In Silico Study of the Effectiveness of 16S rRNA Gene as a Universal Genetic Marker to Identify Closely Related Burkholderia Spp. in Panicle Blight of Rice

The 16S rRNA gene is a housekeeping genetic marker that is available in almost all bacterial species and it is used in bacterial phylogeny and taxonomy studies. In many studies, the 16S rRNA gene is used in identification of certain bacterial species. Being a less conserved genetic marker, certain studies found it is a useful tool to infer the genome-wide similarity levels among the closely related prokaryotic organisms. Thus, this study aimed to compare the variation in the 16S rRNA partial region of Burkholderia spp. that infect the panicle of rice from eight different geographical areas. 58 sequences with total of 688 base pairs (bp) of 16S rRNA gene in B. glumae and B. gladioli were retrieved from public database based on several countries namely United State, Panama, Ecuador, Thailand, China, India, Korea and Malaysia. Then, the data sequences were analysed and validated using MEGAX and ABGD software respectively. The result of phylogenetic tree confirmed that B. glumae and B. gladioli were species that present in the panicle blight of rice. However, Data Analysis in Molecular Biology and Evolution (DAMBE) and Automatic Barcode Gap Discovery (ABGD) software were not able to detect substitution saturation and divergence between B. glumae and B. gladioli respectively based on the 58 sequences of the 16S rRNA partial region. Hence, it proves that 16S rRNA gene is an ineffective genetic marker to be used to differentiate the closely related species of bacteria from similar genus.

Persistence of fecal indicator bacteria and associated genetic markers from wastewater treatment plant effluents in freshwater microcosms

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment.

Differentiating closely affiliated Dehalococcoides lineages by a novel genetic marker identified via computational pangenome analysis

As a group, Dehalococcoides dehalogenate a wide range of organohalide pollutants but the range of organohalide compounds that can be utilized for reductive dehalogenation differs among the Dehalococcoides strains. Dehalococcoides lineages cannot be reliably disambiguated in mixed communities using typical phylogenetic markers, which often confounds bioremediation efforts. Here, we describe a computational approach to identify Dehalococcoides genetic markers with improved discriminatory resolution. Screening core genes from the Dehalococcoides pangenome for degree of similarity and frequency of 100% identity found a candidate genetic marker encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function. This gene exhibits the fewest completely identical amino acid sequences and among the lowest average amino acid sequence identity in the core pangenome. Primers targeting BNR could effectively discriminate between 40 available BNR sequences ( in silico ) and 10 different Dehalococcoides isolates ( in vitro ). Amplicon sequencing of BNR fragments generated from 22 subsurface soil samples revealed a total of 109 amplicon sequence variants, suggesting a high diversity of Dehalococcoides distributed in environment. Therefore, the BNR gene can serve as an alternative genetic marker to differentiate strains of Dehalococcoides in complicated microbial communities. Importance The challenge of discriminating between phylogenetically similar but functionally distinct bacterial lineages is particularly relevant to the development of technologies seeking to exploit the metabolic or physiological characteristics of specific members of bacterial genera. A computational approach was developed to expedite screening of potential genetic markers among phylogenetically affiliated bacteria. Using this approach, a gene encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function was selected and evaluated as a genetic marker to differentiate strains of Dehalococcoides , an environmentally relevant genus of bacteria whose members can transform and detoxify a range of halogenated organic solvents and persistent organic pollutants, in complex microbial communities to demonstrate the validity of the approach. Moreover, many apparently phylogenetically distinct, currently uncharacterized Dehalococcoides were detected in environmental samples derived from contaminated sites.

VITAMIN D AND UROLITHIASIS IN CHILDREN

Background. Urolithiasis is currently one of the topical issues of contemporary urology and medicine in general. This is primarily due to the high prevalence of urolithiasis; according to several population studies it ranges from 3.5 to 9.6%. At the same time, there is a steady increase in its incidence. Therefore, the matter of urolithiasis is one of the most urgent in present-day medicine. Objectives. The aim of the research was to study the content of a polymorphic genetic marker of the vitamin D receptor gene related to development and relapse of urolithiasis in children. Methods. The content of a polymorphic genetic marker of the vitamin D receptor gene related to development and relapse of urolithiasis in 100 children was investigated. Results. The results of the study prove that the vitamin D receptor gene assists in revealing disorders that promote urolithiasis development. Conclusion. Comparative analysis of the frequency of distribution of Fok1 genotypes of the vitamin D receptor gene polymorphism showed that statistical significance of the association (p=0.02) of f allele according to the dominant inheritance model (total Ff+ff genotypes) was established in the group of patients with urolithiasis compare to the corresponding indicator of the control group (63%).

Molecular Markers for Human Sex Determination in Forensic Genetics Analysis

Sex determination is indispensable in forensic anthropology, sexual disorder, and also as part of large-scale genetic population studies. The purpose of this investigation is to determine the human sex from whole blood using multiplex PCR analysis. Blood samples from 75 male and 70 female healthy volunteers were taken from Tikrit city, Iraq. Our study identified a reliable set of three primer locus, namely SRY, ALT1 (internal control) and amelogenin locus. The SRY primer on the Y chromosome showed a 254 bp of PCR product, with 100% accuracy for human male identification. Thus, the pair of SRY primers was considered a strong genetic marker for human sex identification. Amelogenin regions in the Y chromosome showed a true positive band (236 bp) with 100% accuracy on sex identification. Amelogenin regions in X chromosome also showed positive bands (330 bp) in female samples and positive band in male samples except for two samples showed a negative band (null bands). The most obvious finding from this study is that multiplex PCR of ALT1 and SRY is consider as a reliable genetic marker for human sex identification. The research has also shown that amelogenin is good genetic marker for human sex identification.

PABPN1L assemble into “ring-like” aggregates in the cytoplasm of MII oocytes and is associated with female infertility†

Infertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A resent study showed that PABPN1L recruited BTG4 to mRNA 3′-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated an PABPN1L-antibody and found “ring-like” PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed“ring-like” aggregates in vitro. This phenomenon is similar with CCR4–NOT catalytic subunit, CNOT7, when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l −/− and Pabpn1l tm1a/tm1a) simulating the intron1-exon2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l−/− MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and Cnot61 genes are necessary for the deadenylation process and Pabpn1l −/− resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4–NOT-mediated maternal mRNA degradation and PABPN1L variants on intron1-exon2 could be a genetic marker of female infertility. Summary sentence. “Ring-like” PABPN1L aggregates was found in the cytoplasm of MII oocytes and in vitro; intron1-exon2 abnormality of Pabpn1l leads female sterile in mice.