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In this study, we aimed to assess the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on semen parameters. The study comprised 110 sperm volunteers who self-reported SARS-CoV-2 infection from the Human Sperm Bank of the Center for Reproductive Medicine, Shandong University (Jinan, China). The volunteers had normal sperm concentration before infection. Each volunteer provided semen samples before and after infection. We selected 90 days after infection as the cutoff point. Semen parameters within 90 days after infection of 109 volunteers (group A) were compared with semen parameters before infection. Moreover, semen parameters on or after 90 days after infection of 36 volunteers (group B) were compared with semen parameters before infection. Furthermore, based on whether the volunteers had completed the three-dose SARS-CoV-2 vaccination booster, volunteers in group A and B were further divided into two subgroups separately. Semen parameters were compared before and after infection in each subgroup. Our results showed that in this cohort population, the semen quality in volunteers with normal sperm concentrations before infection decreased after SARS-CoV-2 infection within 90 days, while the semen quality returned to preinfection levels after 90 days. The completion of a three-dose SARS-CoV-2 vaccination booster may exert a protective effect on semen quality after infection.

The presence of outliers in signals collected by structural health monitoring systems, caused by sensor failure, equipment malfunction, or transmission interruption, can lead to misjudgments of a structure's working status and damage degree. This study proposes a novel, fast, accurate, and automatic method which are capable of reconstructing signals according to adjacent channels, detecting outliers by amplifying and sorting reconstructing errors, and recovering normal values to the corresponding locations. A parallel reservoir computing-based reconstructor with a decomposition module which purifies frequency components of input for each sub-network is utilized for improved precision. In addition, the adopted local outlier factor algorithm simplifies outlier detection work as simplex threshold comparison. The proposed method is analyzed for its effectiveness in detecting various types of outliers, such as spikes, abnormal segments, external trends, shifting, and baseline drift, using an acceleration dataset from the Shanghai Tower.

Objective: Individual differences were observed in the clinical efficacy of Botulinum toxin A (BoNT-A) in the treatment of the primary Meige syndrome. Our study aimed to explore the potential associations between the clinical efficacy of BoNT-A in the treatment of the primary Meige syndrome and variants of SNAP25, SV2C and ST3GAL2, which are involving in the translocation of the BoNT-A in vivo. Methods: Patients with the primary Meige syndrome treated with BoNT-A were enrolled. Clinical efficacy was evaluated by the maximum improvement rate of motor symptoms and the duration of efficacy. Variants of SNAP25, SV2C and ST3GAL2 were obtained by Sanger sequencing. Another cohort diagnosed with primary cervical dystonia was also enrolled in the replication stage. Results: Among the 104 primary Meige syndrome patients, 80 patients (76.9%) had a good efficacy (the maximum improvement rate of motor symptoms ≥30%) and 24 (23. 1%) had a poor (the maximum improvement rate of motor symptoms

Summary: Knowledge about the impact of different dissolved oxygen (DO) on the composition and function of gut bacteria of aquatic insects is largely unknown. Herein, we constructed freshwater environments with different DOs (hypoxia: 2.50 ± 0.50, normoxia: 7.00 ± 0.50, and hyperoxia: 13.00 ± 0.50 mg/L) where aquatic firefly Aquatica leii larvae lived for three months. Their gut flora was analyzed using the combination of 16S rRNA amplicon sequencing and metagenomics. The results showed no difference in alpha diversity of the gut flora between A. leii inhabiting various DOs. However, the relative abundance of several bacterial lineages presented significant changes, such as Pseudomonas. In addition, bacterial genes with an altered relative abundance in response to various DOs were primarily related to metabolism. The alteration of these functions correlated with the DO change. This is the first to uncover structure of gut flora under various DOs in aquatic insect larvae.

