Restriction Enzymes HhaI Research Articles

Exploring polymorphism's impact on layer production traits: A deep dive into association studies and candidate genes in Kadaknath

This investigation aimed to study the polymorphism and association of important egg production linked candidate genes and layer performances in Kadaknath. The Genomic DNA samples were obtained from blood of 112 birds that were chosen at random. RestrictionFragment Length Polymorphism (RFLP) patterns of three candidate genes viz., cGH, MTNR1C and VIPR-1 was determined using SacI, MboI and HhaI restriction enzymes, respectively. The alleles were separated on 2 % AGE and their molecular sizes were estimated.Two out of the three genes studied i.e. cGH and MTNR1C showed polymorphism however VIPR-1 revealed monomorphic patterns. The different genotypes obtained were then associated with layer performance traits using least-squares analysis of variance. The cGH/SacI genotype was found to have a substantial impact on egg production up to 40 weeks (EP40) and age at sexual maturity (ASM) via association analysis. The cGH gene's AC genotype was linked to the lowest ASM (192.41 ± 11.84 days) and greatest EP40 (36.72 ± 8.86). The EP40 was also affected significantly by MTNR1C/MboI genotypes and birds with AA genotype attained higher body weight at 20 weeks (BW20) and had the highest EP40 (49.29 ± 6.84). The findings suggest that the markers identified can be used in Marker Assisted Selection primarily to select Kadaknath birds for higher BW20 (p ≤ 0.05) and may be used to select birds for lower ASM and higher EP40 with relatively lower level of significance (p ≤ 0.15). It emphasizes on adapting candidate gene PCR-RFLP genotype based selection for faster genetic improvement in layer economic traits of Kadaknath.

Genetic diversity of the PvMSP-3α gene in Plasmodium vivax isolates circulating in the National Capital Region (NCR) of India

Malaria is still a public health problem in tropical countries like India; major malaria parasite species are Plasmodium falciparum and P. vivax. Of which, P. vivax is responsible for ∼40% of the malaria burden at least in the Indian scenario. Unfortunately, there is limited data on the population structure and genetic diversity of P. vivax parasites in India. In this study, we investigated the genetic diversity of P. vivax strains in the South-west district, Delhi and, Nuh district, Haryana [National Capital Region (NCR)], using a polymorphic marker- P. vivax merozoite surface protein-3α (PvMSP-3α) gene. Dried blood spots from microscopically confirmed P. vivax patients were used for investigation of the PvMSP-3α gene. PCR-RFLP was performed on the PvMSP-3α gene to investigate the genotypes and allelic variability with HhaI and AluI restriction enzymes. In total, 40 successfully PCR amplified PvMSP-3α gene segments were subjected to RFLP analysis. Amplified products showed three different base pair size variations viz. genotype A in 31(77.5%), genotype B in 4(10%) and genotype C in 5(12.5%) P. vivax specimens. RFLP with HhaI and AluI revealed 17 (H1-H17) and 25 (A1-A25) allelic variants, respectively. Interestingly, two similar sub-allelic variants, ie. H8 (with HhaI), and A4 (with AluI) clustered within the rural area of Nuh district, Haryana in two samples. With this study, we propose to commission such type of genetic diversity analysis of P. vivax to investigate the circulating genotypes of the parasites from distinct geographical locations across India, that can have significant implications in understanding the population structures of P. vivax.

