Proteus faecis: a potentially pathogenic bacterium isolated from the freshwater Yangtze finless porpoise.

Proteus faecis is a gram-negative facultative anaerobic rod-shaped bacterium capable of swarming motility. It has been isolated from numerous sources such as humans, animals, and refuse and is considered potentially pathogenic towards humans. In this study, bacteria were isolated from the blowhole of a Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in captivity in China. One bacterium, P. faecis porpoise, was isolated and whole genome sequencing done. Biofilm formation, motility and antimicrobial resistance were also investigated. To find putative virulence factors, the genome of P. faecis strain porpoise was compared to the genomic sequences of eight other P. faecis isolates using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) ( https://www.bv-brc.org/ ). The goal of this study was to initially characterize the pathogenicity of this bacterium isolated from a cetacean species using both pathogenomics and conventional approaches.

The intestinal microbiota of a Risso's dolphin (Grampus griseus): possible relationships with starvation raised by macro-plastic ingestion.

Ingesting marine plastics is increasingly common in cetaceans, but little is known about their potential effects. Here, by utilizing 16S rRNA gene sequencing, we profiled the intestinal bacterial communities of a stranded Risso's dolphin (Grampus griseus) which died because of the ingestion of rubber gloves. In this study, we explored the potential relationships between starvation raised by plastic ingestion with the dolphin gut microbiota. Our results showed significant differences in bacterial diversity and composition among the different anatomical areas along the intestinal tract, which may be related to the intestinal emptying process under starvation. In addition, the intestinal bacterial composition of the Risso's dolphin showed both similarity and divergence to that of other toothed whales, suggesting potential roles of both host phylogeny and habitat shaping of the cetacean intestinal microbiome. Perhaps, the microbiota is reflecting a potentially disordered intestinal microbial profile caused by the ingestion of macro-plastics which led to starvation. Moreover, two operational taxonomic units (0.17% of the total reads) affiliated with Actinobacillus and Acinetobacter lwoffii were detected along the intestinal tract. These bacterial species may cause infections in immunocompromised dolphins which are malnourished. This preliminary study profiles the intestinal microbiota of a Risso's dolphin, and provides an additional understanding of the potential relationships between starvation raised by ingesting macro-plastics with cetacean gut microbiota.

OUTER RETINAL TUBULATION IN BIETTI CRYSTALLINE DYSTROPHY ASSOCIATED WITH THE RETINAL PIGMENT EPITHELIUM ATROPHY.

PURPOSE: To determine the prognostic value of outer retinal tubulation (ORT) in the eyes of a Chinese cohort with Bietti crystalline dystrophy (BCD). METHODS: This retrospective, multicenter cohort study enrolled 42 patients with clinically and genetically diagnosed BCD. Eighty eyes with good-quality images of spectral domain optical coherence tomography were included. Demographic details and clinical data were collected. The characteristics of ORT, including prevalence, location, and morphologic characteristics were analyzed. RESULTS: Forty-two patients with BCD harbored potentially CYP4V2 disease-causing mutations. The mutation spectrum comprised 17 unique variants, 9 of which were novel. Fifty-two of these 80 eyes demonstrated evidence of ORT. The incidence of ORT is significantly higher in Stage 2 than other stages ( P < 0.001). ORT was mainly bilateral and located at the margin of the atrophic area of retinal pigment epithelium (RPE), and dynamically changed with the progressive RPE atrophy. The process of RPE atrophy was slower in eyes with ORT ( P = 0.017), with significantly longer intact RPE width in Stage 3 ( P = 0.024). Eyes with ORT had slower vision loss than eyes without ORT ( P = 0.044). CONCLUSION: ORT may be a sign of the onset of RPE atrophy in early-stage BCD and may suggest less risk of rapid progression in late-stage BCD.

A novel microdeletion of 517 kb downstream of the PAX6 gene in a Chinese family with congenital aniridia.

