptible K. pneumoniae carrying CRISPR-Cas systems. In conclusion, we found that blaKPC-2-bearing IncFII plasmids could co-exist with the type I-E* CRISPR-Cas systems in ST15 K. pneumoniae.

IntroductionExtensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle.MethodsIn our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of Microhyla fissipes after TH, cycloheximide (CHX), and TH_CHX treatment.Results and DiscussionWe first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in M. fissipes. Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.

ObjectiveTo investigate the clinical features, diagnosis, and treatment of Chlamydia psittaci (C. psittaci) pneumonia.MethodsWe retrospectively analyzed the clinical data of six patients with C. psittaci pneumonia who were admitted to the Division of Pulmonary and Critical Care Medicine of the Second Hospital of Jiaxing from December 2021 to September 2022.ResultsAll patients reported a fever and other accompanying symptoms, including cough (5/6), chest tightness (1/6), fatigue (2/6), and headache (1/6). Laboratory results showed that all patients had high levels of C-reactive protein (CRP≥70 mg/L), procalcitonin (PCT; 2 patients with PCT levels ≥0.5 ng/L), and erythrocyte sedimentation rate (ESR). Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) levels were elevated in 3/6 and of 2/6 patients, respectively. Chest computed tomography (CT) of most patients showed patchy, high-density shadows with partial consolidation, accompanied by air bronchogram signs and pleural effusion. Six patients were diagnosed with C. psittaci pneumonia using metagenomic next-generation sequencing (mNGS). They showed favorable outcomes following immediate adjustment of the regimen to doxycycline-based therapy and hydration, nutrition, and other follow-up treatments. In the imaging findings obtained at one-two month, the lesions were completely cleared, suggesting a favorable prognosis.ConclusionPatients with C. psittaci pneumonia commonly present sepsis and rapidly progressing disease. Early diagnosis is critical for C. psittaci pneumonia using mNGS, which can lead to favorable prognoses via immediate adjustment therapies.

Introduction: Tadpole tail develops from the tailbud, an apparently homogenous mass of cells at the posterior of the embryo. While much progress has been made in understanding the origin and the induction of the tailbud, the subsequent outgrowth and differentiation have received much less attention, particularly with regard to global gene expression changes. Methods: By using RNA-seq with SMRT and further analyses, we report the transcriptome profiles at four key stages of tail development, from a small tailbud to the onset of feeding (S18, S19, S21 and S28) in Microhyla fissipes, an anuran with a number of advantages for developmental and genetic studies. Results: We obtained 48,826 transcripts and discovered 8807 differentially expressed transcripts (DETs, q < 0.05) among these four developmental stages. We functionally classified these DETs by using GO and KEGG analyses and revealed 110 significantly enriched GO categories and 6 highly enriched KEGG pathways (Protein digestion and absorption; ECM-receptor interaction; Pyruvate metabolism; Fatty acid degradation; Valine, leucine and isoleucine degradation; and Glyoxylate and dicarboxylate metabolism) that are likely critically involved in developmental changes in the tail. In addition, analyses of DETs between any two individual stages demonstrated the involvement of distinct biological pathways/GO terms at different stages of tail development. Furthermore, the most dramatic changes in gene expression profile are those between S28 and any of the other three stages. The upregulated DETs at S28 are highly enriched in “myosin complex” and “potassium channel activity”, which are important for muscle contraction, a critical function of the tail that the animal needs by the end of embryogenesis. Additionally, many DETs and enriched pathways discovered here during tail development, such as HDAC1, Hes1 and Hippo signaling pathway, have also been reported to be vital for the tissue/organ regeneration, suggesting conserved functions between development and regeneration. Conclusion: The present staudy provides a golbal overview of gene expression patterns and new insights into the mechanism involved in anuran tail development and regeneration.