#191 An unusual cause of hypocalcemia in a young patient

Abstract Background and Aims A 33-year-old man visited the Nephrology Department outpatient clinic with abnormal laboratory findings: his serum calcium level was 5.9 mg/dL in a recent health examination. He denied any underlying disease and was on no medications. He complained of a tingling sensation in his hands and of eyelid twitching. On physical examination, he showed Chvostek's sign. His laboratory findings including serum calcium level are shown in Table 1. He did not have any morphological abnormalities, such as a round face, brachydactyly, or dental anomalies. Method Since he showed hypocalcemia, normal 25-hydroxyvitamin D, and elevated parathyroid hormone (PTH) levels, the presence of PTH resistance was presumed. Under strong suspicion of pseudohypoparathyroidism (PHP) in this patient, DNA sequence analysis and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of GNAS and STX16, genetic regions that are often affected by mutations or imprinting changes in PHP, was performed. No pathogenic mutations in GNAS and STX16 were detected by sequencing. Results Results of MS-MLPA of the GNAS locus demonstrated abnormal methylation patterns in the patient after HhaI restriction enzyme treatment. The methylation ratio showed a gain of methylation in the NESP55 region and loss of methylation in the AS, XL, and A/B differentially methylated regions (DMRs) in the patient as compared to a healthy control (Fig. 1). The patient was diagnosed with PHP type 1B (PHP1B) due to the methylation abnormalities in multiple DMRs. His parents both showed normal serum calcium levels; thus, no further genetic testing was conducted. He is on outpatient follow-up with adequate calcium replacement and no specific symptoms. Conclusion PHP is a rare disorder of unknown prevalence. It is characterized by hypocalcemia, hyperphosphatemia, and increased PTH levels. PHP1B is a subtype of PHP characterized by isolated renal PTH resistance, usually without physical abnormalities such as Albright hereditary osteodystrophy or other endocrine abnormalities. PHP1B can be sporadic or familial. The genetic characteristics of most sporadic PHP1B remains unclear, but GNAS imprinting abnormalities and hypomethylation at the A/B DMRs have been reported. Symptoms of PHP1B usually begin in childhood due to low calcium levels and may include numbness, seizures, tetany, cataracts, and dental problems. However, as in our case, specific symptoms may not occur until adulthood. Therefore, when evaluating the cause of hypocalcemia, it is necessary to differentiate PHP1B, even in adults, and particularly in cases with hyperphosphatemia and high PTH levels.

APOE-E4 allele as a potential marker for implantation failure: A comparison between fertile women, ART success and RIF patients.

Apolipoprotein E (APOE) is the most important precursor for the production of steroid hormones and is also involved in regulating the function of steroid hormones, hence playing a significant role in reproductive processes. So, APOE gene expression may be correlated with the implantation process. This study tries to make a better clarification of the correlation between APOE gene polymorphisms and recurrent implantation failure (RIF), where we compared the frequency of APOE polymorphisms in RIF patients, assisted reproductive treatment (ART) success cases and fertile women. In all, 100 women with successful ART who got pregnant (fetal heart rate positive) in their first or second cycle of invitro fertilization or intracytoplasmic sperm injection, 100 infertile RIF cases, and 100 normal fertile control cases with at least one live birth were included in present study. Following DNA extraction, genotypes were determined through polymerase chain reaction-restriction fragment length polymorphism method using HhaI restriction enzyme. Finally, statistical analysis was performed by chi-squared (χ2) test in SPSS software (P < 0.05). The RIF group showed significantly higher frequency for E3/E4 genotype (29%) compared with the other two control groups (fertile = 15%, ART success [ART+] = 13%) (P = 0.007). There was also a significantly higher frequency of the E4 allele in the RIF group (14.5%) compared with both of the control groups (fertile = 7.5%, ART+ = 6.5%) (P = 0.018). APOE4 is correlated with recurrent failure in the process of embryo implantation and, accordingly, it may potentially be considered a possible risk factor to the implantation process. The presence of E4 can be proposed as a predictive indicator in determining the results of assisted reproductive techniques.

A Smart DNA Hydrogel Enables Synergistic Immunotherapy and Photodynamic Therapy of Melanoma.

