X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway.

BACKGROUND: Diabetic foot ulcers (DFU), as severe complications of diabetes mellitus (DM), significantly compromise patient health and carry risks of amputation and mortality. AIM: To offer new insights into the occurrence and development of DFU, focusing on the therapeutic mechanisms of X-Paste (XP) of wound healing in diabetic mice. METHODS: Employing traditional Chinese medicine ointment preparation methods, XP combines various medicinal ingredients. High-performance liquid chromatography (HPLC) identified XP's main components. Using streptozotocin (STZ)-induced diabetic, we aimed to investigate whether XP participated in the process of diabetic wound healing. RNA-sequencing analyzed gene expression differences between XP-treated and control groups. Molecular docking clarified XP's treatment mechanisms for diabetic wound healing. Human umbilical vein endothelial cells (HUVECs) were used to investigate the effects of Andrographolide (Andro) on cell viability, reactive oxygen species generation, apoptosis, proliferation, and metastasis in vitro following exposure to high glucose (HG), while NF-E2-related factor-2 (Nrf2) knockdown elucidated Andro's molecular mechanisms. RESULTS: XP notably enhanced wound healing in mice, expediting the healing process. RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment. HPLC identified 21 primary XP components, with Andro exhibiting strong Nrf2 binding. Andro mitigated HG-induced HUVECs proliferation, metastasis, angiogenic injury, and inflammation inhibition. Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation, with Nrf2 knockdown reducing Andro's proliferative and endothelial protective effects. CONCLUSION: XP significantly promotes wound healing in STZ-induced diabetic models. As XP's key component, Andro activates the Nrf2/HO-1 signaling pathway, enhancing cell proliferation, tubule formation, and inflammation reduction.

Post-translational modifications confer amphotericin B resistance in Candida krusei isolated from a neutropenic patient.

Neutropenia is a common complication in the treatment of hematological diseases and the most common predisposing factor for invasion by fungi, such as Candida krusei. Recent studies have shown that C. krusei, a life-threatening pathogen, has developed resistance to amphotericin B (AMB). However, the mechanisms that led to the rapid emergence of this AMB-resistant phenotype are unclear. In this study, we found the sensitivity for AMB could be promoted by inhibiting histone acyltransferase activity and western blot analysis revealed differences in the succinylation levels of C. krusei isolated from immunocompromised patients and of the corresponding AMB-resistant mutant. By comparative succinyl-proteome analysis, we identified a total of 383 differentially expressed succinylated sites in with 344 sites in 134 proteins being upregulated in the AMB-resistant mutant, compared to 39 sites in 23 proteins in the wild-type strain. These differentially succinylated proteins were concentrated in the ribosome and cell wall. The critical pathways associated with these proteins included those involved in glycolysis, gluconeogenesis, the ribosome, and fructose and mannose metabolism. In particular, AMB resistance was found to be associated with enhanced ergosterol synthesis and aberrant amino acid and glucose metabolism. Analysis of whole-cell proteomes, confirmed by parallel reaction monitoring, showed that the key enzyme facilitating lysine acylation was significantly upregulated in the AMB-resistant strain. Our results suggest that lysine succinylation may play an indispensable role in the development of AMB resistance in C. krusei. Our study provides mechanistic insights into the development of drug resistance in fungi and can aid in efforts to stifle the emergence of AMB-resistant pathogenic fungi.

Genomic analysis of a new type VI secretion system in Vibrio parahaemolyticus and its implications for environmental adaptation in shrimp ponds.

The type VI secretion system (T6SS) in Vibrio spp. is often used to kill heteroclonal neighbors by direct injection of toxic effectors, but its strategies in aquacultural environments receive limited attention. In this study, we conducted genomic analysis for a T6SS-harboring plasmid in V. parahaemolyticus strain VP157. Coculture assays were further conducted to verify its antibacterial function. The results showed that strain VP157 harbored a 132-kb plasmid, pVP157-1, which consists of two fragments: an 87.8-kb fragment identical to plasmid pTJ114-1 and a 44.2-kb T6SS gene cluster with only 4% DNA identity to T6SS1 in the V. parahaemolyticus reference genome. Gene-by-gene analysis of six genes representing core T6SS components suggested that each gene has distinct evolutionary origins. In vitro experimental evolution revealed that pVP157-1 can excise from the VP157 genome with an excision rate of 4%. A coculture assay suggested that strain VP157 had significantly higher antibacterial activity against Bacillus pumilus and V. cholerae than the strain without pVP157-1(VP157∆T6SS). In contrast, a rapid decline was observed for the proportion of VP157∆ T6SS in a mock microbial community, which decreased from 10.7% to 2.1% in 5 days. The results highlighted that the acquisition of T6SS fostered the fitness of V . parahaemolyticus in a complex environment.

