Dideoxy Chain Termination Method Research Articles

Indian and Chinese Origins of West African Village Chickens as Revealed by Mitochondrial DNA Partial D-Loop Regions

The study assessed the maternal genetic origins of West African village chickens using a 920-bp mtDNA D-loop region fragment of 127 samples from four populations (Gambia, Togo, Liberia and Nigeria). The mtDNA D-loop region was amplified following standard PCR protocols, using AV1F2: 5′-AGGACTACGGCTTGAAAAGC-3′ and 5′-TGCTTAAGGTTAATTACTGCTG-3′. as the forward and reverse primers respectively. Sequencing was done using Sanger’s dideoxy chain termination method. In addition, 268 sequence samples harvested from Genbank representing Gallus gallus from Africa, Mediterranean and different regions of Asia, were included in the analysis, bringing the total sample size to 395. 24 polymorphic sites and 13 West African chicken haplotypes (WAC1-13) were detected. The evolutionary relationships amongst studied populations and other African and global chicken populations were investigated using MEGA 7 and NETWORK 4.6 softwares. The results grouped 11 of the haplotypes including the dominant one (WAC1) with India, Pakistan and Egypt into haplogroup E, indicating an Indian sub-continental origin and Egypt as an entry point, while the other haplotypes (WAC8 and 10) clustered with haplogroup B widely believed to be of Chinese matrilineage and reported for the first time in West Africa by this study. Haplogroup B is thought to be a heritage of recent introgression of exotic alleles from commercial lines. The study shows that India and China were most likely geographic origins of the matriarchs of the West African village chickens.

First report of Phakopsora euvitis causing leaf rust disease on grapevine (Vitis labrusca L.) in India.

Grapevine (Vitis labrusca L.), a member of the family Vitaceae and native of North America, is grown as a table grape. During the survey for the grapevine diseases in May 2022, we noticed numerous yellow pustules of rust on lower side of leaves of 'Bangalore Bule' in Nandi village (13°22'59.7"N77°42'33.4"E), Chikkaballapur district of Karnataka state of India. The crop was at maturity stage and the rust disease severity was determined using the scale given by Angelotti et al. (2008), which was up to 10%. The disease symptoms were numerous small raised yellow pustules on the abaxial surface corresponding to adaxial surface chlorotic spots. In severe conditions, spots cover the entire leaf and defoliation occurs. Similar disease symptoms were reported by Ono (2000); Weinert et al. (2003); and Primiano et al. (2017). The pathogenicity test was performed on cuttings of 'Bangalore Bule' grapevine in a glasshouse at 25±1°C. The urediniospores were collected from diseased leaves using a brush, 3×104 ml-1 suspension in distilled water was used for inoculation on the abaxial surface of leaves. Control plants were sprayed with distilled water. The leaves developed symptoms in 15-17 days after inoculation, and the pathogen was confirmed by symptomatology and microscopic observation of urediniospores. Urediniospores were short-pedicellate, sessile, obovoid to obovoid-ellipsoid, and uniformly echinulate with 42.98-32.54 x 31.37-25.15 µm in size. The aecial stage of the Phakopsora has been reported on an alternate host, Meliosma simplicifolia (Hosagoudar 1988). As the internal transcribed spacer (ITS) region offers some utility in the molecular detection of the Phakopsora genus (Rush et al. 2019), the pathogen was confirmed by studying the different regions in the ITS such as ITS1, 5.8S rRNA, and ITS2. Total DNA was extracted from urediniospore mass using the Macherey-Nagel kit (Duren, Germany) by following the manufacturer's protocol. The quantity of isolated DNA was checked using an Qubit 3.0 fluorometer (Invitrogen) before being subjected to polymerase chain reaction (PCR) amplification in a thermocycler (Eppendorf-vapo.protect) using ITS1 and ITS4 primers (IDT, Singapore) targeting ITS1, 5.8S rRNA, and ITS2 regions, and the obtained amplicon (~700 bp) was purified using Macherey- Nagel Nucleospin gel and PCR clean-up kit (Duren, Germany) as per the manufacturer's protocol and sequenced by Sanger's dideoxy chain-termination method [ABI 3730 (48 capillaries) electrophoresis]. The sequence was edited in BioEdit (https://bioedit.software.informer.com/7.2/) and aligned in MUSCLE, and the phylogenetic tree was constructed in MEGA 11 using the neighbor-joining method by following the maximum likelihood criterion (Kumar et al. 2018). The sequence data was deposited at NCBI (accession number OP221661). The basic local alignment search tool (BLAST) search sequence of the isolate Nandi-KA in GenBank revealed 97.91% homology with sequence of Phakopsora sp. (accession number KC815548.1) and 96.87% with Phakopsora euvitis (accession number AB354790.1). Based on disease symptoms, fungal morphology, the pathogenicity test, and ITS sequence, the fungus was identified as Phakopsora euvitis, the pathogen causing grapevine leaf rust disease. Though there are similar disease symptoms observed on grapevine in India (EPPO 2016); the pathogen was not confirmed. To our knowledge, this is the first report of Phakopsora euvitis causing leaf rust disease in grapevine (V. labrusca) in India.

