Dynamic tear meniscus parameters in complete blinking: insights into dry eye assessment.

AIM: To investigate the relationship between dynamic tear meniscus parameters and dry eye using an automated tear meniscus segmentation method. METHODS: The analysis of tear meniscus videos captured within 5s after a complete blink includes data from 38 participates. By processing video data, several key parameters including the average height of the tear meniscus at different lengths, the curvature of the tear meniscus's upper boundary, and the total area of the tear meniscus in each frame were calculated. The effective values of these dynamic parameters were then linearly fitted to explore the relationship between their changing trends and dry eye disease. RESULTS: In 94.74% of the samples, the average height of central tear meniscus increased over time. Moreover, 97.37% of the samples exhibited an increase in the overall tear meniscus height (TMH) and area from the nasal to temporal side. Notably, the central TMH increased at a faster rate compared to the nasal side with the temporal side showing the slowest ascent. Statistical analysis indicates that the upper boundary curvature of the whole tear meniscus as well as the tear meniscus of the nasal side (2, 3, and 4 mm) aid in identifying the presence of dry eye and assessing its severity. CONCLUSION: This study contributes to the understanding of tear meniscus dynamics as potential markers for dry eye, utilizing an automated and non-invasive approach that has implications for clinical assessment.

Antibacterial activity and mechanism of action of bacteriocin LFX01 against Staphylococcus aureus and Escherichia coli and its application on pork model.

Antibacterial activity and mechanism of action of bacteriocins against bacteria that cause pork contamination remain unclear. Here, antibacterial activity of bacteriocin LFX01 against two important indicator strains (i.e., Staphylococcus aureus and Escherichia coli) and its mechanism of action were investigated. The results showed antibacterial activity of LFX01 against growth and biofilm formation of S. aureus_26 (strain 2612:1606BL1486) and E. coli_02 (strain CMCC(B)44102). Additionally, the results demonstrated that LFX01 could decrease cell metabolic activity, disrupt cell membrane permeability and integrity, and trigger leakage of intracellular contents (e.g., K+, ATP, and lactic dehydrogenase). Furthermore, gel retardation showed that LFX01 could bind to the genomic DNA of indicator strains, disrupting DNA structure. These results uncovered mechanism of action of LFX01 against indicator strains from physiological and phenotypic levels. When applied to the surface of fresh pork models, the antibacterial activity of LFX01 against indicator strains was further confirmed. These findings suggested that LFX01 could be a potential pork preservative for controlling foodborne pathogens.

Bacteriophage Lytic Enzyme P9ly as an Alternative Antibacterial Agent Against Antibiotic-Resistant Shigella dysenteriae and Staphylococcus aureus.

Developing new strategies to replace or supplement antibiotics to combat bacterial infection is a pressing task in the field of microbiological research. In this study, we report a lytic enzyme named P9ly deriving from the bacteriophage PSD9 that could infect multidrug-resistant Shigella. This enzyme was identified through whole-genome sequencing of PSD9. The results show that P9ly contains a conserved T4-like_lys domain and belongs to the phage lysozyme family. Recombinant P9ly obtained from protein purification presented biological activity and could digest bacterial cell walls (CW), resulting in the destruction of cell structure and leakage of intracellular components. Furthermore, P9ly exhibited bacteriolytic and bactericidal activity on different strains, especially multidrug-resistant Gram-negative Shigella dysenteriae and Gram-positive Staphylococcus aureus. Additionally, combined use of P9ly with ceftriaxone sodium (CRO) could decrease necessary dose of the antibiotic used and improve the antibacterial effect. In summary, under the current backdrop of extensive antibiotic usage and the continuous emergence of bacterial resistance, this study provides an insight into developing bacteriophage-based antibacterial agents against both Gram-negative and Gram-positive pathogens.

Antibacterial effect of bacteriocin XJS01 and its application as antibiofilm agents to treat multidrug-resistant Staphylococcus aureus infection.