Immunotherapy faces insufficient immune activation and limited immune effectiveness. Herein, we report a smart DNA hydrogel that enables the release of multivalent functional units at the tumor site to enhance the efficacy of immunotherapy. The smart DNA hydrogel was assembled from two types of ultra-long DNA chains synthesized via rolling circle amplification. One DNA chain contained immune adjuvant CpG oligonucleotides and polyaptamers for loading natural killer cell-derived exosomes; the other chain contained multivalent G-quadruplex for loading photodynamic agents. DNA chains formed DNA hydrogel through base-pairing. HhaI restriction endonuclease sites were designed between functional units. Upon stimuli in the tumor sites, the hydrogel was effectively cleaved by the released HhaI and disassembled into functional units. Natural killer cell-derived exosomes played an anti-tumor role, and the CpG oligonucleotide activated antigen-presenting cells to enhance the immunotherapy. Besides the tumor-killing effect of photodynamic therapy, the generated cellular debris acted as an immune antigen to further enhance the immunotherapeutic effect. In a mouse melanoma orthotopic model, the smart DNA hydrogel as a localized therapeutic agent, achieved a remarkable tumor suppression rate of 91.2 %. The smart DNA hydrogel exhibited enhanced efficacy of synergistic immunotherapy and photodynamic therapy, expanding the application of DNA materials in biomedicine.

A female patient carrying a novel DMD mutation with non-random X-chromosome inactivation from a DMD family

ObjectiveTo analyze the clinical phenotype and genetic characteristics of a female proband carrying a novel mutation in the DMD gene with non-random X-chromosome inactivation in a large pedigree with pseudohypertrophic muscular dystrophy.MethodsClinical information of the female proband, her monozygotic twin sister, and other family members were collected. Potential pathogenic variants were detected with Multiplex Ligation-dependent Probe Amplification (MLPA) and whole-exome sequencing (WES). Methylation-sensitive restriction enzyme (HhaI) was employed for X-chromosome inactivation analysis.ResultsThe proband was a female over 5 years old, displayed clinical manifestations such as elevated creatine kinase (CK) levels and mild calf muscle hypertrophy. Her monozygotic twin sister exhibited normal CK levels and motor ability. Her uncle and cousin had a history of DMD. WES revealed that the proband carried a novel variant in the DMD (OMIM: 300,377) gene: NM_004006.3: c.3051_3053dup; NP_003997.2: p.Tyr1018*. In this pedigree, five out of six female members were carriers of this variant, while the cousin and uncle were hemizygous for this variant. X-chromosome inactivation analysis suggested non-random inactivation in the proband.ConclusionThe c.3051_3053dup (p.Tyr1018*) variant in the DMD gene is considered to be the pathogenic variant significantly associated with the clinical phenotype of the proband, her cousin, and her uncle within this family. Integrating genetic testing with clinical phenotype assessment can be a valuable tool for physicians in the diagnosis of progressive muscular dystrophies, such as Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD).

PCR-RFLP metoda u kombinaciji sa elektroforezom na čipu kao alat za određivanje varijabilnosti između važnih vrsta Aspergillus

Aspergillus species are among the most significant producers of aflatoxins, which can contaminate a wide range of agricultural and food products at any stage of production. The aim of this research was to utilize molecular methods to determine and characterize the variability between isolates of standard Aspergillus species. Genomic DNA was isolated from the mycelium of all tested Aspergillus isolates. PCR amplifications were performed using gene-specific primers. The PCR method successfully amplified the ITS1-5.8S rDNA-ITS2 region and portions of the β-tubulin and calmodulin genes of all tested Aspergillus isolates. PCR products obtained after amplification with primer pairs (ITS1/ITS4 and Bt2a/Bt2b), followed by digestion with restriction enzymes HhaI, MwoI, and AlwI in RFLP analysis, facilitated the identification of variability among the studied Aspergillus species. The results of PCR-RFLP analysis on the tested isolates were consistent with those previously obtained through morphological examinations, indicating the effectiveness of this molecular method for identification and determination of variability among important Aspergillus species. The presented molecular method based on PCR-RFLP analysis, due to its advantages such as reproducibility, speed, and high sensitivity, represents a valuable tool for monitoring and controlling contamination by Aspergillus species in the food supply chain. The method described in this study can be successfully used for rapid identification and determination of variability between isolates of Aspergillus species, contributing to improved food safety control and public health.

