Investigation of sex expression profiles and the cantharidin biosynthesis genes in two blister beetles.

Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles, Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase (HMGR; EC:1.1.1.34), which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase (HMGS; EC:2.3.3.10) and isopentenyl-diphosphate Delta-isomerase (IDI; EC:5.3.3.2) were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase (FOHSDR; EC:1.1.1.216) and farnesyl diphosphate synthase (FDPS; EC:2.5.1.1) had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase (ACAT; EC:2.3.1.9) gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.

RNA editing affects cis-regulatory elements and predicts adverse cancer survival.

BACKGROUND: RNA editing exerts critical impacts on numerous biological processes and thus are implicated in crucial human phenotypes, including tumorigenesis and prognosis. While previous studies have analyzed aggregate RNA editing activity at the sample level and associated it with overall cancer survival, there is not yet a large-scale disease-specific survival study to examine genome-wide RNA editing sites' prognostic value taking into account the host gene expression and clinical variables. METHODS: In this study, we solved comprehensive Cox proportional models of disease-specific survival on individual RNA-editing sites plus host gene expression and critical demographic covariates. This allowed us to interrogate the prognostic value of a large number of RNA-editing sites at single-nucleotide resolution. RESULTS: As a result, we identified 402 gene-proximal RNA-editing sites that generally predict adverse cancer survival. For example, an RNA-editing site residing in ZNF264 indicates poor survival of uterine corpus endometrial carcinoma, with a hazard ratio of 2.13 and an adjusted p-value of 4.07 × 10-7 . Some of these prognostic RNA-editing sites mediate the binding of RNA binding proteins and microRNAs, thus propagating their impacts to extensive regulatory targets. CONCLUSIONS: In conclusion, RNA editing affects cis-regulatory elements and predicts adverse cancer survival.

Genomic content of chemosensory receptors in two sister blister beetles facilitates characterization of chemosensory evolution.

BACKGROUND: More than 2500 species belong to the Meloidae family (Coleoptera: Tenebrionoidea), members of which produce the potent defensive blistering agent cantharidin and are commonly known as blister beetles or Spanishflies. Cantharidin has recently been used for cancer therapy. Hycleus cichorii and Hycleus phaleratus have been used in traditional Chinese medicine for more than 2000 years due to their ability to biosynthesize cantharidin. To understand the role of the chemosensory system in beetle evolution, we comparatively analysed the chemosensory receptor families of both blister beetle species and compared them with those of other beetles. RESULTS: We identified 89 odorant receptors (ORs), 86 gustatory receptors (GRs), and 45 ionotropic receptors (IRs) in H. phaleratus and 149 ORs, 102 GRs and 50 IRs in H. cichorii. Nine groups of beetle ORs were recovered, and a similar pattern of ORs in Coleoptera emerged. Two evident expanded clades in Hycleus (Groups 5A and 3) were reconstructed in the phylogenetic tree. Four of eight genes with evidence of positive selection were clustered in the expanded clades of Group 5A. Three, eight and three orthologous pairs of CO2, sugar and fructose receptors, respectively, were identified in both blister beetles. Two evident expanded clades of putative bitter GRs in Hycleus were also found, and the GR in one clade had notably low divergence. Interestingly, IR41a was specifically expanded in blister beetles compared to other insects identified to date, and IR75 was also clearly expanded in both blister beetles based on our phylogenetic tree analysis. Moreover, evidence of positive selection was detected for eight ORs, three GRs and two IRs, half of which were from five duplicate clades. CONCLUSIONS: We first annotated the chemosensory receptor families in a pair of sister beetle genomes (Meloidae: Hycleus), which facilitated evolutionary analysis of chemosensory receptors between sibling species in the Coleoptera group. Our analysis suggests that changes in chemosensory receptors have a possible role in chemical-based species evolution in blister beetles. Future studies should include more species to verify this correlation, which will help us understand the evolution of blister beetles.

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus.

Background: Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. Results: We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Conclusions: Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles.

A novel mitochondrial DNA deletion in a patient with Pearson syndrome and neonatal diabetes mellitus provides insight into disease etiology, severity and progression.

Pearson syndrome (PS) is a rare, mitochondrial DNA (mtDNA) deletion disorder mainly affecting hematopoietic system and exocrine pancreas in early infancy, which is characterized by multi-organ involvement, variable manifestations and poor prognosis. Since the clinical complexity and uncertain outcome of PS, the ability to early diagnose and anticipate disease progression is of great clinical importance. We described a patient with severe anemia and hyperglycinemia at birth was diagnosed with neonatal diabetes mellitus, and later with PS. Genetic testing revealed that a novel mtDNA deletion existed in various non-invasive tissues from the patient. The disease course was monitored by mtDNA deletion heteroplasmy and mtDNA/nucleus DNA genome ratio in different tissues and at different time points, showing a potential genotype-phenotype correlation. Our findings suggest that for patient suspected for PS, it may be therapeutically important to first perform detailed mtDNA analysis on non-invasive tissues at the initial diagnosis and during disease progression.