Multidrug-resistant (MDR) Staphylococcus aureus biofilms have emerged as a serious threat to human health. Recently, the development of antibiotic replacement therapy has gained much attention due to the potential application of bacteriocin. The present study sought to evaluate the antibacterial effect of bacteriocin XJS01 against MDR S. aureus, a previously reported bacteriocin against S. aureus strain 2612:1606BL1486 (S. aureus_26, an MDR strain demonstrated here), and its potential application as an antibiofilm agent. The minimum bactericide concentration of XJS01 against MDR S. aureus_26 was 33.18 µg/mL. XJS01 exhibited excellent storage stability and resistance against acid and reduced the density of established MDR S. aureus_26 biofilm. The hemolytic and HEK293T cytotoxicity activities of XJS01 and the histological analyses in mice confirmed its safety. Moreover, XJS01 effectively disrupted the MDR S. aureus_26 biofilm established on the skin wound surface and reduced the biofilm-isolated bacteria, thereby decreasing the release of pro-inflammatory cytokines and the proliferation of alternatively activated macrophages. Compared to mupirocin, XJS01 exhibited an excellent therapeutic effect on mice skin wounds, confirming it to be a potential alternative to antibiotics.

[Effects of Bupleurum chinense on the diversity of intestinal flora in depressed mice].

Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.

A Novel Bacteriocin Against Shigella flexneri From Lactiplantibacillus plantarum Isolated From Tilapia Intestine: Purification, Antibacterial Properties and Antibiofilm Activity.

Few bacteriocins with antibacterial activity against Shigella flexneri have been reported. Here, a novel bacteriocin (LFX01) produced by Lactiplantibacillus plantarum strain LF-8 from the intestine of tilapia was purified and extensively characterized. LFX01 possesses a molecular weight of 1049.56 Da and an amino acid sequence of I-T-G-G-P-A-V-V-H-Q-A. LFX01 significantly inhibited S. flexneri strain 14 (S. flexneri_14) growth. Moreover, it exhibited excellent stability under heat and acid-base stress, and presented sensitivity to a variety of proteases, such as proteinase K, pepsin, and trypsin. The minimum inhibitory concentration (MIC) of LFX01 against S. flexneri_14 was 12.65 µg/mL, which was smaller than that of most of the previously found bacteriocins. Furthermore, LFX01 significantly inhibited (p < 0.05) S. flexneri_14 cells and decreased their cell viability. In addition, LFX01 could significantly (p < 0.05) inhibit biofilm formation of S. flexneri_14. Scanning electron microscopy analysis presented that the cell membrane permeability of S. flexneri_14 was demolished by LFX01, leading to cytoplasmic contents leakage and cell rupture death. In summary, a novel bacteriocin of lactic acid bacteria (LAB) was found, which could effectively control S. flexneri in both planktonic and biofilm states.

Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions.

Aquatic fireflies are important indicators of the quality of freshwater environments and key models for research on insect adaptation to freshwater environments. For these investigations, gene expression analyses using quantitative real-time PCR are heavily dependent on reliable reference genes. In this study, based on a transcriptome assembly and annotation for the aquatic firefly Aquatica leii at the adult and larval stages, 10 candidate reference genes (α-tubulin, ß-tubulin, ß-actin, EF1A, SDHA, UBQ, GST, GAPDH, RPS31, and RPL13A) were identified for analyses of expression stability. Quantitative real-time PCR analyses for each candidate reference genes in A. leii was conducted for four developmental stages, four adult tissue types, two adult sexes, and two ecological stressors [adults exposed to five temperatures and larvae exposed to four concentrations of benzo(a)pyrene]. Results were evaluated by three independent algorithms (geNorm, NormFinder, and BestKeeper) and one comparative algorithm (RefFinder). The expression stability of candidate reference genes in A. leii differed under various conditions. Reference genes with the most stable expressions levels in different tissues, temperatures, sexes, developmental stages, and concentrations of benzo(a)pyrene were α-tubulin, GST, ß-actin, ß-tubulin, and α-tubulin, respectively. Furthermore, the optimal normalization factors (NFs) for the quantification of the expression levels of target genes by quantitative real-time PCR analyses of A. leii were identified for each experimental group. In particular, NF = 2 for different tissues (α-tubulin + ß-tubulin), different sexes (ß-actin + EF1A), and larvae exposed to different concentrations of benzo(a)pyrene (α-tubulin + EF1A); NF = 3 for developmental stages (GST + GAPDH + SDHA) and adults exposed to different temperatures (ß-tubulin + EFA + GST). In addition, we surveyed the expression profiles of two target genes (CYP3A and CSP8) in larvae exposed to different concentrations of benzo(a)pyrene and in different adult tissues. The results further validated the reliability of the reference genes. The optimal reference genes for various experimental conditions identified in these analyses provide a useful tool for ecological studies of aquatic fireflies.

