Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Enzymatic and Genetic Study in Blood Among Some Children with β – Thalassaemia in Mosul City, Iraq

The present study aimed to demonstrate the extent to which the activity of a number of enzymes and genetic variation of β-globin genes were affected in the blood of 65 children patient with β - thalassemia major of both sexes, their ages ranged between ( 2 – 15 ), who registered in the Thalassemia Center at Ibn Al-Atheer Teaching Hospital for Children in the city of Mosul / Iraq and who are continuing treatment, after they were diagnosed by specialist doctors, as well as 30 healthy children of both sexes with same ages of the patients and it was considered as a control group. The results showed a significant increase (P≤0.05) in the activity of Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), Glucose 6- phosphate dehydrogenase (G6PD) and Adenosine deaminase (ADA) in the serum of treated children patients with β - Thalassaemia major by 73% , 53%, 8%, 9% and 54% respectively, compared with the healthy children group (control group). Also the results showed a significant increase in the activity of G6PD and ADA in the hemolysis of RBC of the same children patients by 7% and 43% compared with control group. When determining the genetic variation of the β-globin gene the result which depends on PCR technique did not show any genetic variation in the size of PCR band ,while the result of the sequencing showed variation in the nucleotides and included converted the nucleotide (A) to (C) in position (250) , change nucleotide (T) to (C) in position (426), replacement (C) nucleotide to (A) in position (623), change (G) nucleotide to (A) in position (630) and replacement (T) to (A) in the position (724), also the result detection three Transversion mutation and two transition mutation in β-globin gene in babies with β-thalassemia

ANALISIS GEN TCF7L2 (Trancription Factor 7 Like 2) PADA KELUARGA PENDERITA DIABETES MELLITUS TIPE 2 KECAMATAN TANGGULANGIN, KABUPATEN SIDOARJO

Diabetes mellitus is a metabolic disorder characterized by not produced insulin or disruption of insulin action. Diabetes mellitus was reported correlated with TCF7L2 gene mutation. This study aimed to analyze the TCF7L2 gene in families with type 2 diabetes. 5 samples were used in this study, the methods flow through DNA Isolation, PCR, and sequencing. Samples were amplified by the PCR technique using primers that were designed to detect the TCF7L2 gene, (F: 5'-GGCTTGATTGTTGATTATGGGC3' and R:5 'TCTGGCACTCAGAGAGT 3'). The amplification and sequencing resulted in 368 bp (located at 103631-103972 bp). We found a transition mutation at C103950T. The polymorphisms found may characteristic in this population because they are not found in other sequences. Keywords : Diabetes mellitus, TCF7L2 gene, mutation

SMARCAD1-mediated recruitment of the DNA mismatch repair protein MutLα to MutSα on damaged chromatin induces apoptosis in human cells

The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.

Sequencing and structure analysis of UBC gene for breast and ‎lung cancer

In the present study, sequencing approach has been adopted for exploring the ‎genetic alteration of sequences for the ubiquitin gene (UBC) in patients of breast and ‎lung cancer and comparing the results with a normal sequence that obtained from NCBI. ‎The aim of this study was to detect for genetic alterations of UBC gene in the breast and ‎lung cancer patients then compare with healthy control subjects, to investigate the ‎association between the mutations at the intron region of the UBC gene and cancer disease, ‎‎40 blood samples were examined from patients with breast and lung cancer aged ranged from (17-65) years, were collected at Al-Amal Hospital of cancer in Baghdad ‎province/Iraq, the period of collecting samples were from October/2018 to January/2019. ‎While twenty-two blood samples from healthy control subjects were collected at ages ‎ranged from(19-59). After DNA extraction, the PCR primer was designed to amplify the ‎region in the UBC gene (part of exon 1 and the whole intron). Here we report the polymorphism of the intron sequence of the UBC gene in Iraqi population as the results of sequencing the PCR amplified products showed three different transition mutation G→A, ‎C→T, T→C in patients with breast cancer were also appeared in healthy control subjects. While nine transition mutations appeared in lung cancer patients, at different locations ‎of the sequence were detected by BLAST tool. ‎

Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin

ABSTRACT Mycoplasma bovis isolates belonging to the sequence type 5 (ST5) group, the dominant group in Japan since 1999, were low susceptible to 16-membered macrolides and tetracyclines and were confirmed to have a guanine-to-adenine transition mutation at position 748 in the 23S rRNA gene (rrl) and adenine-to-thymine transversion mutations at positions 965 and 967 in the 16S rRNA gene (rrs) (Escherichia coli numbering). Moreover, isolates of ST93 and ST155, members of the ST5 group, were low susceptible to lincosamides and azithromycin and showed an adenine-to-guanine transition mutation at position 2059 of rrl. Isolates of ST93 were additionally low susceptible to spectinomycin and showed a cytosine-to-adenine transversion mutation at position 1192 of rrs. Strains of the ST5 group seem to spread to Japan and Europe from North America with imported cows, while strains of ST93 and ST155 originated in Japan. Melting curve analysis using hybridization probes revealed the existence of point mutations involved in decreased susceptibility to macrolides, lincosamides, and spectinomycin, as demonstrated by changes in the melting curve shape and/or decreases in the melting peak temperature, so the susceptibility to these antimicrobials can be assessed on the same day. For decreased susceptibility to fluoroquinolones to exist, nonsynonymous mutations in the DNA gyrase gene (gyrA) and topoisomerase IV gene (parC) had to coexist. The combination of amino acid substitutions of serine at position 83 in gyrA and serine at position 80 in parC resulted in particularly low susceptibility to fluoroquinolones. IMPORTANCE Mycoplasma bovis is the main causal species of bovine mycoplasmal disease and leads to significant economic losses because of its severe symptoms, strong infectivity, and refractoriness. As for mastitis, culling cows with intramammary infections is a general countermeasure to prevent spreading. The conventional antimicrobial susceptibility test for mycoplasma is time-consuming and troublesome, but no quick and easy method for grasping the antimicrobial susceptibility of the causal strain exists at present. Treatment without antimicrobial susceptibility information may be one reason why M. bovis infection is refractory. Detecting a mutation involved in decreased susceptibility to antimicrobial agents of the causal strain makes it possible to easily select suitable antimicrobials for treatment, and this technique will help improve the cure rate and prevent the overuse of ineffective antimicrobial agents. In this study, we developed a technique to quickly and easily assess antimicrobial susceptibility based on the genetic characteristics of M. bovis strains in Japan.

A missense mutation of Ip3r1 in Dp2 mice leads to short-term mydriasis and unfolded protein response in the iris constrictor muscles

ABSTRACTIp3r1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. Mutations in the Ip3r1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele (dilated pupil 2; Dp2) with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation, which is predicted to result in a C1976Y amino acid change in the open reading frame. Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Dp2. The dilated pupil phenotype in Dp2 was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Dp2 mice that did not recover. Apart from the dilated pupil phenotype (mydriasis), there are no other abnormal phenotypes including Ip3r1-related ataxia that may be found. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Dp2 mice represent a valuable self-healing model that may be used to study the therapeutic approach for Ip3r1-related diseases or diseases caused by similar pathomechanisms.

Euplotid: A quantized geometric model of the eukaryotic cell

Life continues to shock and amaze us, reminding us that truth is far stranger than fiction. http://Euplotid.io is a quantized, geometric model of the eukaryotic cell, an attempt at quantifying the incredible complexity that gives rise to a living cell by beginning from the smallest unit, a quanta. Starting from the very bottom we are able to build the pieces which when hierarchically and combinatorially combined produce the emergent complex behavior that even a single celled organism can show. Euplotid is composed of a set of quantized geometric 3D building blocks and constantly evolving dockerized bioinformatic pipelines enabling a user to build and interact with the local regulatory architecture of every gene starting from DNA-interactions, chromatin accessibility, and RNA-sequencing. Reads are quantified using the latest computational tools and the results are normalized, quality-checked, and stored. The local regulatory architecture of each gene is built using a Louvain based graph partitioning algorithm parameterized by the chromatin extrusion model and CTCF-CTCF interactions. Cis-Regulatory Elements are defined using chromatin accessibility peaks which are mapped to Transcriptional Start Sites based on inclusion within the same neighborhood. Deep Neural Networks are trained in order to provide a statistical model mimicking transcription factor binding, giving the ability to identify all Transcription Factors within a given chromatin accessibility peak. By in-silico mutating and re-applying the neural network we are able to gauge the impact of a transition mutation on the binding of any transcription factor. The annotated output can be visualized in a variety of 1D, 2D, 3D and 4D ways overlaid with existing bodies of knowledge such as GWAS results or PDB structures. Once a particular CRE of interest has been identified a Base Editor mediated transition mutation can then be performed in a relevant model for further study.

A Novel Interferon Regulatory Factor 6 Mutation in an Asian Family with Van der Woude Syndrome

Van der Woude syndrome (VWS) is a rare autosomal dominant genetic disorder characterized by orofacial clefting and lip pits. Mutations in the transcription factor interferon regulatory factor 6 gene ( IRF6) have been identified in individuals with VWS. We performed direct sequencing of the gene for molecular investigation of a proband with Bangladeshi-Malay ancestry. A novel transition mutation (c.113T>C), which resulted in an amino acid substitution (p.Ile38Thr) in the deoxyribonucleic acid-binding domain was detected. Testing of family members showed that the mutation segregated with the VWS phenotype for members of her immediate family. Although there is some phenotypic variability, all of the affected members are of the female gender.