BACKGROUND: To identify the disease-causing gene in a Chinese family affected with congenital aniridia. METHODS: Patients underwent systematic ophthalmic examinations such as anterior segment photography, fundus photography, optical coherence tomography, and fundus fluorescein angiography. The proband was screened for pathogenic variants by whole exome sequencing (WES) and copy number variant (CNV) analysis. Real-time quantitative PCR (RT-qPCR) was applied to confirm the CNV results. Breakpoints were identified by long-range PCR followed by Sanger sequencing. RESULTS: All seven members of this Chinese family, including four patients and three normal individuals, were recruited for this study. All patients showed bilateral congenital aniridia with nystagmus, except the son of the proband, who presented with bilateral partial coloboma of the iris. A novel heterozygous deletion (chr11:31,139,019-31,655,997) containing the 3' regulatory enhancers of the PAX6 gene was detected in this family. We also reviewed the reported microdeletions downstream of PAX6 in patients with aniridia. CONCLUSIONS: We identified a novel microdeletion, 517 kb in size located about 133 kb downstream of the PAX6 gene, responsible for congenital aniridia in this Chinese family, which expands the spectrum of aniridia-associated mutations in PAX6.

Identification of Cuproptosis-Related circRNA-miRNA-mRNA Network in Laser-Induced Choroidal Neovascularization Models and in Peripheral Blood Mononuclear Cells of Patients with Neovascular Age-Related Macular Degeneration.

INTRODUCTION: The aims of this study were to investigate the molecular alterations of cuproptosis-related genes and to construct the cuproptosis-related circular RNA (circRNA)-microRNA (miRNA)-mRNA networks in neovascular age-related macular degeneration (nAMD). METHODS: The transcriptional profiles of laser-induced choroid neovascularization (CNV) mouse models and nAMD patient samples were obtained from sequencing and from the GEO database (GSE146887), respectively. The expression levels of ten cuproptosis-related genes (FDX1, DLAT, LIAS, DLD, PDHB, MTF1, CDKN2A, GLS, LIPT1, and PDHA1) were extracted and verified in both mouse CNV models and patient peripheral blood mononuclear cells (PBMCs) samples. The cuproptosis-related circRNA-miRNA-mRNA network was further constructed based on miRNet database, the dataset GSE131646 of small RNA expression profile, and the dataset GSE140178 of circRNA expression profile in mouse CNV models. RESULTS: The significant upregulation of Cdkn2a and Mtf1 and the downregulation of other 5 cuproptosis-related genes were verified in the mouse CNV model, but only CDKN2A significantly upregulated in PBMCs of patients with nAMD. Four miRNAs were detected in the intersection between miRNet prediction and sequencing data: miR-129-5p, miR-129-2-3p, miR-182-5p, and miR-615-3p. There were 9 circRNAs at the intersection of hsa-miR-182-5p and hsa-miR-615-3p predictions, one circRNA predicted by hsa-miR-129-5p and GSE140178 (hsa-circASH1L), and one circRNA predicted by hsa-miR-182-5p and hsa-miR-615-3p (hsa-circNPEPPS). CONCLUSION: This study suggested the repression of cuproptosis in nAMD pathologies and constructed a cuproptosis-related network of 8 cuproptosis-related genes, 4 miRNAs, and 11 circRNAs.

Comprehensive analysis of the PRPF31 gene in retinitis pigmentosa patients: Four novel Alu-mediated copy number variations at the PRPF31 locus.