The impact of mercury on the genome-wide transcription profile of zebrafish intestine.

Mercury is a widely used heavy metal that causes pollution to aquatic environments and severely affects the health of fish. Little is known about how heavy metal pollutants affect fish, particularly for gene expression within important organs such as the intestine. Herein, whole transcriptome sequencing was performed on zebrafish (Danio rerio) intestine tissue after HgCl2 (HGC, 30 µg/L) exposure. A total of 2,257 differentially expressed genes (DEGs) were identified, including 1,788 up- and 469 down-regulated genes. Functional enrichment analysis revealed that these DEGs were primarily related to xenobiotic biodegradation, biomacromolecule metabolism, development, oxidative defense, and immune response. Ten key HGC-responsive DEGs were screened to survey the dynamic changes of expression in response to HGC exposure at different time points, and were also used to validate RNA sequencing data using quantitative real-time PCR (qPCR). Results indicate that the expression of genes encoding UGT1AB, GSTT1B, GSTO1, GSTM2, UGT5G1, GSTT1A, GSTR, GSTM3, GSTA1, and GSTP2 were significantly upregulated in response to the HGC exposure, and potentially help to counteract the adverse effects of HGC. This study provides insight into fish molecular toxicological responses to heavy metals and method on environmental risk assessment.

Colonization of the gut microbiota of honey bee (Apis mellifera) workers at different developmental stages.

The role of the gut microbiome in animal health has become increasingly evident. Although the structure of the gut microbiome of A. mellifera is well known, little is known about the dynamic change across different developmental stages. In this study, we explored the dynamic changes of the gut microbiota of A. mellifera at different developmental stages covering the whole life cycle using high-throughput 16S rRNA gene sequencing. The results indicated that the core (shared) gut microbiota changes significantly among different developmental stages. The diversity of the bacterial community in workers among different ages was significantly different. In addition, by comparing the core gut microbiota among different-aged workers, we found that newly emerged workers had fewer core microbiota. Three genera, Gilliamella, Frischella, and Snodgrassella, were significantly colonized at 1 day poste mergence (dpe); Lactobacillus, Bifidobacterium, Commensalibacter were significantly colonized at 3 dpe and significantly reduced with Gilliamella. Lactobacillus kunkeei and Bartonella were significantly colonized at 12 dpe and were significantly decreased with Lactobacillus helsingborgensis. Commensalibacter and Bifidobacterium were significantly decreased at 25 dpe, and Bacteroides, Escherichia-Shigella, and Porphyromonadaceae were significantly decreased between 19 and 25 dpe. Our results reveal the succession of the gut microbiota of workers from birth to senescence, which provides a theoretical basis for further exploring the roles of gut microbiota during different developmental stages.

The Response of Microbiota Community to Streptococcus agalactiae Infection in Zebrafish Intestine.

Recently, Streptococcus agalactiae has become a major pathogen leading to Streptococcosis. To understand the physiological responses of zebrafish (Danio rerio) to S. agalactiae, the intestinal microbiota composition of the intestine (12 and 24 h post-infection, hpi, respectively) in zebrafish infected with S. agalactiae were investigated. The intestinal bacterial composition was analyzed using PacBio high-throughput full-length 16S rRNA gene sequencing. The most predominant bacteria in the zebrafish intestine were the Fusobacteria phylum and Sphingomonas genus. S. agalactiae infection affected the composition of partially intestinal microbiota. At the species level, the relative abundance of the pathogenic intestinal bacteria Aeromonas veronii, S. agalactiae, and Clostridium tarantellae significantly increased after S. agalactiae infection (p < 0.05), while that of the beneficial intestinal bacteria Bacillus licheniformis, Comamonas koreensis, and Romboutsia ilealis significantly decreased (p < 0.05), showing that S. agalactiae infection aggravates the zebrafish disease through promoting abundance of other intestinal pathogenic bacteria. This study is the first PacBio analyses of the zebrafish intestinal microbiota community under pathogenic infection. Results suggest that the S. agalactiae infection alters the intestinal flora structure in zebrafish.

Comparative transcriptomic analysis provides insights into the response to the benzo(a)pyrene stress in aquatic firefly (Luciola leii).

