Different reference genomes determine different results: Comparing SNP calling in RAD-seq of Engelhardia roxburghiana using different reference genomes.

Advances in next-generation sequencing (NGS) have significantly reduced the cost and improved the efficiency of obtaining single nucleotide polymorphism (SNP) markers, particularly through restriction site-associated DNA sequencing (RAD-seq). Meanwhile, the progression in whole genome sequencing has led to the utilization of an increasing number of reference genomes in SNP calling processes. This study utilized RAD-seq data from 242 individuals of Engelhardia roxburghiana, a tropical tree of the walnut family (Juglandaceae), with SNP calling conducted using the STACKS pipeline. We aimed to compare both reference-based approaches, namely, employing a closely related species as the reference genome versus the species itself as the reference genome, to evaluate their respective merits and limitations. Our findings indicate a substantial discrepancy in the number of obtained SNPs between using a closely related species as opposed to the species itself as reference genomes, the former yielded approximately an order of magnitude fewer SNPs compared to the latter. While the missing rate of individuals and sites of the final SNPs obtained in the two scenarios showed no significant difference. The results showed that using the reference genome of the species itself tends to be prioritized in RAD-seq studies. However, if this is unavailable, considering closely related genomes is feasible due to their wide applicability and low missing rate as alternatives. This study contributes to enrich the understanding of the impact of SNP acquisition when utilizing different reference genomes.

Distinct non-clock-like signatures of the basal cell carcinomas from three sisters with a lethal Gorlin-Goltz syndrome.

BACKGROUND: Gorlin-Goltz syndrome (GS) is an inherited disease characterized by predisposition to basal cell carcinomas (BCCs) and various developmental defects, whose numerous disease-causing PTCH1 mutations have been identified in the hedgehog (Hh) signaling pathway. METHODS: In this study, whole exome sequencing was used to screen for both somatic and germline deleterious mutations in three sisters with a lethal GS. The mutations we found were confirmed by subcloning and Sanger sequencing of the genomic DNA. RNA-seq was performed to profile gene expression in paired BCCs samples and the expression levels for selected genes were validated by quantitative PCR. RESULTS: The clinical and histopathologic features were analyzed for the proband in the three-generation GS family. We identified the insertion mutation PTCH1 c.1341dupA (p. L448Tfs*49), which segregated with BCC phenotype and contributed to the death of two in four patients from a Chinese family with GS. Compared with adjacent non-cancerous tissues (ANCT), four second-hit mutations were found in four of the six pairs of BCC from three patients. Of note, somatic genomic alterations in all six BCC samples were mainly clustered into non-clock-like Signature 7 (ultraviolet mutagenesis) and 11 (related to certain alkylating agents). Both RNA-seq and quantitative RT-PCR confirmed that the mRNA levels of PTCH1 and its effector GLI1 were markedly upregulated in six pairs of BCC samples versus ANCT. CONCLUSIONS: The distinct non-clock-like signatures of BCCs indicated that GS was not a life-threatening illness. The main reasons for untimely death of GS patients were PTCH1 mutation, exposure to intense ultraviolet radiationand the poor economic conditions.

Genome Duplication Increases Meiotic Recombination Frequency: A Saccharomyces cerevisiae Model.

Genetic recombination characterized by reciprocal exchange of genes on paired homologous chromosomes is the most prominent event in meiosis of almost all sexually reproductive organisms. It contributes to genome stability by ensuring the balanced segregation of paired homologs in meiosis, and it is also the major driving factor in generating genetic variation for natural and artificial selection. Meiotic recombination is subjected to the control of a highly stringent and complex regulating process and meiotic recombination frequency (MRF) may be affected by biological and abiotic factors such as sex, gene density, nucleotide content, and chemical/temperature treatments, having motivated tremendous researches for artificially manipulating MRF. Whether genome polyploidization would lead to a significant change in MRF has attracted both historical and recent research interests; however, tackling this fundamental question is methodologically challenging due to the lack of appropriate methods for tetrasomic genetic analysis, thus has led to controversial conclusions in the literature. This article presents a comprehensive and rigorous survey of genome duplication-mediated change in MRF using Saccharomyces cerevisiae as a eukaryotic model. It demonstrates that genome duplication can lead to consistently significant increase in MRF and rate of crossovers across all 16 chromosomes of S. cerevisiae, including both cold and hot spots of MRF. This ploidy-driven change in MRF is associated with weakened recombination interference, enhanced double-strand break density, and loosened chromatin histone occupation. The study illuminates a significant evolutionary feature of genome duplication and opens an opportunity to accelerate response to artificial and natural selection through polyploidization.