Lung and gut microbiomes in pulmonary aspergillosis: Exploring adjunctive therapies to combat the disease.

Species within the Aspergillus spp. cause a wide range of infections in humans, including invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis, and are associated with high mortality rates. The incidence of pulmonary aspergillosis (PA) is on the rise, and the emergence of triazole-resistant Aspergillus spp. isolates, especially Aspergillus fumigatus, limits the efficacy of mold-active triazoles. Therefore, host-directed and novel adjunctive therapies are required to more effectively combat PA. In this review, we focus on PA from a microbiome perspective. We provide a general overview of the effects of the lung and gut microbiomes on the growth of Aspergillus spp. and host immunity. We highlight the potential of the microbiome as a therapeutic target for PA.

Diverse and unique viruses discovered in the surface water of the East China Sea.

BACKGROUND: Viruses are the most abundant biological entities on earth and play import roles in marine biogeochemical cycles. Here, viral communities in the surface water of the East China Sea (ECS) were collected from three representative regions of Yangshan Harbor (YSH), Gouqi Island (GQI), and the Yangtze River Estuary (YRE) and explored primarily through epifluorescence microscopy (EM), transmission electron microscopy (TEM), and metagenomics analysis. RESULTS: The virus-like particles (VLPs) in the surface water of the ECS were measured to be 106 to 107 VLPs/ml. Most of the isolated viral particles possessed a head-and-tail structure, but VLPs with unique morphotypes that had never before been observed in the realm of viruses were also found. The sequences related to known viruses in GenBank accounted for 21.1-22.8% of the viromic datasets from YSH, GQI, and YRE. In total, 1029 viral species were identified in the surface waters of the ECS. Among them, tailed phages turn out to make up the majority of viral communities, however a small number of Phycodnaviridae or Mimiviridae related sequences were also detected. The diversity of viruses did not appear to be a big difference among these three aquatic environments but their relative abundance was geographically variable. For example, the Pelagibacter phage HTVC010P accounted for 50.4% of the identified viral species in GQI, but only 9.1% in YSH and 11.7% in YRE. Sequences, almost identical to those of uncultured marine thaumarchaeal dsDNA viruses and magroviruses that infect Marine Group II Euryarchaeota, were confidently detected in the ECS viromes. The predominant classes of virome ORFs with functional annotations that were found were those involved in viral biogenesis. Virus-host connections, inferred from CRISPR spacer-protospacer mapping, implied newly discovered infection relationships in response to arms race between them. CONCLUSIONS: Together, both identified viruses and unknown viral assemblages observed in this study were indicative of the complex viral community composition found in the ECS. This finding fills a major gap in the dark world of oceanic viruses of China and additionally contributes to the better understanding of global marine viral diversity, composition, and distribution.

Lactobacillus kefiranofaciens, the sole dominant and stable bacterial species, exhibits distinct morphotypes upon colonization in Tibetan kefir grains.

Tibetan kefir grains (TKGs), natural starters for milk fermentation, are believed to comprise diverse microflora of lactic acid and acetic acid bacteria. In order to better understand the bacterial community in TKGs, TKGs that had been cultured continuously either naturally or aseptically for 10 months were subject to analysis using both culture-dependent and various culture-independent methods. Results of DGGE, metagenomics, FISH, qPCR and isolation all demonstrated that Lactobacillus kefiranofaciens is the only dominant and stable bacterial species in TKGs regardless of culture conditions and time. FISH and SEM showed that L. kefiranofaciens exhibited two distinct morphotypes of short rod (3.0 µm in length) and long rod (10.0 µm in length) upon colonization of either the outer surface or inner component of TKGs, providing evidence for its trophic adaptation to the hollow globular grain structure of TKGs. These findings pave ways for further study of the specific symbiotic interaction between L. kefiranofaciens and the dominant Saccharomyces cerevisiae yeast in TKGs in vivo.