Many studies have reported that behavior and bioluminescence of fireflies could be affected by changes in environment conditions. However, little is known about how the deterioration of the aquatic environment affects aquatic fireflies, particularly with respect to molecular responses following exposure to water pollutants, such as benzo(a)pyrene (BaP), which is a key indicator in environmental risk assessment because of the hazards it poses. Here, whole transcriptome sequencing and gene expression analysis were performed on freshwater fireflies (Luciola leii) exposed to BaP (concentration of 0.01 mg/L). Four transcriptomic libraries were constructed for the control and treatment groups, including two biological replicates. From the mixed pools (each pool contains 60 individuals from three time points), a total of 54,282 unigenes were assembled. Furthermore, 329,337 of Single-nucleotide Polymorphisms (SNPs) and 1324 of Simple Sequence Repeats (SSRs) were predicted using bioinformatics, which is useful for the future development of molecular markers. Subsequently, 2414 differently expressed genes (DEGs) were identified in response to BaP stress in comparison to the control, including 1350 up-regulated and 1064 down-regulated DEGs. Functional enrichment showed that these DEGs are primarily related to innate immunity; xenobiotic biodegradation and response, biomacromolecule metabolism, biosynthesis, and absorption. Eight key BaP-responsive DEGs were screened to survey the dynamic changes of expression in response to BaP stress at different time points, and to validate the RNA sequencing data using quantitative real-time PCR. The results indicate that the expression of genes encoding UGT, CYP3A, CYP9, CYP6AS5 and ADHP were induced, while those encoding UGT2B10L, PTGDS, and ALDH were reduced, to participate in response to the BaP exposure and potentially help counteract the adverse effects of BaP. This investigation provides insight into the toxicological response of fireflies to the occurrence of water deterioration.

MicroRNA profile of immune response in gills of zebrafish (Danio rerio) upon Staphylococcus aureus infection.

The gills of fish are large mucosal surfaces that are very important portals for pathogen entry. Investigations have shown that microRNAs (miRNAs) are key regulators of immune response to bacterial infections in the gills of fish; however, how miRNA expression changes in response to infection by Gram-positive bacteria remains largely unknown. To further investigate the immunological role of miRNAs in fish gills under pathogen stress induced by Gram-positive bacterial infection, this study investigated Staphylococcus aureus (SA)-induced changes in the miRNAs levels in gills of adult zebrafish (Danio rerio). miRNA microarrays were used to analyze expression profiles of known miRNA in the gills of zebrafish in response to SA infection and compared these to uninfected control fish. A total of 30 differentially expressed miRNAs (DEMs) were identified. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses were performed. The results indicated that DEM targets were primarily involved in innate immune processes, apoptosis, defense responses, and antibacterial responses. Pathways involving bacterial infection, innate immunity, metabolic process, disease, and apoptosis were mediated by DEMs. Furthermore, real-time quantitative PCR experiments for nine key SA-responsive DEMs that regulated the "SA infection" pathway validated the accuracy of microarray results. Dynamic variations in gene expression were surveyed in detail for these key SA-responsive DEMs for PBS control and at 6, 12, 24, and 48 h after SA challenge in detail. This study provides novel insight into the mechanisms underlying the miRNA regulation during the SA-induced immune response in zebrafish gills, and provides basic knowledge on the innate immune response against Gram-positive bacterial infection in bony fish.

Genome-wide transcriptional response of microRNAs to the benzo(a)pyrene stress in amphioxus Branchiostoma belcheri.

Amphioxus, a cephalochordate found in sand habitats in shallow in-shore seawaters, has been widely used as a model in comparative immunology of chordates. However, the role of microRNAs (miRNAs) in amphioxus under abiotic stress, particularly xenobiotics with strong toxicity, remains largely unknown. Here, a widespread marine contaminant, benzo(a)pyrene (BaP) is used to evaluate its toxic effects on miRNA expression of amphioxus. Six small RNA libraries were sequenced from Branchiostoma belcheri. A total of 144 known and 157 novel miRNAs were identified using deep sequencing and bioinformatics approaches. A total of 58 differentially expressed miRNAs (DEMs) were screened, including 25 up- and 33 down-regulated DEMs under BaP stress. Target genes possibly regulated by DEMs were predicted, and their functional enrichment analyses were performed. Targets of DEMs are primarily involved in xenobiotic and cellular homeostasis, catabolic and transport process. They could be largely linked to nine immune- and toxin detoxification-related pathways, including metabolism of xenobiotics by cytochrome P450, drug metabolism-other enzymes, and drug metabolism-cytochrome P450, etc. Furthermore, quantitative real-time PCR (qRT-PCR) analysis for 12 key BaP-responsive DEMs validates the accuracy of deep sequencing. Experiments were then conducted to investigate their expression responses to BaP stress at different time intervals in detail to further determine their expression dynamic in responses of B. belcheri towards BaP exposure. This study, to the best of our knowledge, investigates the regulatory roles of miRNAs in the toxicological response of amphioxus for the first time, providing valuable information for the protection of lone existing cephalochordate amphioxus.