Amn1 governs post-mitotic cell separation in Saccharomyces cerevisiae.

Post-mitotic cell separation is one of the most prominent events in the life cycle of eukaryotic cells, but the molecular underpinning of this fundamental biological process is far from being concluded and fully characterized. We use budding yeast Saccharomyces cerevisiae as a model and demonstrate AMN1 as a major gene underlying post-mitotic cell separation in a natural yeast strain, YL1C. Specifically, we define a novel 11-residue domain by which Amn1 binds to Ace2. Moreover, we demonstrate that Amn1 induces proteolysis of Ace2 through the ubiquitin proteasome system and in turn, down-regulates Ace2's downstream target genes involved in hydrolysis of the primary septum, thus leading to inhibition of cell separation and clumping of haploid yeast cells. Using ChIP assays and site-specific mutation experiments, we show that Ste12 and the a1-α12 heterodimer are two direct regulators of AMN1. Specifically, a1-α2, a diploid-specific heterodimer, prevents Ste12 from inactivating AMN1 through binding to its promoter. This demonstrates how the Amn1-governed cell separation is highly cell type dependent. Finally, we show that AMN1368D from YL1C is a dominant allele in most strains of S. cerevisiae and evolutionarily conserved in both genic structure and phenotypic effect in two closely related yeast species, K. lactis and C. glabrata.

Diagnostic value of interferon-γ release assay of peripheral blood and pleural effusion for tuberculous pleurisy / 解放军医学杂志

Objective To evaluate the diagnostic value of interferon-γ release assay of blood and pleural effusion for tuberculous pleurisy.Methods Fifty-six adult patients with suspected tuberculous pleurisy were enrolled in our study.The blood and pleural effusion interferon-γ release assay were measured by T-SPOT.TB test in 38 pleural tuberculosis patients and 18 nontuberculous pleurisy controls.The diagnostic sensitivity,specificity,predictive value of T-SPOT.TB in pleural effusion mononuclear cells (PE-MC) and peripheral mononuclear cells (PBMC) were analyzed.Results The sensitivities and specificities,positive predictive values and negative predictive values,respectively,of the PE-MC and PBMC for diagnosing were as follows:86.5％(95％ confidence interval[CI] 71.2％-95.5％) and 100％(95％CI 90.5％-100％);52.9％(95％CI 27.8％-77.0％) and 35.3％(95％CI 14.2％-61.7％);80.0％(95％CI 64.4％-90.9％) and 77.1％(95％CI 62.7％-88.0％);64.3％(95％CI 35.1％-87.2％) and 100％(95％CI 54.1％-100％).By ROC curve analysis,a cut-off value of 47SFC/2.5 × 105 cells in PE-MC showed a sensitivity of 89.2％ and a specificity of 88.2％.Conclusion T-SPOT.TB in PE-MC could be an accurate diagnostic method for tuberculous pleurisy in TB endemic settings.Moreover,47SFC/2.5 × 105 cells might be the optimal cut-off value for diagnosing tuberculous pleurisy.

A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large plant populations.

KEY MESSAGE: This optimized approach provides both a computational tool and a library construction protocol, which can maximize the number of genomic sequence reads that uniformly cover a plant genome and minimize the number of sequence reads representing chloroplast DNA and rRNA genes. One can implement the developed computational tool to feasibly design their own RAD-seq experiment to achieve expected coverage of sequence variant markers for large plant populations using information of the genome sequence and ideally, though not necessarily, information of the sequence polymorphism distribution in the genome. Advent of the next generation sequencing techniques motivates recent interest in developing sequence-based identification and genotyping of genome-wide genetic variants in large populations, with RAD-seq being a typical example. Without taking proper account for the fact that chloroplast and rRNA genes may occupy up to 60 % of the resulting sequence reads, the current RAD-seq design could be very inefficient for plant and crop species. We presented here a generic computational tool to optimize RAD-seq design in any plant species and experimentally tested the optimized design by implementing it to screen for and genotype sequence variants in four plant populations of diploid and autotetraploid Arabidopsis and potato Solanum tuberosum. Sequence data from the optimized RAD-seq experiments shows that the undesirable chloroplast and rRNA contributed sequence reads can be controlled at 3-10 %. Additionally, the optimized RAD-seq method enables pre-design of the required uniformity and density in coverage of the high quality sequence polymorphic markers over the genome of interest and genotyping of large plant or crop populations at a competitive cost in comparison to other mainstream rivals in the literature.