ABSTRACT: Phenyllactic acid (PLA) has been demonstrated to possess antibacterial activity and capacity to prolong food shelf life. However, studies on the performance of PLA in inhibiting Staphylococcus aureus and its effectiveness when applied to dairy products are largely lacking. Here, antibacterial activity (planktonic and biofilm states) of PLA against S. aureus CICC10145 (S. aureus_45) were investigated. The results showed that PLA inhibited growth of S. aureus_45 and formation of S. aureus_45 biofilm. Next, the antibacterial action target of PLA was uncovered from both physiological and phenotypic perspectives. The results showed that PLA decreased cell metabolic activity and cell viability, damaged cell membrane integrity, triggered leakage of intracellular contents (DNA, proteins, and ATP), and caused oxidative stress damage and morphological deformation of S. aureus_45. In practical application, the antibacterial activity of PLA against S. aureus_45 cells was further confirmed in skim milk and cheese as dairy food models, and the antibacterial effects can be adequately maintained during storage for 21 d, at least at 4°C. These findings suggested that PLA could be a potential candidate for controlling S. aureus outgrowth in dairy foods.

Numerous different species of LAB are used in different fields due to their unique characteristics. However, Lacticaseibacillus chiayiensis, a newly established species in 2018, has limited microorganism resources, and lacks comprehensive evaluations of its properties. In this study, L. chiayiensis AACE3, isolated from fermented blueberry, was evaluated by genomic analysis and in vitro assays of the properties. The genome identified genes associated with biofilm formation (luxS, ccpA, brpA), resistance to oxidative stress (tpx, trxA, trxB, hslO), tolerance to acidic conditions (dltA, dltC), resistance to unfavorable osmotic pressure (opuBB, gbuA, gbuB, gbuC), and adhesion (luxS, dltA, dltC). The AACE3 showed 112 unique genes, relative to the other three L. chiayiensis strains. Among them, the presence of genes such as clpP, pepO, and feoA suggests a possible advantage of AACE3 over other L. chiayiensis in terms of environmental adaptation. In vitro evaluation of the properties revealed that AACE3 had robust antibacterial activity against eight common pathogens: Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Salmonella choleraesuis, Shigella flexneri, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In addition, AACE3 showed more than 80% survival rate in all tests simulating gastrointestinal fluid, and it exhibited high antioxidant capacity. Interestingly, the cell culture supernatant was superior to intact organisms and ultrasonically crushed bacterial extracts in all tests of antioxidant capacity. These results suggested that the antioxidant capacity may originate from certain metabolites and extracellular enzymes produced by AACE3. Moreover, AACE3 was a moderate biofilm producer due to the self-agglomeration effect. Taken together, L. chiayiensis AACE3 appears to be a candidate strain for combating the growing incidence of pathogen infections and antioxidant production.

Lactic acid bacteria are generally regarded as alternatives to antibiotics in livestock and poultry farming, especially Lactobacillus strains, which are safe and have probiotic potential. Although Lactobacillus salivarius has long been proposed to be a probiotic, the understanding of the roles of this species is still in its infancy. Here, a strain of L. salivarius CGMCC20700 isolated from the intestinal mucosa of Yunnan black-bone chicken broilers was investigated in the context of its safety and probiotic characteristics by whole-genome sequencing in parallel with phenotypic analysis. Whole-genome sequencing results showed that L. salivarius CGMCC20700 has a single scaffold of 1,737,577 bp with an average guanine-to-cytosine (GC) ratio of 33.51% and 1,757 protein-coding genes. The annotation of Clusters of Orthologous Groups (COG) classified the predicted proteins from the assembled genome as possessing cellular, metabolic, and information-related functions. Sequences related to risk assessment, such as antibiotic resistance and virulence genes, were identified, and the strain was further confirmed as safe according to the results of antibiotic resistance, hemolytic, and acute oral toxicology tests. Two gene clusters of antibacterial compounds and broad-spectrum antimicrobial activity were identified using genome mining tools and antibacterial spectrum tests. Stress resistance genes, active stressor removal genes, and adhesion related genes that were identified and examined with various phenotypic assays (such as stress tolerance tests in acids and bile salts and auto aggregation and hydrophobicity assays). The strain showed a high survival rate in the presence of bile salts and under acidic conditions and exhibited significant auto aggregation capacity and hydrophobicity. Overall, L. salivarius CGMCC20700 demonstrated excellent safety and probiotic potential at both the genomic and physiological levels and can be considered an appropriate candidate probiotic for livestock and poultry farming.

