Direct Sequence Analysis Research Articles

Androgen receptor CAG repeat polymorphism might be a possible cause of familial constitutional delay of growth and puberty.

Induction of puberty in boys with constitutional delay of growth and puberty (CDGP) through a short course of low-dose testosterone therapy indicates the critical interaction between testosterone and the androgen receptor (AR) during the activation and maturation of the hypothalamic-pituitary-gonadal axis at puberty onset. Previous studies have shown an inverse relationship between the CAG repeat length and the transactivation function or expression level of the AR gene. We aimed to investigate whether the AR CAG repeat polymorphism has any implications on pubertal delay. Thirty-three male patients with CDGP were enrolled in the study group, while 53 age-matched healthy individuals who had entered puberty on time were included in the control group. The CAG repeat length was determined through direct DNA sequencing analysis. The median chronological age of boys with CDGP was 14.2 (14.1-14.6) years, compared to 14.2 (13.65-14.8) years for healthy subjects (p = 0.5). In the CDGP group, 22 (66.7%) children had a family history of the condition. There was no significant difference between the groups in terms of AR CAG repeat length (median AR CAG repeat length: 21 (20-24.5) and 20 (20-24), respectively, p = 0.1). However, in boys with CDGP with a similar family history (n = 22), a significantly longer AR CAG repeat length was found compared to the control group (n = 53) (median AR CAG repeat length: 22 (20-25) and 20 (20-24), respectively, p = 0.03). The median AR CAG repeat length in boys without a family history was 21 (20-22) triplets. Although boys with a family history had a slightly longer AR CAG repeat length than those without, the difference was not statistically significant (p = 0.07). Additionally, no significant differences were observed between boys with non-familial CDGP and control subjects (p = 0.8). Furthermore, no significant differences in anthropometric characteristics or hormonal parameters were found when patients with CDGP were categorized by AR CAG repeat length quartiles. This is the first study to investigate the role of AR CAG polymorphism in the etiopathogenesis of CDGP. Our findings suggest that the AR CAG repeat length may be associated with familial CDGP.

Prevalence of hepatitis C virus hypervariable region 1 insertions and their role in antibody evasion.

Chronic hepatitis C virus (HCV) infection afflicts around 50 million people globally, causing ~250,000 deaths yearly. An effective vaccine needs to overcome high viral diversity and HCV's ability to evade neutralizing antibodies (NAbs). Rapid antigenic drift in the N-terminal motif of envelope protein E2, named hypervariable region 1 (HVR1), is critically involved in NAb evasion via an incompletely understood mechanism involving viral entry factors. The canonical length of HVR1 is 27 amino acids, but insertions of 2-4 amino acids was described in patients infected with genotype 1b. We aimed at determining whether HVR1 insertions may be underreported due to extreme HVR1 variability. We observed a 0.7% HVR1 insertion prevalence in routine NGS patient contigs. Thus, we performed direct sequence analysis of E1E2 sequences from 131 HCV infected patients. Interestingly, we observed that 3% of patients harbored viruses (genotype 1a, 2b, 3a) with dominant HVR1 insertions. Insertion of longer non-canonical HVR1s into HCV cell culture recombinants frequently caused loss of fitness. However, culture-viable viruses with HVR1 insertions were fully viable in vivo. Interestingly, in adapted genotype 1b recombinants with HVR1 insertions, we found internal HVR1 deletions, that increased antibody sensitivity, which surprisingly correlated more with reduced LDLr than reduced SR-BI dependency, indicating a role of LDLr in NAb evasion. Conversely, HVR1 insertions had no effect on receptor dependency, however, they modulated epitope-specific NAb sensitivity. HVR1 insertion prevalence and NAb sensitivity modulation indicate they represent a mechanism by which HCV evades emerging NAbs during infection.

Association of rs8670 Polymorphism in the MSX1 Gene With Non-Syndromic Cleft Lip With or Without Cleft Palate in Malay Population.