Prominence of Bioresponsive DNA Nanococoons in Tackling Post-Surgery Cancer Recurrence

Post-surgery cancer recurrence is one of the reasons for increased cancer cases. The effective usage of the enhanced permeability and retention effect of a nanocarrier infused with the bioresponsive release mechanism of checkpoint inhibitors (aPD1 and aCTLA4) can become a boon to mankind. DNA nanococoons (DNCs) comprising cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) with potent immunostimulatory effects can significantly enhance anti-cancer activity. Triglycerylmonostearate (TGMS) with enzymatic cleavage potential at the wound sites of tumor resection, upon caging with restriction enzyme (HhaI) followed by attaching to DNCs, makes the immunotherapy bioresponsive. Hhal-TGMS-DNCs-aPD1 triggered by the inflammation at the wound site undergoes enzymatic cleavage, releases the restriction enzyme, converts DNCs to CpG ODNs sequentially and with sustained aPD1 release exerts an appreciable anti-cancer effect.

Association of vasoactive intestinal peptide receptor 1 gene polymorphism with layer economic traits in Rhode Island Red chicken

The research work was conducted to determine allelic polymorphism of vasoactive intestinal peptide receptor-1 (VIPR-1) gene and its association with layer traits in Rhode Island Red (RIR) chicken. Blood samples were collected from pullets (n=111) belonging to five hatches. Data on body weight at 20 weeks of age (BW20) and layer economic traits, viz. age at sexual maturity (ASM), egg weight at 28 (EW28) and 40 weeks (EW40) of age, and egg production up to 40 weeks of age (EP40)) were recorded. Genomic DNA was isolated by phenol chloroform extraction method. Samples were amplified for product sizes of 364, 434 and 486 bp of VIPR-1 gene. Amplified regions were digested by TaiI, HhaI and TaqI restriction enzymes. Digested fragments of TaiI and HhaI revealed monomorphism, whereas TaqI showed polymorphism. Allelic frequencies for polymorphic locus were 0.98 (A) and 0.02 (B) and genotypic frequencies for AA and AB were 0.96 and 0.04, respectively. The BW20 and layer traits (ASM, EW28, EW40 and EP40) were analysed by least-squares analysis of variance taking sire as random and hatch as fixed effect. Average ASM and egg production up to 40 weeks of age were 135.19±1.15 days and 124.55±1.94 eggs, respectively. Least-square analysis of variance had revealed significant effect of hatch on ASM, EW28 and EP40. However, effect of TaqI-RFLP genotypes was non-significant on layer traits. Further investigations on a larger sample basis are suggested to confirm effect of this mutation on layer traits as molecular marker in RIR chicken.

Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits

Abstract. Setyorini YW, Kurnianto E, Sutopo, Sutiyono. 2023. Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits. Biodiversitas 24: 2898-2905. Luteinizing and follicle-stimulating hormones and their receptors play an important role in the reproductive system’s hormonal activity and physiological function. This study aimed to identify the genotype variation of the Luteinizing Hormone Receptor (LHR) gene exon 11 and the Follicle-stimulating Hormone Receptor (FSHR) gene exon 10 and to elucidate the associating between polymorphism with the milk and reproductive traits in dairy cattle. A total of 100 samples of deoxyribonucleic acid (DNA) from Indonesian Holstein dairy cattle were used in this study. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) was performed for genotyping of the Single Nucleotide Polymorphism (SNP) of the LHR and FSHR genes with the HhaI and AluI restriction enzymes, respectively. The collected phenotype data were milk production, quality, and reproductive trait. In addition, the general linear model procedure in the SAS program was carried out to investigate the association of genotypes with productive and reproductive traits. The results showed that the LHR gene in the population of this study was monomorphic, while the FSHR gene was polymorphic with three genotypes, namely CC, CG, and GG. The sequences of the FSHR gene indicated the presence of a mutation at nucleotide number 2037 that substitutes cytosine for guanine. Cows with the CC genotype showed better parameter service per conception (P&lt;0.05) than the other genotypes but did not correlate with other reproductive parameters and milk traits. Therefore, it was concluded that SNP g.2037C&gt;G polymorphism at FSHR gene exon 10 was identified in the Indonesian Holstein Dairy cattle population and associated with service per conception parameters but not with milk trait.