Retinitis pigmentosa (RP) is a monogenic disease characterized by irreversible degeneration of the retina. PRPF31, the second most common causative gene of autosomal dominant RP, frequently harbors copy number variations (CNVs), but the underlying mechanism is unclear. In this study, we summarized the phenotypic and genotypic characteristics of 18 RP families (F01-F18) with variants in PRPF31. The prevalence of PRPF31 variants in our cohort of Chinese RP families was 1.7% (18/1024). Seventeen different variants in PRPF31 were detected, including eight novel variants. Notably, four novel CNVs encompassing PRPF31, with a proportion of 22.2% (4/18), were validated to harbor gross deletions involving Alu/Alu-mediated rearrangements (AAMRs) in the same orientation. Among a total of 12 CNVs of PRPF31 with breakpoints mapped on nucleotide-resolution, 10 variants (83.3%) were presumably mediated by Alu elements. Furthermore, we described the correlation between the genotypes and phenotypes in PRPF31-related RP. Our findings expand the mutational spectrum of the PRPF31 gene and provide strong evidence that Alu elements of PRPF31 probably contribute to the susceptibility to genomic rearrangement in this locus.

Retinal degeneration in mice lacking the cyclic nucleotide-gated channel subunit CNGA1.

Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B subunits. Biallelic mutations in CNGA1 or CNGB1 genes result in autosomal recessive retinitis pigmentosa (RP). To investigate the pathogenic mechanism of CNG channel-associated retinal degeneration, we developed a mouse model of CNGA1 knock-out using CRISPR/Cas9 technology. We observed progressive retinal thinning and a concomitant functional deficit in vivo as typical phenotypes for RP. Immunofluorescence and TUNEL staining showed progressive degeneration in rods and cones. Moreover, microglial activation and oxidative stress damage occurred in parallel. RNA-sequencing analysis of the retinae suggested down-regulated synaptic transmission and phototransduction as early as 9 days postnatal, possibly inducing later photoreceptor degeneration. In addition, the down-regulated PI3K-AKT-mTOR pathway indicated upregulation of autophagic process, and chaperone-mediated autophagy was further shown to coincide with the time course of photoreceptor death. Taken together, our studies add to a growing body of research exploring the mechanisms of photoreceptor death during RP progression and provide a novel CNGA1 knockout mouse model for potential development of therapies.

MicroRNA-191-5p ameliorates amyloid-ß1-40 -mediated retinal pigment epithelium cell injury by suppressing the NLRP3 inflammasome pathway.

Amyloid ß (Aß) is a crucial component of drusen, the hallmark of the early stage of age-related macular degeneration (AMD), and can cause retinal pigment epithelium (RPE) cell damage through activation of the inflammatory response. MicroRNAs play a critical role in inflammation. However, the mechanism underlying the effect of microRNAs on the NLRP3 inflammasome induced by Aß remains poorly understood. In the present study, we demonstrated that Aß1-40 -mediated RPE damage by inducing a decrease in endogenous miR-191-5p expression. This led to the upregulation of its target gene, C/EBPß. C/EBPß acts as a transcription factor for NLRP3, promotes its transcription, and upregulates the downstream inflammatory factors Caspase-1 and IL-1ß. Correspondingly, overexpression of miR-191-5p alleviated RPE cell injury by suppressing inflammation. The present study elucidates a novel transcriptional regulatory mechanism of the NLRP3 inflammasome. Our findings suggest an anti-inflammatory effect of miR-191-5p in Aß1-40 -induced RPE impairment, shedding light on novel preventive or therapeutic approaches for AMD-associated RPE impairment.

Biofilm formation, antimicrobial assay, and toxin-genotypes of Clostridium perfringens type C isolates cultured from a neonatal Yangtze finless porpoise.

This is a culture-dependent study with the objective of pure culturing and characterizing pathogenic bacteria from the blowhole, lung, stomach and fecal samples of a neonatal crucially endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) that died 27 days after birth. Bacteria were inoculated using a swab onto blood and MacConkey agar plates and representative isolates were identified through 16S rRNA gene sequence analysis. A total of three Clostridium perfringens type C strains from the fecal samples were isolated. Toxin genes, including cpa, cpb and cpb2, were detected by PCR amplification, whereas the etx, iap and cpe genes were not detected. Biofilm formation of the three strains was then examined. Only one strain was capable of biofilm formation. In addition, isolates showed strong resistance against the antibiotics amikacin (3/3), erythromycin (1/3), gentamicin (3/3), streptomycin (3/3), and trimethoprim (3/3), while sensitivity to ampicillin (3/3), bacitracin (3/3), erythromycin (2/3), penicillin G (3/3), and tetracycline (3/3). The results suggested C. perfringens type C could have contributed to the death of this neonatal porpoise.