This study aimed to investigate the association between variants present in the MSX1 gene and the risk of developing non-syndromic cleft lip with or without cleft palate (NSCL±P) among individuals of Malay ethnicity in Malaysia. This case-control study involved 89 patients with NSCL±P and 100 healthy control subjects. Polymerase chain reaction (PCR) was performed on both exon 1 and exon 2 of the MSX1 gene using four pairs of primers. The amplification products were then subjected to denaturing high-pressure liquid chromatography for initial screening, and the presence of a heteroduplex peak was validated using direct sequencing analysis to detect the single-nucleotide polymorphism. Five previously known variations (c.-36G>A, p.Ala30Ala, p.Ala34Gly, p.Gly110Gly, and rs8670: C>T) were detected within the MSX1 gene in both NSCL±P patients and controls.A significant association was found between the rs8670: C>T variant and NSCL±P (p = 0.017; OR: 0.368; 95% CI: 0.152 - 0.893), with this particular single-nucleotide polymorphism present in 20% (20) among controls and 7.9% (7) of the NSCL±P cases. Our data showed a lower incidence of thers8670: C>Tpolymorphism among NSCL±P cases compared to control in this Malay population. However, since this variant is located in the 3'UTR, it could potentially impact the stability ofMSX1mRNA.

De novo Whole-Genome Assembly of the 10-Gigabase Fokienia Hodginsii Genome to Reveal Differential Epigenetic Events Between Callus and Xylem.

Fokienia hodginsii (F. hodginsii), belonging to the genus Fokienia of the Cupressaceae. F. hodginsii has significant application value due to its wood properties and great research value in evolutionary studies as a gymnosperm. However, the genome of F. hodginsii remains unknown due to the large size of gymnosperms genome. Pacific Bioscience sequencing, Hi-C mapping, whole-genome Bisulfite Sequencing (BS-Seq), long-read isoform sequencing (Iso-Seq), direct RNA sequencing (DRS), quantitative proteomics, and metabonomics analysis are employed to facilitate genome assembly, gene annotation, and investigation into epigenetic mechanisms. In this study, the 10G F. hodginsii genome is assembled into 11 chromosomes. Furthermore, 50521 protein-coding genes are annotated and determined that 65% of F. hodginsii genome comprises repetitive sequences. It is discovered that transposable element (TE)-including introns is associated with higher expression. The DNA methylome of F. hodginsii reveals that xylem has a higher DNA methylation level compared to callus. Moreover, DRS reveals the significant alterations in RNA full-length ratio, which potentially associated with poly(A) length (PAL) and alternative polyadenylation (APA). Finally, the morphology measurement and metabonomics analysis revealed the difference of 14 cultivars. In summary, the genomes and epigenetics datasets provide a molecular basis for callus formation in the gymnosperm family.

The Relationship between Intelligence and Mitochondrial DNA Polymorphism at Site of 15925 Base Pairs

Intelligence has a huge influences and is very closely related with human’s life. Because intelligence is indefinable, scholars have specified it through testing tools and named it intelligence quotient (IQ). In measuring IQ, researchers have found that IQ has a wide distribution and tried to find out the reasons by assessing the brain's functional level and environmental and genetic factors. One study reported an association between IQ and mitochondrial DNA (mtDNA) sequence 15,925, and this study investigated the association in Koreans. The number of participants was 57 in the mental retardation group without chromosomal abnormalities and 148 in the normal group, respectively. PCR technique and direct sequencing analysis were used to know participant’s mtDNA sequences. At 15,925 base, all participants had only wild type C and no variant type of m.15,925 C > T. We further studied variants in mtDNA sequences 15,926-15,927. At 15,926 base, the variant type of 15,926 C < T was found in only one case (0.7%) in the control group. At the 15,927 base, variant type of 15,927 G < A was found in 9 cases (6.1%) in the control group and 1 case (1.8%) in the retardation group, respectively. As a result, the polymorphisms of mtDNA sequences at bases 15,925-15,927 had no statistical significance in the Korean control and mental retardation group.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA–protein pull-down assay, six DVG-encoded proteins were identified by LC–MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Molecular Investigation of gyrA Mutations in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus Derived from Diverse Sources