Genotypic distribution of MSTN gene polymorphisms involved in racing performance in Camelus dromedarius

Camel racing is one of the popular sports around the world and is growing rapidly especially in Gulf countries. Camel has adapted itself to harsh and draught climate of desert. ?his quality of camel makes it the best choice in racing industry. Pakistani breed Dromedarius camels are as good as Omani and Sudanese camel breeds in their racing potential. Myostatin (Growth differentiation factor 8) is a protein coded by MSTN gene. Polymorphism in MSTN play a significant role in growth of muscle, increasing fast glycolysis Type IIB muscle fiber, forming tubular aggregates in type IIB fiber by increasing the body strength and racing ability. This gene is also responsible for double muscle phenotype in bovines. In present study, Marecha and Brela camel breeds of Pakistan were studied for genomic characterization of MSTN gene. Blood samples were collected from the healthy animals between the age group of 2-4 years. Genomic DNA was extracted, amplified by using specific sets of primers, purified and sequenced by Sanger?s dideoxy chain termination method. No single nucleotide polymorphisms (SNPs) were found in the exon-3 of MSTN of Marecha & Brela, which shows that the gene is highly conserved among species while phylogenetic data of the dromedarius MSTN gene showed highest similarity with Bos taurus and least similarity with Gallus gallus. Genes that are similar to the camel MSTN are myostatin of Homo sapiens, Pan troglodytes, Bos taurus, Canis lupus, Rattus norvegicus. The significance of this study was to identify the genetic potential of Pakistani camel for racing that will help in the socioeconomic uplift of the local community. It will also help the camel breeders to select the best breed of camel & enhance their genetic potential by using least operating cost. That will in turn provide opportunity to the camel breeders to produce the best breed that will be selected by the trainers for racing that will provide another source of income for the local community and Pakistan?s camels will also be used in the racing industry.

Association of Peroxisome Proliferator-Activated Receptor Gamma 2 Gene Polymorphism with Susceptibility to Diabetic Periodontitis: Experience of a Tertiary Hospital in Fujian Province, China

To investigate the association of the interaction between peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) gene polymorphism and glycosylated hemoglobin (HbAlc) level with susceptibility to diabetic periodontitis (DP). A total of 259 DP patients and 257 patients with type 2 diabetes mellitus (T2DM) treated from August 2017 to April 2020 were enrolled, and another 180 healthy subjects were selected as control group. The HbA1c level of T2DM patients was determined using automatic biochemical analyzer. PPAR-γ2 gene Pro12Ala polymorphism was detected by the dideoxy chain termination method. The risk factors for DP were analyzed by multivariate logistic regression model. The adjusted odds ratio (OR) and 95% confidence interval of PPAR-γ2 gene Pro12Ala polymorphism and HbA1c level for the risk of DP were estimated, and the interaction between Pro12Ala polymorphism and HbA1c level was analyzed. The risk of DP increased in T2DM patients with genotypes Pro12Ala (PA) and Pro12Ala (AA) (OR=2.529, OR=2.594, P<0.01). T2DM patients with 6%≤ HbA1c <8% or HbA1c ≥8.0% had significantly elevated risk of DP (OR=2.046, OR=3.105), and T2DM patients with HbA1c ≥8.0% had higher risk of DP than those with 6%≤ HbA1c <8% (P<0.01). There was a positive interaction between HbA1c level and Pro12Ala (PA) and Pro12Ala (AA) (γ>1). Individuals with genotypes Pro12Ala (PA) or Pro12Ala (AA) are high risk population of DP. The interaction between these genotypes and HbA1c level facilitates the occurrence and development of DP. Measures should be taken to control HbA1c level or to regulate gene expression, so as to prevent DP.

