Theoretical calculation on degradation mechanism of novel copolyesters under CALB enzyme.

Poly(butylene succinate-co-furandicarboxylate) (PBSF) and poly(butylene adipate-co-furandicarboxylate) (PBAF) are novel furandicarboxylic acid-based biodegradable copolyesters with great potential to replace fossil-derived terephthalic acid-based copolyesters such as poly(butylene succinate-co-terephthalate) (PBST) and poly(butylene adipate-co-terephthalate) (PBAT). In this study, quantum chemistry techniques after molecular dynamics simulations are employed to investigate the degradation mechanism of PBSF and PBAF catalyzed by Candida antarctica lipase B (CALB). Computational analysis indicates that the catalytic reaction follows a four-step mechanism resembling the ping-pong bibi mechanism, with the initial two steps being acylation reactions and the subsequent two being hydrolysis reactions. Notably, the first step of the hydrolysis is identified as the rate-determining step. Moreover, by introducing single-point mutations to expand the substrate entrance tunnel, the catalytic distance of the first acylation step decreases. Additionally, energy barrier of the rate-determining step is decreased in the PBSF system by site-directed mutations on key residues increasing hydrophobicity of the enzyme's active site. This study unprecedently show the substrate binding pocket and hydrophobicity of the enzyme's active site have the potential to be engineered to enhance the degradation of copolyesters catalyzed by CALB.

Genomic alterations in retinoblastoma tumors of Argentine patients.

INTRODUCTION: Retinoblastoma is initiated by inactivation of RB1 gene, but additional alterations may be required for tumor progression. Substitution and INDEL variants in different genes, aside RB1, are infrequent, while large copy number variants (CNVs) like gains on 1q, 2p, 6p and loss on 16q are common, they include oncogenes or tumor suppressors and are typical of retinoblastoma. AIM: To provide the molecular profile that is useful for prognosis and understanding of retinoblastoma development. METHODS: To identify genomic variants in six retinoblastoma tumors whole exome sequencing and informatic analysis were performed. RESULTS: RB1 was the only gene with nonsense or frameshift mutations. SNVs in other 11 genes were missense and at non-canonical splice-sites, all nonpathogenic. CNVs, similar to those reported, were identified in all retinoblastoma tumors. The most frequent were 1q gain and 16q loss. Additionally, deletions were identified on 13q, including RB1 gene, and on the X chromosome, including BCOR gene, the most frequently mutated, after RB1, in retinoblastoma. The number of CNVs detected in each tumor was between 1 and 7, depending on the age at diagnosis. CONCLUSION: The analysis of genomic alterations in retinoblastoma is useful to understand the severity of tumor progression and to apply appropriate treatments.

PARP inhibitors in ovarian cancer: Mechanisms, resistance, and the promise of combination therapy.

Current approaches to treating ovarian cancer focus mainly on surgical cytoreduction and chemotherapy using platinum-based drugs, while newer methods such as immunotherapy are being investigated to enhance treatment outcomes. Treating ovarian cancer is complicated by challenges such as late-stage detection, tumor diversity, and limited treatment choices. Therefore, innovative strategies such as precision medicine and targeted therapies like PARPi (Poly ADP-Ribose Polymerase inhibitors) are increasingly necessary. The article highlights the significance of an innovative therapeutic approach focusing on PARPi in revolutionizing ovarian cancer treatment and improving patient outcomes. It covers the basic knowledge of PARP, its structure, and its function in DNA repair. It further emphasizes how inhibiting PARP can help in treating ovarian cancer. It elaborates on the mechanism of action of PARPi. It covers the clinical trials governing PARPi and the combination of drugs used with PARPi. It mentions how the resistance is developed to PARPi and the strategies to overcome the resistance developed.

[Molecular pathogenesis of adult T-cell leukemia/lymphoma].