MicroRNA-708-5p acts as a therapeutic agent against metastatic lung cancer.

MicroRNAs (miRNAs) have recently been recognized as targets for anti-metastatic therapy against cancer malignancy. Development of effective miRNA mediated therapies remains a challenge to both basic research and clinical practice. Here we presented the evidence for a miR-708-5p mediated replacement therapy against metastatic lung cancer. Expression of miR-708-5p was substantially reduced in metastatic lung cancer samples and cancer cell lines when compared to non-metastatic counterparts. Expression of the miRNA suppressed cell survival and metastasis in vitro through its direct target p21, and inhibited the PI3K/AKT pathway and stem cell-like characteristics of lung cancer cells. Systemic administration of this miRNA in a mouse model of NSCLC using polyethylenimine (PEI)-mediated delivery of unmodified miRNA mimics induced tumor specific apoptosis. It also effectively protected the tested animals from developing metastatic malignancy without causing any observed toxicity. The findings strongly support miR-708-5p as a novel and effective therapeutic agent against metastatic malignancy of non-small cell lung cancer.

Inhibitory effect of total saponins of Panax notoginseng on rat bone marrow mesenchymal stem cell apoptosis / 生理学报

The study was aimed to investigate the effects of total saponins of Panax notoginseng (tPNS) on cobalt chloride (CoCl2)-induced apoptosis of rat bone marrow mesenchymal stem cells (rBMSCs) and the underlying mechanism. rBMSCs were isolated by density gradient centrifugation from Sprague Dawley (SD) rats. After being incubated with different concentrations of tPNS (1, 10, 100 μg/mL) for 48 h, the rBMSCs were stained with EdU and PI for proliferation and cell cycle assay, respectively. CoCl2 group was treated with 300 μmol CoCl2 for 24 h, and different concentrations tPNS groups were treated with 300 μmol CoCl2 plus 1, 10 or 100 μg/mL tPNS. After Annexin V-FITC/PI staining, flow cytometry was applied to measure the cell apoptosis. For mitochondrial membrane potential assay, rhodamine123 and Hoechst33342 staining were used. qRT-PCR was applied to analyze gene expression of Bcl-2 family. The results showed that the proliferation rates of the three concentrations tPNS groups were all higher than that of the control group (all P < 0.05). Compared with control group, only 100 μg/mL tPNS group exhibited increased cell percentage of S and G2 phase. Compared with that in control group (without CoCl2), the apoptotic rate was increased by 14.2% in CoCl2 group. And the apoptotic rates were reduced by 14.4%, 12.8% and 13.9% in three concentrations tPNS groups, compared with that in CoCl2 group (all P < 0.01). CoCl2 could decrease the mitochondrial membrane potential, while different concentrations of tPNS reversed the inhibitory effect of CoCl2. Bcl-2 and Bcl-xl mRNA expressions in all tPNS groups were higher than those in CoCl2 group (all P < 0.05). Moreover, 10 and 100 μg/mL tPNS groups showed lower ratios of Bax/Bcl-2, compared with CoCl2 group. The results suggest that tPNS protects the rBMSCs against CoCl2-induced apoptosis through improving the cell mitochondrial membrane potential, up-regulating the expressions of anti-apoptosis genes Bcl-2 and Bcl-xl, and reducing the Bax/Bcl-2 gene expression ratio.

3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision model, which postulates a clash between simultaneous transcriptional processes occurring on opposite DNA strands.

Mechanism of HLA-G participation in inhibiting lymphocyte proliferation by amniotic mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the inhibitory mechanism of human amniotic mesenchymal stem cells (HAMSC) on lymphocyte proliferation and to validate the participation of the nonclassic human leukocyte antigen (HLA) class I molecule (HLA-G) in immunosuppressive action of HAMSC. HAMSC were isolated from fetal membranes of human placentas, and were cultured and expanded. The phenotypes of HAMSC were identified by flow cytometry, at same time the HLA-G levels on membrane surface and in cytoplasm were detected by flow cytometry. The soluble HLA-G (sHLA-G) level in HAMSC supernatants was determined by ELISA, MTT assay was used to examine the effect of mixed cultured HAMSC on proliferation of lymphocytes. The results showed that both surface and cytoplasm of HAMSC expressed HLA-G, the average rates of HLA-G expression on surface and in cytoplasm were (16.75 ± 3.871)% and (39.14 ± 4.274)%, respectively. The sHLA-G level in cell culture supernatant was 5.2 ng/ml. After HAMSC and culture supernatants were added in the MLR, the inhibitory rate on lymphocyte proliferation increased obviously, meanwhile the inhibitory rate on lymphocyte proliferation decreased when the HLA-G antibody was added in MLR. It is concluded that the surface and cytoplasm of HAMSC express HAL-G, at same time HAMSC secrete the HLA-G to supernatants of culture. The HLA-G is one of critical factors inhibiting immuno-function of HAMSC. This study contributes to improve the clinical therapeutic trails for using the HAMSC to prevent rejection.