A Unique Genotype of Pseudohypoaldosteronism Type 1b in a Highly Consanguineous Population.

ContextPseudohypoaldosteronism (PHA) is a condition in which serum aldosterone level is normal or elevated but its action is deficient.ObjectiveThis study describes the molecular genetics of PHA 1b in the highly consanguineous population of 2 Arabian Gulf countries, Saudi Arabia and Oman.MethodsThis study enrolled 22 patients from 13 unrelated families (2 families with 5 patients from Oman and 11 families with 17 patients from Saudi Arabia). All of these patients had presented within the first 10 days of life with nausea and vomiting, hyponatremia, hyperkalemia, and hypotension. We isolated DNA from peripheral blood and PCR-sequenced all exons and exon-intron boundaries of SCNN1A and, if negative, SCNN1B and SCNN1G using the Dideoxy Chain termination method.ResultsWe found a total of 8 mutations in 13 families as follows: 6 mutations in SCNN1A, 1 in SCNN1B, and 1 in SCNN1G. All of these mutations were novel except one. SCNN1A mutations were: c.1496A>G, p.Q499R (novel) in 1 patient; c.1453C>T, p.Q485X (novel) in 1 patient; c.1322_1322delA, p.N441Tfs*41 (novel) in 2 patients of 1 family; c.876 + 2 delGAGT (novel) in 3 patients of 1 family; c.203_204 delTC, p.I68Tfs*76 (a known mutation) in 8 patients of 5 families; and whole SCNN1A gene deletion (novel) in 2 patients of 2 families. In addition, a nonsense SCNN1B mutation c.1694C>A, p.S565X (novel) was found in 3 siblings from 1 Omani family, and an SCNN1G deletion mutation c.527_528 delCA, p.T176Rfs*9 (novel) in 2 siblings from another Omani family.ConclusionWe characterized a unique genotype of PHA 1b with several novel gene structure–disrupting mutations in SCNN1A, SCNN1B, and SCNN1G in a highly consanguineous population.

Clinical Application of Circulating Tumor DNA in Diffuse Large B-Cell Lymphoma--Review

Diffuse large B-cell lymphoma (DLBCL) as an aggressive lymphoma, there has not a good molecular marker to assess the therapeutic efficacy and prognosis of the disease. As compared with the traditional deteation method, it was found that the circulating tumor DNA (ctDNA) can be used as a non-invasive specific biomarker which can dynamically provide the information about the lymphoma. ctDNA in DLBCL can be obtained by dideoxy chain termination method combined with PCR, so as to detect genetic markers, targeted sequences of gene which is related to lymphoma; the digital PCR (dPCR) for lymphoma somatic mutations and the detection of abnormal methylation; ctDNA is closely related to the diagncsis, therapeutic efficiency and prognosis of DLBCL, thus ctDNA can be used for the early detection, mid-term and prognostic monitoring in DLBCL, which makes ctDNA have a broad clinical applied prospect.