Tri-allelic pattern of short tandem repeats identifies the murderer among identical twins and suggests an embryonic mutational origin.

Monozygotic twins can be co-identified by genotyping of short tandem repeats (STRs); however, for distinguishing them, STR genotyping is ineffective, especially in the case of murder. Here, a rarely occurring tri-allelic pattern in the vWA locus (16, 18, 19) was identified only in the DNA of one identical twin, which could help to exonerate the innocent twin in a murder charge. This mutation was defined as primary through genotyping of the family and could be detected in blood, buccal and semen samples from the individual; however, two alternative allele-balanced di-allelic patterns (16, 18 or 16, 19) were detected in hair root sheath cells. Such a kind of segregation indicates a one-step mutation occurs in cell mitosis, which is after embryonic zygote formation and during the early development of the individual after the division of the blastocyte. Sequencing revealed the insertion between the allele 18 and 19 is a repeat unit of TAGA/TCTA (plus/minus strand), which belongs to "AGAT/ATCT"-based core repeats identified from all tri-allelic pattern reports recorded in the STR base and a detailed model was proposed for STR repeat length variation caused by false priming during DNA synthesis. Our model illustrates the possible origination of allele-balanced and unbalanced tri-allelic pattern, clarifies that the genotypes of parent-child mismatches, aberrant di-allelic patterns, and type 1 or 2 tri-allelic patterns should be considered as independent, but interconnected forms of STR mutation.

Female specific association between NNMT gene and schizophrenia in a Han Chinese population.

Accumulating evidence has shown that alterations in one carbon metabolism might play an important role in the pathogenesis of schizophrenia (SZ). Nicotinamide-N-methyltransferase (NNMT) is one of the key enzymes of one-carbon metabolism. To examine whether NNMT gene was associated with SZ in Han Chinese population, we selected seven single nucleotide polymorphisms (SNPs) in NNMT gene, and investigated its association with SZ from a cohort of 42 SZ patients and 86 healthy controls by Mass-ARRAY technology. Statistical analyses revealed that one (rs694539) of the SNPs in the female subgroup showed significant difference between SZ patients and controls both in genotypic (p= 0.0170) and allelic frequencies (p = 0.0059). We also found that the frequency of haplotype 'A G G C T C T' in the female patients was significantly higher than in controls (p=0.0015). Our results suggest that NNMT rs694539 may have a role in the etiology of SZ in a Han Chinese female population.

Mitochondrial haplogroups and hypervariable region polymorphisms in schizophrenia: a case-control study.

Previous studies have detected associations between mitochondrial haplogroups and schizophrenia (SZ). However, no study has examined the relationship between major mitochondrial DNA (mtDNA) haplogroups and SZ in the Chinese population. The aim of this study was to assess the association between mtDNA haplogroups and SZ genesis in the Chinese Han population. We used a case-control study and sequenced the mtDNA hypervariable regions (HVR1, HVR2, and HVR3) in the Han population. We analyzed mtDNA haplogroups and HVR polymorphisms in 298 SZ patients and 298 controls. The haplotypes were classified into 10 major haplogroups: A, B, CZ, D, F, G, M, N, N9a, and R. Statistical analysis revealed that only N9a showed a nominally significant association with protection from SZ [1.68% vs. 6.38%, p=0.004, OR=0.251 (0.092-0.680); after adjustment for age and sex: p=0.006, OR=0.246 (0.090-0.669)]. Three HVR polymorphisms were found to be nominally significantly different between subjects with SZ and controls, and all except one (m.204T>C) are linked to the N9a haplogroup. Our results indicate that mtDNA haplogroup N9a might be a protective factor for SZ.

The distribution of genetic diversity of KIR genes in the Chinese Mongolian population.

Killer cell immunoglobulin-like receptors (KIRs) are expressed on natural killer cells and as such regulate their response against infection and malignancy. KIR genes are variable in gene content and type, which results in different KIR haplotypes, and can be used to discriminate individuals and populations from different regions or ethnic groups. In the present study, we represent the first report on the KIR gene frequency and content diversities of 14 KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and 2 pseudogenes (KIR3DP1 and 2DP1) in the Chinese Mongolian population. The 16 detected KIR genes were all observed. All the individuals were typed positive for the four framework genes KIR3DL3, 3DL2, 2DL4 and the pseudogene KIR3DP1, as well as for the pseudogene KIR2DP1. The observed carrier gene frequencies (OF) of the other KIR genes ranged from 16% at the KIR2DL2 locus to 93% at the KIR3DL1 locus. Over all, 48 different gene profiles were found in the study population and the most commonly observed KIR gene profile with a frequency of 14% consisted of KIR2DL4, 3DL2, 3DL3, 2DP1, 3DP1, 2DL1, 2DL3 and 3DL1 which belongs to the AA genotype. Principal component analysis (PCA) and the dendrogram illustrated the genetic distances between our study population and previously published populations from other ethnic groups or regions. The results of the present study show that the KIR gene family is highly polymorphic and can be a valuable tool for enriching the Chinese ethnical gene information resources, for anthropological studies, as well as for KIR gene related disease research.