Molecular detection and phylogeography of the hypervirulent subtype of &lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt; Beijing 14717-15

Aim. To develop a method for detecting Mycobacterium tuberculosis strains belonging to highly lethal, hypervirulent, multidrug-resistant Beijing 14717-15-cluster to be applied to screen for M. tuberculosis retrospective collections obtained from various regions of Russia. The collection included M. tuberculosis DNA samples obtained during population studies. Spoligotyping and genotyping of 24 variable number tandem repeats loci were performed according to standard protocols. Phylogenetic analysis was based on the whole genome sequencing data. Polymorphism at the genomic position 2423040 A G, specific for the Beijing 14717-15 cluster, was detected using PCR-RFLP and HhaI restriction enzyme. The bioinformatic and phylogenetic analysis of whole genome sequencing data for 205 strains of the early ancient sublineage of the M. tuberculosis Beijing genotype showed that strains with the VNTR 14717-15 profile (according to MIRU-VNTRplus.org) and those close to it were grouped into one monophyletic cluster of related strains, which we defined as Beijing 14717-15-cluster. Among the SNPs specific for the 14717-15 cluster, we chose a functionally neutral SNP at the genomic position 2423040 A G (Rv2161c Val33Ala) and developed a method for its detection in the PCR-RFLP format. Next, we applied this method to screening DNA collections from the regions of the European and Asian parts of Russia and Asian countries. The data demonstrate the presence of strains of the Beijing 14717-15 cluster only in the Asian region of Russia peaking in Buryatia (18%). We have developed a method for detection of the hypervirulent and highly lethal genetic cluster M. tuberculosis Beijing 14717-15 based on detection of the specific mutation in the Rv2161c gene using PCR followed by HhaI restriction of the PCR product and agarose gel electrophoresis to discriminate between wild-type and mutant alleles. The advantages of the proposed method are the speed, simple and unambiguous data interpretation, opportunity to detect contamination and mixed infection, suitability for efficient analysis of large collections of M. tuberculosis strains.

Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor.

Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.

OPTIMIZING GENOTYPING PROCEDURE AND DETERMINING FREQUENCY OF SINGLE NUCLEOTIDE POLYMORPHISM RS7799039 BELONGING TO LEP GENE IN FOUR-YEAR-OLD BOYS IN HANOI

The LEP (leptin) gene plays an important role in the regulation of food intake and energy metabolism. The single nucleotide polymorphism (SNP) rs7799039 located in the promoter of the LEP gene may affect the risk of obesity and other diseases such as diabetes, cardiovascular disease, cancer, etc. due to altering the gene expression. The objective of this study is to find out the optimum procedure for genotyping SNP rs7799039 by using the polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method and determine the allele and genotype frequencies of this SNP in 61 four-year-old boys with normal nutritional status in Hanoi. The sequence of primers has been designed as follows: forward primer 5'-tttcctgtaattttcccatgag-3' and reverse primer 5'-aaagcaaagacaggcataaaa-3'. The thermal cycle for the PCR reaction includes initiation at 94 °C (3 minutes), 34 cycles of denaturation at 94 °C (30 seconds), annealing at 56 °C (30 seconds), extension at 72 °C (30 seconds), final extension at 72 °C (8 minutes), and then keep the products chilling at 4 °C. HhaI restriction enzyme was used to distinguish A and G alleles of rs7799039. PCR products are incubated with 0.5 µl HhaI restriction enzyme for 15 minutes at 37°C and then the products of restriction enzyme digestion are separated by gel electrophoresis. Genotype frequencies of our studied population are 62.3% AA, 31.1% AG, and 6.6% GG (P HWE = 0.452).