SLC22A1 rs622342 Polymorphism Predicts Insulin Resistance Improvement in Patients with Type 2 Diabetes Mellitus Treated with Metformin: A Cross-Sectional Study

Background Metformin is the most widely used oral antidiabetic agent and can reduce insulin resistance (IR) effectively. Organic cation transporter 1 (encoded by SLC22A1) is responsible for the transport of metformin, and ataxia-telangiectasia-mutated (ATM) is a gene relating to the DNA repair and cell cycle control. The aim of this study was to evaluate if the genetic variants in SLC22A1 rs622342 and ATM rs11212617 could be effective predictors of islet function improvement in patients with type 2 diabetes mellitus (T2DM) on metformin treatment. Methods This cross-sectional study included 111 patients with T2DM treated with metformin. Genotyping was performed by the dideoxy chain-termination method. The homeostatic indexes of IR (HOMA-IR) and beta-cell function (HOMA-BCF) were determined according to the homeostasis model assessment. Results Fasting plasma glucose (FPG) levels, HbA1c levels, and HOMA-IR were significantly higher in patients with the rs622342 AA genotype than in those with C allele (P < 0.05). However, these significant differences were not observed between rs11212617 genotype groups. Further data analysis revealed that the association between the rs622342 polymorphism and HOMA-IR was gender related, and so was rs11212617 polymorphism and HOMA-BCF. HOMA-IR was significantly higher in males with rs622342 AA genotype than in those with C allele (P=0.021), and HOMA-BCF value was significantly higher in females carrying rs11212617 CC genotype than in those with A allele (P=0.038). The common logarithm (Lg10) of HOMA-BCF was positively correlated with the reciprocal of HbA1c (r = 0.629, P < 0.001) and negatively associated with Lg10 FPG (r = −0.708, P < 0.001). Conclusions The variant of rs622342 could be a predictor of insulin sensitivity in patients with T2DM treated with metformin. The association between the rs622342 polymorphism and HOMA-IR and the association between the rs11212617 polymorphism and HOMA-BCF were both gender related.

Comparative analysis of apicoplast genomes of Babesia infective to small ruminants in China

BackgroundBabesiosis is an economically important disease caused by tick-borne apicomplexan protists of the genus Babesia. Most apicomplexan parasites, including Babesia, have a plastid-derived organelle termed an apicoplast, which is involved in critical metabolic pathways such as fatty acid, iron-sulphur, haem and isoprenoid biosynthesis. Apicoplast genomic data can provide significant information for understanding and exploring the biological features, taxonomic and evolutionary relationships of apicomplexan parasites, and identify targets for anti-parasitic drugs. However, there are limited data on the apicoplast genomes of Babesia species infective to small ruminants.MethodsPCR primers were designed based on the previously reported apicoplast genome sequences of Babesia motasi Lintan and Babesia sp. Xinjiang using Illumina technology. The overlapped apicoplast genomic fragments of six ovine Babesia isolates were amplified and sequenced using the Sanger dideoxy chain-termination method. The full-length sequences of the apicoplast genomes were assembled and annotated using bioinformatics software. The gene contents and order of apicoplast genomes obtained in this study were defined and compared with those of other apicomplexan parasites. Phylogenetic trees were constructed on the concatenated amino acid sequences of 13 gene products using MEGA v.6.06.ResultsThe results showed that the six ovine Babesia apicoplast genomes consisted of circular DNA. The genome sizes were 29,916–30,846 bp with 78.7–81.0% A + T content, 29–31 open reading frames (ORF) and 23–24 transport RNAs. The ORFs encoded four DNA-directed RNA polymerase subunits (rpoB, rpoCl, rpoC2a and rpoC2b), 13 ribosomal proteins, one elongation factor TU (tufA), two ATP-dependent Clp proteases (ClpC) and 7–11 hypothetical proteins. Babesia sp. has three more genes than Babesia motasi (rpl5, rps8 and rpoB). Phylogenetic analysis showed that Babesia sp. is located in a separate clade. Babesia motasi Lintan/Tianzhu and B. motasi Ningxian/Hebei were divided into two subclades.ConclusionsTo our knowledge, this study is the first to elucidate the whole apicoplast genomic structural features of six Babesia isolates infective to small ruminants in China using Sanger sequencing. The data provide useful information confirming the taxonomic relationships of these parasites and identifying targets for anti-apicomplexan parasite drugs.