A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei.

The causative agent of shrimp AHPND was identified as specific Vibrio parahaemolyticus strains, which harbor a virulent plasmid that contains the toxic genes pirA and B (pirAB). Herein, a Vibrio bacterium was isolated from shrimp in Shanghai. This bacterium was identified as Vibrio owensii using 16S rRNA gene phylogeny, whole genome sequencing and comparative analysis. The V. owensii cells are rod-shape (1.86 ±â€¯0.15 µm) with a single polar flagellum (4 µm). In addition, V. owensii form mauve colonies with jagged edges on CHROMagar plates. The pirAB genes on the plasmid revealed 100% sequence similarity to that of AHPND V. parahaemolyticus, and the encoded proteins were detected in the culture media. Subculture of V. owensii showed that the pirAB genes are unstable, and their loss rate is approximately 22% and reaches a dynamic equilibrium after the fifth generation. Upon immersion bioassay, the cumulative mortality of V. owensii (pirAB+)-infected shrimp was up to 100% within 4 days, and typical AHPND clinical signs were observed. Approximately 105 CFU/hepatopancreas of V. owensii cells were observed in the pirAB+-infected shrimp based on both culture-dependent and -independent assay. Our results indicate that the expression of pirAB in the V. owensii strain is responsible for AHPND.

Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species.

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease caused by pirAB toxins encoded by a plasmid found in Vibrio parahaemolyticus. The pirAB toxins are the homologs of the Photorhabdus insect-related (Pir) toxins. Here, we report the complete sequences of the AHPND-causing plasmid isolated from V. owensii, as well as those of its 11 siblings (pVH family). In addition, we also included 13 related plasmids (pVH-r family) without the pirAB genes isolated from a variety of species within the Vibrio Harveyi clade. Furthermore, the pirAB-Tn903 composite transposon was identified in pVH, and both ends of the transposon appeared to have inserted simultaneously into the ancestor plasmid at different sites. The homologue counterparts of pirAB were also detected in a non-pVH plasmid in V. campbellii. Taken together, our results provide novel insights into the acquisition and evolution of pirAB as well as related plasmids in the Vibrio Harveyi clade.

Characterization and prevalence of a novel white spot syndrome viral genotype in naturally infected wild crayfish, Procambarus clarkii, in Shanghai, China.

White spot syndrome virus (WSSV) infection is commonly detected by vp28-qPCR assay in wild crayfish, Procambarus clarkii, a widespread crustacean species in the aquatic environment in China. The virions of crayfish WSSV have been isolated and purified. Based on TEM observation, they exhibited morphological structures that are identical to known WSSV. In addition, the WSSV major envelope protein VP28 was observed based on Western blot analysis of the total structural proteins of crayfish WSSV. PCR amplification and sequencing analyses of variable regions of ORF14/15, ORF23/24 and ORF94, along with viral genomic sequencing and phylogenomic analysis, indicated that the crayfish WSSV, named WSSV-CN-Pc, represents a new WSSV genotype. Intramuscular injection bioassay revealed that WSSV-CN-Pc was as virulent as the WSSV Taiwan strain. The WSSV-CN-Pc exhibited characteristics of a dominant genotype, with high infection load (107-108 WSSV/mg) and high prevalence (91.7%, 110 of 120 crayfish samples) observed in the surveyed wild crayfish. WSSV-CN-Pc was also detected, with similar infection pattern as observed in crayfish, in farmed Litopenaeus vannamei shrimp that shared similar ecological niches with the sampled crayfish. Our results indicated that there was horizontal transmission of WSSV-CN-Pc between crayfish and shrimp in nature. Our findings also implicated that crayfish and shrimp farming should be integrated and managed with cautions in order to reduce the risk of spread and circulation of WSSV in the aquatic environment.

Novel Virophages Discovered in a Freshwater Lake in China.