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I–K–K–V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25–121 °C and pH 2–10. It significantly reduced cell viability of bacterial pathogens (p

ABSTRACT: Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 μg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.

As a fish unique to Yunnan Province in China, Sinocyclocheilus grahami hosts abundant potential probiotic resources in its intestinal tract. However, the genomic characteristics of the probiotic potential bacteria in its intestine and their effects on S. grahami have not yet been established. In this study, we investigated the functional genomics and host response of a strain, Lactobacillus salivarius S01, isolated from the intestine of S. grahami (bred in captivity). The results revealed that the total length of the genome was 1,737,623 bp (GC content, 33.09%), comprised of 1895 genes, including 22 rRNA operons and 78 transfer RNA genes. Three clusters of antibacterial substances related genes were identified using antiSMASH and BAGEL4 database predictions. In addition, manual examination confirmed the presence of functional genes related to stress resistance, adhesion, immunity, and other genes responsible for probiotic potential in the genome of L. salivarius S01. Subsequently, the probiotic effect of L. salivarius S01 was investigated in vivo by feeding S. grahami a diet with bacterial supplementation. The results showed that potential probiotic supplementation increased the activity of antioxidant enzymes (SOD, CAT, and POD) in the hepar and reduced oxidative damage (MDA). Furthermore, the gut microbial community and diversity of S. grahami from different treatment groups were compared using high-throughput sequencing. The diversity index of the gut microbial community in the group supplemented with potential probiotics was higher than that in the control group, indicating that supplementation with potential probiotics increased gut microbial diversity. At the phylum level, the abundance of Proteobacteria decreased with potential probiotic supplementation, while the abundance of Firmicutes, Actinobacteriota, and Bacteroidota increased. At the genus level, there was a decrease in the abundance of the pathogenic bacterium Aeromonas and an increase in the abundance of the potential probiotic bacterium Bifidobacterium. The results of this study suggest that L. salivarius S01 is a promising potential probiotic candidate that provides multiple benefits for the microbiome of S. grahami.

Background: Pemetrexed plus platinum doublet chemotherapy regimen remains to be the standard first-line treatment for lung adenocarcinoma patients. However, few biomarkers can be used to identify potential beneficiaries with maximal efficacy and minimal toxicity. This study aimed to explore potential biomarker models predictive of efficacy and toxicity after pemetrexed plus platinum chemotherapy based on metabolomics profiling. Methods: A total of 144 patients who received at least two cycles of pemetrexed plus platinum chemotherapy were enroled in the study. Serum samples were collected before initial treatment to perform metabolomics profiling analysis. Logistic regression analysis was performed to establish prediction models. Results: 157 metabolites were found to be differentially expressed between the response group and the nonresponse group. A panel of Phosphatidylserine 20:4/20:1, Sphingomyelin d18:1/18:0, and Phosphatidic Acid 18:1/20:0 could predict pemetrexed and platinum chemotherapy response with an Area Under the Receiver Operating Characteristic curve (AUROC) of 0.7968. 76 metabolites were associated with hematological toxicity of pemetrexed plus platinum chemotherapy. A panel incorporating triglyceride 14:0/22:3/22:5, 3-(3-Hydroxyphenyl) Propionate Acid, and Carnitine C18:0 was the best predictive ability of hematological toxicity with an AUROC of 0.7954. 54 differential expressed metabolites were found to be associated with hepatotoxicity of pemetrexed plus platinum chemotherapy. A model incorporating stearidonic acid, Thromboxane B3, l-Homocitrulline, and phosphoinositide 20:3/18:0 showed the best predictive ability of hepatotoxicity with an AUROC of 0.8186. Conclusions: This study established effective and convenient models that can predict the efficacy and toxicity of pemetrexed plus platinum chemotherapy in lung adenocarcinoma patients before treatment delivery.

