Compensatory mutations potentiate constructive neutral evolution by gene duplication.

The functions of proteins generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicated and evolved a homodimeric enzyme to determine whether and how this could happen. We identified hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when coexpressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

Replication properties of a contemporary Zika virus from West Africa.

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa.

Implementing Multifactorial Risk Assessment with Polygenic Risk Scores for Personalized Breast Cancer Screening in the Population Setting: Challenges and Opportunities.

Risk-stratified breast screening has been proposed as a strategy to overcome the limitations of age-based screening. A prospective cohort study was undertaken within the PERSPECTIVE I&I project, which will generate the first Canadian evidence on multifactorial breast cancer risk assessment in the population setting to inform the implementation of risk-stratified screening. Recruited females aged 40-69 unaffected by breast cancer, with a previous mammogram, underwent multifactorial breast cancer risk assessment. The adoption of multifactorial risk assessment, the effectiveness of methods for collecting risk factor information and the costs of risk assessment were examined. Associations between participant characteristics and study sites, as well as data collection methods, were assessed using logistic regression; all p-values are two-sided. Of the 4246 participants recruited, 88.4% completed a risk assessment, with 79.8%, 15.7% and 4.4% estimated at average, higher than average and high risk, respectively. The total per-participant cost for risk assessment was CAD 315. Participants who chose to provide risk factor information on paper/telephone (27.2%) vs. online were more likely to be older (p = 0.021), not born in Canada (p = 0.043), visible minorities (p = 0.01) and have a lower attained education (p < 0.0001) and perceived fair/poor health (p < 0.001). The 34.4% of participants requiring risk factor verification for missing/unusual values were more likely to be visible minorities (p = 0.009) and have a lower attained education (p ≤ 0.006). This study demonstrates the feasibility of risk assessment for risk-stratified screening at the population level. Implementation should incorporate an equity lens to ensure cancer-screening disparities are not widened.

Differential outcomes of re-stratified high-risk prostate cancer patients treated with external beam radiation therapy plus high-dose-rate brachytherapy boost.

Purpose: We report outcomes of high-risk prostate cancer (PCa) patients, initially classified according to a 3-tier NCCN classification system, treated with external beam radiation therapy (EBRT) and high-dose-rate brachytherapy boost (HDR-BT). Patients were analyzed based on a re-stratification of their risk grouping using CAPRA score and a newer 5-tier NCCN classification. Material and methods: 471 high-risk PCa patients treated with EBRT, HDR-BT, and androgen deprivation therapy (ADT) between 1999 and 2018 were included. Competing risk survival analyses to compare individuals with CAPRA scores < 6 vs. ≥ 6 for biochemical relapse (BCR) and metastasis incidence were conducted. Also, overall survival (OS) for both groups using Kaplan-Meier analysis was assessed. The same analyses were repeated using a 5-tier NCCN stratification comparing those classified as high-risk vs. very high-risk patients. Results: The median age was 71 years, and the median follow-up period was 72 months. The whole cohort received an EQD2 of 74 Gy or greater, with a median EQD2 of 106.89 Gy. Both a CAPRA score ≥ 6 and belonging to the NCCN very high-risk group were associated with BCR, with subdistribution hazard ratios (sHRs) of 3.04 (p = 0.015) and 2.53 (p = 0.013), respectively. For metastasis incidence, both the CAPRA and NCCN groups had similar sHRs of 2.60 (p = 0.094) and 2.71 (p = 0.037), respectively. For 10-year OS, patients with CAPRA score ≥ 6 and belonging to the NCCN very high-risk group presented similar HRs of 2.11 (p = 0.005) and 2.10 (p = 0.002). Conclusions: We showed that high-risk PCa patients classified according to the 3-tier NCCN system benefit from further stratification using the CAPRA score or the 5-tier NCCN stratification method. Patients with a CAPRA score ≥ 6 or classified as very high-risk demonstrate a higher hazard of BCR, metastasis, and death. These patients might benefit from further intensification of their investigations and treatment, based on ongoing research.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

Protein functions generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicate and evolve an homodimeric enzyme to examine if and how this could happen. We identify hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when co-expressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

The Cross-Scale Association between Pathomics and Radiomics Features in Immunotherapy-Treated NSCLC Patients: A Preliminary Study.

BACKGROUND: Recent advances in cancer biomarker development have led to a surge of distinct data modalities, such as medical imaging and histopathology. To develop predictive immunotherapy biomarkers, these modalities are leveraged independently, despite their orthogonality. This study aims to explore the cross-scale association between radiological scans and digitalized pathology images for immunotherapy-treated non-small cell lung cancer (NSCLC) patients. METHODS: This study involves 36 NSCLC patients who were treated with immunotherapy and for whom both radiology and pathology images were available. A total of 851 and 260 features were extracted from CT scans and cell density maps of histology images at different resolutions. We investigated the radiopathomics relationship and their association with clinical and biological endpoints. We used the Kolmogorov-Smirnov (KS) method to test the differences between the distributions of correlation coefficients with the two imaging modality features. Unsupervised clustering was done to identify which imaging modality captures poor and good survival patients. RESULTS: Our results demonstrated a significant correlation between cell density pathomics and radiomics features. Furthermore, we also found a varying distribution of correlation values between imaging-derived features and clinical endpoints. The KS test revealed that the two imaging feature distributions were different for PFS and CD8 counts, while similar for OS. In addition, clustering analysis resulted in significant differences in the two clusters generated from the radiomics and pathomics features with respect to patient survival and CD8 counts. CONCLUSION: The results of this study suggest a cross-scale association between CT scans and pathology H&E slides among ICI-treated patients. These relationships can be further explored to develop multimodal immunotherapy biomarkers to advance personalized lung cancer care.

Induction of programmed cell death (apoptosis) by dengue virus in vitro and in vivo

Dengue is a human disease wich may be fatal in its hemorrhagic form. How dengue virus and hostspecified factors underlie virulence and pathogenesis is poorly undrestood. An inmunological disorder is thougth to be involved in dengue physilogical symptoms. Whether the immune response is deleterious or beneficial to the host remains a matther of debate. In this review, we summarized developments in research on viral pathogenesis in the context of apoptosis triggered by dengue virus infection. Apoptosis, an active process of cell destruction, is one of the importand consequences of dengue virus infection in vitro and in vivo. Dengue virus replication induces apoptosis in mouse neurons and human hepatocytes. The ability to activate this genetically programmed cell death pathway is dependent on both viral and cellular determinants