A WD-repeat protein stabilizes ORC binding to chromatin.

Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.

[Comparative analysis of deterioration products in bio-waste oil and vegetable oils].

OBJECTIVE: To analyze the difference of deterioration products in bio-waste oil and vegetable oils. METHODS: The changes of species and abundance of deterioration products were analyzed through observing the differences in Raman spectra during the process of deterioration and refining. RESULTS: The deterioration contents produced during heating, cooking, frying and wasting, instead of storage, were significantly more abundant than normal contents. Through the refining process, the deterioration products abundance was reduced in vegetable oils while increased in bio-waste oils. CONCLUSION: Due to the distinct deteriorating processes, the species and abundance of deterioration products are remarkably different in bio-waste oil and vegetable oils. The deterioration products in vegetable oils would be mostly removed, but those in bio-waste oils are concentrated instead of eliminated during the refining procedure.

Social behavioral profiling by unsupervised deep learning reveals a stimulative effect of dopamine D3 agonists on zebrafish sociality.

It has been a major challenge to systematically evaluate and compare how pharmacological perturbations influence social behavioral outcomes. Although some pharmacological agents are known to alter social behavior, precise description and quantification of such effects have proven difficult. We developed a scalable social behavioral assay for zebrafish named ZeChat based on unsupervised deep learning to characterize sociality at high resolution. High-dimensional and dynamic social behavioral phenotypes are automatically classified using this method. By screening a neuroactive compound library, we found that different classes of chemicals evoke distinct patterns of social behavioral fingerprints. By examining these patterns, we discovered that dopamine D3 agonists possess a social stimulative effect on zebrafish. The D3 agonists pramipexole, piribedil, and 7-hydroxy-DPAT-HBr rescued social deficits in a valproic-acid-induced zebrafish autism model. The ZeChat platform provides a promising approach for dissecting the pharmacology of social behavior and discovering novel social-modulatory compounds.

A zebrafish model of combined saposin deficiency identifies acid sphingomyelinase as a potential therapeutic target.

Sphingolipidoses are a subcategory of lysosomal storage diseases (LSDs) caused by mutations in enzymes of the sphingolipid catabolic pathway. Like many LSDs, neurological involvement in sphingolipidoses leads to early mortality with limited treatment options. Given the role of myelin loss as a major contributor toward LSD-associated neurodegeneration, we investigated the pathways contributing to demyelination in a CRISPR-Cas9-generated zebrafish model of combined saposin (psap) deficiency. psap knockout (KO) zebrafish recapitulated major LSD pathologies, including reduced lifespan, reduced lipid storage, impaired locomotion and severe myelin loss; loss of myelin basic protein a (mbpa) mRNA was progressive, with no changes in additional markers of oligodendrocyte differentiation. Brain transcriptomics revealed dysregulated mTORC1 signaling and elevated neuroinflammation, where increased proinflammatory cytokine expression preceded and mTORC1 signaling changes followed mbpa loss. We examined pharmacological and genetic rescue strategies via water tank administration of the multiple sclerosis drug monomethylfumarate (MMF), and crossing the psap KO line into an acid sphingomyelinase (smpd1) deficiency model. smpd1 mutagenesis, but not MMF treatment, prolonged lifespan in psap KO zebrafish, highlighting the modulation of acid sphingomyelinase activity as a potential path toward sphingolipidosis treatment.

Pathogenetic characterization of a Micrococcus luteus strain isolated from an infant.