Polygenic molecular architecture underlying non-sexual cell aggregation in budding yeast.

Cell aggregation in unicellular organisms, induced by either cell non-sexual adhesion to yield flocs and biofilm, or pheromone-driving sexual conjugation is of great significance in cellular stress response, medicine, and brewing industries. Most current literatures have focused on one form of cell aggregation termed flocculation and its major molecular determinants, the flocculation (FLO) family genes. Here, we implemented a map-based approach for dissecting the molecular basis of non-sexual cell aggregation in Saccharomyces cerevisiae. Genome-wide mapping has identified four major quantitative trait loci (QTL) underlying nature variation in the cell aggregation phenotype. High-resolution mapping following up with knockout and allele replacement experiments resolved the QTL into the underlying genes (AMN1, RGA1, FLO1, and FLO8) or even into the causative nucleotide. Genetic variation in the QTL genes can explain up to 46% of phenotypic variation of this trait. Of these genes, AMN1 plays the leading role, differing from the FLO family members, in regulating expression of cell clumping phenotype through inducing cell segregation defect. These findings provide novel insights into the molecular mechanism of how cell aggregation is regulated in budding yeast, and the data will be directly implicated to understand the molecular basis and evolutionary implications of cell aggregation in other fungus species.

Induction of dendritic cells with multidrug resistance from K562/MDR1 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To induce the differentiation of K562/MDR1 cells into dendritic cells (DC) with multidrug resistance property.</p><p><b>METHODS</b>K562/MDR1 cells and K562 cells were cultured in the presence of GM-CSF and IL-4 to generate DC and matured by TNF-α. On d14 K562/MDR1-DC and K562-DC cells were harvested and the expressions of CD1a, CD83, CD80, CD86, HLA-ABC and HLA-DR were assessed by flow cytometry (FCM). The antigen presentation function of K562/MDR1-DC and K562-DC was determined by allogenic mixed lymphocyte reaction (Allo-MLR). The expression of P-glycoprotein and the intracellular accumulation of daunorubicin (DNR) were detected by FCM. The sensitivity of K562/MDR1-DC and K562-DC cell to vincristine, adriamycin was measured using MTT assay.</p><p><b>RESULTS</b>Both K562/MDR1 and K562 cells were differentiated into dendritic cells in the presence of cytokine cocktails, showing the morphologic and immunophenotypic characteristics of DC. K562/MDR1-DC more markedly enhanced proliferation of allogeneic lymphocytes in MLR than K562-DC. High level expression of P-glycoprotein and efflux of DNR were demonstrated in K562/MDR1-DC. K562/MDR1-DC showed multidrug resistance property, with higher IC(50) to VCR and ADM than that of K562-DCs.</p><p><b>CONCLUSION</b>K562/MDR1 cells can be differentiated into DC with the presence of cytokines, the induced K562/MDR1-DC cells express high level of P-glycoprotein and acquire the multidrug resistance property.</p>

Clinical observation on abdominal cluster-needling combined with sacro-iliac-needling for treatment of chronic pelvic inflammation / 中国针灸

<p><b>OBJECTIVE</b>To observe therapeutic effect of abdominal cluster-needling combined with sacro-iliac-needling on chronic pelvic inflammation.</p><p><b>METHODS</b>One hundred and ten cases of pelvic inflammation were randomly divided into a treatment group and a control group. The treatment group of 70 cases were treated by abdominal cluster-needling combined with sacro-iliac-needling; the control group of 40 cases were treated by oral administration of Fuyankang tablet. Their therapeutic effects were compared.</p><p><b>RESULTS</b>The cured rate and the total effective rate were 75.7% and 97.1% in the treatment group, and 37.5% and 85.0% in the control group, respectively, with significant difference between the two groups (P < 0.01, P < 0.05).</p><p><b>CONCLUSION</b>The therapeutic effect of abdominal cluster-needling combined with sacro-iliac-needling is significantly better than that in the control group for chronic pelvic inflammation.</p>