Interaction among sheath diseases complex of rice and ribosomal DNA analysis for the differentiation of Rhizoctonia solani, R. oryzae and R. oryzae-sativae

An experiment was conducted in the laboratory and field to study the interaction among three sheath diseases of rice namely sheath blight, sheath spot and aggregated sheath spot and their causal agents, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. In addition to RFLP-PCR analysis and DNA sequencing of three sheath disease species, antagonism among the species was studied using dual culture technique. The results showed that antagonist, R. solani, inhibited radial growth against test fungi (R. oryzae and R. oryzae-sativae) from 37.08-46.49%. Disease incidence and severity of sheath blight was not reduced when R. solani was inoculated with either R. oryzae, R. oryzae-sativae, or a combination of the two. However, the incidence and severity of sheath spot and aggregated sheath spot were inhibited significantly when either R. oryzae or R. oryzae-sativae was inoculated with R. Solani. The highest percentage of relative lesion height and infected tiller was found at maturity followed by hard dough and flowering stage. Average yield losses ranged from 13.99-18.62 % when R. solani inoculated with both of R. oryzae-sativae and R. oryzae or any of them while R. solani alone caused 19.15% loss. DNA sequence analysis differentiated R. solani, R. oryzae and R. oryzae-sativae and phylogenetic tree revealed the close similarity of the species to the isolates of Philippines, Japan and Malaysia, respectively. In HhaI restriction enzyme, fragments 375 and 345 bp were specific for R. solani and 375, 235, and 120 bp was for R. oryzae-sativae which showed identical pattern across the isolates of two species.

Applicability of rpoB Gene for PCR-RFLP based Discrimination of Bifidobacterial Species Isolated from Human and Animal Sources

Bifidobacteria are widely used as probiotics for their application in the development of functional food and prophylactic therapy. This has necessitated the development of a molecular approach for the genera to be widely identified up to species and subspecies level. In the current study, PCR-RFLP of the partial RNA polymerase β-subunit (rpoB) gene fragment was evaluated for differential identification of Bifidobacterium species. The rpoB gene partial sequences of 575 bp were amplified from 93 previously identified isolates collected from various sources of human and animal origin along with 12 standard reference strains. The PCR amplified products were digested with three restriction endonucleases HhaI, HinfI and BanI separately. Dendrograms constructed from the patterns of HhaI, were found to be more discriminatory and successfully differentiated all the twelve species and also at sub-species level in between B. longum subsp. longum and B. longum subsp. infantis. However, B. adolescentis and B. pseudocatenulatum group clusters were not separated and represented by one group. The groups were further discriminated by HinfI restriction digestion. A separate combination thereof may be used for inferring the classification of bifidobacterial species targeted on rpoB PCR-RFLP analysis. To our knowledge, this work is the first report based on use of rpoB PCR-RFLP for discrimination of the isolates of genus Bifidobacterium and also provides insights into specific advantages of this method over hsp60 PCR-RFLP in differentiating B. longum subsp. longum and B. longum subsp. infantis.

Clinical profiling and screening for HNF4α and GCK gene mutations in Kashmiri patients with maturity-onset diabetes of the young (MODY)

