Epidemiology of Plasmodium vivax in Duffy negatives and Duffy positives from community and health centre collections in Ethiopia.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Deep Learning Techniques to Characterize the RPS28P7 Pseudogene and the Metazoa-SRP Gene as Drug Potential Targets in Pancreatic Cancer Patients.

The molecular explanation about why some pancreatic cancer (PaCa) patients die early and others die later is poorly understood. This study aimed to discover potential novel markers and drug targets that could be useful to stratify and extend expected survival in prospective early-death patients. We deployed a deep learning algorithm and analyzed the gene copy number, gene expression, and protein expression data of death versus alive PaCa patients from the GDC cohort. The genes with higher relative amplification (copy number >4 times in the dead compared with the alive group) were EWSR1, FLT3, GPC3, HIF1A, HLF, and MEN1. The most highly up-regulated genes (>8.5-fold change) in the death group were RPL30, RPL37, RPS28P7, RPS11, Metazoa_SRP, CAPNS1, FN1, H3-3B, LCN2, and OAZ1. None of their corresponding proteins were up or down-regulated in the death group. The mRNA of the RPS28P7 pseudogene could act as ceRNA sponging the miRNA that was originally directed to the parental gene RPS28. We propose RPS28P7 mRNA as the most druggable target that can be modulated with small molecules or the RNA technology approach. These markers could be added as criteria to patient stratification in future PaCa drug trials, but further validation in the target populations is encouraged.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil.

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Dental caries prevalence and severity positively associate with AMY1 gene copy number.

OBJECTIVE: To establish the possible relation between total caries (TC) and caries severity (CS) with the AMY1 gene copy number (AMY1GCN). MATERIALS AND METHODS: This was an observational, cross-sectional, population-based, and association study with 303 participants. Each participant underwent a complete anamnesis and stomatological check-up, and peripheral blood was obtained to extract gDNA. TC and CS were determined as the number of caries at the dental exploration and the number of dental surfaces affected by caries, respectively, and AMY1GCN was determined by qPCR. RESULTS: We found an elevated caries prevalence (92.7%); TC and CS were 8 ± 10 and 10 ± 13 (median ± IR). There were higher TC and CS in those participants with AMY1GCN above the mean value (0.02 and 0.01 p values, respectively). A positive correlation between TC and CS with AMY1GCN (0.11 and 0.125 r values, 0.03 and 0.01 p values, respectively) was found, in addition to an association between TC and CS with AMY1GCN (1.5 and 1.6 OR values, 0.48 and 0.26 p values, respectively). CONCLUSION: TC and CS were positively related to the AMY1GCN. CLINICAL RELEVANCE: Dental caries has a high prevalence and a multifactorial etiology and has been related to a genetic component. Indeed, the salivary enzyme alpha-amylase could play a significant role in caries susceptibility, considering that its codifying gene (AMY1) can show variation in its gene copy number. This can be considered an important factor for the development of caries at a genetic level.

Comparison of Plasmodium Vivax Infections in Duffy Negatives From Community and Health Center Collections in Ethiopia.

Background: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 4.2 million annual cases and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and P. falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. Methods: A total of 9,580 and 4,667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centers. Results: Among the community-based cross-sectional samples, infection rate of P. vivax among the Duffy positives was 2-22 fold higher than among the Duffy negatives. Parasite positivity rate was 10-50 fold higher in Duffy positive than Duffy negatives among samples collected from the health center settings and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. P. vivax parasitemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. Conclusions: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centers. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

The genome of Anoplarchus purpurescens (Stichaeidae) reflects its carnivorous diet.