Aim: Peritoneal dialysis is a common renal replacement method for end-stage renal disease. Long-term peritoneal dialysis leads to peritoneal dialysis-related peritoneal fibrosis, which leads to a cessation of treatment. Calpain is a protein belonging to calcium-dependent endopeptidase family and plays an important role in extracellular matrix remodeling. Here, we evaluated the effect of calpain in peritoneal dialysis-related peritoneal fibrosis.Methods: We established two animal models of peritoneal fibrosis and inhibited the activity of Calpain, and then collected peritoneal tissue to evaluate the progress of fibrosis and the changes of Calpain and β-catenin. We obtained Rat peritoneal mesothelial cells and Human peritoneal mesothelial cell line and stimulated with TGF-β to produce extracellular matrix. Next we inhibited Calpain activity or reduced Calpain9 expression, and then assessed changes in extracellular matrix and β-catenin.Results: Inhibition of calpain activity attenuated chlorhexidine glucose and peritoneal dialysis-induced peritoneal thickening and β-catenin expression in mice. In addition, compared with the control group, when primary rat peritoneal mesothelial cells or human peritoneal mesothelial cells were treated with transforming growth factor beta, down-regulation of calpain activity inhibited the expression of Fibronectin and Collagen I, and increased the expression of E-cadherin. These changes could be adjusted after silencing calpain9. Finally, calpain9 deficiency was associated with down-regulation of Fibronectin and β-catenin in human peritoneal mesothelial cells.Conclusion: Our results suggest that calpain9 may be a key molecule in mediating peritoneal dialysis-related peritoneal fibrosis.

Microorganisms play as fundamental contributors to maintain hosts’ fitness, which can be shaped by external environment. Moreover, symbiotic microbiome also varied within species (e.g., between sexes and developmental stages). However, we still need more studies to quantify whether the intraspecific variation patterns of symbiotic microbes can be modified with the change of environment. The Chinese giant salamander (CGS; Andrias davidianus) is a Critically Endangered species. Despite quantitative captive bred individuals were released to rebuild wild populations, the effectiveness is limited. More importantly, no studies have revealed the adaptation of released CGSs to the complex field conditions. In the present study, we explored whether reintroduction can reshape the intraspecific variations of symbiotic microbiota in captive bred CGSs using high-throughput amplicon sequencing of the16S rRNA gene. We found no significant difference of symbiotic microbiome in captive bred males and females, but released males and females differed significantly in skin microbiome. Juveniles had higher diversity of microbial symbiont than adults in hatchery, but lower diversity in field. Moreover, dominant bacterial taxa differed between juveniles and adults in both hatchery and field. Importantly, this symbiotic microbiome variations within species can be modified (alpha and beta diversity, and community composition) when captive bred individuals were released to the field. Overall, we observed a lower alpha diversity and higher relative abundance of Chryseobacterium, Plesiomonas, and Acinetobacter in the bacterial community of captive bred individuals. Instead, higher alpha diversity of symbiotic microbiota and higher relative abundance of S24-7 and Lactobacillus was detected in released individuals. These modifications may associate with the change of living environment, as well as the specific behavior within CGSs (e.g., movement patterns and foraging activities). Future studies can incorporate other approaches (e.g., blood physiology) to better evaluate the growth and health of reintroduced CGSs.

BackgroundPigment regression is an intriguing phenomenon that can be caused by disorders in melanin metabolism or endocrine regulation, or by autoimmune disorders. Albino animals serve as excellent models for the study of the genetic determination of morphology, particularly the evolution of and molecular mechanisms underlying chromatophore-related diseases in animals and humans.Material and MethodsThe artificial culture of Andrias davidianus, the largest extant amphibian, is flourishing in China due to the great ecological and economic value of this animal. Approximately 0.1% of individuals express an albino phenotype accompanied by delayed somatic growth and mortality at early developmental stages. In this study, brain and skin transcriptomics were conducted to study the underlying molecular basis of the phenotype.ResultsThe results indicated decreased transcription of genes of melanin synthesis. Interestingly, MHC I isotypes and immune-related pathways accounted for the primary transcriptional differences between groups, suggesting that the albino phenotype represents a systematic immune problem to a far greater extent than a pigmentation defect. Albino individuals exhibited shifted transcription of MHC I isotypes, and the albino-specific isotype was characterized by increased charges and decreased space in the antigen- binding pocket, implying a drastic change in antigen specificity and a potential risk of autoimmune disorders.ConclusionThese results suggest an association between the albino phenotype and MHC I variants in A. davidianus, which could serve as a convenient model for vitiligo or other autoimmune diseases.