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.

Abstract Dietary shifts can alter the relative availability of different nutrients and are therefore associated with metabolic adaptation in animals. The Coccinellidae (ladybirds) exhibits three major types of feeding habits and provides a useful model to study the effects of dietary changes on the evolution of mitogenomes, which encode proteins directly involved in energy metabolism. Here, mitogenomes of three coccinellid species were newly sequenced. These data were combined with other ten previously sequenced coccinellid mitogenomes to explore the relationship between mitogenome evolution and diets. Our results indicate that mitogenomic data can be effectively used to resolve phylogenetic relationships of Coccinellidae. Strong codon usage bias in coccinellid mitogenomes was predominantly determined by nucleotide composition. The 13 mitochondrial protein‐coding genes (PCGs) globally evolved under negative constraints, with some PCGs showing a stronger purifying selection. Six PCGs (nad3, nad4L, and nad5 from Complex I; cox1 and cox3 from Complex IV; and atp6 from Complex V) displayed signs of positive selection. Of these, adaptive changes in cox3 were potentially associated with metabolic differences resulting from dietary shifts in Coccinellidae. Our results provide insights into the adaptive evolution of coccinellid mitogenomes in response to both dietary shifts and other life history traits.

Objective: To objective of the study was to investigate whether serum brain-derived neurotrophic factor (BDNF) levels are associated with the severity of restless legs syndrome (RLS) in Parkinson's disease (PD).Methods: A total of 249 PD patients with (n = 53) and without RLS (n = 196) and 326 age-matched controls were included in this study. All the serum BDNF levels of the participants were measured. The International Restless Legs Syndrome Study Group Rating Scale (IRLSSG-RS) was administered for the severity of RLS. The severity of PD patients were assessed by the Unified PD Rating Scale (UPDRS) and the Hoehn and Yahr (H-Y) stage.Results: The prevalence of RLS was significantly higher in PD patients (21.3%) than in the controls group (7.4%) (p < 0.05). The IRLSSG-RS score in PD patients with RLS (16.25 ± 5.24) was significantly increased than in controls with RLS (12.08 ± 3.99) (p < 0.01). The serum BDNF levels were significantly decreased in PD patients with RLS than in PD patients without RLS, controls without RLS, and controls with RLS (p < 0.001). BDNF levels were negatively associated with IRLSSG-RS in both PD patients with RLS and controls with RLS group (both p < 0.01). Multiple regression analysis confirmed that in either PD with RLS or controls with RLS group, BDNF was an independent contributor to IRLSSG-RS (both p < 0.01).Conclusions: Decreased serum BDNF levels may be involved in the pathophysiology of RLS in PD, suggesting that it may serve as a potential blood biomarker of diagnostic value for RLS in PD.

Mitochondria are the leading organelle for energy metabolism. The toxic effects of environmental toxicants on mitochondrial morphology, energy metabolism, and their determination of cell fate have already been broadly studied. However, minimal research exists on effects of environmental toxicants such as pesticides on mitochondrial energy metabolism at in vitro subcellular level, particularly from an omics perspectives (e.g., metabolomics). Here, German cockroach (Blattella germanica) was fed diets with (0.01 and 0.001 mg/mL) and without abamectin, and highly purified fat body mitochondria were isolated. Swelling measurement confirmed abnormal mitochondrial swelling caused by abamectin stress. The activity of two key mitochondrial energy metabolism-related enzymes, namely succinic dehydrogenase and isocitrate dehydrogenase, was significantly affected. The metabolomic responses of the isolated mitochondria to abamectin were analyzed via untargeted liquid chromatography/mass spectrometry metabolomics technology. Fifty-two differential metabolites (DMs) were identified in the mitochondria between the 0.001 mg/mL abamectin-fed and the control groups. Many of these DMs were significantly enriched in pathways involved in ATP production and energy consumption (e.g., oxidative phosphorylation, TCA cycle, and pentose phosphate pathway). Nineteen of the DMs were typically related to energy metabolism. This study is valuable for further understanding mitochondrial toxicology under environmental toxicants, particularly its subcellular level.