Purpose: To explore the clinical characteristics of Micrococcus luteus bloodstream infection in an infant and characterize the phenotype and genotype of the isolated strains, as well as seek suitable infection models for assessing virulence. Methods: Clinical data was collected from an infant patient diagnosed with M. luteus bloodstream infection. Metagenomic sequencing was performed on the isolated blood sample. The strain was isolated and underwent mass spectrometry, biochemical tests, antibiotic susceptibility assays, and whole-genome sequencing. The Galleria mellonella infection model was used to assess M. luteus virulence. Results: Patient responded poorly to cephalosporins, but recovered after Linezolid treatment. Metagenomic sequencing identified M. luteus as the predominant species in the sample, confirming infection. They were identified as M. luteus with a high confidence level of 98.99% using mass spectrometry. The strain showed positive results for Catalase, Oxidase, and Urea tests, and negative results for Mannose, Xylose, Lactose, Mannitol, Arginine, and Galactose tests, consistent with the biochemical profile of M. luteus reference standards. M. luteus susceptibility to 15 antibiotics was demonstrated and no resistance genes were detected. Predicted virulence genes, including clpB, were associated with metabolic pathways and the type VI secretion system. The infection model demonstrated dose-dependent survival rates. Conclusion: The infant likely developed a bloodstream infection with M. luteus due to compromised immunity. Although the isolated strain is sensitive to cephalosporin antibiotics and has low pathogenicity in infection models, clinical treatment with cephalosporins was ineffective. Linezolid proved to be effective, providing valuable guidance for future clinical management of such rare infections.

Distribution of drug-resistant genes in alveolar lavage fluid from patients with psittacosis and traceability analysis of causative organisms.

Background: Chlamydia psittaci is a small bacterium often found in birds, including poultry, and domesticated mammals, which causes psittacosis (or parrot fever) in humans. Different strains of C. psittaci respond variably to antibiotics, suggesting a possible risk of antibiotic resistance. In general, different genotypes of C. psittaci have relatively stable hosts and different pathogenicity. Methods: Macrogenomic sequencing was performed using nucleic acids extracted from psittacosis patients' alveolar lavage fluid samples and analyzed for genetic variability and antibiotic resistance genes. Nucleic acid amplification sequences specific to the core coding region of the C. psittaci ompA gene were used, and a phylogenetic tree was constructed with C. psittaci genotypic sequences from other sources, including Chinese published sources. The C. psittaci found in each patient were genotyped by comparing ompA gene sequences. In addition, to better illustrate the relationship between genotype and host of C. psittaci, 60 bird fecal samples were collected from bird-selling stores for screening and C. psittaci typing. Results: Macrogenomic sequence alignment revealed the presence of resistance genes in varying abundance in samples from all three patients, including C. psittaci resistance gene sequences from two patients that matched those previously published on NCBI. Based on ompA genotyping, two patients were infected with C. psittaci genotype A and one patient was infected with genotype B. All five C. psittaci-positive samples obtained from bird-selling stores were genotype A. Both genotypes are reported to be infectious to humans. The host origin of the samples and the previously reported main sources of each genotype suggested that all but one of the C. psittaci genotype A in this study were derived from parrots, while genotype B was probably derived from chickens. Conclusion: The presence of bacterial resistance genes in psittacosis patients may affect the efficacy of clinical antibiotic therapy. Focusing on the developmental progression of bacterial resistance genes and differences in the therapeutic efficacy may facilitate effective treatment of clinical bacterial infections. Pathogenicity genotypes (e.g., genotype A and genotype B) are not limited to one animal host, suggesting that monitoring the development and changes of C. psittaci may help prevent transmission to humans.

Top2a promotes the development of social behavior via PRC2 and H3K27me3.

Little is understood about the embryonic development of sociality. We screened 1120 known drugs and found that embryonic inhibition of topoisomerase IIα (Top2a) resulted in lasting social deficits in zebrafish. In mice, prenatal Top2 inhibition caused defects in social interaction and communication, which are behaviors that relate to core symptoms of autism. Mutation of Top2a in zebrafish caused down-regulation of a set of genes highly enriched for genes associated with autism in humans. Both the Top2a-regulated and autism-associated gene sets have binding sites for polycomb repressive complex 2 (PRC2), a regulatory complex responsible for H3K27 trimethylation (H3K27me3). Moreover, both gene sets are highly enriched for H3K27me3. Inhibition of the PRC2 component Ezh2 rescued social deficits caused by Top2 inhibition. Therefore, Top2a is a key component of an evolutionarily conserved pathway that promotes the development of social behavior through PRC2 and H3K27me3.