Genome-wide gene expression analysis reveals novel insights into the response to nitrite stress in gills of Branchiostoma belcheri.

Amphioxus has been widely used as a model for the comparative immunology of vertebrates. Studies have reported that gene expression changes in the amphioxus gill in response to biotic stress, such as microbial and their mimic challenge, but little is known about how gene expression is affected by abiotic stress in the marine environment, such as nitrite. A lack of information regarding gene expression response to abiotic stress hinders a comprehensive understanding of gill defense response in amphioxus. Here, RNA sequencing was used to carry out gene expression profiling analyses of Branchiostoma belcheri gills under nitrite stress. Six libraries were created for the control and treatment groups, including three biological replicates. In total, 2416 differently expressed genes (DEGs) were detected in response to nitrite stress, of which 1522 DEGs were up-regulated in the treatment group in comparison to the control, while the remaining 894 DEGs were down-regulated genes. Functional enrichment revealed that these DEGs are primarily involved in disease, innate immunity, xenobiotic biodegradation and metabolism, and biomolecular processes and apoptosis. We screened 11 key nitrite-responsive DEGs to detect their expression responses to nitrite stress at different time points, and validate the sequencing data using real time quantitative PCR. The results indicated that the expression of gene encoding CYP3A, POD, CASPR1, GST, MAO, DDH, and XDH/XO were induced, while those encoding MRC, GT, DNASE1L, and RIPK5 were reduced, to participate in the anti-nitrite response. This study provides a useful resource for research of molecular toxicology in amphioxus under environmental stress.

Identification of circular RNAs and their altered expression under poly(I:C) challenge in key antiviral immune pathways in amphioxus.

Amphioxus is a key model for studying comparative immunity of vertebrates. Circular RNA (circRNA), as RNAs with a circular structure, has received little attention until recently, where several studies have reported that circRNA expression changes are involved in the immune response in animals. However, circRNA and its immune role in amphioxus have not been previously studied. Here, circRNAs in Chinese amphioxus (Branchiostoma belcheri) were sequenced, and 1859 circRNAs were identified using two algorithms (find_circ and CIRI). The analysis of miRNA target sites on circRNAs showed that 332 circRNAs may function as miRNA sponges. Furthermore, we identified circRNAs that were conserved between B. belcheri and vertebrates, tracing the origin of these circRNAs within chordates. Additionally, in combination with several key antiviral immune (poly(I:C), pIC) pathways identified in our previous B. belcheri studies, nine circRNAs potentially involved in these pathways were identified using bioinformatic predictions. Among these nine circRNAs, eight were selected to examine their expression response in B. belcheri challenged by pIC in comparison to control using real-time quantitative PCR. The results showed that four circRNAs were induced as part of the antiviral response against pIC, while expression of two circRNAs was decreased, and the expression levels of the remaining two were not significantly altered after pIC challenge. This work is the first to identify circRNAs and reveal their antiviral role in amphioxus. Therefore, it opens a new window to explore the comparative immunology of circRNAs in chordates and the regulatory roles of circRNAs in antiviral immunity in amphioxus.

Characterization of ladybird Henosepilachna vigintioctopunctata transcriptomes across various life stages.

Henosepilachna vigintioctopunctata is a vegetable pest that has spread worldwide. It belongs to the Coccinellidae family, whose members exhibit remarkable diversity, both in terms of their diets and the colored spots that appear on the elytra in the adult stage. Transcriptomic data from H. vigintioctopunctata at different life stages would be useful for further investigating the genetic basis of this dietary diversity and the formation of the colored spots in ladybird beetles, as well as revealing the population dynamics of H. vigintioctopunctata, which could be useful in pest control. Here, we generated a comprehensive RNA-seq data set (a total of ~24 Gb of clean data) for H. vigintioctopunctata by sequencing samples collected at different life stages. We characterized the transcriptomes of each of the four life stages (egg, larva, pupa, adult) and generated a high-coverage pool by combining all the RNA-seq reads. Furthermore, we identified a catalog of simple sequence repeat (SSR) markers. This represents the first study to collect transcriptome data from all life stages of a ladybird beetle.