Epidemiological characterization of Clonorchis sinensis infection in humans and freshwater fish in Guangxi, China.

BACKGROUND: Clonorchiasis is a widespread yet neglected foodborne disease with over 85% of all cases found in China. Guangxi province, located in southeastern China, ranks among the highest endemic provinces. We explore the epidemiological status and determinants of Clonorchis sinensis (C. sinensis) infection in humans and freshwater fish in Guangxi, China. METHODS: Data on C. sinensis infection in humans from January 2008 to December 2017were extracted from the China Information System for Disease Control and Prevention. An active surveillance of C. sinensis infection in fish was conducted in 2016-2017. County level data including potential environmental, social-economical and behavioral determinants was also collected. Univariate and multivariate logistic regression models were used to explore the determinants of C. sinensis infection in humans and fish. Simple and multiple zero-inflated Poisson regression models were fit to assess the associated factors of clonorchiasis in humans at the county level. RESULTS: Totally, 4526 C. sinensis cases were reported between 2008 and 2017, with an annual prevalencerate of 0.96/100,000 persons. Of 101 counties in Guangxi, 97 reported at least 1 case. Among 2,098 fish samples, 203 (9.7%) from 70 counties contained C. sinensis. The rate was higher in small fish including Pseudorasbora parva (45.3%), Misgurnus anguillicaudatus (41.2%), Hemicculter leuciclus (34.5%), unclassified small fishes (30.9%), Cyprinidae (20.0%), Cirrhinus molitorella (16.4%), Carassius auratus (13.6%) and Cyprinus carpio (13.3%), while it was lower in fish species that are usually used in preparing raw fish dishes including Ctenopharyngodon idellus (3.6%), Spinibarbus denticulatus (3.7%), Monopterus albus (6.4%), Cyprinus carpio (4.4%), Oreochromis mossambicus (3.3%) and Spualiobarbus Curriculus (6.6%). The C. sinensis infection in fish was only associated with fish species. The estimated human clonorchiasis prevalence at the county level was positively associated with raw fish consumption habits and certain rivers. CONCLUSIONS: Clonorchiasis is highly prevalent in both humans and freshwater fish in Guangxi. Environmental, social-economic and behavioral determinants contribute to the high prevalence as well as the significant differential distribution by county. Regular surveillance should be implemented for clonorchiasis to demonstrate the change in epidemiology and burden, which will benefit the design of interventions.

High historical variability weakens the effects of current climate differentiation on microbial community dissimilarity and assembly.

Understanding the influences of global climate change on soil microbial communities is essential in evaluating the terrestrial biosphere's feedback to this alarming anthropogenic disturbance. However, little is known about how intra-site historical climate variability can mediate the influences of current climate differences on community dissimilarity and assembly. To fill this gap, we examined and disentangled the interactive effects of historical climate variability and current climate differences on the soil bacterial community dissimilarity and stochasticity of community assembly among 143 sites from 28 forests across eastern China. We hypothesize that the relative importance of stochasticity and community dissimilarity are related to historical climate variability and that an increasing sum of intra-site historical variability enhances stochasticity while reduces dissimilarity between two communities. To test our hypothesis, we statistically controlled for covariates between sites including differences in soil chemistry, plant diversity, spatial distance, and seasonal climate variations at annual timescales. We observed that an increase in inter-site current climate differences led to a reduced impact of stochasticity in community assembly and a pronounced divergence between communities. In stark contrast, when communities were subjected to a high level of intra-site historical climate fluctuation, the observed impact incurred from current climate differences was substantially weakened. Moreover, the influence of increased historical variability was consistent along the gradient of current temperature differences between sites. However, effects induced by historical fluctuation in precipitation were disproportional and only evident when small inter-site differences were observed. Consequently, if the prior climate variability is ignored, especially regarding environmental factors like temperature, we assert that the influence current climate differentiation has on regulating community dissimilarity and assembly stochasticity will be underestimated. Together, our findings highlight the importance and need of explicitly controlling the mean and the historical variability of climate factors for the next "generation" of climate change experiments to come.