Digestion is driven by digestive enzymes and digestive enzyme gene copy number can provide insights on the genomic underpinnings of dietary specialization. The "Adaptive Modulation Hypothesis" (AMH) proposes that digestive enzyme activity, which increases with increased gene copy number, should correlate with substrate quantity in the diet. To test the AMH and reveal some of the genetics of herbivory vs carnivory, we sequenced, assembled, and annotated the genome of Anoplarchus purpurescens, a carnivorous prickleback fish in the family Stichaeidae, and compared the gene copy number for key digestive enzymes to that of Cebidichthys violaceus, a herbivorous fish from the same family. A highly contiguous genome assembly of high quality (N50 = 10.6 Mb) was produced for A. purpurescens, using combined long-read and short-read technology, with an estimated 33,842 protein-coding genes. The digestive enzymes that we examined include pancreatic α-amylase, carboxyl ester lipase, alanyl aminopeptidase, trypsin, and chymotrypsin. Anoplarchus purpurescens had fewer copies of pancreatic α-amylase (carbohydrate digestion) than C. violaceus (1 vs. 3 copies). Moreover, A. purpurescens had one fewer copy of carboxyl ester lipase (plant lipid digestion) than C. violaceus (4 vs. 5). We observed an expansion in copy number for several protein digestion genes in A. purpurescens compared to C. violaceus, including trypsin (5 vs. 3) and total aminopeptidases (6 vs. 5). Collectively, these genomic differences coincide with measured digestive enzyme activities (phenotypes) in the two species and they support the AMH. Moreover, this genomic resource is now available to better understand fish biology and dietary specialization.

Replication mechanisms of circular ssDNA plant viruses and their potential implication in viral gene expression regulation.

The replication of members of the two circular single-stranded DNA (ssDNA) virus families Geminiviridae and Nanoviridae, the only ssDNA viruses infecting plants, is believed to be processed by rolling-circle replication (RCR) and recombination-dependent replication (RDR) mechanisms. RCR is a ubiquitous replication mode for circular ssDNA viruses and involves a virus-encoded Replication-associated protein (Rep) which fulfills multiple functions in the replication mechanism. Two key genomic elements have been identified for RCR in Geminiviridae and Nanoviridae: (i) short iterative sequences called iterons which determine the specific recognition of the viral DNA by the Rep and (ii) a sequence enabling the formation of a stem-loop structure which contains a conserved motif and constitutes the origin of replication. In addition, studies in Geminiviridae provided evidence for a second replication mode, RDR, which has also been documented in some double-stranded DNA viruses. Here, we provide a synthesis of the current understanding of the two presumed replication modes of Geminiviridae and Nanoviridae, and we identify knowledge gaps and discuss the possibility that these replication mechanisms could regulate viral gene expression through modulation of gene copy number.

Genomic insights into linezolid-resistant Enterococci revealed its evolutionary diversity and poxtA copy number heterogeneity.

OBJECTIVES: This study aimed to determine the molecular mechanisms of linezolid-resistant enterococci (LRE) in swine slaughterhouses in China and apply the "One Health" perspective to analyse the evolutionary dynamics of poxtA-positive E. faecium in clinical and non-clinical settings worldwide. METHODS: The phenotypic and genomic characteristics of multiple LRE isolates were systematically investigated using antimicrobial susceptibility testing, transfer assays, evolutionary experiments, quantitative RT-PCR assays, whole-genome sequencing, and bioinformatics analyses. RESULTS: Swine faeces served as a significant reservoir for LRE isolates, and optrA and poxtA were the primary contributors to linezolid resistance. Co-occurrence network analysis revealed a significant interconnection between optrA and several other ARGs. The poxtA copy number heterogeneity and polymorphism were initially observed in E. faecium parental and evolved isolates. The poxtA-carrying tandem repeat region exhibits high mobility and has undergone extensive duplication owing to linezolid pressure. The poxtA copy number varies from four copies on the plasmid of E. faecium IC25 to 11 copies on the plasmid and six copies on the chromosome in the evolved isolate IC25-50_poxtA. Furthermore, phylogenetic analysis of 185 poxtA-positive E. faecium strains worldwide found that one isolate from a French patient in 2018 shared only two SNPs with CC17 E. faecium isolates IC25 and IC7-2 from this study, highlighting the potential global transmission of CC17 poxtA-positive E. faecium between humans and animals. CONCLUSION: This study identified amplification of poxtA as a response of E. faecium to linezolid pressure. Phylogenetic analysis shed light on the potential global transmission of hospital-associated CC17 poxtA-positive E. faecium in clinical and non-clinical settings.

A review of the molecular mechanisms of acaricide resistance in mites and ticks.