The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout

BackgroundViral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence).MethodsOverlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection.ResultsComplete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0–10% mortality).ConclusionsWhen chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.

Methylenetetrahydrofolate Reductase Gene Polymorphism C677T is Associated with Increased Risk of Coronary Heart Disease in Chinese Type 2 Diabetic Patients

Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus (T2DM). Previous researches report that methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms might influence the occurrence of coronary heart disease (CHD) in T2DM patients. The purpose of this study was to evaluate whether MTHFR C677T and A1298C mutations are associated with the risk of CHD in T2DM patients. Methods A total of 197 subjects with T2DM were studied, of which 95 patients with CHD. The genotypes of MTHFR C677T and A1298C were analyzed by using dideoxy chain-termination method, and compared between patients with CHD and those without CHD. Results We found that the frequency of the 677T allele was significantly higher in T2DM patients with CHD than those without CHD (P=0.011). However, there was no significant difference in any of the examined haplotypes between T2DM patients with and without CHD. Furthermore, the 677T allele was associated with a higher risk of CHD development in diabetic patients with lower homocysteine (Hcy) levels (≤15 μmol/L) (P=0.006), while no effect of MTHFR gene polymorphism on the incidence of CHD was found in patients with higher Hcy levels (>15 μmol/L) (P=0.491). Conclusion The MTHFR C677T gene polymorphism is associated with the risk of CHD of diabetic patients and could be used as an effective marker for CHD in Chinese diabetic populations with normal Hcy levels.

Molecular biology study of RHD gene in Chinese Han voluntary blood donors with RhD-negative blood type

Objective To explore the RHD gene structure of Chinese Han voluntary blood donors with RhD-negative blood type, and to conduct a molecular biological analysis of a newly discovered RHD gene mutation. Methods From 2016 to 2017, a total of 85 Chinese Han voluntary blood donors who participated in blood donation in Shandong Blood Center were selected as the subject by randomized digital table. Real-time fluorescence quantitative (RQ)-PCR method was used to detect exon 7 of RHD gene in DNA samples from 85 blood donors. The multiplex PCR-sequence specific primer (SSP) method was used to detect exons 3 to 7 and 9 of RHD gene in DNA samples from these 85 blood donors. The RHD gene in DNA samples from 30 blood donors whose DNA samples expressed RHD gene exon 7 were sequenced using a fluorescently labeled Sanger dideoxy chain termination method. The procedure of this study is in accordance with the requirements of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each blood donor. Results ① In 85 blood donors with RhD-negative blood type, DNA samples of 30 donors had exon 7 of RHD gene, and of 55 blood donors had deletion of exon 7 of RHD gene.② Among the 30 donors with RHD gene exon 7, the DNA samples of 29 donors had exons 3 to 7 and 9 of the RHD gene. Exon 3, 4, 5, 7 and 9 of RHD gene were present in DNA sample of one donor, but exon 6 was deleted. DNA samples from 55 donors without RHD gene exon 7, were deletion of RHD gene exons 3 to 7 and 9. ③ Among the 30 blood donors with RHD gene exon 7, 29 DNA samples had one c. 1227G>A point mutation on RHD gene exon 9. There was a non-sense mutation in c. 933C>A on RHD gene exon 6 in one blood donor′s DNA sample with deletion of the exon 6 of RHD gene. This mutation produced a stop codon, resulting in inconsistent multiplex PCR-SSP and DNA sequencing results of RHD gene in this blood donor. Conclusions The deletion of RHD gene is the main molecular mechanism that causes the RhD-negative phenotype of Chinese Han people. The RHD gene c. 1227G>A mutation is a major feature of Del phenotype. A new c. 933C>A mutation in the RHD gene results in a RhD-negative phenotype. Key words: Rh-Hr blood system; RhD-negativity; Red cells; Immunohematology; Immunogenetics

Mutational analysis of rare subtypes of congenital adrenal hyperplasia in a highly inbred population