Rhodosporidium kratochvilovae strain YM25235 is a cold-adapted oleaginous yeast strain that can grow at 15°C. It is capable of producing polyunsaturated fatty acids. Here, we used the Nanopore Platform to first assemble the R. kratochvilovae strain YM25235 genome into a 23.71 Mb size containing 46 scaffolds and 8,472 predicted genes. To explore the molecular mechanism behind the low temperature response of R. kratochvilovae strain YM25235, we analyzed the RNA transcriptomic data from low temperature (15°C) and normal temperature (30°C) groups using the next-generation deep sequencing technology (RNA-seq). We identified 1,300 differentially expressed genes (DEGs) by comparing the cultures grown at low temperature (15°C) and normal temperature (30°C) transcriptome libraries, including 553 significantly upregulated and 747 significantly downregulated DEGs. Gene ontology and pathway enrichment analysis revealed that DEGs were primarily related to metabolic processes, cellular processes, cellular organelles, and catalytic activity, whereas the overrepresented pathways included the MAPK signaling pathway, metabolic pathways, and amino sugar and nucleotide sugar metabolism. We validated the RNA-seq results by detecting the expression of 15 DEGs using qPCR. This study provides valuable information on the low temperature response of R. kratochvilovae strain YM25235 for further research and broadens our understanding for the response of R. kratochvilovae strain YM25235 to low temperature.

The egg-pathogenic fungus Purpureocillium lilacinum parasitizes on nematode eggs, and thus, it is used as a good biocontrol agent against plant root-knot nematodes. However, little is known about the transcriptional response of P. lilacinum while infecting nematode eggs. This study presents the whole transcriptome sequencing of P. lilacinum and transcriptome-wide gene expression analysis of P. lilacinum upon infecting the eggs of Meloidogyne incognita compared to non-infecting controls. A transcriptomic library of P. lilacinum was used as reference gene set and six transcriptomic libraries of the non-infecting control and P. lilacinum infecting M. incognita eggs were constructed, respectively, comprising three biological replicates of each. A total of 1,011 differently expressed genes (DEGs) were identified in the infecting samples, including 553 up-regulated and 458 down-regulated genes compared to the non-infecting control samples. Furthermore, functional enrichment analysis exhibited that these DEGs were primarily involved in oxidative phosphorylation, oxidoreductase activity, and metabolic processes. Fifteen DEGs were randomly selected to verify the RNA sequencing results through quantitative real-time polymerase chain reaction (qPCR). The study focused on P. lilacinum genes that were strongly expressed upon infecting M. incognita eggs. These DEGs were primarily involved in detoxification, parasitic behavior, and nutritional utilization. This study contributes significantly to the understanding of the molecular mechanisms underlying the parasitic action of P. lilacinum on nematode eggs and provides a valuable genetic resource for further research on parasitic behavior of P. lilacinum. Notably, this study examined the transcriptomics of P. lilacinum infecting M. incognita eggs at only one time point. Since there were fungi at different stages of the infection process at that time point, the transcriptional profiles are not precisely examining one specific stage in this process.

Abstract Background To compare neurocognitive functioning of Type 1 diabetic mellitus (T1DM) and healthy adults, and explore risk factors of cognitive dysfunction of T1DM patients, especially the association between cognitive impairment and diabetic peripheral neuropathy (DPN). Methods Seventy T1DM (age: 32.17 ± 9.57 yr., duration: 8.99 ± 7.02 yr) patients and 48 healthy volunteers were included. All subjects received evaluation of MMSE and MoCA scales. Cognitive function of T1DM patients was evaluated in different cognitive domains. Risk factors affecting cognitive function were further explored. Results Three patients with educational level ≤ 6-year were excluded from final analysis. Scores of both MMSE (28.4 ± 1.7 vs. 29.1 ± 1.0, P = 0.005) and MoCA scales (25.9 ± 2.7 vs.27.1 ± 2.4, P = 0.017) in T1DM group were lower than that in control group. For MMSE scale, scores of orientation (9.60 ± 0.79 vs.9.87 ± 0.39, P

