Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Clinical value of FAT1 mutations to indicate the immune response in colorectal cancer patients.

Immunotherapy is currently approved for CRC whose tumors have high MSI-H. To find additional biomarkers for immunotherapy in CRC, targeted sequencing was performed on tumor tissues from a discovery cohort of 161 CRC patients. Validation cohorts from the cBioPortal were also used for survival and tumor cell infiltration analyses. The FAT1-mutated CRC group often co-occurred with MSI events and displayed a higher tumor mutational burden compared to the FAT1 wild-type CRC. Overall survival was higher in patients with FAT1 mutations than in patients with wild type FAT1. The altered PI3K-AKT pathway and immune pathways were enriched in the FAT1-mutated CRC. A higher infiltration rate of immune cells including CD4+ T cells, CD8+ T cells, macrophages M1 and regulatory T cells were also observed in the colorectal tumors with FAT1 mutation compared to tumors with wild type FAT1. The results showed that CRC patients with FAT1 mutations exhibited an immunotherapy-favorable profile.

Decreased expression of ATP-binding cassette protein G1 promotes abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

BACKGROUND: Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). ATP-binding cassette protein G1 (ABCG1) is crucial in mediating the outflow of cholesterol, phosphatidylcholine and sphingomyelin and reducing intracellular lipid accumulation. OBJECTIVE: This study aimed to evaluate whether ABCG1 participates in the abnormal adipogenesis of chondrocytes in osteoarthritic cartilage of temporomandibular joint. METHODS: Eight-week-old female rats were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical (IHC) staining, and qRT-PCR were performed. Primary condylar chondrocytes of rats were transfected with ABCG1 shRNA or overexpression lentivirus and then stimulated with fluid flow shear stress (FFSS). Cells were collected for oil red O staining, immunofluorescence staining, and qRT-PCR analysis. RESULTS: Abnormal adipogenesis, characterized by increased expression of Adiponectin, CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4) and Perilipin1, was enhanced in the degenerative cartilage of TMJ OA in rats with UAC, accompanied by decreased expression of ABCG1. After FFSS stimulation, we observed lipid droplets in the cytoplasm of cultured cells with increased expression of Adiponectin, Cebpα, Fabp4 and Perilipin1 and decreased expression of ABCG1. Knockdown of Abcg1 induced abnormal adipogenesis and differentiation of condylar chondrocytes. Overexpression of ABCG1 alleviated the abnormal adipogenesis and differentiation of condylar chondrocytes induced by FFSS. CONCLUSIONS: Abnormal adipogenesis of chondrocytes and decreased ABCG1 expression were observed in degenerative cartilage of TMJ OA. ABCG1 overexpression effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration.

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration.

Macrophages and neutrophils are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, which presumably facilitate the regrowth of injured tissues. Taking advantage of the regeneration abilities of zebrafish and the newly generated leukocyte-specific zebrafish lines with labeling of both leukocyte lineages, we assessed the behaviors and functions of neutrophils and macrophages during tail fin regeneration. Live imaging showed that within 6 hours post amputation, the inflammatory stage, neutrophils were the primary cells scavenging apoptotic bodies and small cell debris, although they had limited phagocytic capacity and quickly underwent apoptosis. From 6 hours post amputation on, the resolution and regeneration stage, macrophages became the dominant scavengers, efficiently resolving inflammation and facilitating tissue remodeling and regrowth. Ablation of macrophages but not neutrophils severely impaired the inflammatory resolution and tissue regeneration, resulting in the formation of large vacuoles in the regenerated fins. In contrast, removal of neutrophils slightly accelerates the regrowth of injured fin. Our study documents the differing behaviors and functions of macrophages and neutrophils during tissue regeneration.

Irf8 regulates macrophage versus neutrophil fate during zebrafish primitive myelopoiesis.

In vertebrates, myeloid cells comprise polymorphonuclear and mononuclear lineages that arise from 2 successive waves of development: a transitory primitive wave giving rise to limited myeloid cells during embryonic stage and a definitive wave capable of producing myeloid cells throughout the fetal and adult life. One key unresolved question is what factors dictate polymorphonuclear versus mononuclear lineage fates during myelopoiesis. Here we show that during zebrafish embryogenesis interferon regulatory factor-8 (irf8) is expressed specifically in macrophages but not neutrophils. Suppression of Irf8 function in zebrafish causes a depletion of macrophages and an enhanced output of neutrophils but does not affect the overall number, proliferation, and survival of primitive myeloid cells. These data indicate that the skewed myeloid lineage development in Irf8 knockdown embryos results from a cell-fate switching. Such a conclusion is further supported by the observation showing that overexpression of Irf8 promotes macrophage formation at the expense of neutrophil development. Genetic epistasis analysis reveals that Irf8 acts downstream of Pu.1 but is insufficient to promote macrophage development in the absence of Pu.1. Our findings demonstrate that Irf8 is a critical determinant for neutrophil versus macrophage fate choice during zebrafish primitive myelopoiesis.