AimMaturity-onset Diabetes of Young (MODY) is a monogenic form of diabetes affecting 1–5% of young (often ≤25 years) diabetic patients exhibiting an autosomal dominant mode of inheritance. Considering the significance of genetic polymorphisms in a variety of diseases, this study aimed to determine the association between HNF4α and GCK gene polymorphisms and the risk of MODY in the Kashmir community, as well as their clinical differences. MethodThe study was conducted on clinically confirmed MODY patients (n = 50), and age and gender-matched controls (25 T1DM and 25 non-diabetic) recruited from the endocrinology department of the hospital, for evaluating the HNF4α and GCK mutation. Under standard conditions, PCR-mediated amplification was done to evaluate the respective exons. Preliminary mutations were detected using restriction enzymes (BfaI and HhaI), which were then followed by sequencing of representative samples. The diabetic history, clinical and biochemical data were obtained after proper consent. ResultsOur data revealed no association of HNF4α (exon7) and GCK (exon8) gene mutation with MODY disease susceptibility in the Kashmiri population. On diagnosis, no MODY patient was given immediate insulin; instead, metformin (68%) or sulphonyl-urea (28%) and dietary changes (4%) were recommended. Later in life, 54% of MODY patients develop insulin dependency. The MODY probability was calculated to be 73.88% (±4.56). HbA1c levels were lower [7.48% (±1.64)] than in T1DM [9.17(±2.29%)]. ConclusionsYoung early-onset diabetic patients were able to keep their HbA1c and blood glucose levels stable with a modified diet and metformin/sulphonyl-urea, but they may become insulin-dependent in the future, as seen in our study. As a result, prompt diagnosis and management are essential for avoiding complications. Furthermore, no HNF4α (exon7) or GCK (exon 8) mutations were found in MODY patients or T1DM/healthy non-diabetic controls.

Ultrasensitive DNA methyltransferase activity sensing and inhibitor evaluation with highly photostable upconversion nanoparticle transducer.

Sensitive and accurate detection of DNA methyltransferase (MTase) is conducive to the understanding of the fundamental biological processes related to DNA methylation, clinical disease diagnosis, and drug discovery. Herein, a new fluorescence transducer based on Förster resonance energy transfer (FRET) between the donor upconversion nanoparticles (UCNPs) and the efficient acceptor gold nanorods (AuNRs) for MTase activity analysis and its inhibitor screening is presented. A double-strand DNA linker between UCNPs and AuNRs could be digested by restriction endonuclease HhaI, preventing the FRET process and recovering the upconversion luminescence (UCL) intensity. With the treatment of MTase, the cutting site was disturbed by the methylation of cytosine, blocking the enzyme digestion. The transducer presented here showed an excellent analytical performance toward MTase M.HhaI in the concentration range 0.08~24UmL-1 with a detection limit of 0.057UmL-1 calculated according to the UCL intensity changes at 656nm excited by 980nm CW laser, which is superior to most of the reported methods. Furthermore, the as-fabricated transducer also demonstrated high testing and screening capability toward enzyme inhibitors' evaluation. The method takes the advantage of low background fluorescence of UCNPs to improve the accuracy of the measurement, which can be developed as a general strategy for the analysis of various disease-related methyltransferase activity and their corresponding inhibitors, offering a promising strategy for high-performance diagnosis, high-efficient drug exploitation, and treatment effectiveness evaluation.

G1 point mutation in growth differentiation factor 9 gene affects litter size in Sudanese desert sheep.

Background and Aim:Sudanese desert sheep encompass different sheep breeds named according to the different Sudanese tribes that rear them such as the Dubasi, Shugor, and Watish sheep. The objectives of this study were to screen for G1 point mutation in the polymorphic growth differentiation factor 9 (GDF9) gene, investigate its association with litter size, and construct the phylogeny of the different tribal breeds that belong to the Sudanese Desert sheep tribal types.Materials and Methods:Genomic DNA was extracted from whole blood of three tribal Desert sheep breeds (Dubasi, Watish, and Shugor) using the guanidine chloride method. Polymerase chain reaction-restriction fragment length polymorphism with HhaI restriction enzyme and sequencing techniques was used for genotyping the GDF9 locus for possible mutations associated with litter size in the three desert sheep tribal types.Results:G1 mutation in GDF9 caused the replacement of Arginine by Histidine at residue 87. The wild type allele (A) had the highest frequency, whereas the mutant type allele (a) had the lowest in all the sequenced subtypes. The genotype frequencies of the wild type ewes (AA) were higher than the heterozygous (Aa) and the mutant type (aa) frequencies in the three studied desert sheep types. No significant differences were found in the allele frequency between the three tribal types. Litter size was significantly influenced by the genotypes of GDF9 gene, parities, and subtypes (p≤0.01, 0.01, and 0.05, respectively). In the Watish sheep type, heterozygous sheep in their second parity recorded the highest litter size. Sequence alignment of GDF9 gene samples with the database entry indicated that all three tribal types were similar and identical to the reference sequence. The phylogenetic tree revealed that Shugor is the common ancestor of the studied types and Watish is more closely related to Shugor than Dubasi. This result mi ght partly explain the lower reproductive performance of Dubasi compared to Watish and Shugor.Conclusion:The presence of one copy of GDF9 gene increased litter size in the studied Sudanese Desert sheep. This locus may be used as a biomarker for litter size improvement through genotypic selection and allele or gene introgression.