The intestine is the largest mucosal tissue in fish and an important antibacterial site. The pathogenic Streptococcus agalactiae (Gram-positive) is a major bacterial threat to fish intestine health. However, antibacterial molecular mechanisms in the fish intestine, particularly gene expression at genomic scale, in response to S. agalactiae challenge remains largely unknown. With the purpose of investigating this issue, this study performed RNA-seq for control and pathogenic S. agalactiae-challenged intestines of zebrafish. A total of 952 differentially expressed genes (DEGs) were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that DEGs were primarily related to immunity, metabolism, and enzyme activity. These include complement and coagulation cascades, cytokine-cytokine receptor interaction, and metabolic pathways. In particular, the Staphylococcus aureus (Gram-positive bacteria) infection pathway was detected in the KEGG pathway enrichment analysis. The RNA-seq results were validated by assessing the expression levels of 12 key genes of this pathway and 10 randomly selected DEGs using quantitative real-time PCR. Expression patterns of 12 key genes in the Staphylococcus aureus infection pathway were further surveyed across 0, 6, 12, 24, and 48 h after S. agalactiae challenge. It is worth noting that 53 immune related genes, belonging to 9 categories, were systematically identified in response to S. agalactiae challenge, such as antigen processing and presentation (e.g., mr1l, tnfa, and cd4), chemokine and receptors (e.g., ccr2, ccr6, and cxcl18b), and complement and coagulation cascades (e.g., cd59, c3, and c6). This study provides valuable genomic information for better comprehension of genes and signaling pathways involved in the intestinal antibacterial response in zebrafish, pointing out genes potentially involved in the intestinal antibacterial response, and affording relevant information to be applied in further fish disease control.

Distinct expression of the miRNAs has rarely been explored in basal cell carcinoma (BCC) of skin, and the regulatory role of miRNAs in BCC development remains quite opaque. Here, we collected control tissues from adjacent noncancerous skin (n=15; control group) and tissues at tumor centers from patients with cheek BCC (n=15; BCC group) using punch biopsies. After six small RNA sequencing- (sRNA-seq-) based miRNA expression profiles were generated for both BCC and controls, including three biological replicates, we conducted comparative analysis on the sRNA-seq dataset, discovering 181 differentially expressed miRNAs (DEMs) out of the 1,873 miRNAs in BCCs. In order to validate the sRNA-seq data, expression of 15 randomly selected DEMs was measured using the TaqMan probe-based quantitative real-time PCR. Functional analysis of predicted target genes of DEMs in BCCs shows that these miRNAs are primarily involved in various types of cancers, immune response, epithelial growth, and morphogenesis, as well as energy production and metabolism, indicating that BCC development is caused, at least in part, by changes in miRNA regulation for biological and disease processes. In particular, the “basal cell carcinoma pathways” were found to be enriched by predicted DEM targets, and regulatory relationships between DEMs and their targeted genes in this pathway were further uncovered. These results revealed the association between BCCs and abundant miRNA molecules that regulate target genes, functional modules, and signaling pathways in carcinogenesis.

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, β-tubulin, β-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, β-actin, β-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 + β-tubulin), different sexes (β-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 (β-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.

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.