ContextApart from 21 Hydroxylase deficiency, other subtypes of congenital adrenal hyperplasia (CAH) are rare. We studied the clinical features and molecular genetics of a relatively large series of patients with CYP17A1, HSD3β2 and StAR deficiencies. Patients and methodsWe studied 21 patients including 7 patients with CYP17A1, 10 patients with HSD3β2 and 4 patients with StAR deficiencies. For mutation detection, we isolated DNA from peripheral leucocytes, amplified genes of interest using polymerase chain reaction and directly sequenced the amplicons using Dideoxy Chain Termination method. ResultsRegardless of their karyotype, patients with CYP17A1 deficiency presented with normally looking external female genitalia and were raised as females. Hypertension and hypokalemia were prominent features in 4 of 7 patients. Two missense (p.R416H, p.R239Q) and 2 non-sense (p.Y329X, p.Y329X) mutations were found in these 7 cases. In 3 unrelated families with 10 affected siblings with HSD3β2 mutations, two non-sense mutations were found (p.Q334X, p.R335X). 46XY patients with HSD3β2 deficiency presented with ambiguous genitalia while 46XX patients presented with normal female external genitalia. Adrenal crisis was common in patients with both karyotypes. In the 4 patients with StAR deficiency, both genetic male and female patients presented with normally looking female external genitalia and adrenal crisis. One previously reported missense mutation (p.R182H) was found in 3 unrelated patients and a novel non-sense mutation (p.Q264X) in the fourth patient. ConclusionsThese cases of rare subtypes of CAH illustrate the heterogeneous phenotypic and genetic features of these subtypes and add unique novel mutations to the previously known ones.

Sequence Analysis and Phylogenetic Relationship of Myostatin Gene of Bikaneri Camel (Camelus dromedarius)

The draught potential of the Bikaneri camel depends on the gene affecting the muscular growth. Myostatin (MSTN) or growth and differentiation factor (GDF8) gene is the major regulator of myogenesis and skeletal muscle growth in mammals. Genomic DNA was isolated from whole blood of randomly selected Bikaneri camels (n=6) through spin column method. The MSTN exon-2 region of 375 bp was amplified using primers designed from homologous regions of MSTN gene sequence (GenBank accession number. DQ167575). The PCR amplified fragments of all the animals were sequenced through Sanger dideoxy chain termination method. The sequence of MSTN exon-2 gene was submitted to NCBI GenBank database to which an accession number KX863740 was assigned. A Neighborhood Joining (NJ) phylogenetic tree was constructed based on the lowest Bayesian Information Content (BIC) value. Sequence comparison of MSTN exon-2 gene of Bikaneri camel with homologous regions of goat, sheep, Algerian camel, buffalo, horse and pig revealed more than 95% homology. The sequence information generated for MSTN gene of Bikaneri camel would help in better understanding of growth traits and could support in conservation of dwindling camel population in Rajasthan.

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 → Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 → Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

Novel single nucleotide polymorphisms (SNPs) of CYP2W1 gene in Chinese Uygur and Han populations

Cytochrome P450 2W1 (CYP2W1) is expressed specially in certain cancers and could metabolize some substances into cytotoxic antitumor drugs in Caucasian ethnic population. To investigate the genetic polymorphism of CYP2W1 in Chinese, all the nine exons and exon–intron junctions were sequenced by dideoxy chain termination method among 385 Chinese subjects (including 223 Han and 162 Uygur). The present results showed that 40 single nucleotide polymorphisms (SNPs) were detected (14 in the exons and 26 in the introns), 10 were novel variants of which in Chinese. There were 7 novel SNPs in the exons and other 3 novel SNPs in the introns. Four of the 6 novel non-synonymous variations in exons, 131T > C (Leu44Pro), 1289C > A (Ala88Glu), 2027G > A (Arg187Gln) and 5070C > T (Thr383Met) were computationally predicted to affect CYP2W1 protein function, in spite of these variants were heterozygotes. Moreover, the allele frequencies in 6 known SNPs including CYP2W1*2 (2008G > A) and CYP2W1*3 (173A > C) were analyzed, which were significantly lower in Chinese Han (2.9% and 0.0%, respectively) and Uygur (5.2% and 0.0%, respectively) individuals, than those reported previously in Caucasians (9.1% and 33.1%, respectively, P < 0.05). These data provide useful information on the pharmacogenetic studies of CYP2W1 among Chinese and other ethnic populations.

