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Efficient DNA lesion repair is crucial for cell survival, especially within actively transcribed DNA regions that contain essential genetic information. Additionally, DNA breaks in regions of active transcription are prone to generating insertions and deletions, which are hallmark features of cancer genomes. Cockayne syndrome protein B (CSB) is the sole ATP-dependent chromatin remodeler that is essential for coupling DNA repair pathways with transcription, leading to more efficient DNA repair in regions of active transcription. CSB is best known for its essential function in transcription-coupled nucleotide excision repair (TC-NER), a process that rapidly removes helix-distorting DNA lesions that stall RNA polymerase II, such as those created by chemotherapeutic platinum compounds and UV irradiation. In addition to NER, CSB has also been reported to couple homologous recombination to transcription. Most recently, CSB has also been shown to couple single-strand DNA break repair to transcription. In this review, we will discuss the overlapping and distinct mechanisms by which CSB couples these different DNA repair pathways to transcription. We will also discuss how these CSB functions may account for Cockayne syndrome and the emerging roles of CSB as an innovative target for cancer therapy. Full article

Diagnostic testing plays a key role in a surveillance program as diagnostic testing aims to accurately determine the infection or disease status of an individual animal. Diagnostic assays for AIV can be categorized into four broad types: tests for detecting the virus, its antigen, its genomic material, and antibodies to the virus. Virus characterization almost always follows virus detection. The present article surveys the current literature on the goals, principles, test performance, advantages, and disadvantages of these diagnostic assays. Virus isolation can be achieved using embryonating eggs or cell cultures in a lab setting. Virus antigens can be detected by antigen-capturing immunoassays or tissue immunoassays. Viral RNA can be detected by PCR-based assays (gel-based reverse transcription–polymerase chain reaction (RT-PCR), or probe or SYBR^® Green-based real-time RT-PCR), loop-mediated isothermal amplification, in situ hybridization, and nucleic acid sequence-based amplification. Antibodies to AIV can be detected by ELISA, agar gel immunodiffusion, hemagglutination inhibition, and microneutralization. Avian influenza virus can be characterized by hemagglutination inhibition, neuraminidase inhibition, sequencing (dideoxynucleotide chain-termination sequencing, next-generation sequencing), genetic sequence-based pathotype prediction, and pathogenicity testing. Novel and variant AIVs can be recognized by DNA microarrays, electron microscopy, mass spectroscopy, and Biological Microelectromechanical Systems. A variety of diagnostic tests are employed in AIV surveillance and monitoring. The choice of their use depends on the goal of testing (fit for purpose), the time of testing during the disease, the assay target, the sample matrix, assay performance, and the advantages and disadvantages of the assay. The article concludes with authors’ perspective of the use of diagnostic assays in the surveillance and monitoring of AIV in poultry. Full article

Background: Variability in recanalization success during endovascular treatment for acute ischemic stroke (AIS) has led to increased interests in thrombus composition and associated cellular materials. While evidence suggests that bacteria may influence thrombus characteristics, limited data exist on microbiological profiles of thrombi in stroke patients. Objectives: Characterization of bacterial communities present in thrombi of AIS patients undergoing mechanical thrombectomy, providing insights into microbial contributions to stroke pathogenesis and treatment outcomes. Methods: Thrombi were collected from 20 AIS patients. After extracting metagenome, 16S rDNA sequencing was performed. Bioinformatic analysis included taxonomy and diversity assessments. The presence of bacterial DNA and viable bacteria in thrombi was validated using polymerase chain reaction (PCR) and bacterial culturing followed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis, respectively. Results: 16S rDNA was amplified in 19/20 thrombi (95%). Analysis identified a diverse microbial community, with Corynebacterium spp. as the most prevalent genus, followed by Staphylococcus spp., Bifidobacterium spp., Methylobacterium spp., and Anaerococcus spp. Alpha diversity analyses (Shannon index: 4.0–6.0 and Simpson index: 0.8–1.0) revealed moderate to high microbial diversity across samples; beta diversity demonstrated distinct clustering, indicating inter-patient variability in microbial profiles. PCR confirmed the presence of DNA specific to dominant bacterial taxa identified through sequencing. Culturing showed the presence of Staphylococcus epidermidis and Enterococcus faecalis in some clots as identified through MALDI analysis. Conclusions: This study shows bacterial communities present in AIS patients’ thrombi, suggesting a potential link between microbial signatures and thrombus characteristics. Full article