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T-cell malignancy caused by human T-cell leukemia virus type-1 (HTLV-1) infection. Genetic alterations are thought to contribute to the pathogenesis of ATLL alongside HTLV-1 products such as Tax and HBZ. Several large-scale genetic analyses have delineated the entire landscape of somatic alterations in ATLL, which is characterized by frequent alterations in T-cell receptor/NF-κB pathways and immune-related molecules. Notably, up to one-fourth of ATLL patients harbor structural variations disrupting the 3'-UTR of the PD-L1 gene, which facilitate escape of tumor cells from anti-tumor immunity. Among these alterations, PRKCB and IRF4 mutations, PD-L1 amplification, and CDKN2A deletion are associated with poor prognosis in ATLL. More recently, several single-cell transcriptome and immune repertoire analyses have revealed phenotypic features of premalignant cells and tumor heterogeneity as well as virus- and tumor-related changes of the non-malignant hematopoietic pool in ATLL. Here we summarize the current understanding of the molecular pathogenesis of ATLL, focusing on recent progress made by genetic, epigenetic, and single-cell analyses. These findings not only provide a deeper understanding of the molecular pathobiology of ATLL, but also have significant implications for diagnostic and therapeutic strategies.

[Uveal Melanoma: Molecular and Genetic Mechanisms of Development and Therapeutic Approaches].

Uveal melanoma (UM) is a neuroectodermal tumor that results from malignant transformation of melanocytes in the eye uvea, including the iris, the ciliary body, and the choroid. UM accounts for 5% of all melanoma cases and is extremely aggressive with half of the UM patients developing metastases within the first 1-2 years after tumor development. Molecular mechanisms of UM carcinogenesis are poorly understood, but are known to differ from those of skin melanoma. Activating mutations of the GNAQ and GNA11 genes, which code for the large G protein subunits Gq and G11, respectively, are found in 90% of UM patients. The Gaq/PKC/MAPK signaling pathway is a main signaling cascade that leads to the transformation of melanocytes of the uveal tract, and major regulators of the cascade provide targets for the development of drugs. Metastatic UM (MUM) is most often associated with mutations of BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. A combination of a commercial expression test panel of 15 genes and a mutation panel of 7 genes, supplemented with data on the size of the primary tumor, is highly efficient in predicting the risk of metastasis. The risk of metastasis determines the choice of therapy and the patient follow-up regimen. However, no systemic therapy for MUM has been developed to date. New drugs undergoing clinical trials are mostly targeted drugs designed to inhibit the protein products of mutant genes or immunotherapeutic agents designed to stimulate the immune response against specific antigens. In addition to these approaches, potential therapeutic targets of epigenetic regulation of UM development are considered in the review.

Cytotoxic Impact of Naringenin-Loaded Solid Lipid Nanoparticles on RIN5F Pancreatic ß Cells via Autophagy Blockage.

BACKGROUND: Autophagy plays a crucial role in modulating the proliferation of cancer diseases. However, the application of Naringenin (Nar), a compound with potential benefits against these diseases, has been limited due to its poor solubility and bioavailability. OBJECTIVE: This study aimed to develop solid lipid nanoparticles (Nar-SLNs) loaded with Nar to enhance their therapeutic impact. METHODS: In vitro experiments using Rin-5F cells exposed to Nar and Nar-SLNs were carried out to investigate the protective effects of Nar and its nanoformulation against the pancreatic cancer cell line of Rin-5F. RESULTS: Treatment with Nar and Nar-SLN led to an increase in autophagic markers (Akt, LC3, Beclin1, and ATG genes) and a decrease in the level of miR-21. Both Nar and Nar-SLN treatments inhibited cell proliferation and reduced the expression of autophagic markers. Notably, Nar-SLNs exhibited greater efficacy compared to free Nar. CONCLUSION: These findings suggest that SLNs effectively enhance the cytotoxic impact of Nar, making Nar-SLNs a promising candidate for suppressing or preventing Rin-5F cell growth.

Unlocking the tumor-immune microenvironment in osteosarcoma: insights into the immune landscape and mechanisms.