Background: Fluoroquinolones are the most effective antibiotics against Staphylococcus aureus isolates. In hospitals, excessive use of antibiotics has led to the emergence of highly resistant strains of S. aureus isolates. Objective: The aim of this study was to detect the mutations that occur in the gyrA gene encoding for DNA gyrase, which is one of the targets for fluoroquinolone resistance. Methods: Fifty clinical isolates were diagnosed as S. aureus according to molecular and bacteriological methods. The susceptibility tests were performed on all bacterial isolates by the disc diffusion method using methicillin and six fluoroquinolone antibiotics. Results: Out of fifty isolates, twelve were resistant to methicillin and all six antibiotics (nalidixic acid, lomefloxacin, ciprofloxacin, norfloxacin, ofloxacin, and levofloxacin). From the fifty isolates, 12 were resistant, 3 were intermediate, and 38 were sensitive to three or more tested antibiotics. The resistance of S. aureus isolates was also confirmed by the minimum inhibitory concentration test. The main sources of isolates were burns (10%), nose (16) wounds (8%), operation room (10%), ear (20%), urine (8%), skin (6%), and throat (22%). Twelve resistant isolates were used to examine the mutations in the gyrA gene. A direct sequence analysis found eight mutations in the gyrA gene; these mutations included 2 (25% missense mutations), 1 (12.5%) deletion mutation, and 5 (62.5%) silent mutations at various sites. Conclusion: gyrA mutations resulting from the excessive use of antibiotics may be one of the mechanisms leading to fluoroquinolone resistance.

Frequency of the 'Asia type' DEL with weak D phenotype in Chinese.

DEL individuals account for 9-30% of serological RhD negative population in east Asia and majority of them carrying the RHD*DEL1 allele are referred to as 'Asia type' DEL individuals. There is a lack of data on the molecular basis for 'Asia type' DELs with weak RhD phenotype. Therefore, the aim of this study is to unveil 'Asia type' DELs by elucidating the genetic background and analyzing the serological results METHODS: With a microplate typing protocol, RhD characterization was performed in samples from one million blood donors collected at Chengdu blood center during the period from 2019 to 2022. RhD confirmatory test was performed by direct antiglobulin test and indirect antiglobulin test with five anti-D reagents to detect RhD variants. Molecular characterization of samples categorized as RhD variants was studied by using direct genomic DNA sequencing and RHD zygosity analysis, followed by adsorption and elution tests conduced for samples carrying RHD*DEL1 allele to confirm the presence of RhD antigens on the red cells RESULTS: We reported here 21 RhD variant samples were detected by micro-column gel agglutination assay with IgG anti-D antibodies. Moreover, the agglutination reaction was stronger with IgG anti-D reagents in micro-column gel card than with IgM/IgG blended anti-D antibodies. Each of the 21 samples carried the RHD*DEL1 allele, which indicated that they were 'Asia type' DEL. Of the 21 'Asia type' DEL samples, 9 samples were detected to be RHD+/ RHD+ homozygotes, whereas the other 12 samples were RHD+/RHD- hemizygotes. Among the samples phenotyped for RhCE, seven were CCee and four were Ccee. In this study, DEL samples carrying RHD*DEL1 showed weak RhD phenotype with some anti-D reagents in RhD confirmatory test, which suggest that a serology strategy using several anti-D reagents may be helpful to detect this 'Asia type' DEL. Further studies are needed to elucidate whether the 'Asia type' DELs with weak RhD phenotype have stronger antigenicity and could cause serious transfusion reaction.