Background: Bladder cancer (BC) is a heterogeneous malignancy, and predicting response to immune checkpoint inhibitors (ICIs) remains a challenge. Herein, we investigate a high-risk bladder tumor, which developed during anti-BRAF/MEK therapy for a concurrent advanced BRAF-V600E-positive malignant melanoma (MM) and subsequently underwent complete spontaneous necrosis following Nivolumab immunotherapy, only to recur thereafter while still under the same treatment. This unique scenario provided an opportunity to investigate the roles of BRAF gene mutations in BC pathogenesis, respectively, of PD-L1 expression in immunotherapy response prediction. Methods: We retrospectively analyzed BC specimens obtained via transurethral resection at two critical time-points: prior to the complete spontaneous necrosis under Nivolumab (prenecrosis) and after tumor recurrence postnecrosis (postnecrosis). The BRAF gene mutation status was evaluated using quantitative polymerase chain reaction (qPCR). PD-L1 expression was assessed by immunohistochemistry (IHC), quantified using the combined positive score (CPS), and a cutoff of ≥10 for positivity. Results: Neither pre- nor postnecrosis BC samples harbored BRAF gene mutations. Prenecrosis PD-L1 expression (CPS = 5) indicated a minimal likelihood of response to immunotherapy. However, complete spontaneous necrosis occurred under Nivolumab, followed by recurrence with further reduced PD-L1 expression (CPS = 1). Conclusions: The complete BC regression challenges the conventional role of PD-L1 as a sole predictive biomarker for immunotherapy. This study also highlights the potential role of BRAF/MEK inhibitors in BC oncogenesis and underscores the need for alternative biomarkers, such as tumor mutation burden (TMB) and circulating tumor DNA (ctDNA), to guide treatment selection in BC better. Full article

Background: The nuclear-encoded enzyme polymerase gamma (Pol-γ) is crucial in the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence numerous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SDs) and are mainly autosomal-recessively inherited. Clinical manifestations include muscle weakness and fatigue, and severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with seizures, liver failure, or intractable epilepsy. Here, we analyzed fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SDs, sensory ataxic neuropathy, and infantile muscular atrophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative in human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first-degree relative (the mother of the patient), an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. Assessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass, and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles, without further progression. The patient also reported fatigue and muscle pain after physical activity, with no sensory deficits. Extensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carried the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first-degree relative (his mother) who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction in mtDNA content (~50%) and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was reduced by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as determined by fluorescence microscopy and mitochondrial staining. Conclusions: Our findings suggest that the c.678G>C; p.(Gln226His) variant, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first-degree relative (but with an identical mitochondrial genome), the pathogenicity of c.678G>C; p.(Gln226His) of POLG should be confirmed in future studies, in particular, in conjunction with other POLG-variants. Full article

Background: The pyrrolobenzodiazepine (PBD) dimer SJG-136 reached Phase II clinical trials in ovarian cancer and leukaemia in the UK and USA in the 2000s. Several structural analogues of SJG-136 are currently in clinical development as payloads for Antibody-Drug Conjugates (ADCs). There is growing evidence that PBDs exert their pharmacological effects through inhibition of transcription factors (TFs) in addition to arrest at the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. Hence, PBDs can be used to target specific DNA sequences to inhibit TFs as a novel anticancer therapy. Objective: To explore the ability of SJG-136 to bind to the cognate sequences of transcription factors using a previously described HPLC/MS method, to obtain preliminary mechanistic evidence of its ability to inhibit transcription factors (TF), and to determine its effect on TF-dependent gene expression. Methods: An HPLC/MS method was used to assess the kinetics and thermodynamics of adduct formation between the PBD dimer SJG-136 and the cognate recognition sequence of the TFs NF-κB, EGR-1, AP-1, and STAT3. CD spectroscopy, molecular dynamics simulations, and gene expression analyses were used to rationalize the findings of the HPLC/MS study. Results: Notable differences in the rate and extent of adduct formation were observed with different DNA sequences, which might explain the variations in cytotoxicity of SJG-136 observed across different tumour cell lines. The differences in adduct formation result in variable downregulation of several STAT3-dependent genes in the human colon carcinoma cell line HT-29 and the human breast cancer cell line MDA-MB-231. Conclusions: SJG-136 can disrupt transcription factor-mediated gene expression, which contributes to its exceptional cytotoxicity in addition to the DNA-strand cleavage initiated by its ability to crosslink DNA. Full article