The Arachnida subclass of Acari comprises many harmful pests that threaten agriculture as well as animal health, including herbivorous spider mites, the bee parasite Varroa, the poultry mite Dermanyssus and several species of ticks. Especially in agriculture, acaricides are often used intensively to minimize the damage they inflict, promoting the development of resistance. Beneficial predatory mites used in biological control are also subjected to acaricide selection in the field. The development and use of new genetic and genomic tools such as genome and transcriptome sequencing, bulked segregant analysis (QTL mapping), and reverse genetics via RNAi or CRISPR/Cas9, have greatly increased our understanding of the molecular genetic mechanisms of resistance in Acari, especially in the spider mite Tetranychus urticae which emerged as a model species. These new techniques allowed to uncover and validate new resistance mutations in a larger range of species. In addition, they provided an impetus to start elucidating more challenging questions on mechanisms of gene regulation of detoxification associated with resistance.

Evaluation of C4 Gene Copy Number in Pediatric Acute Neuropsychiatric Syndrome.

Pediatric acute-onset neuropsychiatric syndrome (PANS) is an abrupt-onset neuropsychiatric disorder. PANS patients have an increased prevalence of comorbid autoimmune illness, most commonly arthritis. In addition, an estimated one-third of PANS patients present with low serum C4 protein, suggesting decreased production or increased consumption of C4 protein. To test the possibility that copy number (CN) variation contributes to risk of PANS illness, we compared mean total C4A and total C4B CN in ethnically matched subjects from PANS DNA samples and controls (192 cases and 182 controls). Longitudinal data from the Stanford PANS cohort (n = 121) were used to assess whether the time to juvenile idiopathic arthritis (JIA) or autoimmune disease (AI) onset was a function of total C4A or C4B CN. Lastly, we performed several hypothesis-generating analyses to explore the correlation between individual C4 gene variants, sex, specific genotypes, and age of PANS onset. Although the mean total C4A or C4B CN did not differ in PANS compared to controls, PANS patients with low C4B CN were at increased risk for subsequent JIA diagnosis (hazard ratio = 2.7, p value = 0.004). We also observed a possible increase in risk for AI in PANS patients and a possible correlation between lower C4B and PANS age of onset. An association between rheumatoid arthritis and low C4B CN has been reported previously. However, patients with PANS develop different types of JIA: enthesitis-related arthritis, spondyloarthritis, and psoriatic arthritis. This suggests that C4B plays a role that spans these arthritis types.

Genomic characterisation of de novo EGFR copy number gain and its impact on the efficacy of first-line EGFR-tyrosine kinase inhibitors for EGFR mutated non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation generally respond well to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). However, genomic characterisation of de novo EGFR copy number gain (CNG) and its impact on the efficacy of first-line EGFR-TKIs remains unclear. METHODS: This multicenter, retrospective and real-world study included two cohorts that enroled EGFR mutant NSCLC patients. EGFR CNG was tested by next-generation sequencing of untreated tissue specimens. Cohort 1 detected the impact of EGFR CNG on first-line EGFR-TKIs treatment, and cohort 2 explored the genomic characterisation. RESULTS: Cohort 1 enroled 355 patients from four cancer centres between January 2013 and March 2022. The patients were divided into three groups, included the EGFR non-CNG, EGFR CNG, and EGFR uncertain-CNG. No significant difference in progression-free survival (PFS) was found between the three groups (10.0 months vs. 10.8 months vs. 9.9 months, respectively, p = 0.384). Furthermore, the overall response rate was not statistically significant in the EGFR CNG group compared to the EGFR non-CNG or uncertain arm (70.3% vs. 63.2% vs. 54.5%, respectively, p = 0.154). Cohort 2 included 7876 NSCLC patients with 16.4% showing EGFR CNG. Gene mutations such as TP53, IKZF1, RAC1, MYC, MET, CDKN2A/B and alterations of the metabolic-related and ERK signalling pathway were significantly associated with patients with EGFR CNG compared to those without. CONCLUSIONS: De novo EGFR CNG had no effect on the efficacy of first-line EGFR-TKI treatment in EGFR mutant NSCLC patients, and tumours with EGFR CNG had more complex genomic profiles than those without.

Prognostic Values of Gene Copy Number Alterations in Prostate Cancer.