FRI532 Thyroid Hormone Resistance: A Possible New Variant In The THRB Gene

Abstract Disclosure: A. Siddiqui: None. Z. Khan: None. T. Ahmed: None. K. Selk: None. Background: Resistance to the Thyroid Hormone (RTH) is caused by reduced target tissue response to thyroid hormone, resulting in the loss of the normal feedback mechanism that suppresses secretion of thyrotropin (TSH). Most cases of RTH are familial, usually inherited in an autosomal dominant manner, with over 100 different pathogenic variants having been described. Clinical Case: A 45-year-old male with history of attention deficit disorder presented for management of hypothyroidism. The patient was Anti-TPO positive, consistent with Hashimoto's thyroiditis and he was treated with varying doses of levothyroxine. Labs on 100 mcg levothyroxine showed a TSH of 7.07 (0.35-4.94 uIU/mL), free t4 1.86 (0.7-1.48 ng/dL), and free t3 4.41 (2.3-4.2 pg/mL). This initially prompted concern for a TSHoma. An alpha subunit level was found to be 0.3 ng/dL and pituitary MRI was within normal limits. Given continued persistent thyroid hormone elevations, further testing for human anti-mouse antibody interference was performed and also unrevealing. The patient continued to report feeling tired and fatigued, with multiple joint pains and inability to focus or concentrate. His levothyroxine was increased to 125 mcg per day. However, even after the adjustment, he still did not notice any change in his symptoms. A trial of addition of liothyronine 5mcg daily was also performed but he self-discontinued this as he did note any improvement. Overall, his findings were highly suspicious for thyroid hormone resistance. Genetic testing for a thyroid hormone resistance beta (THRB) mutation was performed. Direct sequence analysis of the coding region and splice-sites of the THRB gene identified one copy of the c.1368G>T (p.Leu456Phe) variant of uncertain significance. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. However, the THRB c.1368G>T (p.Leu456Phe) variant has not been described in online databases or published literature. It also has not been reported in large, multi-ethnic general populations. Conclusion: This is the first case to our knowledge demonstrating the THRB c.1368G>T (p.Leu456Phe) variant gene as likely giving rise to RTH. Further testing of family members has been recommended to help clarify the clinical significance of this finding. Presentation: Friday, June 16, 2023

TNFα rs1800629 Polymorphism and Response to Anti-TNFα Treatment in Behçet Syndrome: Data from an Italian Cohort Study.

Tumor Necrosis Factor-alpha (TNFα) rs1800629 (-308G>A) is a single nucleotide polymorphism (SNP) related to variable responses to anti-TNFα therapy. This therapy is efficient in severe and refractory manifestation of Behçet syndrome (BS), an auto-inflammatory systemic vasculitis. We investigated (1) the association between rs1800629 genotypes and responses to therapy and (2) the correlation between SNP and clinical patterns in a cohort of 74 BS Italian patients receiving anti-TNFα therapy with a follow-up of at least 12 months. The rs1800629 was genotyped through amplification, direct sequencing and bioinformatics analyses. The rs1800629 GG and GA genotypes were assessed as predictors of outcomes dividing the patients between therapy responders and non-responders. The rs1800629 GG and GA genotypes were found, respectively, in 59/74 (79.7%) and 15/74 BS patients (21.3%) (p < 0.05). We identified 16/74 (21.9%) non-responder patients, of which 9/16 (56.3%) showed the GG genotype and 7/16 (43.7%) the GA genotype. A total of 50/58 (86.2%) responder patients showed the GG genotype, and 8/58 (13.8%) the GA genotype (p < 0.05). The percentage of non-responder females (68.8%) was significantly higher than non-responder males (31.2%) (p < 0.05). No correlation between SNP and clinical patterns was observed. To successfully include rs1800629 as a predictive biomarker of TNFα inhibitor response, genome-wide association studies in larger, well-characterised cohorts are required.

Identıfıcatıon of KRAS Mutatıons in Colorectal Carcınoma Patıents at Dr. M. Djamıl Hospıtal, West Sumatra-Indonesıa

Kirsten rat sarcoma viral oncogene (KRAS) gene mutations lead to abnormal activation of the RAS signalling pathway and have been associated with poor prognosis and resistance to some therapeutics. This study aimed to identify mutation characteristics of the KRAS genes codon 12 and 13 in colorectal cancer patients in West Sumatra. KRAS mutations were analyzed in 20 DNA of colorectal cancer patients' tissue samples by using polymerase chain reaction (PCR) with specific primer and direct sequencing analysis. Our findings showed five samples (25%) with mutated KRAS at codons 12 and 13 (including three samples with GGT→GAT, one sample with GGT→GTT in codon 12, and one sample with GGC→GAC in codon 13). In conclusion, we found two variations of amino acid changes at codon 12 (G12D and G12V) and one at codon 13 (G13D). More research with many samples is required to obtain conclusive data on the relationship between these gene mutations and colorectal cancer response to therapy and prognosis.