Novel mutations in the genes TGM1 and ALOXE3 underlying autosomal recessive congenital ichthyosis

Ichthyoses are clinically characterized by scaling or hyperkeratosis of the skin or both. It can be an isolated condition limited to the skin or appear secondarily with involvement of other cutaneous or systemic abnormalities. The present study investigated clinical and molecular characterization of three consanguineous families (A, B, C) segregating two different forms of autosomal recessive congenital ichthyosis (ARCI). Linkage in three consanguineous families (A, B, C) segregating two different forms of ARCI was searched by typing microsatellite and single nucleotide polymorphism marker analysis. Sequencing of the two genes TGM1 and ALOXE3 was performed by the dideoxy chain termination method. Genome-wide linkage analysis established linkage in family A to TGM1 gene on chromosome 14q11 and in families B and C to ALOXE3 gene on chromosome 17p13. Subsequently, sequencing of these genes using samples from affected family members led to the identification of three novel mutations: a missense variant p.Trp455Arg in TGM1 (family A); a nonsense variant p.Arg140* in ALOXE3 (family B); and a complex rearrangement in ALOXE3 (family C). The present study further extends the spectrum of mutations in the two genes involved in causing ARCI. Characterizing the clinical spectrum resulting from mutations in the TGM1 and ALOXE3 genes will improve diagnosis and may direct clinical care of the family members.

Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing.

Aim:To study the major histocompatibility complex (MHC) Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and nucleotide sequencing techniques.Materials and Methods:DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR) software.Results:TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE), however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison.Conclusion:The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI), both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

Safety and efficacy analysis on modified FOLFOXIRI as first-line treatment for advanced colorectal cancer in China-single center experience

To evaluate the safety and preliminary efficacy of modified FOLFOXIRI (combination of reducing dosage irinotecan, oxaliplatin and fluorouracil) in first-line treatment for patients with metastatic colorectal cancer. A total of 53 patients with advanced colorectal cancer receiving modified FOLFOXIRI regimen were recruited continuously from January 2010 to January 2014. Safety profile was recorded based on NCI Common Terminology Criteria for Adverse Events version 3.0 (NCI-CTCAE v3.0). Objective response was evaluated by Response Evaluation Criteria in Solid Tumors version1.1 (RECIST 1.1) after administration of at least 4 cycles chemotherapy. Kras and Braf gene sequencing was tested by dideoxy chain-termination method. Relation between efficacy and two genes was examined. Among 53 patients, no treatment-related mortality was presented. The rate of grade 3 to 4 adverse event was 32.1% (17/53), including neutropenia 13.2%(7/53), anemia 11.3% (6/53) and fatigue 9.4% (5/53). Overall response rate (ORR) and disease control rate (DCR) were respectively 65.9% (29/44) and 90.0% (40/44). Radical resection rate (R0) was 29.5% (13/44). Efficacy of mFOLFOXIRI regimen plus targeting therapy was assessed in 44 patients. mFOLFOXIRI regimen plus targeting therapy achieved an ORR of 72.7% (8/11), which was higher than the ORR 65.9% (21/33) of triplet regimen alone, but the difference was not statistically significant (P=0.198). Paraffin specimens of 48 colorectal cancer cases were tested. Twenty-one cases were Kras mutant (43.75%), 3 cases were Braf mutant (6.25%). There were no significant differences between two groups (P>0.05). Reducing dosage mFOLFOXIRI can be safely used in advanced colorectal cancer and can achieve promising results in terms of short term efficacy.