Case Report: Cancer spectrum and genetic characteristics of a de novo germline POLD1 p.L606M variant-induced polyposis syndrome.

Germline variations in the DNA polymerase genes, POLE and POLD1, can lead to a hereditary cancer syndrome that is characterized by frequent gastrointestinal polyposis and multiple primary malignant tumors. However, because of its rare occurrence, this disorder has not been extensively studied. In this report, we present the case of a 22-year-old female patient who had been diagnosed with gastrointestinal polyposis, breast fibroadenoma, multiple primary colorectal cancers, and glioblastoma (grade IV) within a span of 4 years. Next-generation sequencing analysis revealed a germline variant in POLD1 (c.1816C>A; p.L606M). In silico analysis using protein functional predicting software, including SIFT, Polyphen, GERP++, and CADD, further confirmed the pathogenicity of POLD1 p.L606M (classified as ACMG grade Class 4). In line with polymerase deficiency, both rectal cancer and glioblastoma tissues exhibited a high tumor mutation burden, with 16.9 muts/Mb and 347.1 muts/Mb, respectively. Interestingly, the patient has no family history of cancer, and gene examination of both parents confirms that this is a de novo germline variant. Therefore, molecular screening for POLD1 may be necessary for patients with such a cancer spectrum, regardless of their family history.

Premature ovarian insufficiency: a review on the role of oxidative stress and the application of antioxidants.

Normal levels of reactive oxygen species (ROS) play an important role in regulating follicular growth, angiogenesis and sex hormone synthesis in ovarian tissue. When the balance between ROS and antioxidants is disrupted, however, it can cause serious consequences of oxidative stress (OS), and the quantity and quality of oocytes will decline. Therefore, this review discusses the interrelationship between OS and premature ovarian insufficiency (POI), the potential mechanisms and the methods by which antioxidants can improve POI through controlling the level of OS. We found that OS can mediate changes in genetic materials, signal pathways, transcription factors and ovarian microenvironment, resulting in abnormal apoptosis of ovarian granulosa cells (GCs) and abnormal meiosis as well as decreased mitochondrial Deoxyribonucleic Acid(mtDNA) and other changes, thus accelerating the process of ovarian aging. However, antioxidants, mesenchymal stem cells (MSCs), biological enzymes and other antioxidants can delay the disease process of POI by reducing the ROS level in vivo.

GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

AIMS: Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear. METHODS: Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis. RESULTS: Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice. CONCLUSION: Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453-464.

The Application of Complementary and Alternative Medicine in Polycystic Ovary Syndrome Infertility.

Polycystic ovary syndrome (PCOS) is a lifelong reproductive endocrine disease, which is the most common cause of anovular infertility. Modern medicine mainly treats infertile patients with PCOS by improving living habits, ovulation induction therapy, and assisted reproductive technology (ART), but the effect is not satisfied. Complementary alternative medicine (CAM) has conspicuous advantages in the treatment of PCOS infertility due to its good clinical efficacy, wide mechanism of action, and no obvious adverse reactions, but its safety and effectiveness in the treatment of PCOS infertility have not been proved. Based on the existing clinical and experimental studies, this paper looks for the therapeutic effect and the mechanism behind it, and explores the safety and effectiveness of its treatment in PCOS infertility, in order to provide reference for future clinical treatment and experimental research.

Structure-Guided Discovery of Potent Antifungals that Prevent Ras Signaling by Inhibiting Protein Farnesyltransferase.

Infections by fungal pathogens are difficult to treat due to a paucity of antifungals and emerging resistances. Next-generation antifungals therefore are needed urgently. We have developed compounds that prevent farnesylation of Cryptoccoccus neoformans Ras protein by inhibiting protein farnesyltransferase with 3-4 nanomolar affinities. Farnesylation directs Ras to the cell membrane and is required for infectivity of this lethal pathogenic fungus. Our high-affinity compounds inhibit fungal growth with 3-6 micromolar minimum inhibitory concentrations (MICs), 4- to 8-fold better than Fluconazole, an antifungal commonly used in the clinic. Compounds bound with distinct inhibition mechanisms at two alternative, partially overlapping binding sites, accessed via different inhibitor conformations. We showed that antifungal potency depends critically on the selected inhibition mechanism because this determines the efficacy of an inhibitor at low in vivo levels of enzyme and farnesyl substrate. We elucidated how chemical modifications of the antifungals encode desired inhibitor conformation and concomitant inhibitory mechanism.