Prospective validation of VEGF and eNOS polymorphisms as predictors of first-line bevacizumab efficacy in patients with metastatic colorectal cancer

Bevacizumab (Bev) plus chemotherapy is a standard first-line treatment in metastatic colorectal cancer (mCRC), however to date no predictive factors of response have been identified. Results of our previous analysis on patients enrolled in a randomized prospective phase III multicenter study (ITACa study) showed a predictive value of Vascular Endothelial Growth Factor (VEGF) polymorphism (VEGF + 936), a 27-nucleotide variable number tandem repeat (VNTR) of the endothelial nitric oxide synthase (eNOS) gene and eNOS + 894 polymorphism. mCRC patients, treated with Bev plus chemotherapy, were included in this prospective validation trial. eNOS + 894G > T was analyzed by Real time PCR, while the eNOS VNTR and VEGF + 936C > T were determined by standard PCR and direct sequencing analysis. These polymorphisms were assessed in relation to progression-free survival (PFS), overall survival (OS) and objective response rate (ORR). These three polymorphisms were not predictive of PFS (p 0.91, 0.59 and 0.09, respectively), and OS (p 0.95, 0.32 and 0.46, respectively). Moreover, the haplotype analyses did not confirm what was found in our previous study; patients bearing a specific haplotype of eNOS had not significantly improved outcomes. This prospective study failed to validate the predictive impact of eNOS and VEGF polymorphisms for response to Bev plus first-line chemotherapy in mCRC patients.

Genetic diversity of Helicobacter pylori type IV secretion system cagI and cagN genes and their association with clinical diseases

A number of cagPAI genes in the Helicobacter pylori genome are considered the most evolved genes under a diversifying selection and evolutionary pressure. Among them, cagI and cagN are described as a part of the two different-operon of cagPAI that are involved in the T4SS machinery, but the definite association of these factors with clinical manifestations is still unclear. A total of 70 H. pylori isolates were obtained from different gastroduodenal patients. All isolates were examined for the presence of primary H. pylori virulence genes by PCR analysis. Direct DNA sequence analysis was performed for the cagI and cagN genes. The results were compared with the reference strain. The cagI, cagN, cagA, cagL, vacA s1m1, vacA s1m2, vacA s2m2, babA2, sabA, and dupA genotypes were detected in 80, 91.4, 84, 91.4, 32.8, 42.8, 24.4, 97.1, 84.3, and 84.3% of the total isolates, respectively. The most variable codon usage in cagI was observed at residues 20–25, 55–60, 94, 181–199, 213–221, 241–268, and 319–320, while the most variable codon usage in CagN hypervariable motif (CagNHM) was observed at residues 53 to 63. Sequencing data analysis of cagN revealed a hypothetical hexapeptide motif (EAKDEN/K) in residues of 278–283 among six H. pylori isolates, which needs further studies to evaluate its putative function. The present study demonstrated a high prevalence of cagI and cagN genes among Iranian H. pylori isolates with gastroduodenal diseases. Furthermore, no significant correlation between cagI and cagN variants and clinical diseases was observed in the present study. However, all patients had a high prevalence of cagPAI genes including cagI, cagN, cagA, and cagL, which indicates more potential role of these genes in disease outcome.

Identification of a novel B allele with a c.145C>T variant.

The authors have disclosed no conflicts of interest. Figure S1: Direct sequence analysis of the partial exon 3 region in the ABO gene of the proband. Direct sequencing revealed the heterozygous sequence (C and T) at nucleotide 145. Figure S2: Direct sequence analysis of the partial exon 3 region in the ABO gene of the proband's father. Direct sequencing revealed the heterozygous sequence (C and T) at nucleotide 145. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