Comparative transcriptomic analysis provides insights into antibacterial mechanisms of Branchiostoma belcheri under Vibrio parahaemolyticus infection.

Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus (Branchiostoma belcheri) infected with Vibrio parahaemolyticus (V. p.) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p.-infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p.-infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p. TREATMENT: Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri. This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus.

A Phylogenomic Framework and Divergence History of Cephalochordata Amphioxus.

Amphioxus, or cephalochordates, are often used as the living invertebrate proxy of vertebrate ancestors and are widely used as evolutionary biology models of chordates. However, their phylogeny, divergence history, and speciation characteristics remain poorly understood, and phylogenomic studies to explore these problems lacking entirely from the literature. Here, we determined a new transcriptome of Branchiostoma japonicum. Combined with mass sequences of all other 18 species, a 19-way phylogeny was constructed via multiple methods (ML, BI, PhyloBayes, and ASTRAL), consistently supporting a phylogeny of [(B. belcheri + B. japonicum) + (B. lanceolatum + B. floridae) + Asymmetron lucayanum] in amphioxus. Congruent phylogenetic signals were found across mitochondrial genes, 12S RNA, and complete mitochondrial genomes according to previous reports, indicating that 12S RNA may have potential as a molecular marker for phylogenetic analysis in amphioxus. Molecular dating analysis indicated a radiation of the cephalochordates during the Cretaceous (∼104-61 million years ago), supporting an association between the diversification and speciation of cephalochordates with continental drift and associated changes in their respective habitats during this time. The identified functional enrichment analysis for species-specific domains indicated that their function mainly involves immune response, apoptosis, and lipid metabolism and utilization, signaling that pathogens and changes of energy requirements are an important driving force for amphioxus speciation. This study represents the first large-scale phylogenomic analysis of most major amphioxus genera based on phylogenomic data, providing a new perspective on both phylogeny and divergence speciation of cephalochordates.

Outcome Following the Treatment of Ventriculitis Caused by Multi/Extensive Drug Resistance Gram Negative Bacilli; Acinetobacter baumannii and Klebsiella pneumonia.

Introduction: CNS ventriculitis is a serious complication following an intracranial insult that demands immediate treatment with broad-spectrum antibiotics in a critical care setting. Infections due to multi/extensive drug resistance (MDR/XDR) microorganisms are very challenging, which may demand an additional approach to the ongoing practice; intravenous and intraventricular administration of antibiotics. Aim: To study the efficacy and safety of thorough ventricular irrigation followed by daily intraventricular antibiotic administration in patients with MDR/XDR ventriculitis. Materials and Methods: A retrospective analysis was done on 19 inpatients with ventriculitis caused by Acinetobacter baumannii (AB) or Klebsiella pneumonia (KP), at Shanghai Tenth People's Hospital from January 2016 to October 2017. We reviewed our experience; the role of thorough ventricular irrigation with Colistin mixed normal saline, followed by intraventricular Colistin therapy. Treatment outcomes were evaluated based on the clinical symptoms, Cerebro-Spinal Fluid (CSF) culture, laboratory findings and complications. Results: A total of 19 patients were included (15 males and 4 females), with a mean age in years of 51, which ranged from 18-67. Fourteen patients had Acinetobacter baumannii (AB) and 5 had Klebsiella pneumoniae (KP). The average CSF sterilization period following ventricular irrigation and intraventricular Colistin was 6 days. Sixteen patients (84%) were cured, and 3 patients (15%) died during the course of the treatment. Conclusion: In addition to Intraventricular Colistin, thorough ventricular irrigation could increase the cure rate up to 84% in patients suffering from MDR/XDR CNS ventriculitis.

Cloning, sequencing and analysis of the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus.

According to the conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGSGLAV, IGSGLV, IGSIA, IGSG and IGSA; while the strain CG021 has the same types of IGSs except lacking IGSA. Among these five IGS types, IGSGLAV is the biggest type, including the gene cluster of tRNAGlu-tRNALys-tRNAAla-tRNAVal; IGSGLV includes that of tRNAGlu-tRNALys-tRNAVal; IGSAG, tRNAAla-tRNAGlu; IGSIA, tRNAIle-tRNAAla; IGSG, tRNAGlu and IGSA, tRNAAla. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non- coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.