Interference and disposition of hemoglobin variants on HbA1c testing results

Objective To analyze the causes of the HbA1c results discordant with blood glucose measurements, evaluate the interference degree of hemoglobin variants on the HbA1c results achieved by the method of ion exchange high performance liquid chromatography (IE-HPLC), and discuss the measures should be taken by the lab. Methods Four cases with inconsistency between the results of HbA1c with IE-HPLC method and those of blood glucose were collected. The dideoxy chain termination method was used for hemoglobin gene sequencing and immunoturbidimetric method (TINIA method) for detection of HbA1c. Hemoglobin electrophoresis analysis of whole blood samples was conducted with hemoglobin capillary electrophoresis method. Results There were 2 patients with Hb J-Bangkok .The genotypes were β41-42/βJ-Bangkok and βN/βJ-Bangkok, the accounts of Hb J-Bangkok were 93.9% and 52.4%, respectively.Three was one patient with Hb E.The genotype was βN/βE, the accounts of Hb E was 23.6%.There was one patient with Hb G-Taipei.The genotype was βN/βG-Taipei, the accounts of Hb G-Taipei was 39.4%. In the Hb J-Bangkok patients, the interferences of HbA1c results were found in both IE-HPLC method and TINIA method. In the rest 2 patients, the interferences were found in IE-HPLC method, but not in TINIA method. Conclusions β-thalassemia combined with hemoglobin variants including Hb J-Bangkok, Hb J-Bangkok, Hb E and Hb G-Taipei has interference to varying degrees on the detection of HbA1c with IE-HPLC. In these cases, the lab should use other methods not affected by Hb variants for HbA1c detection or alternative indicators to monitor glucose levels. （Chin J Lab Med,2014,37:123-126） Key words: Hemoglobin A, Glycosylated; Hemoglobins; Variation (genetics); Chromatography, high pressure liquid

First Report of Root-Knot Nematode Meloidogyne arenaria on Atractylodis macrocephalae in China.

Atractylodis macrocephalae is an important Chinese herbal medicine plant and its rhizome is of high medicinal value. In recent years, a severe decline in yield has been observed in Bozhou City (China's largest A. macrocephalae producing area), Anhui Province, China. A survey for plant-parasitic nematodes was conducted in this area from June to September 2011. Stunted plants displayed chlorotic or necrotic lower leaves near the ground part by the growth reduction; examination of the roots of stunted plants revealed the presence of galls typical of infestation by root knot nematode. Root nodules were found on the tap and lateral roots caused the fleshy tap root deformity. The incidence of diseased plants was estimated to be 45%, and yield loss was quantified as 43.5%. Nematodes were extracted from the root samples as previously described (4) and identified by morphology, enzyme analysis, and molecular characterization. Morphology of the female perineal patterns and measurements of the second-stage juveniles (J2s) matched those of the original description of Meloidogyne arenaria. Enzyme analysis of the esterase (Est) phenotype was also typical of the AII phenotype in M. arenaria (2). DNA was extracted according to a modified protocol (1), and the rDNA-internal transcribed spacer (ITS1_5.8S_ITS2) region was amplified with universal primers V5367 (5'-TTGATTACGTCCCTGCCCTTT-3') and 26S (5'-TTTCACTCGCCGTTACTAAGG-3'). PCR yielded a fragment of 764 bp and the purified product was sequenced by Sanger's dideoxy chain termination method (ABI3730). Sequences were identical to that of M. arenaria in GenBank (Accession No. AF387092) (3). Amplification of the D2/D3 fragments of the 28S RNA with universal primers D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') yielded a PCR fragment of 758 bp. These sequences were also identical to that of M. arenaria in GenBank (Accession No. AF435803). For further confirmation, amplification of the IGS region with universal primers 5S (5'-TTAACTTGCCAGATCGGACG-3') and 18S (5'-TCTAATGAGGGAACCAGCTACTA-3') yielded a PCR fragment of 713 bp. These sequences were 99.64% homologous to that of M. arenaria (GenBank Accession No. MAU42342). To our knowledge, this is the first report of M. arenaria species on A. macrocephalae in China. The fleshy tap root of A. macrocephalae is the main edible part of the plant, and the damage caused by root knot nematode will potentially reduce the yield and quality of this herb.