Osteosarcoma has a unique tumor microenvironment (TME), which is characterized as a complex microenvironment comprising of bone cells, immune cells, stromal cells, and heterogeneous vascular structures. These elements are intricately embedded in a mineralized extracellular matrix, setting it apart from other primary TMEs. In a state of normal physiological function, these cell types collaborate in a coordinated manner to maintain the homeostasis of the bone and hematopoietic systems. However, in the pathological condition, i.e., neoplastic malignancies, the tumor-immune microenvironment (TIME) has been shown to promote cancer cells proliferation, migration, apoptosis and drug resistance, as well as immune escape. The intricate and dynamic system of the TIME in osteosarcoma involves crucial roles played by various infiltrating cells, the complement system, and exosomes. This complexity is closely associated with tumor cells evading immune surveillance, experiencing uncontrolled proliferation, and facilitating metastasis. In this review, we elucidate the intricate interplay between diverse cell populations in the osteosarcoma TIME, each contributing uniquely to tumor progression. From chondroblastic and osteoblastic osteosarcoma cells to osteoclasts, stromal cells, and various myeloid and lymphoid cell subsets, the comprehensive single-cell analysis provides a detailed roadmap of the complex osteosarcoma ecosystem. Furthermore, we summarize the mutations, epigenetic mechanisms, and extracellular vesicles that dictate the immunologic landscape and modulate the TIME of osteosarcoma. The perspectives of the clinical implementation of immunotherapy and therapeutic approaches for targeting immune cells are also intensively discussed.

Poly(Adenosine Diphosphate Ribose) Polymerase (PARP) Inhibitors in the Treatment of Advanced Ovarian Cancer: A Narrative Review.

Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have appeared as a revolutionary approach to treating advanced ovarian cancer, particularly in patients with breast cancer (BRCA) mutations and homologous recombination deficiency (HRD). This narrative review explores PARP inhibitors' clinical efficiency, safety, and changing role in this context. PARP inhibitors, such as olaparib, niraparib, or rucaparib, provide considerable benefits regarding progression-free survival expansion and overall outcomes improvement in first-line maintenance and recurrent settings. The underlying mechanisms, patient selection criteria, and resistance patterns are discussed, alongside insights into combination therapies to overcome resistance and enhance therapeutic efficacy. Ongoing clinical trials and future potential for personalized therapy approaches using PARP inhibitors for advanced ovarian cancer are also highlighted. However, despite these drugs' phenomenal ability to revolutionize treatment protocols for such cancer types, several challenges remain: toxicity management, cost, and development of resistance will require more research to optimize their use or broaden patient populations who can benefit from them.

Molecular profiling of pre- and post- 5-azacytidine myelodysplastic syndrome samples identifies predictors of response.

Treatment with the hypomethylating agent 5-azacytidine (AZA) increases survival in high-risk (HR) myelodysplastic syndrome (MDS) patients, but predicting patient response and overall survival remains challenging. To address these issues, we analyzed mutational and transcriptional profiles in CD34+ hematopoietic stem/progenitor cells (HSPCs) before and following AZA therapy in MDS patients. AZA treatment led to a greater reduction in the mutational burden in both blast and hematological responders than non-responders. Blast and hematological responders showed transcriptional evidence of pre-treatment enrichment for pathways such as oxidative phosphorylation, MYC targets, and mTORC1 signaling. While blast non-response was associated with TNFa signaling and leukemia stem cell signature, hematological non-response was associated with cell-cycle related pathways. AZA induced similar transcriptional responses in MDS patients regardless of response type. Comparison of blast responders and non-responders to normal controls, allowed us to generate a transcriptional classifier that could predict AZA response and survival. This classifier outperformed a previously developed gene signature in a second MDS patient cohort, but signatures of hematological responses were unable to predict survival. Overall, these studies characterize the molecular consequences of AZA treatment in MDS HSPCs and identify a potential tool for predicting AZA therapy responses and overall survival prior to initiation of therapy.

Structure-guided mutations in CDRs for enhancing the affinity of neutralizing SARS-CoV-2 nanobody.