Searching for putative virulence factors in the genomes of Shewanella indica and Shewanella algae.

Bacterial pathogens are a major threat to both humans and animals worldwide. It is crucial to understand the mechanisms of various disease processes at the molecular level. Shewanella species are widespread in the environment and some are considered as emerging opportunistic human and marine mammal pathogens. In this study, putative virulence factors on the genome of Shewanella indica BW, a bacterium isolated from the Bryde's whale (Balaenoptera edeni), were determined. Additionally, for comparative purposes, putative virulence factors from two other S. indica and ten S. algae strains were also determined using the Pathosystems Resource Integration Center (PATRIC) pipeline. We confirmed the presence of previously reported virulence factors and we are proposing several new candidate virulence factors. Interestingly, the putative virulence factors were very similar between the two species with the exception of microbial collagenase which was present in all S. algae genomes, but absent in all S. indica genomes.

Identification of a novel RPGR mutation associated with X-linked cone-rod dystrophy in a Chinese family.

BACKGROUND: Cone-rod dystrophy (CORD) is a group of inherited retinal dystrophies, characterized by decreased visual acuity, color vision defects, photophobia, and decreased sensitivity in the central visual field. Our study has identified a novel pathogenic variant associated with X-linked cone-rod dystrophy (XLCORD) in a Chinese family. METHODS: All six family members, including the proband, affected siblings, cousins and female carriers, have underwent thorough ophthalmic examinations. The whole exome sequencing was performed for the proband, followed by Sanger sequencing for spilt-sample validation. A mammalian expression vector (AAV-MCS) with mutated retinitis pigmentosa GTPase regulator (RPGR) sequence was expressed in HEK293 T cells. The mutated protein was verified by Western blotting and immunohistochemistry. RESULTS: A novel mutation in the RPGR gene (c.2383G > T, p.E795X) is identified to be responsible for CORD pathogenesis. CONCLUSIONS: Our findings have expanded the spectrum of CORD-associated mutations in RPGR gene and serve as a basis for genetic diagnosis for X-linked CORD.

An improved method for establishment of murine retinal detachment model and its 3D vascular evaluation.

Retinal detachment (RD) results in disruption of retinal physiology and visual function. Although surgical intervention has been well-developed to restore the retinal anatomic structure, post-op progression of visual function decline is prominent in a large proportion of patients. Therefore, the establishment of a disease model that accurately mimics RD pathogenesis is crucial to mechanistic study and drug screening. General protocols to induce RD in mice are frequently associated with complications leading to model instability and reduced reproducibility. In this study, we established a stable and reproducible mice RD model with a detached area of over 90% and rare complications. Briefly, the modified method was realized by vitreous humor extraction to reduce intraocular pressure, followed by directly-visible hyaluronic acid injection into subretinal space. The detachment of retina was confirmed by fundus photography, and progressive thinning of the outer nuclear layer (ONL) was determined by HE staining. Apoptotic signals were prominent in the ONL. Consistently, visual function was significantly compromised as determined by ERG. Moreover, retinal vasculature appeared to remodel and acquired winding, twisted and dilated structures illustrated by 3D reconstruction. In addition, activation of Müller cells and microglia, and infiltration of blood-derived macrophages were detected locally. Collectively, we have established a modified protocol to model RD with increased stability, reproducibility and fewer complications, and 3D high-resolution imaging and reconstruction of vasculature could provide new tools to evaluate this model.