The Qinghai–Tibet Plateau (QTP) is the highest and largest mountain plateau in the world, which has become a focus area of amphibian biodiversity research and conservation, depending on its large number of endemic and threatened species. Among the 58 families of Anura, only a few species of four families (Megophryidae, Bufonidae, Dicroglossidae, Ranidae) are distributed in QTP. Revealing the genetic diversity of these species is crucial for research on their environmental adaptability and biodiversity conservation. Chromosome rearrangements are a critical source of genetic variation, which is recognized as a driver of speciation, providing the genetic material for differentiation and environmental adaptation of amphibians. Here, we identified the karyotypes of 10 species of the above families from the QTP. The karyotypes of these species were obtained from new sites that were not previously reported. Among them, the karyotypes of D. himalayanus and tetraploid B. zamdaensis were reported for the first time. In particular, the ploidy of B. zamdaensis from Zanda, China, was found to be distinctly different from the ploidy from Spiti River, India. This indicates that they have presented species differentiation and supports the multiple and complicated polyploidization events in the Bufotes toads. Furthermore, the different locations of the secondary constriction between the Weixi and Zhongdian populations of O. xiangchengensis support that there is a karyotypic variation between the two subspecies (O. xiangchengensis xiangchengensis vs. O. xiangchengensis deqinicus). A series of chromosomal variations may have facilitated the rapid evolution of amphibians in the QTP, and our study will provide support for further research on amphibian genetic diversity and biodiversity conservation.

Abstract The studies of climatic‐niche shifts over evolutionary time accompanied by key morphological innovations have attracted the interest of many researchers recently. We applied ecological niche models (ENMs), ordination method (environment principal component analyses; PCA‐env), combined phylogenetic comparative methods (PCMs), and phylogenetic generalized least squares (PGLS) regression methods to analyze the realized niche dynamics and correspondingly key morphological innovations across clades within Scutiger boulengeri throughout their distributions in Qinghai–Tibet Plateau (QTP) margins of China. Our results show there are six clades in S. boulengeri and obvious niche divergences caused by niche expansion in three clades. Moreover, in our system, niche expansion is more popular than niche unfilling into novel environmental conditions. Annual mean temperature, annual precipitation, and precipitation of driest month may contribute to such a shift. In addition, we identified several key climatic factors and morphological traits that tend to be associated with niche expansion in S. boulengeri clades correspondingly. We found phenotypic plasticity [i.e., length of lower arm and hand (LAHL), hind‐limb length (HLL), and foot length (FL)] and evolutionary changes [i.e., snout–vent length (SVL)] may together contribute to niche expansion toward adapting novel niche, which provides us a potential pattern of how a colonizing toad might seed a novel habitat to begin the process of speciation and finally adaptive radiation. For these reasons, persistent phylogeographic divisions and accompanying divergences in niche occupancy and morphological adaption suggest that for future studies, distinct genetic structure and morphological changes corresponding to each genetic clade should be included in modeling niche evolution dynamics, but not just constructed at the species level.

We provide the first distribution pattern of the Microhyla heymonsi group based on available molecular and morphological data collected from East and Southeast Asia. Our analyses show a high level of genetic diversity in the M. heymonsi group with nine distinct lineages from China, Myanmar, Vietnam, Laos, Malaysia, Thailand, Cambodia, as well as Singapore, and Indonesia. The study also reveals the discovery of two new species in Vietnam, Microhyla hmongorum sp. nov. from Lai Chau Province and Microhyla xodangorum sp. nov. from Kon Tum Province. When comparing the 12S–16S rRNA gene, the genetic divergence between Microhyla xodangorum and other congeners of the Microhyla heymonsi group ranges from 7.5-- to 8.9% (M. cf. heymonsi) and approximately 8.4% between the new species and M. heymonsi s. str. from Taiwan, China. The genetic divergence between Microhyla hmongorum and its congeners ranges from 4.5–5.6% (M. cf. heymonsi) to 8.7% (Microhyla xodangorum). These new findings bring the total number of known species in the genus Microhyla to 48 and the recorded species of Microhyla from Vietnam to 14.