Whilst risk prediction for individual prostate cancer (PCa) cases is of a high priority, the current risk stratification indices for PCa management have severe limitations. This study aimed to identify gene copy number alterations (CNAs) with prognostic values and to determine if any combination of gene CNAs could have risk stratification potentials. Clinical and genomic data of 500 PCa cases from the Cancer Genome Atlas stable were retrieved from the Genomic Data Commons and cBioPortal databases. The CNA statuses of a total of 52 genetic markers, including 21 novel markers and 31 previously identified potential prognostic markers, were tested for prognostic significance. The CNA statuses of a total of 51/52 genetic markers were significantly associated with advanced disease at an odds ratio threshold of ≥1.5 or ≤0.667. Moreover, a Kaplan-Meier test identified 27/52 marker CNAs which correlated with disease progression. A Cox Regression analysis showed that the amplification of MIR602 and deletions of MIR602, ZNF267, MROH1, PARP8, and HCN1 correlated with a progression-free survival independent of the disease stage and Gleason prognostic group grade. Furthermore, a binary logistic regression analysis identified twenty-two panels of markers with risk stratification potentials. The best model of 7/52 genetic CNAs, which included the SPOP alteration, SPP1 alteration, CCND1 amplification, PTEN deletion, CDKN1B deletion, PARP8 deletion, and NKX3.1 deletion, stratified the PCa cases into a localised and advanced disease with an accuracy of 70.0%, sensitivity of 85.4%, specificity of 44.9%, positive predictive value of 71.67%, and negative predictive value of 65.35%. This study validated prognostic gene level CNAs identified in previous studies, as well as identified new genetic markers with CNAs that could potentially impact risk stratification in PCa.

Improvement of recombinant L-Asparaginase production in Pichia pastoris.

Pichia pastoris is a successful expression system that is frequently preferred in the secretion of proteins for both basic research and industrial purposes. In this study, recombinant Rhizomucor miehei (RmASNase) L-asparaginase was produced in Pichia pastoris. The impact of gene copy number on increasing protein production was examined with six clones harboring various gene copy numbers (1-5 and 5 +). The results demonstrated that the clone with three copies of the expression cassette integrated had the highest production level. Also, biochemical characterization of the enzyme was performed. It was determined that the optimum pH and temperature values of the purified enzyme were pH 7.0 and 50 °C, respectively. Stability analyses of the enzyme showed that it maintains its activity of 80% in the pH range of 5-9 and 67% in the temperature range of 20-50 °C. Ca+2 and Mn+2 ions increased the enzyme activity to 121% and 138%, respectively. In future studies, it is also possible to improve the activity and stability values of the enzyme with advanced molecular techniques and to increase production efficiency by producing at fermenter scale and under optimum conditions.

Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

Grapevine genome analysis demonstrates the role of gene copy number variation in the formation of monoterpenes.

Volatile organic compounds such as terpenes influence the quality parameters of grapevine through their contribution to the flavour and aroma profile of berries. Biosynthesis of volatile organic compounds in grapevine is relatively complex and controlled by multiple genes, the majority of which are unknown or uncharacterised. To identify the genomic regions that associate with modulation of these compounds in grapevine berries, volatile metabolic data generated via GC-MS from a grapevine mapping population was used to identify quantitative trait loci (QTLs). Several significant QTLs were associated with terpenes, and candidate genes were proposed for sesquiterpene and monoterpene biosynthesis. For monoterpenes, loci on chromosomes 12 and 13 were shown to be associated with geraniol and cyclic monoterpene accumulation, respectively. The locus on chromosome 12 was shown to contain a geraniol synthase gene (VvGer), while the locus on chromosome 13 contained an α-terpineol synthase gene (VvTer). Molecular and genomic investigation of VvGer and VvTer revealed that these genes were found in tandemly duplicated clusters, displaying high levels of hemizygosity. Gene copy number analysis further showed that not only did VvTer and VvGer copy numbers vary within the mapping population, but also across recently sequenced Vitis cultivars. Significantly, VvTer copy number correlated with both VvTer gene expression and cyclic monoterpene accumulation in the mapping population. A hypothesis for a hyper-functional VvTer allele linked to increased gene copy number in the mapping population is presented and can potentially lead to selection of cultivars with modulated terpene profiles. The study highlights the impact of VvTPS gene duplication and copy number variation on terpene accumulation in grapevine.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam.