NEAT1/hsa-mir-98-5p/MAPK6 axis is involved in non-small-cell lung cancer development.

Long noncoding RNAs (lncRNAs) or microRNAs belong to the two most important noncoding RNAs and they are involved in a lot of cancers, including non-small-cell lung cancer (NSCLC). Therefore, currently, we focused on the biological and clinical significance of lncRNA nuclear enriched abundant transcript 1 (NEAT1) and hsa-mir-98-5p in NSCLC. It was observed that NEAT1 was upregulated while hsa-mir-98-5p was downregulated respectively in NSCLC cell lines compared to human normal lung epithelial BES-2B cells. Inhibition of NEAT1 can suppress the progression of NSCLC cells and hsa-mir-98-5p can reverse this phenomenon. Bioinformatics search was used to elucidate the correlation between NEAT1 and hsa-mir-98-5p. Additionally, a novel messenger RNA target of hsa-mir-98-5p, mitogen-activated protein kinase 6 (MAPK6), was predicted. Overexpression and knockdown studies were conducted to verify whether NEAT1 exhibits its biological functions through regulating hsa-mir-98-5p and MAPK6 in vitro. NEAT1 was able to increase MAPK6 expression and hsa-mir-98-5p mimics can inhibit MAPK6 via downregulating NEAT1 levels. We speculated that NEAT1 may act as a competing endogenous lncRNA to upregulate MAPK6 by attaching hsa-mir-98-5p in lung cancers. Taken these together, NEAT1/hsa-mir-98-5p/MAPK6 is involved in the development and progress in NSCLC. NEAT1 could be recommended as a prognostic biomarker and therapeutic indicator in NSCLC diagnosis and treatment.

Putative virulence factors of Plesiomonas shigelloides.

Plesiomonas shigelloides is a Gram-negative rod-shaped bacterium which has been isolated from humans, animals and the environment. It has been associated with diarrhoeal disease in humans and various epizootic diseases in animals. In this study P. shigelloides strains were isolated from the faecal material of a captive Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in semi-natural conditions in China. Plesiomonas shigelloides strain EE2 was subjected to whole genome sequencing. The draft genome was then compared to the genome sequences of ten other P. shigelloides isolates using the Pathosystems Resource Integration Center pipeline. In addition to several virulence factors which have been previously reported, we are proposing new candidate virulence factors such as a repeats-in-toxin protein, lysophospholipase, a twin-arginine translocation system and the type VI secretion effector Phospholipase A1.

Assembly and characterization of the MHC class I region of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) is the sole freshwater subspecies of N. asiaeorientalis and is now critically endangered. Major histocompatibility complex (MHC) is a family of highly polymorphic genes that play an important immunological role in antigen presentation in the vertebrates. Currently, however, little is known about MHC region in the genome of the YFP, which hampers conservation genetics and evolutionary ecology study using MHC genes. In this work, a nucleotide sequence of 774,811 bp covering the YFP MHC class I region was obtained by screening a YFP bacterial artificial chromosome (BAC) library, followed by sequencing and assembly of positive BAC clones. A total of 45 genes were successfully annotated, of which four were MHC class I genes. There are high similarities among the four YFP MHC class I genes (>94%). Divergence in the coding region of the four YFP MHC class I genes is mainly localized to exons 2 and 3, which encode the antigen-binding sites of MHC class I genes. Additionally, comparison of the MHC structure in YFP to those of cattle, sheep, and pig showed that MHC class I genes are located in genome regions with regard to the conserved genes, and the YFP contains the fewest MHC class I genes among these species. This is the first report characterizing a cetacean MHC class I region and describing its organization, which would be valuable for further investigation of adaptation in natural populations of the YFP and other cetaceans.

Isolation of culturable aerobic bacteria and evidence of Kerstersia gyiorum from the blowhole of captive Yangtze finless porpoises.