Scrub typhus is a mite-borne, re-emerging public health problem in India, particularly in Tamil Nadu, South India. More than 40 serotypes of Orientia tsutsugamushi have been documented worldwide. However, the information on the circulation of its molecular sub-types in India is scanty. A retrospective study was conducted among serologically confirmed cases of scrub typhus. DNA isolated from blood was screened by a nested polymerase chain reaction (nPCR) targeting the GroEL and the 56 kDa type-specific antigen (TSA) genes. Out of 59 samples, 14 partial fragments of GroEL and the twelve 56 kDa genes were PCR-amplified and DNA-sequenced. The neighbor-joining (NJ) analysis indicated three distinct phylogenetic clades, including a novel genotype designated as Ot-Thanjavur-Tamil Nadu (Ot-TJTN, 9 nos. 64.3%); Karp-like (4 nos. 28.6%); and Kuroki-Gilliam type (1 no. 7.1%). Also, phylogenetic analysis of twelve 56 kDa variable domains (VDΙ-ΙΙΙ) of TSA gene sequences revealed a distinctive new genotypic cluster of eight samples (66.6%), and the remaining four (33.4%) were Karp-like genotypes. The Simplot analysis for the similarity and event of recombination testing elucidated the existence of the new genotype of the Ot-TJTN cluster, which was undescribed so far, in the Kato and TA716 lineages. The significant findings recommend further studies to understand the ongoing transmission dynamics of different O. tsutsugamushi strains in vector mites, rodent hosts, and humans in this region. Full article

There are two forms of DNA polymerase δ in human cells, Pol δ4 and Pol δ3, which differ based on their possession of the p12 subunit. The degradation of p12 has emerged as an important regulatory mechanism that controls the generation of Pol δ3. The underlying importance of this system lies in the altered enzymatic properties of the two forms of Pol δ engendered by the influence of p12. We briefly review how the balance of these two forms is regulated through the degradation of p12. We focus on the roles of Pol δ4, whose cellular functions are less well known. This is significant because recent studies show that this is the form engaged in the homology-dependent repair of double-strand breaks. We consider new horizons for future research into this system and their potential involvement in tumorigenesis. Full article

The Replicon Theory has guided the way experiments into DNA replication have been designed and interpreted for 60 years. As part of the related, explanatory package guiding experiments, it is thought that the timing of the cell cycle depends in some way on a critical mass for initiation, Mi, as licensed by a variety of macromolecules and molecules reflecting the state of the cell. To help in the re-interpretation of this data, we focus mainly on the roles of DnaA, RNA polymerase, SeqA, and ribonucleotide reductase in the context of the “nucleotypic effect”. Full article

Harmful blooms of the dinoflagellate Cochlodinium polykrikoides (Margalefidinium polykrikoides) had detrimental aquacultural and economic effects globally, and to reduce the damage caused by these blooms, early biomonitoring and quantitative analysis of C. polykrikoides are of the utmost importance. Here, for the detection of C. polykrikoides using quantitative real-time polymerase chain reactions, we developed specific primers targeting the large subunit ribosomal DNA (LSU rDNA) and evaluated their applicability in the field during the occurrence of a C. polykrikoides bloom. The specific primers not only accurately detected C. polykirkoides but also had a detection performance comparable with that obtained using microscopic observations. Accordingly, we developed a system that can be used in the field and applied when red tides occur, with accurate results being obtained more than five times more rapidly than those obtained based on microscopic analysis. Collectively, our findings indicate that the C. polykrikoides bloom detection system developed in this study can be applied to rapidly detect and accurately quantify C. polykrikoides in environmental samples. Data obtained using this system could be used as a basis for developing prompt monitoring and warning systems for the early detection of C. polykrikoides blooms in the field. Full article