Association between two key SNPs on chromosome 12p13 and ischemic stroke in Chinese Han population.

OBJECTIVE: Genome-wide single nucleotide polymorphism (SNP) association studies recently identified two SNPs (rs11833579 and rs12425791) on chromosome 12p13 that are associated with ischemic stroke (IS) in Caucasian or Black persons from America and the Netherlands. Our aim was to determine whether these SNPs were associated with IS in Chinese Han population. METHODS: We used a case-control study involving 648 IS patients and 648 age-matched, sex-matched, and ethnicity-matched non-IS controls from two ethnic populations and determined the genotypes of two polymorphisms by TaqMan SNP genotyping assays to assess any links with IS. RESULTS: Significant allelic association was identified between rs11833579 and IS in the Han population (odds ratio=1.27, 95% confidence interval=1.08-1.49). One risk haplotype (A-G; odds ratio=1.52, 95% confidence interval=1.21-1.92) was identified in the Han population. Genotypic association analysis demonstrated that rs11833579 confers susceptibility to IS only in a recessive model (P=0.004) rather in additive model. However, the association between rs12425791 and IS was insignificant in Chinese Han population. CONCLUSION: The A allele of SNP rs11833579 on chromosome 12p13 may play a role in mediating susceptibility to IS in the Han Chinese population in a recessive model. The A-G haplotype is also significantly associated with higher IS risk in the Han Chinese population. However, larger populations are warranted to validate our findings.

Rapid Mounting of Zebrafish Larvae for Brain Imaging.

Brain imaging requires mounting of zebrafish larvae in a vertical position, but anesthetized or fixed larvae tend to fall on their sides without external support. Current solution is to manually hold individual larva until liquid agarose solidifies, which is time consuming, labor intensive, and unfriendly to beginners. We developed a method to form larva-shaped slots in agarose gel using a computer numerical controlled manufactured mold. Each slot nearly perfectly fits a larva in its upright position, and larvae can be easily mounted by inserting into the slots. On average, each larva can be mounted in <1 min using this method.

The association of functional polymorphisms of IL-6 gene promoter with ischemic stroke: analysis in two Chinese populations.

Polymorphisms of G-572C and G-174C in the interleukin-6 (IL-6) promoter can affect both the transcription and secretion of IL-6 and may be involved in inflammation related to and the pathogenesis of ischemic stroke (IS). However, whether IL-6 polymorphisms are indeed risk factors for IS remains controversial. We recruited 748 Chinese IS patients diagnosed by magnetic resonance imaging (MRI) within 24h of symptom onset and 748 normal healthy controls from two ethnic populations and performed two case-control studies in order to assess the nature of the polymorphisms of IL-6 and any links with IS. Common polymorphic loci in the IL-6 gene promoter were determined by TaqMan SNP genotyping assays. Multivariate logistic regression analysis was used to examine the association between IL-6 genotypes and a diagnosis of IS. We found that the C allele frequency at the -174 promoter region of IL-6 was extremely low in both IS patients and controls in both ethnic groups. The G allele of the promoter single nucleotide polymorphism (SNP) G-572C was more common in IS subjects than controls (P=0.004, corrected for multiple testing) in the Han population but not in the Uyghur population. GC carriage therefore increased the risk of IS in the Han ethnic group (OR 1.45, 95% CI 1.13-1.86). In addition, the differences in GG and GC frequency between the two ethnic populations were significant. The C allele frequency at the -174 promoter region of IL-6 was rare in Chinese IS patients and controls from either ethnic group. We conclude that IL-6-572GC may be an independent risk factor for IS in the Chinese Han population.

Viral RNA level, serum antibody responses, and transmission risk in recovered COVID-19 patients with recurrent positive SARS-CoV-2 RNA test results: a population-based observational cohort study.