GENETIC POLYMORPHISMS OF HLA-G GENE IN RHEUMATOID ARTHRITIS

This study was aimed to evaluate the influence of Human Leukocyte Antigens- G (HLA-G) gene polymorphisms on the risk of rheumatoid arthritis (RA). In the present study, blood samples were collected from 100 rheumatoid arthritis patients and 100 healthy controls. Serum levels of HLA-G in both patients and controls were measured using ELISA kit which was shown a higher significant differences (p&lt; 0.05) in comparison between them (539.3 ng/ml and 139.3ng/ml) respectively. The frequency of the allele and genotypes in the patient groups and control groups were determined using Polymerase Chain Reaction (PCR) for 14 bp polymorhisms in exon 8 (rs66554220) while direct sequencing analysis was done to determine exon 2 (rs1130355) (G 1850 A) SNPs of human leucocyte antigens- G (HLA- G). There was statistically significant difference in all genotypes of HLA-G14-bp of exon 8 in RA patients in comparison with controls. The probability of homozygote genotype of insertion was (0.006), homozygote genotype of deletion was (0.003) and for heterozygote of insertion was (0.000) in comparison between RA patients and controls. From the results of odd ratio (OR) found that existence of homozygotes of insertion give protection effect from disease O.R= 0.44 while heterozygotes of deletion act as risk factor for disease O.R= 3.31. Homozygotes of deletion had no effect because was not found in patients groups. From the sequencing results of exon 2 of HLA-G gene found the mutation occur in position 1850 G&gt;A which give three genotypes (GG, AG and AA). Calculations of genotypes frequency for exon 2 showed that GG and AA genotypes was associated with the disease with p- value (0.000) for both genotypes. While AG genotype showed no association with disease p value (0.449).From the results of odd ratio found that GG and AA of exon 2 gave protection effect from disease O.R( 0.29 and 0.76 ) respectively, while AG genotype acted as risk factor for disease O.R= 4.0.

Long-term durability of sustained virologic response for hepatitis C virus infection in solid organ transplant recipients receiving direct-acting antivirals

Data are limited regarding the long-term durability of sustained virologic response (SVR) in solid organ transplant recipients who achieve SVR12 with direct-acting antivirals (DAAs) for hepatitis C virus (HCV). We reported the virologic outcomes in 42 recipients who received DAAs for acute or chronic HCV infection after heart, liver, and kidney transplantation. After achieving SVR12, all recipients received HCV RNA surveys at SVR24, and biannually until the last visit. If HCV viremia was detected during the follow-up period, direct sequencing and phylogenetic analysis were performed to confirm late relapse or reinfection. Sixteen (38.1%), 11 (26.2%), and 15 (35.7%) patients underwent heart, liver and, kidney transplantation. Thirty-eight (90.5%) received sofosbuvir (SOF)-based DAAs. No recipients had late relapse or reinfection after a median (range) of post-SVR12 follow-up 4.0 (1.0-6.0) years. We demonstrate that the durability of SVR in solid organ transplant recipients is excellent once SVR12 is achieved with DAAs.

Mitochondrial DNA variants in a cohort from Argentina with suspected Leber's hereditary optic neuropathy (LHON).

The present study investigates the spectrum and analysis of mitochondrial DNA (mtDNA) variants associated with Leber hereditary optic neuropathy (LHON) in an Argentinean cohort, analyzing 3 LHON-associated mitochondrial genes. In 32% of the cases, molecular confirmation of the diagnosis could be established, due to the identification of disease-causing variants. A total of 54 variants were observed in a cohort of 100 patients tested with direct sequencing analysis. The frequent causative mutations m.11778G>A in MT-ND4, m.3460G>A in MT-ND1, and m.14484T>C in MT-ND6 were identified in 28% of the cases of our cohort. Secondary mutations in this Argentinean LHON cohort were m.11253T>C p.Ile165Thr in MT-ND4, identified in three patients (3/100, 3%) and m.3395A>G p.Tyr30Cys in MT-ND1, in one of the patients studied (1%). This study shows, for the first time, the analysis of mtDNA variants in patients with a probable diagnosis of LHON in Argentina. Standard molecular methods are an effective first approach in order to achieve genetic diagnosis of the disease, leaving NGS tests for those patients with negative results.