Amphioxus is the closest living invertebrate proxy of the vertebrate ancestor. Systematic gene identification and expression profile analysis of amphioxus organs are thus important for clarifying the molecular mechanisms of organ function formation and further understanding the evolutionary origin of organs and genes in vertebrates. The precise regulation of microRNAs (miRNAs) is crucial for the functional specification and differentiation of organs. In particular, those miRNAs that are expressed specifically in organs (OSMs) play key roles in organ identity, differentiation, and function. In this study, the genome-wide miRNA transcriptome was analyzed in eight organs of adult amphioxus Branchiostoma belcheri using deep sequencing. A total of 167 known miRNAs and 23 novel miRNAs (named novel_mir), including 139 conserved miRNAs, were discovered, and 79 of these were identified as OSMs. Additionally, analyses of the expression patterns of eight randomly selected known miRNAs demonstrated the accuracy of the miRNA deep sequencing that was used in this study. Furthermore, potentially OSM-regulated genes were predicted for each organ type. Functional enrichment of these predicted targets, as well as further functional analyses of known OSMs, was conducted. We found that the OSMs were potentially to be involved in organ-specific functions, such as epidermis development, gonad development, muscle cell development, proteolysis, lipid metabolism, and generation of neurons. Moreover, OSMs with non–organ-specific functions were detected and primarily include those related to innate immunity and response to stimuli. These findings provide insights into the regulatory roles of OSMs in various amphioxus organs.

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.

We determined the complete mitogenome of Pyrrhocoris tibialis (Hemiptera: Heteroptera: Pyrrhocoridae) to better understand the diversity and phylogeny within Pentatomomorpha, which is the second largest infra-order of Heteroptera. Gene content, gene arrangement, nucleotide composition, codon usage, ribosomal RNA (rRNA) structures, and sequences of the mitochondrial transcription termination factor were well conserved in Pyrrhocoroidea. Different protein-coding genes have been subject to different evolutionary rates correlated with the G + C content. The size of control regions (CRs) was highly variable among mitogenomes of three sequenced Pyrrhocoroidea species, with the P. tibialis CR being the largest. All the transfer RNA genes found in Pyrrhocoroidea had the typical clover leaf secondary structure, except for trnS1 (AGN), which lacked the dihydrouridine arm and possessed an unusual anticodon stem (9 bp vs. the normal 5 bp). A total of three different phylogenetic relationships among the five super-families of Pentatomomorpha were obtained using three analytical methods (MrBayes and RAxML under site-homogeneous models and PhyloBayes under a site-heterogeneous CAT + GTR model) and two mitogenomic datasets (nucleotides and amino acids). The tree topology test using seven methods statistically supported a phylogeny of (Aradoidea + (Pentatomoidea + (Lygaeoidea + (Pyrrhocoroidea + Coreoidea)))) as the best topology, as recognized by both RAxML and MrBayes based on the two datasets.

Objective. To assess the therapeutic efficacy and safety of botulinum toxin type A (BTX-A) for treating idiopathic trigeminal neuralgia (ITN) in patients ≥80 years old. Methods. Selected patients n=43 with ITN, recruited from the neurology clinic and inpatient department of the Second Affiliated Hospital of Soochow University between August 2008 and February 2014, were grouped by age, one subset n=14 ≥80 years old and another n=29

The family Miridae is one of the most species-rich families of insects. To better understand the diversity and evolution of mirids, we determined the mitogenome of Lygus pratenszs and re-sequenced the mitogenomes of four mirids (i.e., Apolygus lucorum, Adelphocoris suturalis, Ade. fasciaticollis and Ade. lineolatus). We performed a comparative analysis for 15 mitogenomic sequences representing 11 species of five genera within Miridae and evaluated the potential of these mitochondrial genes as molecular markers. Our results showed that the general mitogenomic features (gene content, gene arrangement, base composition and codon usage) were well conserved among these mirids. Four protein-coding genes (PCGs) (cox1, cox3, nad1 and nad3) had no length variability, where nad5 showed the largest size variation; no intraspecific length variation was found in PCGs. Two PCGs (nad4 and nad5) showed relatively high substitution rates at the nucleotide and amino acid levels, where cox1 had the lowest substitution rate. The Ka/Ks values for all PCGs were far lower than 1 (