Managing recovered COVID-19 patients with recurrent-positive SARS-CoV-2 RNA test results is challenging. We performed a population-based observational study to characterize the viral RNA level and serum antibody responses in recurrent-positive patients and evaluate their viral transmission risk. Of 479 recovered COVID-19 patients, 93 (19%) recurrent-positive patients were identified, characterized by younger age, with a median discharge-to-recurrent-positive length of 8 days. After readmission, recurrent-positive patients exhibited mild (28%) or absent (72%) symptoms, with no disease progression. The viral RNA level in recurrent-positive patients ranged from 1.8 to 5.7 log10 copies/mL (median: 3.2), which was significantly lower than the corresponding values at disease onset. There are generally no significant differences in antibody levels between recurrent-positive and non-recurrent-positive patients, or in recurrent-positive patients over time (before, during, or after recurrent-positive detection). Virus isolation of nine representative specimens returned negative results. Whole genome sequencing of six specimens yielded only genomic fragments. 96 close contacts and 1,200 candidate contacts of 23 recurrent-positive patients showed no clinical symptoms; their viral RNA (1,296/1,296) and antibody (20/20) tests were negative. After full recovery (no longer/never recurrent-positive), 60% (98/162) patients had neutralizing antibody titers of ≥1:32. Our findings suggested that an intermittent, non-stable excretion of low-level viral RNA may result in recurrent-positive occurrence, rather than re-infection. Recurrent-positive patients pose a low transmission risk, a relatively relaxed management of recovered COVID-19 patients is recommended.

FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries.

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Differentially expressed genes between female and male adult Anopheles anthropophagus.

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

The zebrafish subcortical social brain as a model for studying social behavior disorders.

Social behaviors are essential for the survival and reproduction of social species. Many, if not most, neuropsychiatric disorders in humans are either associated with underlying social deficits or are accompanied by social dysfunctions. Traditionally, rodent models have been used to model these behavioral impairments. However, rodent assays are often difficult to scale up and adapt to high-throughput formats, which severely limits their use for systems-level science. In recent years, an increasing number of studies have used zebrafish (Danio rerio) as a model system to study social behavior. These studies have demonstrated clear potential in overcoming some of the limitations of rodent models. In this Review, we explore the evolutionary conservation of a subcortical social brain between teleosts and mammals as the biological basis for using zebrafish to model human social behavior disorders, while summarizing relevant experimental tools and assays. We then discuss the recent advances gleaned from zebrafish social behavior assays, the applications of these assays to studying related disorders, and the opportunities and challenges that lie ahead.

Effect of polymorphisms of endothelial nitric oxide synthase on ischemic stroke: a case-control study in a Chinese population.

BACKGROUND: Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) plays a key role in vascular regulation and atherosclerosis, therefore, eNOS may be a candidate gene for ischemic stroke (IS). However, it is still controversial whether eNOS polymorphisms are a risk factor for IS. METHODS: Three polymorphisms of the eNOS gene (-922A/G, -786T/C, 894G/T) were determined by using TaqMan SNP genotyping assay in 309 Chinese patients with IS and 309 Chinese controls. RESULTS: The frequency of eNOS -922 G allele was significantly higher in the patients than the controls (12.14% vs 8.09%, P=0.018). The distribution of eNOS genotypes differed insignificantly between the 2 groups. The frequency of the eNOS -786 CC genotype was higher in the patients than the controls (OR=3.819, P=0.029). With respect to -922A/G, the AG+GG genotype increased the risk for IS (OR=1.523, P=0.047). After adjustment for confounding factors, the odds ratios of -786 CC and -922 variant genotype (AG+GG) for IS were 4.580 and 1.656, respectively. However, haplotype analysis revealed the frequencies of Hap4 (GCG) and Hap7 (GCT) were significantly higher in the patients than the controls (P=0.035, 0.042). CONCLUSIONS: eNOS -922A/G and -786T/C may affect the susceptibility to IS and certain haplotypes of the eNOS gene may be associated with a higher susceptibility to IS.

[Study on immuno-effect with GRA4 or SAG2 gene recombinant BCG vaccine of Toxoplasma gondii].