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic congener of dioxin and has serious long-term effects on the environment and human health. Pyruvate Kinase L/R (PKLR) gene expression levels and gene variants are associated with pyruvate kinase enzyme deficiency, which has been identified as the cause of several diseases linked to dioxin exposure. In this study, we estimated PKLR gene copy number and gene expression levels using real-time quantitative PCR (RT-qPCR) assays, genotyped PKLR SNP rs3020781 by Sanger sequencing, and quantified plasma pyruvate kinase enzyme activity in 100 individuals exposed to Agent Orange/Dioxin near Bien Hoa and Da Nang airfields in Vietnam and 100 healthy controls. The means of PKLR copy numbers and PKLR gene expression levels were significantly higher, while pyruvate kinase enzyme activity was significantly decreased in Agent Orange/Dioxin-exposed individuals compared to healthy controls (P < 0.0001). Positive correlations of PKLR gene copy number and gene expression with 2,3,7,8-TCDD concentrations were observed (r = 0.2, P = 0.045 and r = 0.54, P < 0.0001, respectively). In contrast, pyruvate kinase enzyme activity was inversely correlated with 2,3,7,8-TCDD concentrations (r = -0.52, P < 0.0001). PKLR gene copy number and gene expression levels were also inversely correlated with pyruvate kinase enzyme activity. Additionally, PKLR SNP rs3020781 was found to be associated with 2,3,7,8-TCDD concentrations and PKLR gene expression. In conclusion, PKLR copy number, gene expression levels, and pyruvate kinase enzyme activity are associated with 2,3,7,8-TCDD exposure in individuals living in Agent Orange/Dioxin-contaminated areas.

The triterpenoid saponin content difference is associated with the two type oxidosqualene cyclase gene copy numbers of Pulsatilla chinensis and Pulsatilla cernua.

Pulsatilla chinensis is an important medicinal herb, its dried radix is used to treat the inflammation since ancient China. Triterpenoid saponins are proved to be the main active compounds of Pulsatilla genus. The triterpenoid saponin contents vary widely in different Pulsatilla species. But no enzyme involved in the triterpenoid saponin biosynthetic pathway was identified in Pulsitilla genus. This seriously limits the explanation of the triterpene content difference of Pulsatilla species. In this article, we obtained two oxidosqualene cyclase (OSC) genes from P. chinensis and P. cernua by touchdown PCR and anchored PCR. These two OSCs converted 2,3-oxidosqualene into different triterpenoids. The OSC from P. cernua is a monofunctional enzyme for ß-amyrin synthesis, while the OSC from P. chinensis is a multifunctional enzyme for lupeol and ß-amyrin synthesis, and the lupeol is the main product. Then we identified the 260th amino acid residue was the key site for the product difference by gene fusion and site-directed mutant technology. When the 260th amino acid residue was tryptophan (W260) and phenylalanine (F260), the main catalysate was ß-amyrin and lupeol, respectively. Then we found that the expression of these two genes was strongly correlated with the lupeol-type and ß-amyrin-type triterpenoid contents in P. cernua and P. chinensis. Finally, we found the gene copy number difference of these two genotypes leaded to the triterpenoid diversity in P. cernua and P. chinensis. This study provides useful information for the molecular breeding and quality improvement of P. chinensis and a molecular marker to identify the P. chinensis decoction pieces.

The complement system and human autoimmune diseases.

Genetic deficiencies of early components of the classical complement activation pathway (especially C1q, r, s, and C4) are the strongest monogenic causal factors for the prototypic autoimmune disease systemic lupus erythematosus (SLE), but their prevalence is extremely rare. In contrast, isotype genetic deficiency of C4A and acquired deficiency of C1q by autoantibodies are frequent among patients with SLE. Here we review the genetic basis of complement deficiencies in autoimmune disease, discuss the complex genetic diversity seen in complement C4 and its association with autoimmune disease, provide guidance as to when clinicians should suspect and test for complement deficiencies, and outline the current understanding of the mechanisms relating complement deficiencies to autoimmunity. We focus primarily on SLE, as the role of complement in SLE is well-established, but will also discuss other informative diseases such as inflammatory arthritis and myositis.