Chronic Voluntary Alcohol Consumption Alters Promoter Methylation and Expression of Fgf-2 and Fgfr1

Alcohol abuse accounts for 3.3 million deaths annually, rendering it a global health issue. Recently, fibroblast growth factor 2 (FGF-2) and its target, fibroblast growth factor receptor 1 (FGFR1), were discovered to positively regulate alcohol-drinking behaviors in mice. We tested whether alcohol intake and withdrawal alter DNA methylation of Fgf-2 and Fgfr1 and if there is a correlation regarding mRNA expression of these genes. Blood and brain tissues of mice receiving alcohol intermittently over a six-week period were analyzed using direct bisulfite sequencing and qRT-PCR analysis. Assessment of Fgf-2 and Fgfr1 promoter methylation revealed changes in the methylation of cytosines in the alcohol group compared with the control group. Moreover, we showed that the altered cytosines coincided with binding motives of several transcription factors. We also found that Fgf-2 and Fgfr1 gene expression was significantly decreased in alcohol-receiving mice compared with control littermates, and that this effect was specifically detected in the dorsomedial striatum, a brain region involved in the circuitry of the reward system. Overall, our data showed alcohol-induced alterations in both mRNA expression and methylation pattern of Fgf-2 and Fgfr1. Furthermore, these alterations showed a reward system regional specificity, therefore, resembling potential targets for future pharmacological interventions.

Comparison of fecal microbiota of SPF and non-SPF Beagle dogs.

Microbial colonization of animal intestine impacts host metabolism and immunity. The study was aimed to investigate the diversity of the intestinal microflora in specific pathogen free (SPF) and non-SPF Beagle dogs of different ages by direct sequencing analysis of the 16S rRNA gene. Stool samples were collected from four non-SPF and four SPF healthy Beagle dogs. From a total of 792 analyzed Operation taxonomic units, four predominant bacterial phyla were identified: Firmicutes (75.23%), Actinobacteria (10.98%), Bacteroidetes (9.33%), and Proteobacteria (4.13%). At the genus level, Streptococcus, Lactobacillus, and Bifidobacterium were dominated. Among which, Alloprevotella, Prevotella_9, and Faecalibacterium were presented exclusively in non-SPF beagles, with potentially anti-inflammatory capability, which could protect non-SPF beagles from complex microbial environment. The number and diversity of intestinal flora for non-SPF Beagle dogs were the highest at birth and gradually decreased with growth, whereas the results for the SPF beagle samples were the opposite, with the number and diversity of intestinal microbiota gradually increases as beagles grow. In a nutshell, the microbial complexity of the rearing environment can enrich the gut microbiota of beagles, many of which are anti-inflammatory microbiota with the potential to increase the adaptability of the animal to the environment. However, the gut microbiota of SPF beagles was more sensitive to environmental changes than that of non-SPF beagles. This study is of great significance for understanding the bionomics of intestinal microflora in non-SPF and SPF beagles, improving the experimental accuracy in scientific research.

The Role of CD1 Gene Polymorphism in the Genetic Susceptibility to Spondyloarthropathies in the Moroccan Population and the Possible Cross-Link with Celiac Disease.

Spondyloarthropathies (SpA) are a group of chronic inflammatory disorders usually affecting the axial spine and asymmetrical peripheral joints. Strong evidence links genetic and environmental factors to SpA pathogenesis. The HLA-B27 is the most important genetic factor associated with SpA. Nevertheless, the involvement of other HLA and non-HLA loci has been also reported. Some patients with SpA may also manifest features of celiac disease (CeD), thus suggesting a genetic overlap across these autoimmune diseases. Recently, CD1 glycoproteins, a class of molecules able to bind and present non peptidic antigens to T cells, aroused interest for their contribution to the pathogenesis of CeD. Therefore, to evaluate whether functional polymorphisms of CD1A and E genes also influence susceptibility to SpA, we analyzed 86 patients from Morocco affected by SpA and 51 healthy controls, using direct sequencing analysis. An increase of CD1E*01/01 homozygous genotype (p = 0.046) was found in SpA, compared with controls. CD1E*01/01 genotype was associated particularly to patients with sacroiliac joints/spine/peripheral joints pain (p = 0.0068), while a decrease of CD1E*01/02 genotype was evidenced compared to controls (p = 0.0065). Results from haplotypes analysis demonstrated that CD1A*02-E*02 decreased the risk of SpA, while CD1A*02-E*01 increased risk to develop disease. Our data indicate a relationship between CD1 genes and susceptibility to SpA in the Moroccan population and suggest the existence of shared genetic risk loci across SpA and CeD that might be useful to explain common pathogenetic features and define novel therapeutic strategies.