Abstract Background The association between serum advanced oxidation protein products (AOPP) and mortality risk remains equivocal. We aimed to assess the correlation of serum AOPP levels with the risk of all-cause mortality in hemodialysis (HD) patients. Methods A total of 1394 maintenance HD patients with complete data on AOPP and related parameters were included from China Collaborative Study on Dialysis (CCSD), a multi-center, prospective cohort study. The primary outcome was all-cause mortality, the secondary outcome was cardiovascular disease (CVD) mortality. Results During a median follow-up duration of 5.2 years (IQR, 2.1–5.4), all-cause mortality occurred in 492 (31.4%) participants. Overall, there was a reversed L-shaped association between serum AOPP and all-cause mortality in HD patients (P for nonlinearity = 0.04), with an inflection point at 87 µmol/L. Accordingly, there was no significant association between serum AOPP and all-cause mortality (per SD increment; HR, 0.94; 95%CI, 0.84, 1.05) in participants with AOPP

The Microhyla heymonsi species complex from central Vietnam was examined, and based upon morphological and molecular evidence, two new species are described. The discovery of Microhyla daklakensis sp. nov. and Microhyla ninhthuanensis sp. nov. brings the total number of known species in the genus to 46 and the species number of Microhyla in Vietnam to 13. The Truong Son Range harbors the highest diversity of the genus Microhyla with 11 recorded species so far. However, this apparent micro-endemic diversity is at risk because of habitat loss by deforestation, which highlights the necessity of further research leading to improved conservation measures.

The reintroduction of captive-bred individuals is a primary approach to rebuild the wild populations of the Chinese giant salamander (Andrias davidianus), the largest extant amphibian species. However, the complexity of the wild habitat (e.g., diverse microorganisms and potential pathogens) potentially threatens the survival of reintroduced individuals. In this study, fresh (i.e., containing environmental microbiota) or sterilized river sediments (120°C sterilized treatment) were added to the artificial habitats to treat the larvae of the Chinese giant salamander (control group—Cnt: 20 individuals, treatment group 1 with fresh river sediments—T1: 20 individuals, and treatment group 2 with sterilized river sediments—T2: 20 individuals). The main objective of this study was to test whether this procedure could provoke their wild adaptability from the perspective of commensal microbiotas (skin, oral cavity, stomach, and gut) and larvae transcriptomes (skin, spleen, liver, and brain). Our results indicated that the presence of habitat sediments (whether fresh or sterilized) reshaped the oral bacterial community composition. Specifically, Firmicutes decreased dramatically from ~70% to ~20–25% (mainly contributed by Lactobacillaceae), while Proteobacteria increased from ~6% to ~31–36% (mainly contributed by Gammaproteobacteria). Consequently, the proportion of antifungal operational taxonomic units (OTUs) increased, and the function of oral microbiota likely shifted from growth-promoting to pathogen defense. Interestingly, the skin microbiota, rather than the colonization of habitat microbiota, was the major source of the pre-treated oral microbiota. From the host perspective, the transcriptomes of all four organs were changed for treated individuals. Specifically, the proteolysis and apoptosis in the skin were promoted, and the transcription of immune genes was activated in the skin, spleen, and liver. Importantly, more robust immune activation was detected in individuals treated with sterilized sediments. These results suggested that the pathogen defense of captive-bred individuals was improved after being treated, which may benefit their survival in the wild. Taken together, our results suggested that the pre-exposure of captive-bred Chinese giant salamander individuals to habitat sediments could be considered and added into the reintroduction processes to help them better adapt to wild conditions.

The molecular basis enabling the adaptation of animals to spatially heterogeneous environments is a critical clue for understanding the variation, formation, and maintenance of biodiversity in the context of global climate change. Mountain dragons (Agamidae: Diploderma) thrive in the Hengduan Mountain Region, a biodiversity hotspot and a typical spatially heterogeneous environment. Here, we compare the liver and muscle metabolome and gut microbiome of 11 geographical populations from three Diploderma species (D. iadinum, D. yulongsense, and D. vela) after 7 days acclimation in the same laboratory conditions. Amino acid metabolism, particularly the products of the glutathione cycle, accounted for major interspecies variations, implying its significance in genetic differentiation among mountain dragons. Notably, the cold-dwelling D. vela and D. yulongense populations tended to have higher glycerophosphate, glycerol-3-phosphocholine, and kinetin levels in their liver, higher carnosine levels in their muscle, and higher Lachnospiraceae levels in their gut. Phylogeny, net primary productivity (NPP), and the temperature had the highest explanation rate to the variations in muscle metabolome, liver metabolome, and gut microbiome, respectively, suggesting heterogeneity of biological systems in response to climatic variations. Therefore, we suggested that the organ heterogeneity in environmental responsiveness might be substantial for mountain dragons to thrive in complicated environments.