TP53 Co-Mutational Features and NGS-Calibrated Immunohistochemistry Threshold in Gastric Cancer.

PURPOSE: TP53 is the most frequently mutated gene in gastric cancer and it can be potentially used for gastric cancer diagnosis and screening. However, standardized clinical approaches that could accurately and cost-effectively detect TP53 mutations in gastric cancer are largely lagged behind. PATIENTS AND METHODS: We conducted next-generation sequencing (NGS) analysis of 425 cancer-related genes in 42 gastric cancer patients in our cohort. A 1313-patient cohort derived from the cBioPortal database was used for validation. We performed immunohistochemistry (IHC) staining with four commonly used p53 antibodies, and the NGS results were used as the gold standard to optimize the IHC threshold for each antibody. RESULTS: By NGS analysis, we found that around 80% of gastric cancer patients in our cohort harbored TP53 alterations. Genetic alterations of BRCA1/2 or KMT2B were mostly exclusive with TP53 mutations, so were the MSI status or low grade of tumors. These results were further validated using the data from cBioPortal. We then used the NGS-derived TP53 status to optimize four commonly used IHC antibodies for detecting TP53 mutations. We showed that all antibodies could achieve more than 93% accuracy when proper IHC positivity thresholds were used, especially for the SP5 antibody that could reach 100% sensitivity and specificity with the 20% threshold. CONCLUSION: Our results indicated that exclusivity between TP53 and BRCA mutations could be potentially used as a cost-effective way to predict BRCA status. Also, setting proper IHC thresholds for each specific antibody is critical to accurately detect TP53 mutations and facilitate disease diagnosis.

Genomic Profiling Identified Novel Prognostic Biomarkers in Chinese Midline Glioma Patients.

BACKGROUND: Molecular characteristics are essential for the classification and grading of gliomas. However, diagnostic classification of midline glioma is still debatable and substantial molecular and clinical heterogeneity within each subgroup suggested that they should be further stratified. Here, we studied the mutation landscape of Chinese midline glioma patients in hope to provide new insights for glioma prognosis and treatment. METHODS: Tissue samples from 112 midline glioma patients underwent next-generation sequencing targeting 425 cancer-relevant genes. Gene mutations and copy number variations were investigated for their somatic interactions and prognostic effect using overall survival data. Pathway-based survival analysis was performed for ten canonical oncogenic pathways. RESULTS: We identified several currently established diagnostic and prognostic biomarkers of glioma, including TP53 (33%), EGFR (26%), TERT (24%), PTEN (21%), PIK3CA (14%), ATRX (14%), BRAF (13%), and IDH1/2 (6%). Among all genetic aberrations with more than 5% occurrence rate, six mutations and three copy number gains were greatly associated with poor overall survival (univariate, P < 0.1). Of these, TERT mutations (hazard ratio [HR], 3.00; 95% confidence interval [CI], 1.37-6.61; P = 0.01) and PIK3CA mutations (HR, 2.04; 95% CI, 1.08-3.84; P = 0.02) remained significant in multivariate analyses. Additionally, we have also identified a novel MCL1 amplification (found in 31% patients) as a potential independent biomarker for glioma (multivariate HR, 2.78; 95% CI, 1.53-5.08; P < 0.001), which was seldom reported in public databases. Pathway analyses revealed significantly worse prognosis with abnormal PI3K (HR, 1.81; 95% CI, 1.12-2.95; P = 0.01) and cell cycle pathways (HR, 1.97; 95% CI, 1.15-3.37; P = 0.01), both of which stayed meaningful after multivariate adjustment. CONCLUSIONS: In this study, we discovered shorter survival in midline glioma patients with PIK3CA and TERT mutations and with abnormal PI3K and cell cycle pathways. We also revealed a novel prognostic marker, MCL1 amplification that collectively provided new insights and opportunities in understanding and treating midline gliomas.

Sensing and Processing of DNA Interstrand Crosslinks by the Mismatch Repair Pathway.

DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutSα (MSH2-MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutSα functions as an ICL sensor. MutSα recruits MutLα and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication.