Polymerase Gene Research Articles

Genomic and Pathologic Characterization of the First FAdV-C Serotype 4 Isolate from Black-Necked Crane.

Fowl adenoviruses (FAdVs) are distributed worldwide in poultry and incriminated as the etiological agents for several health problems in fowls, and are capable of crossing species barriers between domestic and wild fowls. An FAdV strain was, for the first time, isolated from black-necked crane in this study, and was designated as serotype 4 Fowl aviadenovirus C (abbreviated as BNC2021) according to the phylogenetic analysis of its DNA polymerase and hexon gene. The viral genomic sequence analysis demonstrated that the isolate possessed the ORF deletions that are present in FAdV4 strains circulating in poultry fowls in China and the amino acid mutations associated with viral pathogenicity in the hexon and fiber 2 proteins. A viral challenge experiment with mallard ducks demonstrated systemic viral infection and horizontal transmission. BNC2021 induced the typical clinical signs of hepatitis-hydropericardium syndrome (HHS) with swelling and inflammation in multiple organs and showed significant viral replication in all eight organs tested in the virus-inoculated ducks and their contactees at 6 dpi. The findings highlight the importance of surveillance of FAdVs in wild birds.

Acute bee paralysis virus field isolates from apiaries suffering colony losses in Türkiye

Multiple viral infections are one of the main drivers of honey bee colony losses and threaten the sustainability of colony health. The Acute bee paralysis virus (ABPV) is one of the common honey bee viruses that can induce overt and/or covert infections in bee colonies. Globally more knowledge is needed about its effects leading to colony losses. ABPV-related pathology includes paralytic, trembling bees, unable to fly and, in some cases, ABPV causes the death of the bee colonies. The molecular phylogenetic of ABPV isolates obtained from apiaries suffering colony losses and/or with CCD-like complaints in Türkiye was performed in this study. The isolates’ structural and non-structural gene regions were analyzed phylogenetically and compared according to sampling years and apiaries. According to the results, the nucleotide identity of the RNA-dependent RNA polymerase (RdRp) gene in Turkish ABPV field isolates is more conserved than the capsid gene. The evaluation of molecular differentiation among the ABPV field isolates can be used for determining the origin and relatedness of ABPV strains and periodic monitoring of ABPV infections in apiaries.

The development of a novel quantitative assay for the detection of convert mortality nodavirus (CMNV) in Litopenaeus vannamei

Due to the viral covert mortality disease (VCMD), a new disease brought on by covert mortality nodavirus (CMNV), the Litopenaeus vannamei aquaculture industry in China and Southeast Asian countries have experienced major economic losses over the past ten years. A rapid and accurate on-site pre-detection plays as the first step and a critical part in the efficient control of VCMD spreads and outbreaks. For the rapid and accurate on-site detection of CMNV, a novel real-time quantitative assay based on quantitative recombinase polymerase amplification (qRPA) was developed in this study. By optimizing the optimum primer pairs based on the conserved RNA-dependent RNA polymerase (RdRp) genes, appropriate reaction temperatures, and probe dosages. It was possible to perform the CMNV-qRPA test at 42 °C in 20 min without exhibiting any cross-reactivity with the four major aquatic infections. Then its ability was compared to real-time quantitative PCR (qPCR) as well as nested PCR assays for detection of CMNV. As results of the sensitivity tests, the limit of detection (LOD) for the CMNV-qRPA, CMNV nested PCR, and CMNV qPCR assays were 5.5 × 102 copies μL−1, 5.5 copies μL−1 and 5.5 × 102 copies μL−1, respectively. Based on these results, it is demonstrated that the CMNV-qRPA assay developed in this study is both sensitive and specific for the rapid on-site pre-diagnosis of CMNV. Those findings were also demonstrated the application potential of CMNV-qRPA assay for efficiently preventing and controlling the spread and outbreak of VCMD in the aquaculture industry and application prospect of this diagnosis method in other related fields.

Designing of rapid assay for the detection of RdRp/Orf1ab specific to SARS-CoV-2

SARS-CoV-2 is still threat and mostly used detection method is real time reverse transcriptase polymerase chain reaction (rRT-PCR) for the open reading frame (Orf1ab), RNA-dependent RNA polymerase (RdRp), nucleocapsid (N) and envelope (E) genes of virus. However, rRT-PCR may have false negative rate for the nucleic acid detection. Since the RdRp/Orf1ab has high sensitivity for the molecular detection, two sandwich models, Model 1A-Model 1B, based on hybridization on lateral flow assay (LFA) were designed here and applied with the synthetic and clinical samples of RdRp/Orf1ab. In this purpose colloidal gold nanoparticles (AuNPs) were used as label. Membranes having different flow rate, three oligonucleotide probe concentrations and running buffers were used. Although synthetic target sequence was recognized by all the LFAs, PCR products obtained from either the synthetic plasmid DNA or oro/nasopharyngeal swabs were detected by Model 1 A using W12 membrane. Designed strip assays detected the RdRp/Orf1ab of the clinical samples as 100% sensitivity and specifity. It means that they might be used for the detection of virus and can be modified for the recognition of mutant genes of virus. These findings also demonstrated the importance of membranes, sandwich models, probe concentrations and sample contents for developing LFAs for viral detection.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCRand western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

Implications of Translesion DNA Synthesis Polymerases on Genomic Stability and Human Health.

Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health.

Fast and highly sensitive screening of PCR products of severe acute respiratory syndrome coronavirus 2 by voltage‐programmed capillary gel electrophoretic separation

AbstractFast and highly sensitive detection has become critical to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, fast analysis tends to reduce the sensitivity and accuracy of such method. This study is to investigate a fast and highly sensitive screening method for detecting polymerase chain reaction (PCR) products of SARS‐CoV‐2 through voltage‐programmed capillary gel electrophoretic separation. By separating and detecting the PCR products of RNA‐dependent RNA polymerase (RdRp) and spike (S) genes, we have diagnosed coronavirus disease 2019 (COVID‐19) cases within 4 min using such method with high reliability and accuracy, indicating at least a 7.5‐fold faster than previous COVID‐19 diagnostic methods. The detection limits of RdRp and S genes fragments were 1.34–3.46 pM. The developed method can be used to analyze PCR products with specific DNA sizes as well as detect SARS‐CoV‐2 mutations without losing resolution. Furthermore, it can also be used for the fast analysis of DNA molecules related with various infectious diseases.

Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases.
 The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases.
 Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively.
 Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB).
 Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits.

Porcine Sapovirus in Northern Vietnam: Genetic Detection and Characterization Reveals Co-Circulation of Multiple Genotypes.

Porcine sapovirus (PoSaV) has been reported in many countries over the world, which may cause gastroenteritis symptoms in pigs with all ages. There has been no report on PoSaV infection in Vietnam up to now. In this study, a total of 102 samples were collected from piglets, fattening pigs, and sows with diarrhea in several cities and provinces in northern Vietnam. The PoSaV genome was examined using polymerase chain reaction (PCR). Sequencing of the partial RNA-dependent RNA polymerase (RdRp) gene sequences (324 bp) was performed. Of the 102 tested samples, 10 (9.8%) and 7/20 (35%) were detected as positive for the PoSaV RdRp gene using the PCR method at the individual and farm levels, respectively. Genetic analysis of the partial RdRp gene region of about 324 bp indicated that the nucleotide identity of the current 10 Vietnamese viral strains ranged from 61.39% to 100%. Among the 10 strains obtained, 8 belonged to genotype III and the remaining 2 strains were clustered in genotype VIII. The Vietnamese genotype III viruses formed two sub-clusters. The Vietnamese PoSaV strains were closely related to PoSaVs reported in South Korea, Venezuela, and the Netherlands. This research was the first to describe PoSaV infection in northern Vietnam during 2022-2023.

Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.).

Herpesviruses have been detected in bat species from several countries, with a limited number of studies examining herpesviruses in Pteropus spp. (flying foxes) and no investigation of herpesviruses in Australian flying foxes. We examined the presence and prevalence of herpesviruses in the four mainland Australian flying fox species. A nested PCR targeting highly conserved amino acid motifs in the DNA polymerase (DPOL) gene of herpesviruses was used to analyze 564 samples collected from 514 individual Pteropus scapulatus, Pteropus poliocephalus, Pteropus alecto, and Pteropus conspicillatus. The prevalence of herpesvirus DNA in blood, urine, oral, and fecal swabs from the four species was 17% in P. scapulatus, 11% in P. poliocephalus, 10% in P. alecto, and 9% in P. conspicillatus (31% in P. conspicillatus spleen tissue). Five putative novel herpesviruses were detected. Following PCR amplicon sequence analysis, four of the herpesviruses grouped phylogenetically with the gammaherpesviruses, with nucleotide identities between 79% and 90% to gammaherpesviruses from Asian megabats. A betaherpesvirus was detected in P. scapulatus with 99% nucleotide identity to the partial DPOL gene sequence of an Indonesian fruit bat betaherpesvirus. This study lays the foundation for future epidemiology research of herpesviruses in Australian Pteropus spp. and adds to the discussion of hypotheses surrounding the evolutionary epidemiology of bat-borne viruses on a global scale.