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC50 values of ∼0.03 µM, as compared to the native nanobody (IC50 ∼0.77 µM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

Unveiling the Complexity of cis-Regulation Mechanisms in Kinases: A Comprehensive Analysis.

Protein cis-regulatory elements (CREs) are regions that modulate the activity of a protein through intramolecular interactions. Kinases, pivotal enzymes in numerous biological processes, often undergo regulatory control via inhibitory interactions in cis. This study delves into the mechanisms of cis regulation in kinases mediated by CREs, employing a combined structural and sequence analysis. To accomplish this, we curated an extensive dataset of kinases featuring annotated CREs, organized into homolog families through multiple sequence alignments. Key molecular attributes, including disorder and secondary structure content, active and ATP-binding sites, post-translational modifications, and disease-associated mutations, were systematically mapped onto all sequences. Additionally, we explored the potential for conformational changes between active and inactive states. Finally, we explored the presence of these kinases within membraneless organelles and elucidated their functional roles therein. CREs display a continuum of structures, ranging from short disordered stretches to fully folded domains. The adaptability demonstrated by CREs in achieving the common goal of kinase inhibition spans from direct autoinhibitory interaction with the active site within the kinase domain, to CREs binding to an alternative site, inducing allosteric regulation revealing distinct types of inhibitory mechanisms, which we exemplify by archetypical representative systems. While this study provides a systematic approach to comprehend kinase CREs, further experimental investigations are imperative to unravel the complexity within distinct kinase families. The insights gleaned from this research lay the foundation for future studies aiming to decipher the molecular basis of kinase dysregulation, and explore potential therapeutic interventions.

Landscape of C-MET overexpression in non-small cell lung cancer: a large-scale study of clinicomolecular features and prognosis based on Chinese data.

Background: Real-world data on C-MET protein overexpression in non-small cell lung cancer (NSCLC) patients, particularly among the Asian Chinese population, are limited. Objectives: This study aimed to evaluate the clinicomolecular characteristics and prognosis of C-MET overexpression in Chinese NSCLC patients, focusing on those with positive C-MET overexpression (immunohistochemistry (IHC) 3+). Design: A retrospective and observational study. Methods: Data were collected from NSCLC patients diagnosed at the First Affiliated Hospital of Guangzhou Medical University between November 2006 and April 2021. We identified C-MET overexpression using IHC and C-MET overexpression positivity was defined as IHC 3+ with ⩾50% tumor cells. Additionally, patient genotypes were collected for subgroup analysis. Results: Data from 9785 NSCLC patients were collected. C-MET (-) accounted for 5% (503/9785), C-MET (+) for 27% (2654/9785), C-MET (++) for 36% (3464/9785), and C-MET (+++) for 32% (3164/9785). Genetic testing was available for 4326 patients. Wild-type was observed in 37% (1591 cases), with epidermal growth factor receptor (EGFR) abnormalities being the most common at 49% (2127 cases). Positive C-MET overexpression correlated significantly with women (p < 0.001), early-stage (p = 0.003), adenocarcinoma (p < 0.001), and driver mutations (p < 0.001). Patients with anaplastic lymphoma kinase (ALK) alterations had a higher occurrence of C-MET overexpression positivity (57.1%). Positive C-MET overexpression was significantly associated with EGFR (p < 0.001), ALK (p < 0.001), and KRAS alterations (p = 0.024). Compared to C-MET overexpression (IHC 0), C-MET overexpression (IHC 2+) (hazard ratio (HR) = 0.455, p < 0.001) and C-MET overexpression (IHC 3+) (HR = 0.569, p < 0.001) were correlated with better overall survival in overall NSCLC patients, especially for C-MET overexpression (IHC 2+). Conclusion: Our study elucidates the clinicomolecular characteristics and prognosis of C-MET overexpression in NSCLC patients, particularly those with positive C-MET overexpression (IHC 3+). This provides insight into the prevalence of C-MET overexpression in Chinese NSCLC patients and offers a basis for considering C-MET overexpression as a prognostic and predictive marker in NSCLC.