Virophages are small double-stranded DNA viruses that are parasites of giant DNA viruses that infect unicellular eukaryotes. Here we identify a novel group of virophages, named Dishui Lake virophages (DSLVs) that were discovered in Dishui Lake (DSL): an artificial freshwater lake in Shanghai, China. Based on PCR and metagenomic analysis, the complete genome of DSLV1 was found to be circular and 28,788 base pairs in length, with a G+C content 43.2%, and 28 predicted open reading frames (ORFs). Fifteen of the DSLV1 ORFs have sequence similarity to known virophages. Two DSLV1 ORFs exhibited sequence similarity to that of prasinoviruses (Phycodnaviridae) and chloroviruses (Phycodnaviridae), respectively, suggesting horizontal gene transfer occurred between these large algal DNA viruses and DSLV1. 46 other virophages-related contigs were also obtained, including six homologous major capsid protein (MCP) gene. Phylogenetic analysis of these MCPs showed that DSLVs are closely related to OLV (Organic Lake virophage) and YSLVs (Yellowstone Lake virophages), especially to YSLV3, except for YSLV7. These results indicate that freshwater ecotopes are the hotbed for discovering novel virophages as well as understanding their diversity and properties.

Draft Genome Sequence of Vibrio owensii Strain SH-14, Which Causes Shrimp Acute Hepatopancreatic Necrosis Disease.

We sequenced Vibrio owensii strain SH-14, which causes serious acute hepatopancreatic necrosis disease (AHPND) in shrimp. Sequence analysis showed a large extrachromosomal plasmid, which encoded pir toxin genes and shared highly sequence similarity with the one observed in AHPND-causing Vibrio parahaemolyticus strains. The results suggest that this plasmid appears to play an important role in shrimp AHPND.

Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

Fine structure of Tibetan kefir grains and their yeast distribution, diversity, and shift.

Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species--Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S. cerevisiae and Lactobacillus bacteria in TKGs.

Efficient purification and concentration of viruses from a large body of high turbidity seawater.

Marine viruses are the most abundant entities in the ocean and play crucial roles in the marine ecological system. However, understanding of viral diversity on large scale depends on efficient and reliable viral purification and concentration techniques. Here, we report on developing an efficient method to purify and concentrate viruses from large body of high turbidity seawater. The developed method characterizes with high viral recovery efficiency, high concentration factor, high viral particle densities and high-throughput, and is reliable for viral concentration from high turbidity seawater. Recovered viral particles were used directly for subsequent analysis by epifluorescence microscopy, transmission electron microscopy and metagenomic sequencing. Three points are essential for this method:•The sampled seawater (>150 L) was initially divided into two parts, water fraction and settled matter fraction, after natural sedimentation.•Both viruses in the water fraction concentrated by tangential flow filtration (TFF) and viruses isolated from the settled matter fraction were considered as the whole viral community in high turbidity seawater.•The viral concentrates were re-concentrated by using centrifugal filter device in order to obtain high density of viral particles.

Diversity of virophages in metagenomic data sets.

Virophages, e.g., Sputnik, Mavirus, and Organic Lake virophage (OLV), are unusual parasites of giant double-stranded DNA (dsDNA) viruses, yet little is known about their diversity. Here, we describe the global distribution, abundance, and genetic diversity of virophages based on analyzing and mapping comprehensive metagenomic databases. The results reveal a distinct abundance and worldwide distribution of virophages, involving almost all geographical zones and a variety of unique environments. These environments ranged from deep ocean to inland, iced to hydrothermal lakes, and human gut- to animal-associated habitats. Four complete virophage genomic sequences (Yellowstone Lake virophages [YSLVs]) were obtained, as was one nearly complete sequence (Ace Lake Mavirus [ALM]). The genomes obtained were 27,849 bp long with 26 predicted open reading frames (ORFs) (YSLV1), 23,184 bp with 21 ORFs (YSLV2), 27,050 bp with 23 ORFs (YSLV3), 28,306 bp with 34 ORFs (YSLV4), and 17,767 bp with 22 ORFs (ALM). The homologous counterparts of five genes, including putative FtsK-HerA family DNA packaging ATPase and genes encoding DNA helicase/primase, cysteine protease, major capsid protein (MCP), and minor capsid protein (mCP), were present in all virophages studied thus far. They also shared a conserved gene cluster comprising the two core genes of MCP and mCP. Comparative genomic and phylogenetic analyses showed that YSLVs, having a closer relationship to each other than to the other virophages, were more closely related to OLV than to Sputnik but distantly related to Mavirus and ALM. These findings indicate that virophages appear to be widespread and genetically diverse, with at least 3 major lineages.