Multiple disciplines can help to discover cryptic species and resolve taxonomic confusions. The Asian horned toad genus Megophrys sensu lato as a diverse group was proposed to contain dozens of cryptic species. Based on molecular phylogenetics, morphology, osteology, and bioacoustics data, the species profiles of Megophrys toads in the eastern corner of Himalayas in Medog County, Tibet Autonomous Region, China was investigated. The results indicated that this small area harbored at least four Megophrys species, i.e., M. medogensis, M. pachyproctus, Megophrys zhoui sp. nov., and Megophrys yeae sp. nov., the latter two being described in this study. Additionally, the mitochondrial DNA trees nested the low-middle-elevation and high-elevation groups of M. medogensis into a monophyletic group, being in discordance with the paraphyletic relationship between them revealed in the nuclear DNA trees. The findings highlighted the underestimated biodiversity in Himalayas, and further indicated that the Megophrys toads here have been probably experienced complicated evolutionary history, for example, introgression between clades or incomplete lineage sorting and niche divergences in microhabitats. Anyway, it is urgent for us to explore the problems because these toads are suffering from increasing threats from human activities and climatic changes.

Abstract Background Metamorphic climax is the crucial stage of amphibian metamorphosis responsible for the morphological and functional changes necessary for transition to a terrestrial habitat. This developmental period is sensitive to environmental changes and pollution. Understanding its metabolic basis and requirements is significant for ecological and toxicological research. Rana omeimontis tadpoles are a useful model for investigating this stage as their liver is involved in both metabolic regulation and fat storage. Results We used a combined approach of transcriptomics and metabolomics to study the metabolic reorganization during natural and T3-driven metamorphic climax in the liver and tail of Rana omeimontis tadpoles. The metabolic flux from the apoptotic tail replaced hepatic fat storage as metabolic fuel, resulting in increased hepatic amino acid and fat levels. In the liver, amino acid catabolism (transamination and urea cycle) was upregulated along with energy metabolism (TCA cycle and oxidative phosphorylation), while the carbohydrate and lipid catabolism (glycolysis, pentose phosphate pathway (PPP), and β-oxidation) decreased. The hepatic glycogen phosphorylation and gluconeogenesis were upregulated, and the carbohydrate flux was used for synthesis of glycan units (e.g., UDP-glucuronate). In the tail, glycolysis, β-oxidation, and transamination were all downregulated, accompanied by synchronous downregulation of energy production and consumption. Glycogenolysis was maintained in the tail, and the carbohydrate flux likely flowed into both PPP and the synthesis of glycan units (e.g., UDP-glucuronate and UDP-glucosamine). Fatty acid elongation and desaturation, as well as the synthesis of bioactive lipid (e.g., prostaglandins) were encouraged in the tail during metamorphic climax. Protein synthesis was downregulated in both the liver and tail. The significance of these metabolic adjustments and their potential regulation mechanism are discussed. Conclusion The energic strategy and anabolic requirements during metamorphic climax were revealed at the molecular level. Amino acid made an increased contribution to energy metabolism during metamorphic climax. Carbohydrate anabolism was essential for the body construction of the froglets. The tail was critical in anabolism including synthesizing bioactive metabolites. These findings increase our understanding of amphibian metamorphosis and provide background information for ecological, evolutionary, conservation, and developmental studies of amphibians.

The toad genus Oreolalax is widely distributed in southwest China and northern Vietnam. A new species of the genus is described from Sichuan Province, China. Phylogenetic analyses based on the mitochondrial 12S rRNA and 16S rRNA gene sequences supported the new species as an independent clade clustered into the clade also containing O. nanjiangensis and O. chuanbeiensis. The new species can be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 51.2–64.2 mm in males); head broad; tympanum hidden; interorbital region with dark triangular pattern; belly with marbling; male lacking spines on lip margin; spiny patches on chest small with thick sparse spines in male; nuptial spines thick and sparse; tibio-tarsal articulation reaching beyond nostril when leg stretched forward; toe webbing at base.