BRCA2 mutations in familial breast cancer with prostate cancer: a case report and literature review.

Prostate cancer (PCa) is the second most common tumor in men globally. Its etiology has been attributed to multiple factors, including age and ethnicity, with family history identified as a significant risk factor. The role of family history in prostate cancer risk appears to be more extensive than previously thought, with evidence suggesting that prostate cancer and breast cancer may occur concurrently within families. BRCA2 mutations have been linked to an increased risk of prostate cancer, particularly in patients diagnosed with early-onset disease. It is estimated that BRCA2 mutations account for approximately 5% of familial prostate cancer cases. It is noteworthy that cases of prostate cancer in patients with BRCA2 mutations are rare in clinical practice. Here we report a case of prostatitis carcinoma with a mutation in the BRCA2 gene in a patient who underwent robotic-assisted radical prostatectomy for prostatitis carcinoma after medication was not effective. Genetic testing of him, his son, and his daughter showed that they all had mutations in this gene, and it is noteworthy that the type of BRCA2 mutation in his son has never been reported before, which is rare in clinical practice.

Key structural role of a conserved cis-proline revealed by the P285S variant of soybean serine hydroxymethyltransferase 8.

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life.  SHMT is a pyridoxal 5'-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site.  Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8.  P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode.  We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution.  Crystal structures at 1.9 - 2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an a-helix and shifts a turn region into the active site.  This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ~180° with concomitant loss of conserved enzyme-PLP interactions.  A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ~10 residues.  These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function.

Features and efficacy of triple-targeted therapy for patients with EGFR-mutant non-small-cell lung cancer with acquired BRAF alterations who are resistant to epidermal growth factor receptor tyrosine kinase inhibitors.

BACKGROUND: The recommended first-line treatment for advanced epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients is EGFR-tyrosine kinase inhibitors (EGFR-TKIs). BRAF alterations have been identified as resistance mechanisms. We aimed to identify features of and subsequent treatment strategies for such patients. PATIENTS AND METHODS: We conducted a systematic literature review of NSCLC patients harboring acquired BRAF alterations. Additionally, BRAF-altered NSCLC patients who progressed from EGFR-TKIs at West China Hospital of Sichuan University were screened. Patient characteristics, treatment options, and outcomes were analyzed. RESULTS: A total of 104 patients were included, 2 of whom came from our center. Seventy-five patients (72.1%) harbored BRAF mutations (57 class I mutations, 7 class II mutations, 9 class III mutations, and 2 non-class I-III mutations), and 29 (27.9%) harbored BRAF fusions. Eighteen patients received triple-targeted therapy, including prior EGFR-TKIs plus dabrafenib and trametinib, and 23 patients received other treatments. The median progression-free survival was significantly longer in patients receiving triple-targeted therapy than in those receiving other treatments (8.0 versus 2.5 months, P < 0.001). Similar findings were observed in patients with BRAF mutations (9.0 versus 2.8 months, P = 0.004), particularly in those with BRAF class I mutations (9.0 versus 2.5 months, P < 0.001). A potential benefit was also observed among patients with BRAF fusions (5.0 versus 2.0 months, P = 0.230). Twenty patients (48.8%) experienced adverse events. Dose reduction of RAF or MEK inhibitor was required in five patients (12.2%). Five patients (12.2%) permanently discontinued treatment (three on triple-targeted therapy; one on prior EGFR-TKI plus vemurafenib; one on prior EGFR-TKI plus trametinib). CONCLUSIONS: BRAF alterations, specifically BRAF mutations and BRAF fusions, facilitate resistance to EGFR-TKIs. Triple-targeted therapy is effective and safe for patients with EGFR-mutant NSCLC with acquired BRAF alterations, mainly among patients with BRAF class I mutations and potentially in patients with BRAF fusions.

An automated commercial open access assay for detection of Mycoplasma genitalium macrolide resistance.