Allelic polymorphism in exon 1 of GDF9 and exon 2 of BMP15 genes and its impact on litter size at lambing in Iran-Black sheep

The main purpose of this study was to assess the presence of the previously reported single nucleotide polymorphisms (SNPs) in the sheep growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes and their association with litter size at lambing in Iran-Black sheep breed. Blood samples were taken from 120 Iran-Black ewes. DNA extraction was conducted using a modified salting-out method. DNA fragments with sizes of 462bp and 141bp for the GDF9 and BMP15 genes were amplified using PCR with specific primers, respectively. The PCR-RFLP approach was adopted for detecting the genotypes. The results indicated that the SNP in the exon 2 of BMP15 is a monomorphic locus in Iran-Black sheep. However, the substitution of G to A nucleotide was determined in the GDF9 locus. Digestion of the 462bp PCR product from exon 1 of theGDF9 using the HhaI restriction enzyme produced fragments of 52, 156, and 254bp. However, DNA fragments containing the A nucleotide yielded only two fragments (52 and 410bp). The heterozygous animals for this mutation in GDF9 locus had fragments of all four sizes (52, 156, 254, and 410bp). The frequency (0.75) of the wild type allele (+) in GDF9 locus was higher than the frequency (0.25) of mutant allele (G). The observed frequencies for the GG, G+ and ++ genotypes were 0.05, 0.40 and 0.55, respectively. The association results indicated that the mutation of GDF9 gene has a substantial impact on lambing rate and the Iran-Black ewes with the GG and G+ genotypes had higher lambing rate than those with the ++ genotype. Thus, a gene assisted selection program to improve lambing rate in this breed can be designed based on the GDF9 gene mutation. Key words: BMP15, GDF9, Litter Size, Iran-Black Sheep

Infection and molecular identification of ascaridoid nematodes from the important marine food fish Japanese threadfin bream Nemipterus japonicus (Bloch) (Perciformes: Nemipteridae) in China.

The Japanese threadfin bream N. japonicus (Bloch) (Perciformes: Nemipteridae) is an important marine food fish in Asia. However, our present knowledge of the occurrence of its nematode parasites is still limited. In the present study, the species composition and infection rate of ascaridoid nematodes in N. japonicus from the South China Sea, were studied for the first time. Five ascaridoid species, namely Anisakis typica (L3), Hysterothylacium amoyense (L3), Hysterothylacium sp. IV-A (L3), adult of H. thalassini and Raphidascaris lophii (L3), were identified using integrative taxonomy. Hysterothylacium amoyense was the most prevalent species (prevalence 47.2%, mean intensity 14.9±17.1). Hysterothylacium thalassini and R. lophii were reported in the Japanese threadfin bream for the first time. Two different genotypes of A. typica (overall prevalence of 3.4%; mean intensity 1.7±0.9) were found in the South China Sea for the first time. The unique restriction polymorphism patterns of three species of Hysterothylacium are provided for rapid diagnosis. Our present results indicate that RFLP analysis of ITS region, using the restriction enzymes HhaI and RsaI, represents a simple and practical method for large-scale surveys of Hysterothylacium for seafood industry.