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and threatened human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, but how this clone evolves in vivo then adapts to the host and facilitates dissemination remains largely unknown. Here, the genomic dynamics of 4 ST11-CRKP isolates, which were sequentially collected from the urine of a patient with initial serious scrotal abscess and finally recovered without effective medication, were analyzed. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4 to 24 mutations, among which 94% to 100% of them were synonymous or intergenic mutations. During 47 days of antibiotics therapy, CRKP underwent adaptive evolution, including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by a deletion within the ramR ribosomal binding site, which has been described by us previously. On the other hand, mutations associated with two genes, acyltransferase (act) and ompK26, resulted in the attenuation phenotype of ST11-CRKP. act deficiency reduced the capsular polysaccharide (CPS) production, enhanced biofilm formation, weakened capsular protection, and decreased induction of proinflammatory cytokines. Further RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK, and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of the upstream repressor binding site. This study presents a within-host adaption of ST11-CRKP and suggests an important role of CPS in the adaptive evolution of virulence and persistence of CRKP. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life-threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intraabdominal infection, liver abscess, and meningitis. CRKP infection is the leading cause of high mortality in hospitals. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility is increasingly identified. Understanding how this clone has evolved is crucial for developing strategies to control its further dissemination. The significance of our research is the identification of the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP that facilitate persistence and dissemination. Furthermore, our study also highlights the importance of monitoring the within-host evolution of pathogens during the treatment and developing interventions to minimize the potential impact of host adaptation on human health.

Ischemic stroke is a common cause of death and disability worldwide, which leads to serious neurological and physical dysfunction and results in heavy economic and social burdens. For now, timely and effective dissolution of thrombus, and ultimately improvement in the recovery of neurological functions, is the treatment strategy focus. Recently, many studies have reported that transcranial ultrasound stimulation (TUS), as a non-invasive method, can dissolve thrombus, improve cerebral blood circulation, and exert a neuroprotective effect post-stroke. TUS can promote functional recovery and improve rehabilitation efficacy among patients with ischemic stroke. This mini-review summarizes the potential mechanism and limitation of TUS in stroke aims to provide a new strategy for the future treatment of patients with ischemic stroke.

Buffalo and cow milk have a very different composition in terms of fat, protein, and total solids. For a better knowledge of such a difference, the milk metabolic profiles and characteristics of metabolites was investigated in Italian Mediterranean buffaloes and Chinese Holstein cows were investigated by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in this study. Totally, 23 differential metabolites were identified to be significantly different in the milk from the two species of which 15 were up-regulated and 8 down-regulated in Italian Mediterranean buffaloes. Metabolic pathway analysis revealed that 4 metabolites (choline, acetylcholine, nicotinamide and uric acid) were significantly enriched in glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, glycine, serine and threonine metabolism, as well as purine metabolism. The results provided further insights for a deep understanding of the potential metabolic mechanisms responsible for the different performance of Italian Mediterranean buffaloes’ and Chinese Holstein cows’ milk. The findings will offer new tools for the improvement and novel directions for the development of dairy industry.

Global climate change is considered to be one of the main threats to organisms. As poikilothermic animals, amphibians are in particular sensitive because they cannot adapt to the dramatic climate change through active physiological regulation. Using 104 representative species, the present study conducted an assessment of amphibians vulnerability to climate change in China through the combination of two approaches. Specifically, 18 vulnerability criteria belonging to five categories (i.e., thermal tolerance, individual reproductive, population diffusion and diversity, food and habitat, and climate conditions) were first selected and scored based on literatures and experts opinions. Species were then ranked into three levels of climate change vulnerability (i.e., high, moderate, and low) by calculating vulnerability scores and conducting natural breaks analyses, as well as performing a principal coordinate analysis (PCoA) and k-means cluster analyses, respectively. To integrate the two results, a matrix with the ranks from each result was developed to produce a final integrated list. Our results indicated that the 104 amphibian species were classified into three types by natural breaks, with 54 low vulnerable species, 41 moderately vulnerable species, and nine highly vulnerable species. Based on the results of PCoA and k-means cluster analyses, five species were highly vulnerable, 38 species were moderately vulnerable, and 61 species were low vulnerable. The combination of the two ranks suggested that 36 species such as Hyla tsinlingensis and Liangshantriton taliangensis were of low vulnerability, 54 species such as Echinotriton chinhaiensis and Hynobius chinensis were of moderate vulnerability, and 14 species such as Ichthyophis kohtaoensis and Zhangixalus prasinatus were of high vulnerability. Overall, our results indicated that climate change could have strong potential effects on amphibians in China. And the highly vulnerable species such as Ichthyophis kohtaoensis, Zhangixalus prasinatus, and Theloderma corticale should be the priority in future conservation activities.