Gynaephora (Lepidoptera Erebidae: Lymantriinae) is a small genus, consisting of 15 nominated species, of which eight species are endemic to the Qinghai-Tibetan Plateau (QTP). In this study, we employed both mitochondrial and nuclear loci to infer a molecular phylogeny for the eight QTP Gynaephora spp. We used the phylogeny to estimate divergence dates in a molecular dating analysis and to delimit species. This information allowed us to investigate associations between the diversification history of the eight QTP species and geological and climatic events. Phylogenetic analyses indicated that the eight QTP species formed a monophyletic group with strong supports in both Bayesian and maximum likelihood analyses. The low K2P genetic distances between the eight QTP species suggested that diversification occurred relatively quickly and recently. Out of the eight species, five species were highly supported as monophyletic, which were also recovered by species delimitation analyses. Samples of the remaining three species (G. aureata, G. rouergensis, and G. minora) mixed together, suggesting that further studies using extensive population sampling and comprehensive morphological approaches are necessary to clarify their species status. Divergence time estimation results demonstrated that the diversification and speciation of Gynaephora on the QTP began during the late Miocene/early Pliocene and was potentially affected by the QTP uplift and associated climate changes during this time.

Insect mitochondrial genome (mitogenome) are the most extensively used genetic information for molecular evolution, phylogenetics and population genetics. Pentatomomorpha (>14,000 species) is the second largest infraorder of Heteroptera and of great economic importance. To better understand the diversity and phylogeny within Pentatomomorpha, we sequenced and annotated the complete mitogenome of Corizus tetraspilus (Hemiptera: Rhopalidae), an important pest of alfalfa in China. We analyzed the main features of the C. tetraspilus mitogenome, and provided a comparative analysis with four other Coreoidea species. Our results reveal that gene content, gene arrangement, nucleotide composition, codon usage, rRNA structures and sequences of mitochondrial transcription termination factor are conserved in Coreoidea. Comparative analysis shows that different protein-coding genes have been subject to different evolutionary rates correlated with the G+C content. All the transfer RNA genes found in Coreoidea have the typical clover leaf secondary structure, except for trnS1 (AGN) which lacks the dihydrouridine (DHU) arm and possesses a unusual anticodon stem (9 bp vs. the normal 5 bp). The control regions (CRs) among Coreoidea are highly variable in size, of which the CR of C. tetraspilus is the smallest (440 bp), making the C. tetraspilus mitogenome the smallest (14,989 bp) within all completely sequenced Coreoidea mitogenomes. No conserved motifs are found in the CRs of Coreoidea. In addition, the A+T content (60.68%) of the CR of C. tetraspilus is much lower than that of the entire mitogenome (74.88%), and is lowest among Coreoidea. Phylogenetic analyses based on mitogenomic data support the monophyly of each superfamily within Pentatomomorpha, and recognize a phylogenetic relationship of (Aradoidea + (Pentatomoidea + (Lygaeoidea + (Pyrrhocoroidea + Coreoidea)))).

BACKGROUND: Mounting evidence shows that urate may become a biomarker of Parkinson's disease (PD) diagnosis and prognosis and a neuroprotectant candidate for PD therapy. However, the cellular and molecular mechanisms underlying its neuroprotective actions remain poorly understood. RESULTS: In this study, we showed that urate pretreatment protected dopaminergic cell line (SH-SY5Y and MES23.5) against 6-hydroxydopamine (6-OHDA)- and hydrogen peroxide- induced cell damage. Urate was found to be accumulated into SH-SY5Y cells after 30 min treatment. Moreover, urate induced NF-E2-related factor 2 (Nrf2) accumulation by inhibiting its ubiquitinationa and degradation, and also promoted its nuclear translocation; however, it did not modulate Nrf2 mRNA level or Kelch-like ECH-associated protein 1 (Keap1) expression. In addition, urate markedly up-regulated the transcription and protein expression of γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) and heme oxygenase-1 (HO-1), both of which are controlled by Nrf2 activity. Furthermore, Nrf2 knockdown by siRNA abolished the intracellular glutathione augmentation and the protection exerted by urate pretreatment. CONCLUSION: Our findings demonstrated that urate treatment may result in Nrf2-targeted anti-oxidant genes transcription and expression by reducing Nrf2 ubiquitination and degradation and promoting its nuclear translocation, and thus offer neuroprotection on dopaminergic cells against oxidative stresses.