Genetic polymorphisms of 15 STR loci in Chinese Han population living in Xi'an city of Shaanxi Province.

Fifteen autosomal STRs loci were analyzed from a population sample of 446 unrelated healthy individuals of Chinese Han population residing in Xi'an city of Shaanxi Province using a multiplex PCR system. We report allele frequencies distribution and statistical parameters for all STR loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA. The observed genotype frequencies and expected of genotype frequencies were evaluated by chi(2)-test and the Fisher exact tests. Chi-Square test showed all STR loci were in Hardy-Weinberg equilibrium (p<0.05). Our studied population data were compared with the previously published population data of other ethnics or areas. After comparing, significant differences were found between Chinese Han population in Xi'an and Korean, Tibetan, Uigur, Chinese Han population in Guangdong Province, Hui population, Chinese Salar, Dongxiang and Tu ethnic minority living in Qinghai Province of China at some loci. The forensic parameters from the data showed high values. The data in the present study can be used greatly for routine forensic application in the region of Xi'an city.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

[Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene].

AIM: To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene. METHODS: The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot. RESULTS: The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramatically in mRNA and at protein level. CONCLUSION: The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.

Silencing of CXCR4 inhibits the proliferation, adhesion, chemotaxis and invasion of salivary gland mucoepidermoid carcinoma Mc3 cells in vitro.

Mucoepidermoid carcinoma is the most common malignant tumor in salivary glands and high-grade mucoepidermoid carcinoma is often accompanied with poor prognosis. Many recent research works demonstrated that stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor-4 (CXCR4) interaction was critical for metastasis of various cancers. In this study, the immunoexpression of CXCR4 in human salivary gland mucoepidermoid carcinoma in different grades was detected by immunohistochemical analysis and the expression of CXCR4 and its ligand SDF-1 in mucoepidermoid carcinoma MEC-1 cell line and its highly metastatic clone Mc3 was examined by RT-PCR, flow cytometry and immunocytochemical analysis. It was found that CXCR4 was over-expressed in Mc3 cell line and SDF-1 was expressed in both cell lines at a nearly equal level. We further constructed CXCR4-shRNA expression vector to stably transfect Mc3 cells. We found that silencing of endogenous CXCR4 gene expression in Mc3 cells resulted in inhibition of the proliferation, adhesion, chemotaxis and invasion of Mc3 cells in vitro. This study implies that CXCR4 molecule is a potential factor controlling the proliferation and metastasis of Mc3 cells.

[Expression of a candidate human Era binding protein A19 and the preparation of its polyclonal antibody].

AIM: To express a candidate hEra binding protein A19 in Escherichia coli and to prepare anti-A19 antibody. METHODS: A19 gene was amplified by PCR from the plasmid containing A19 gene and was cloned into the expression vector pGEX-4T3 which was then transformed into E.coli. The A19 protein was expressed under IPTG induction. Antiserum was prepared by immunizing rabbits with the expressed A19 protein. The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: The expressed A19 accounted for about 30.2% of total bacterial protein. The titer of the antiserum was about 1:4 000. Western blot analysis indicated that the antiserum had high specificity. CONCLUSION: A19 fusion protein was highly expressed. The specific anti-A19 antiserum was prepared successfully.

[Expression of human Fab antibody against keratin in E.coli and its renaturation].

AIM: To express Fd fragment and L chain of human anti-keratin Fab in E.coli BL21(DE3) respectively and obtain human anti-keratin Fab by renaturation in-vitro. METHODS: Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab were subcloned into vector pET32a respectively. The recombinant plasmids pETL and pETFd were transformed into E.coli BL21(DE3) and induced to express with IPTG. Fd fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. The renatured Fab was characterized by SDS-PAGE, Western blot and ELISA. RESULTS: Fd fragment and L chain of human anti-keratin Fab were efficiently expressed. ELISA and Western blot showed that the renatured Fab could bind with human keratin. CONCLUSION: The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.

[Epidemiological investigation of Taenia saginata asiatica in Duyun, Guizhou and detection of amino acids and elements of adult worms].

OBJECTIVE: To study epidemiological factors of taeniasis and to detect amino acid and element components of adult worms in Duyun of Guizhou Province. METHODS: 1. Traditional methods were used for epidemiological investigation. 2. Automatic amino acid analyzer and bioassay were applied for the detection. RESULTS: Among 70 persons with clinical symptoms, 25 patients (24 men and 1 woman) were found to have adult taenia worms in their faeces after taking Areca catechu L. and other drugs. Sixteen amino acids and 12 elements were determined in adult worms. CONCLUSION: Duyun area in Guizhou is a highly endemic area of taeniasis. The pathogenic parasite is identified as Taenia saginata asiatica. Its clinical symptoms are similar to that of Taenia saginata saginata.