Azithromycin, a macrolide antibioticum, is the first-line treatment for Mycoplasma genitalium (MG), but resistant MG is an increasing problem. Macrolide resistance-mediated mutations (MRM) has been linked to point mutations in region V of the MG 23S rRNA gene. We have evaluated an open access analyzer (Panther Fusion, Hologic Inc) for detectability of MRM (mutations A2071G and A2072G) and MG wild type (WT) in clinical samples. Also, the agreement of the Panther Fusion assay results with a corresponding established In-house MRM-WT PCR (ABI 7500) was calculated. Left over material from 55 clinical samples positive for MG by the Aptima test (Hologic) based on transcription-mediated amplification (TMA), collected from January to February 2023 in Region Skåne, Sweden, was analyzed. Specific amplification curves were generated for positive controls of MG mutations (A2071G and A2072G) and WT by the Panther Fusion assay. The limit of detection (LOD) was 5.3 copies/mL for WT, 8.1 copies/mL for mutation A2071G, and 81 copies/mL for mutation A2072G. The overall concordance was 91% between the Panther Fusion and the In-house PCR (Kappa 0.621, 95% CI; 0.327-0.914) for detection of WT or MRM in MG-positive clinical samples. The Panther Fusion detected MRM in 20% (11/55) and WT in 62% (34/55) of the samples. The corresponding In-house PCR results were 25% (14/55) and 65% (36/55). In summary, the Panther Fusion assay demonstrated detection of low copy number of MRM and WT of MG. Among clinical samples substantial agreement between the Panther Fusion and In-house PCR results was observed. Integrating MG-analysis (TMA) and MRM-WT assay on the Panther platform could make MRM testing more readily available. However, the Panther Fusion had a lower success rate (82% vs 90%) for macrolide susceptibility testing, hence testing with a complementary method should be considered for samples where neither WT nor MRM MG are detectable.

Colorectal Cancer Brain Metastasis With Concomitant KRAS and BRAF Mutations: A Case Report.

Colorectal cancer (CRC) brain metastasis (BM) is a rare but aggressive manifestation of the disease, with poor prognosis and limited treatment options. Although brain metastases are more commonly associated with primary tumors located in the lung, skin, and breast, their occurrence in colorectal cancer is uncommon. Genetic mutations are highly important in tumor progression, and mutations in KRAS and BRAF genes are key drivers in colorectal cancer. However, the concurrent presence of both mutations is exceedingly rare. This case report presents a unique instance of colorectal cancer brain metastasis harboring both KRAS and BRAF mutations, highlighting its clinical significance and therapeutic challenges. We present the case of a 62-year-old male patient diagnosed with brain metastasis (in the cerebellum and right parietal lobe) who presented to the hospital with neurological symptoms. He underwent a CT imaging investigation that revealed multiple tumors. Subsequent biopsies confirmed the diagnosis of brain metastasis, with histological characteristics consistent with colonic adenocarcinoma. Tests also revealed aberrant expression of both KRAS and BRAF mutations. This case highlights the importance of considering brain metastases in colorectal cancer patients due to their detrimental effects on prognosis and survival rates. Additionally, the simultaneous presence of BRAF and KRAS mutations, in this case, adds an extra layer of complexity and severity.

Large scale analysis of the SARS-CoV-2 main protease reveals marginal presence of nirmatrelvir-resistant SARS-CoV-2 Omicron mutants in Ontario, Canada, December 2021-September 2023.