Gold nanostructures modified carbon-based electrode enhanced with methylene blue for point-of-care COVID-19 tests using isothermal amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) and RNA-dependent RNA polymerase (RdRP) genes were detected via electrochemical measurements using a screen-printed carbon electrode (SPCE) (3-electrode system) coupled with a battery-operated thin-film heater based on the loop-mediated isothermal amplification (LAMP) technique. The working electrodes of the SPCE sensor were decorated with synthesized gold nanostars (AuNSs) to obtain a large surface area and improve sensitivity. The LAMP assay was enhanced using a real-time amplification reaction system to detect the optimal target genes (E and RdRP) of SARS-CoV-2. The optimized LAMP assay was performed with diluted concentrations (from 0 to 109 copies) of the target DNA using 30 μM of methylene blue as a redox indicator. Target DNA amplification was conducted for 30 min at a constant temperature using a thin-film heater, and the final amplicon electrical signals were detected based on cyclic voltammetry curves. Our electrochemical LAMP analysis of SARS-CoV-2 clinical samples showed an excellent correlation with the Ct value of real-time reverse transcriptase-polymerase chain reaction, indicating successful validation of results. A linear relationship between the peak current response and the amplified DNA was observed for both genes. The AuNS-decorated SPCE sensor with the optimized LAMP primer enabled accurate analysis of both SARS-CoV-2-positive and -negative clinical samples. Therefore, the developed device is suitable for use as a point-of-care test DNA-based sensor for the diagnosis of SARS-CoV-2.

Isolation and Identification of Salmosamonella typhi from clinical samples with molecular detection of O-antigen encoded genes

Abstract 
 Antibiotic treatment of S.typhi is difficult as compared to treatment of acute infection. Antibiotic resistance carried against S.typhi by using 6 kinds of antibiotics from different classes, their results showed that all isolates were high resistance to Ampicillin (99%), Gentamicin (98%), Amikacin (79%) and less resistances Trimethoprim (55%) , Imipenem (60%) and Ceftriaxone(66%) .
 The present study focused on the molecular detection of Wzx flippase, Wzy polymerase genes in some Salmonella typhi isolates, Samples were collected from typhoid patients by classical lab work. Antibiotics susceptibility were investigated using disc diffusion method. The DNA and molecular Wzx flippase, Wzy polymerase were implemented using specific primers. The results showed that there was 33.33% having Wzy gene. The Wzx gene didn’t observed in any Salmonella isolates. The present study concluded that there was an important of the genetic diversity of O-antigen encoded genes included Wzx and Wzy which may be effect in typhoid diagnosis and treatment types.

Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory element analyses in sunflower (Helianthus annuus).

RNA interference (RNAi) regulates a variety of eukaryotic gene expressions that are engaged in response to stress, growth, and the conservation of genomic stability during developmental phases. It is also intimately connected to the post-transcriptional gene silencing (PTGS) process and chromatin modification levels. The entire process of RNA interference (RNAi) pathway gene families mediates RNA silencing. The main factors of RNA silencing are the Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. To the best of our knowledge, genome-wide identification of RNAi gene families like DCL, AGO, and RDR in sunflower (Helianthus annuus) has not yet been studied despite being discovered in some species. So, the goal of this study is to find the RNAi gene families like DCL, AGO, and RDR in sunflower based on bioinformatics approaches. Therefore, we accomplished an inclusive in silico investigation for genome-wide identification of RNAi pathway gene families DCL, AGO, and RDR through bioinformatics approaches such as (sequence homogeneity, phylogenetic relationship, gene structure, chromosomal localization, PPIs, GO, sub-cellular localization). In this study, we have identified five DCL (HaDCLs), fifteen AGO (HaAGOs), and ten RDR (HaRDRs) in the sunflower genome database corresponding to the RNAi genes of model plant Arabidopsis thaliana based on genome-wide analysis and a phylogenetic method. The analysis of the gene structure that contains exon-intron numbers, conserved domain, and motif composition analyses for all HaDCL, HaAGO, and HaRDR gene families indicated almost homogeneity among the same gene family. The protein-protein interaction (PPI) network analysis illustrated that there exists interconnection among identified three gene families. The analysis of the Gene Ontology (GO) enrichment showed that the detected genes directly contribute to the RNA gene-silencing and were involved in crucial pathways. It was observed that the cis-acting regulatory components connected to the identified genes were shown to be responsive to hormone, light, stress, and other functions. That was found in HaDCL, HaAGO, and HaRDR genes associated with the development and growth of plants. Finally, we are able to provide some essential information about the components of sunflower RNA silencing through our genome-wide comparison and integrated bioinformatics analysis, which open the door for further research into the functional mechanisms of the identified genes and their regulatory elements.

Protein expression systems combining a flavonoid-inducible promoter and T7 RNA polymerase in Bacillus subtilis.

Recombinant protein production must be tightly controlled when overproduction adversely affects the host bacteria. We developed a flavonoid-inducible T7 expression system in Bacillus subtilis using the qdoI promoter to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene controlled by the T7 promoter in a multicopy plasmid, we confirmed that this expression system is tightly regulated by flavonoids, such as quercetin and fisetin. Altering the qdoI promoter for T7 pol control to its hybrid derivative increased the expression level by 6.6-fold at maximum values upon induction. However, faint expression leakage was observed under a noninducing condition. Therefore, the two expression systems with the original qdoI promoter and the hybrid construct can be used selectively, depending on the high control accuracy or production yield required.

C-myc modulates the replication of RGNNV via glutamine-mediated ATP production in grouper fin cells

C-myc is a proto-oncogene that plays an important role in a variety of diseases. There were a lot of research on the correlation between C-myc and human viruses. However, the study about C-myc related to aquatic species virus is very limited. In the present study, the qRT-PCR, cellular immunofluorescence and western blotting determination data reported that C-myc and glutaminase (GLS) genes were significantly upregulated when grouper fin cells (GF-1) were infected with red grouper nervous necrosis virus (RGNNV). After knocking down the C-myc gene, the mRNA and protein levels of GLS, capsid protein (CP) and RNA polymerase (RdRp) of RGNNV were significantly reduced in RGNNV-infected GF-1 cells and the overexpression of the C-myc gene remarkably promoted these genes, which indicated that the replication of the virus and GLS gene were positively regulated by C-myc in RGNNV-infected GF-1 cells. In addition, supplementation of exogenous ATP can partially restore viral replication when RGNNV-infected GF-1 cells were cultured in glutamine-free medium, which confirmed that the glutamine was decomposed into ATP to provide energy for viral replication. Further studies confirmed that overexpression of C-myc can increase the content of ATP in normal cells. To sum up, these data suggested that activation of C-myc gene affected viral replication by regulating GLS expression to drive glutamine dissolution.

Towards understanding host–pathogen dynamics of cetacean poxvirus: attainable approach through the application of a repetitive non-invasive skin sampling in bottlenose dolphins (Tursiops truncatus) under human care

Nowadays, zoos and aquariums, along with the constant advancement of sociocultural moral values, are proactively committed to ensuring and safeguarding cetacean health standards. This entails developing new approaches to health assessments by embracing minimally invasive sampling methods and enhanced animal handling and management, among other aspects. Hence, in the present survey, to appraise skin diseases, the implementation of cytology cell samplers as a non-invasive skin sampling device on 18 bottlenose dolphins housed in two facilities in the Canary Islands during the months of April, October, and December 2019 was performed to isolate cetacean poxvirus in tattoo-like lesions through a real-time PCR-based method using the DNA polymerase gene. Samples were repeatedly collected over time from eleven tattoo-like lesions and from apparently healthy skin to serve as a control for all study animals. From a total of 55 skin samples, detection of the poxvirus was attained in 31 (56.36%); specifically, on 20 of 21 samples collected from tattoo-like lesions (95.23%) and on 11 of 34 samples acquired from apparently healthy skin (32.35%). Correspondingly, the current study constitutes the first report of the isolation of cetacean poxvirus in skin samples without macroscopical signs of tattoo lesions in cetaceans. Likewise, ten of the eleven dolphins that showed tattoo lesions housed in Facility 1 were positive for tattoo skin disease, while four dolphins held in Facility 2 were positive for cetacean poxvirus without ever showing clinical evidence of the disease. This raises the question of whether this pathogen can produce latent infections and whether progression of the disease may depend on environmental stimuli, viral load, or the good health/immunological status of individual animals. Accordingly, further scientific research on cetaceans under human care could provide the knowledge, skills, and resources to understand the host–pathogen dynamics of cetacean poxviruses and their effect on cetaceans’ health.

Tracing the evolutionary history of hepatitis B virus genotype H endemic to Mexico.

Hepatitis B virus (HBV) spreads efficiently among all human populations worldwide. HBV is classified into ten genotypes (A to J) with their geographic distribution and clinical features. In Mexico, HBV genotype H is the leading cause of hepatitis B and has been detected in indigenous populations, suggesting that HBV genotype H may be native to Mexico. However, little is known about the evolutionary history of HBV genotype H. Thus, we aimed to determine the age of HBV genotype H in Mexico using molecular dating techniques. Ninety-two HBV sequences of the reverse transcriptase (RT) domain of the polymerase gene (~1,251 bp) were analyzed; 48 were genotype H, 43 were genotype F, and the oldest HBV sequence from America was included as the root. All sequences were aligned, and the most recent common ancestor (TMRCA) time was calculated using the Bayesian Skyline Evolutionary Analysis. Our results estimate a TMRCA for the genotype H in Mexico of 2070.9 (667.5-4489.2) years before the present (YBP). We identified four major diversification events in genotype H, named H1, H2, H3, and H4. The TMRCA of H1 was 1213.0 (253.3-2638.3) YBP, followed by H2 1175.5 (557.5-2424.2) YBP, H3 949.6 (279.3-2105.0) YBP, and H4 1230.5 (336.3, 2756.7) YBP. We estimated that genotype H diverged from its sister genotype F around 8140.8 (1867.5-18012.8) YBP. In conclusion, this study found that genotype H in Mexico has an estimated age of 2070.9 (667.5-4489.2) YBP and has experienced at least four major diversification events since then.

Complete oxidative degradation of diclofenac via coupling free radicals and oxygenases of a micro/nanostructured biogenic Mn oxide composite from engineered Pseudomonas sp. MB04R-2

Oxidative degradation can effectively degrade aromatic emerging contaminants (ECs). However, the degradability of lone inorganic/biogenic oxides or oxidases is typically limited when treating polycyclic ECs. Herein, we report a dual-dynamic oxidative system comprising engineered Pseudomonas and biogenic Mn oxides (BMO), which completely degrades diclofenac (DCF), a representative halogen-containing polycyclic EC. Correspondingly, recombinant Pseudomonas sp. MB04R-2 was constructed via gene deletion and chromosomal insertion of a heterologous multicopper oxidase cotA, allowing for enhanced Mn(II)-oxidizing activity and rapid formation of the BMO aggregate complex. Additionally, we characterized it as a micro/nanostructured ramsdellite (MnO2) composite using multiple-phase composition and fine structure analyses. Furthermore, using real-time quantitative polymerase chain reaction, gene knockout, and expression complementation of oxygenase genes, we demonstrated the central and associative roles of intracellular oxygenases and cytogenic/BMO-derived free radicals (FRs) in degrading DCF and determined the effects of FR excitation and quenching on the DCF degradation efficiency. Finally, after identifying the degraded intermediates of 2H-labeled DCF, we constructed the DCF metabolic pathway. In addition, we evaluated the degradation and detoxification effects of the BMO composite on DCF-containing urban lake water and on biotoxicity in zebrafish embryos. Based on our findings, we proposed a mechanism for oxidative degradation of DCF by associative oxygenases and FRs.

Relationship Between FKBP5 Polymorphisms at Rs1334894, Rs9394309, and Rs6912833 Loci, and Dyslipidemia in Pediatric Nephrotic Syndrome

Background: This study aimed to investigate the relationship between rs1334894, rs9394309, and rs6912833 polymorphisms in <I>FKBP5</I> and hyperlipidemia in nephrotic syndrome (NS). Methods: The case group included 74 children with primary NS, while the control group included 76 healthy children. Polymerase chain reaction and gene sequencing were used to detect polymorphisms at the rs1334894, rs9394309, and rs6912833 loci of <I>FKBP5,</I> in children with NS. Clinical data of total cholesterol (CHOL), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and plasma albumin were collected. Correlations between <I>FKBP5</I> polymorphisms and serum lipid levels were analyzed during the active period of the disease. Results: A significant difference in LDL levels between the AA and TA genotypes was observed at the rs6912833 locus of <I>FKBP5</I> in the case group, but no statistical difference in CHOL, TG, and HDL levels between these genotypes was found. No statistically significant differences in LDL levels between the AA and TA genotypes, and among the steroid-sensitive, -dependent, and -resistant groups were noted. Moreover, no statistically significant differences in the CHOL, TG, HDL, and LDL levels were observed between TT and TC genotypes at the rs1334894 locus. Conclusion: The AA genotype at the rs6912833 locus of <I>FKBP5</I> may be correlated with plasma lipid levels (LDL) in PNS patients.