OBJECTIVE: To compare the immuno-protection induced by the recombinant BCG vaccine of Toxoplasma gondii GRA4 gene (rBCG-GRA4) and SAG2 gene (rBCG-SAG2) in BALB/c mice. METHODS: 108 SPF BALB/c mice were divided into 6 groups: PBS, BCG, rBCG, rBCG-GRA4, rBCG-SAG2 and rBCG-GRA4+SAG2, each with 18 mice. Each mouse was injected by 100 microl corresponding materials for 2 times. Blood was taken from tail vein before inoculation. 4,6 and 8 weeks after inoculation, spleen was moved and blood was taken from orbit vein of 3 mice from each group for the detection of cytokines, IgG and IgM antibodies, T lymphocyte subgroups and transformation efficiency. 3 weeks after the last inoculation, 9 mice from each group were challenged intraperitoneally with 50 tachyzoites of T. gondii RH strain and their survival time was observed. RESULTS: rBCG vaccine of T. gondii induced immune response. The value of CD3+ CD4+/CD3+CD8+ of group BCG-GRA4+SAG2 was the highest (14.06+/-1.17) in the 4th week; the IgG titer in the BCG-GRA4+SAG2 group was the highest (0.18+/-0.02) in the 6th week and the IgM titer in the BCG-SAG2 group was the highest (0.82+/-0.05) in the 8th week. The average survival time of the mice in BCG-SAG2 group was about 8.61 days after challenged with tachyzoites, and that of the PBS control group, 7.33 days. The average survival time in the 3 immunized groups was one day longer than that of the control. CONCLUSION: The rBCG vaccine of T. gondii shows certain immuno-protection in mice.

An Integrated Control Strategy Takes Clonorchis sinensis Under Control in an Endemic Area in South China.

Clonorchis sinensis is an important foodborne zoonosis worldwide and prevalent in China for more than 2000 years. According to the experience of controlling Schistosoma japonica, China started to establish the integrated control strategy for C. sinensis in endemic areas. Lou village, the largest village in Shenzhen city in South China was taken as a pilot site. This longitudinal study assessed the infection status of C. sinensis among people and intermediate hosts from 2006 to 2014 in Lou village. After a continuous intervention with the integrated control strategy, the prevalence of C. sinensis decreased significantly to 2.01% in 2014. The infection intensity also reduced significantly with eggs per gram varying from 45.6 ± 3.4 in 2010 to 21.7 ± 1.6 in 2012. There is also a statistically significant decrease of the prevalence of C. sinensis metacercariae in fish hosts from 16.51% in 2008 before the intervention to 5.33% in 2014. All the old-styled toilets were replaced by sanitary ones with a harmless processing design in 2014. No viable parasite eggs were detected in stool samples from the reconstructed toilets. Health education played an important role in changing the eating habits among the local residents, with a significant decrease in the prevalence of eating raw fish from 91.99% in 2008 to 59.87% in 2014. The evaluation suggested that the integrated strategy we have performed in Lou village is effective in controlling the C. sinensis infection and maintaining the infection rate at a lower level, which can be promoted in other endemic areas.

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

Correlation of the content of hepatitis B core antigen in peripheral blood mononuclear cells with HBV virus load.

The dysfunction of peripheral blood mononuclear cell (PBMC) plays an important role in hepatitis B virus (HBV) chronic infection and tolerance. This study is aimed to explore the changes of expression and distribution of Hepatitis B virus core antigen (HBcAg) in the PBMCs of patients infected with chronic hepatitis B virus by using confocal laser scanning microscopy (CLSM). The levels of HBcAg in PBMCs were correlated with the HBV load in serum, and the change of HBcAg distribution in different PBMC organelles may represent various HBV infection states. HBcAg was mainly distributed in the nuclei of PBMC in the cases of immune tolerance and no inflammatory activity. Taken together, our data suggest that the measurement of HBcAg and its distribution in PBMCs using CLSM may serve as an alternative approach to monitor HBV load and the immune states of HBV infection with ease of using and improved sensitivity.