Background: In response to the COVID-19 pandemic, a new oral antiviral called nirmatrelvir-ritonavir (PaxlovidTM) was authorized for use in Canada in January 2022. In vitro studies have reported mutations in Mpro protein that may be associated with the development of nirmatrelvir resistance. Objectives: To survey the prevalence, relevance and temporal patterns of Mpro mutations among SARS-CoV-2 Omicron lineages in Ontario, Canada. Methods: A total of 93,082 Mpro gene sequences from December 2021 to September 2023 were analyzed. Reported in vitro Mpro mutations were screened against our database using in-house data science pipelines to determine the nirmatrelvir resistance. Negative binomial regression was conducted to analyze the temporal trends in Mpro mutation counts over the study time period. Results: A declining trend was observed in non-synonymous mutations of Mpro sequences, showing a 7.9% reduction (95% CI: 6.5%-‬9.4%; p<0.001) every 30 days. The P132H was the most prevalent mutation (higher than 95%) in all Omicron lineages. In vitro nirmatrelvir-resistant mutations were found in 3.12% (n=29/929) Omicron lineages with very low counts, ranging from one to 19. Only two mutations, A7T (n=19) and M82I (n=9), showed temporal presence among the BA.1.1 in 2022 and the BQ.1.2.3 in 2022, respectively. Conclusion: The observations suggest that, as of September 2023, no significant or widespread resistance to nirmatrelvir has developed among SARS-CoV-2 Omicron variants in Ontario. This study highlights the importance of creating automated monitoring systems to track the emergence of nirmatrelvir-resistant mutations within the SARS-CoV-2 virus, utilizing genomic data generated in real-time.

Frequency and spectrum of mutations in human sperm measured using duplex sequencing correlate with trio-based de novo mutation analyses.

De novo mutations (DNMs) are drivers of genetic disorders. However, the study of DNMs is hampered by technological limitations preventing accurate quantification of ultra-rare mutations. Duplex Sequencing (DS) theoretically has < 1 error/billion base-pairs (bp). To determine the DS utility to quantify and characterize DNMs, we analyzed DNA from blood and spermatozoa from six healthy, 18-year-old Swedish men using the TwinStrand DS mutagenesis panel (48 kb spanning 20 genic and intergenic loci). The mean single nucleotide variant mutation frequency (MF) was 1.2 × 10- 7 per bp in blood and 2.5 × 10- 8 per bp in sperm, with the most common base substitution being C > T. Blood MF and substitution spectrum were similar to those reported in blood cells with an orthogonal method. The sperm MF was in the same order of magnitude and had a strikingly similar spectrum to DNMs from publicly available whole genome sequencing data from human pedigrees (1.2 × 10- 8 per bp). DS revealed much larger numbers of insertions and deletions in sperm over blood, driven by an abundance of putative extra-chromosomal circular DNAs. The study indicates the strong potential of DS to characterize human DNMs to inform factors that contribute to disease susceptibility and heritable genetic risks.

Concomitant BRAF V600E and NRAS Q61R mutations in the same thyroid nodule: a case report.

Background: Papillary thyroid cancer (PTC) is the most common type of well-differentiated endocrine malignancy. Generally, thyroid nodules with multiple oncogenic mutations are uncommon with an occurrence which may be related to more aggressive biological behavior of tumors. RET/PTC rearrangement, RAS, and BRAF mutations are considered to be mutually exclusive in PTC. Concomitant RET/PTC, RAS, or BRAF mutations have been documented, although the impact of these mutations for tumor growth and survival is debated. Case Description: Here we present a rare case of woman 46 years old with a neck mass and thyroid nodule classified as TIR5 on cytological examination. We found contemporary BRAF p.(Val600Glu) [p.(V600E); c.1799T>A] and NRAS p.(Gln61Arg) [p.(Q61R); c.182A>G] mutations in morphologically different areas within the same lobe (the right one); The two lesions show different morphology. The mutated BRAF lesion showed morphological characteristics compatible with classic papillary carcinoma. The mutant NRAS lesion shows morphological features compatible with follicular variant papillary carcinoma. To the best of our knowledges, this is the first time that such mutations, which are normally mutually exclusive, have been detected at the same time. Conclusions: The finding of synchronous mutations is a rare occurrence suggesting for intratumoral heterogeneity (ITH) even in PTC. Patients with multiple mutations have a clinical worse prognosis, generally characterized by an aggressive thyroid cancer, which may influence the surgical treatment, chemotherapy, and BRAF V600E mutation-targeting therapy.