Bacterial respiratory illnesses are problematic in aquatic mammals such as the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP), which is now at a critically endangered status. Yet little is known about the bacteria inhabiting the respiratory tract of YFPs. In this study, we preliminarily characterized the culturable aerobic bacteria from blow samples of captive YFPs. The bacterial diversity was assessed through cultivation by direct exhalation onto Columbia blood agar plates and identification of representative isolates through 16S rRNA gene sequence analysis. In total, eleven bacterial species belonging to four phyla Proteobacteria (71 %), Firmicutes (25 %), Bacteroidetes (3 %) and Actinobacteria (1 %) were identified. Most of these isolates were opportunistic pathogens found in respiratory illnesses in humans and animals. We also reported the first case of Kerstersia gyiorum isolated from an animal. This work provides a preliminary assessment of the bacteria present in the respiratory tract of captive YFPs, which will be an important first step in elucidating the roles of normal microbiota in maintaining respiratory health of YFPs. This study also points out the necessity of future long-term monitoring of blowhole microorganisms in the YFPs and making emergency preparedness plans for respiratory tract infections. These measures can aid in assessing the pathogenic risk of the critically endangered YFP populations.

Fecal Bacterial Composition of the Endangered Yangtze Finless Porpoises Living Under Captive and Semi-natural Conditions.

Intestinal microbiota is essential to the health and physiology of host animals. We undertook the first microbiological study of the fecal bacterial composition from critically endangered (CR) Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis; YFPs) living under captive and semi-natural conditions using both high-throughput sequencing method and 16S rRNA gene clone library method. As determined by high-throughput sequencing of V3-V4 regions of the 16S rRNA gene, semi-natural samples harbored 30 and 36 operational taxonomic units (OTUs), which was more than the 22 and 27 OTUs detected from YFPs living in captivity. In captive YFPs Firmicutes was the predominant phylum, whereas this was Proteobacteria for YFPs living in semi-nature conditions. This suggests habitat-specific fecal bacterial composition of YFPs. Plesiomonas spp. and Aeromonas spp., which are potentially pathogenic, were identified in all the feces. Bacterial diversity from one porpoise living in captivity was also determined by constructing a 16S rRNA gene clone library and only 1 phylum was identified. High-throughput sequencing was more effective at determining the bacterial diversity compared to the 16S rRNA gene clone library. This study provides important information for the management and conservation of the CR YFPs.

Mixed lineage leukemia 5 (MLL5) protein regulates cell cycle progression and E2F1-responsive gene expression via association with host cell factor-1 (HCF-1).

Trithorax group proteins methylate lysine 4 of histone 3 (H3K4) at active gene promoters. MLL5 protein, a member of the Trithorax protein family, has been implicated in the control of the cell cycle progression; however, the underlying molecular mechanism(s) have not been fully determined. In this study, we found that the MLL5 protein can associate with the cell cycle regulator "host cell factor" (HCF-1). The interaction between MLL5 and HCF-1 is mediated by the "HCF-1 binding motif" (HBM) of the MLL5 protein and the Kelch domain of the HCF-1 protein. Confocal microscopy showed that the MLL5 protein largely colocalized with HCF-1 in the nucleus. Knockdown of MLL5 resulted in reduced cell proliferation and cell cycle arrest in the G1 phase. Moreover, down-regulation of E2F1 target gene expression and decreased H3K4me3 levels at E2F1-responsive promoters were observed in MLL5 knockdown cells. Additionally, the core subunits, including ASH2L, RBBP5, and WDR5, that are necessary for effective H3K4 methyltransferase activities of the Trithorax protein complexes, were absent in the MLL5 complex, suggesting that a distinct mechanism may be used by MLL5 for exerting its H3K4 methyltransferase activity. Together, our findings demonstrate that MLL5 could associate with HCF-1 and then be recruited to E2F1-responsive promoters to stimulate H3K4 trimethylation and transcriptional activation, thereby facilitating the cell cycle G1 to S phase transition.