In a recent clinical trial, beetroot juice supplementation for 14 days yielded positive effects on systemic inflammation in adults with long COVID. Here, we explored the relationship between circulating markers of mitochondrial quality and inflammation in adults with long COVID as well as the impact of beetroot administration on those markers. We conducted secondary analyses of a placebo-controlled randomized clinical trial testing beetroot juice supplementation as a remedy against long COVID. Analyses were conducted in 25 participants, 10 assigned to placebo (mean age: 40.2 ± 11.5 years, 60% women) and 15 allocated to beetroot juice (mean age: 38.3 ± 7.7 years, 53.3% women). Extracellular vesicles were purified from serum by ultracentrifugation and assayed for components of the electron transport chain and mitochondrial DNA (mtDNA) by Western blot and droplet digital polymerase chain reaction (ddPCR), respectively. Inflammatory markers and circulating cell-free mtDNA were quantified in serum through a multiplex immunoassay and ddPCR, respectively. Beetroot juice administration for 14 days decreased serum levels of interleukin (IL)-1β, IL-8, and tumor necrosis factor alpha, with no effects on circulating markers of mitochondrial quality control. Significant negative associations were observed between vesicular markers of mitochondrial quality control and the performance on the 6 min walk test and flow-mediated dilation irrespective of group allocation. These findings suggest that an amelioration of mitochondrial quality, possibly mediated by mitochondria-derived vesicle recycling, may be among the mechanisms supporting improvements in physical performance and endothelial function during the resolution of long COVID. Full article

The polymerase chain reaction (PCR) is a molecular biology tool with diverse applications in the aquatic sciences. Classical PCR is a nonquantitative method that can be used to detect target DNA sequences that are characteristic of particular microbial taxa but cannot determine their concentrations in water samples. Various quantitative forms of PCR have been developed to remove this limitation. Of these, the two that currently are used most widely are real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Several outlines of the mathematical and statistical basis of these methods for estimating target sequence concentrations are available in the literature, but we are aware of no thorough and rigorous derivation of the theoretical underpinnings of either. The purpose of this review is to provide such derivations, and to identify and compare the main strengths and weaknesses of the two methods. We find that both estimation methods are sound, provided careful attention is paid to specific details that differ between the two. With qPCR, it is especially important to reduce any significant PCR inhibition by sample constituents and to properly fit the standard curve to heteroskedastic calibration data. With ddPCR, it is important to ensure that the value of the mean droplet volume used in calculating concentrations is correct for the particular combination of droplet generator and master mix used. The advantages of qPCR include lower instrument and per-sample costs, a shorter turnaround time for obtaining results, a higher upper limit of quantification, and a wider dynamic range. The advantages of ddPCR include freedom from dependence on a standard curve, an inherently lower sensitivity to PCR inhibitors, a lower limit of quantification, a simpler theoretical basis, and simpler data analysis. We suggest qPCR often will be preferable in laboratory studies where investigators have significant control over the range of target sequence concentrations in samples, concentrations are sufficiently high so proper calibration does not require standards with concentrations low enough to exhibit exaggerated variability in the threshold cycle, and no significant inhibition is present, or more generally, in studies where funding levels do not permit the higher cost of instrumentation and supplies required by ddPCR or where the shorter turnaround time for qPCR is essential. If sufficient funds are available, ddPCR often will be preferable when the ability to quantify low concentrations is important, especially if inhibitors are likely to be present at concentrations that are problematic for qPCR. Full article

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are few effective treatments available to treat infection and KSHV oncogenesis. Disrupting the KSHV infectious cycle would diminish the viral spread. The KSHV lytic phase and production of new virions require efficient copying and packaging of the KSHV genome. KSHV encodes its own lytic DNA replication machinery, including the processivity factor (PF-8), which presents itself as an attractive target for antiviral development. We characterized PF-8 at the single molecule level using transmission electron microscopy to identify key molecular interactions that mediate viral DNA replication initiation. Our results indicate that PF-8 forms oligomeric ring structures (tetramer, hexamer, and/or dodecamer) similar to the related Epstein–Barr virus processivity factor (BMRF1). Our DNA positional mapping revealed high-frequency binding locations of PF-8 within the lytic origin of replication (OriLyt). A multi-variable analysis of PF-8 DNA-binding activity with three mutant OriLyts provides new insights into the mechanisms that PF-8 associates with viral DNA and complexes to form multi-ring-like structures. Collectively, these data enhance the mechanistic understanding of the molecular interactions (protein–protein and protein-DNA) of an essential KSHV DNA replication protein. Full article

Background/Objectives: Short tandem repeat expansions are the most common cause of inherited neurological diseases. These disorders are clinically and genetically heterogeneous, such as in myotonic dystrophy and spinocerebellar ataxia, and they are caused by different repeat motifs in different genomic locations. Major advances in bioinformatic tools used to detect repeat expansions from short read sequencing data in the last few years have led to the implementation of these workflows into next generation sequencing pipelines in healthcare. Here, we aimed to evaluate the clinical utility of analysing repeat expansions through exome sequencing in a large cohort of genetically undiagnosed patients with neurological disorders. Methods: We here analyse 27 disease-causing DNA repeats found in the coding, intronic and untranslated regions in 12,496 exomes in patients with a range of neurogenetic conditions. Results: We identified—and validated by polymerase chain reaction—29 repeat expansions across a range of loci, 48% (n = 14) of which were diagnostic. We then analysed the genotyping performance across all repeat loci and found that, despite high coverage in most repeats in coding regions, some loci had low genotyping rates, such as those that cause spinocerebellar ataxia 2 (ATXN2, 0.1–8.4%) and Huntington disease (HTT, 0.2–58.2%), depending on the capture kit. Conversely, while most intronic repeats were not genotyped, we found a high genotyping rate in the intronic locus that causes spinocerebellar ataxia 36 (NOP56, 30.1–98.3%) and in the one that causes myotonic dystrophy type 1 (DMPK, myotonic dystrophy type 1). Conclusions: We show that the key factors that influence the genotyping rate of repeat expansion loci analysis are the sequencing read length and exome capture kit. These results provide important information about the performance of exome sequencing as a genetic test for repeat expansion disorders. Full article

Hypoxia due to stroke is a major cause of neuronal damage, leading to loss of cognition and other brain functions. Sevoflurane preconditioning improves recovery after hypoxia. Hypoxia interferes with protein expression at the translational level; however, its effect on mRNA levels for neuronal protein kinase and anti-apoptotic genes is unclear. To investigate the link between sevoflurane preconditioning and gene expression, hippocampal slices were treated with 4% sevoflurane for 15 min, a 5 min washout, 10 min of hypoxia, and 60 min of recovery. We used quantitative PCR to measure mRNA levels in the CA1 region of rat hippocampi. The mRNA levels for specific critical proteins were examined, as follows: Protein kinases, PKCγ (0.22), PKCε (0.38), and PKMζ (0.55) mRNAs, and anti-apoptotic, bcl-2 (0.44) and bcl-xl (0.41), were reduced 60 min after hypoxia relative to their expression in tissue not subjected to hypoxia (set to 1.0). Sevoflurane preconditioning prevented the reduction in PKMζ (0.88 vs. 1.0) mRNA levels after hypoxia. Pro-apoptotic BAD mRNA was not significantly changed after hypoxia, even with sevoflurane preconditioning (hypoxia 0.81, sevo hypoxia 0.84 vs. normoxia 1.0). However, BAD mRNA was increased by sevoflurane in non-hypoxic conditions (1.48 vs. 1.0), which may partially explain the deleterious effects of volatile anesthetics under certain conditions. The DNA repair enzyme poly ADP-ribose polymerase 1 (PARP-1) was increased by sevoflurane in tissue not subjected to hypoxia (1.23). PARP-1 mRNA was reduced in untreated tissue after hypoxia (0.21 vs. 1.0); sevoflurane did not improve PARP-1 after hypoxia (0.27). Interestingly, the mRNA level of the cognitive kinase PKMζ, a kinase essential for learning and memory, was the only one protected against hypoxic downregulation by sevoflurane preconditioning. These findings correlate with previous studies that found that sevoflurane-induced improvement of neuronal survival after hypoxia was dependent on PKMζ. Maintaining mRNA levels for critical proteins may provide an important mechanism for preserving neuronal function after stroke. Full article