Cooperativity of c-MYC with Krüppel-Like Factor 6 Splice Variant 1 induces phenotypic plasticity and promotes prostate cancer progression and metastasis.

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

Researching COVID to enhance recovery (RECOVER) tissue pathology study protocol: Rationale, objectives, and design.

IMPORTANCE: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. METHODS: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes. DISCUSSION: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC.

ACE2 and TMPRSS2 distribution in the respiratory tract of different animal species and its correlation with SARS-CoV-2 tissue tropism.

A wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.

Cellular mechanisms associated with sub-optimal immune responses to SARS-CoV-2 bivalent booster vaccination in patients with Multiple Myeloma.

BACKGROUND: The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. METHODS: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. FINDINGS: We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. INTERPRETATION: Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Master Transcription Factor Reprogramming Unleashes Selective Translation Promoting Castration Resistance and Immune Evasion in Lethal Prostate Cancer.

Signaling rewiring allows tumors to survive therapy. Here we show that the decrease of the master regulator microphthalmia transcription factor (MITF) in lethal prostate cancer unleashes eukaryotic initiation factor 3B (eIF3B)-dependent translation reprogramming of key mRNAs conferring resistance to androgen deprivation therapy (ADT) and promoting immune evasion. Mechanistically, MITF represses through direct promoter binding eIF3B, which in turn regulates the translation of specific mRNAs. Genome-wide eIF3B enhanced cross-linking immunoprecipitation sequencing (eCLIP-seq) showed specialized binding to a UC-rich motif present in subsets of 5' untranslated regions. Indeed, translation of the androgen receptor and major histocompatibility complex I (MHC-I) through this motif is sensitive to eIF3B amount. Notably, pharmacologic targeting of eIF3B-dependent translation in preclinical models sensitizes prostate cancer to ADT and anti-PD-1 therapy. These findings uncover a hidden connection between transcriptional and translational rewiring promoting therapy-refractory lethal prostate cancer and provide a druggable mechanism that may transcend into effective combined therapeutic strategies. SIGNIFICANCE: Our study shows that specialized eIF3B-dependent translation of specific mRNAs released upon downregulation of the master transcription factor MITF confers castration resistance and immune evasion in lethal prostate cancer. Pharmacologic targeting of this mechanism delays castration resistance and increases immune-checkpoint efficacy. This article is featured in Selected Articles from This Issue, p. 2489.

Molecular Detection of Candida auris Using DiaSorin Molecular Simplexa® Detection Kit: A Diagnostic Performance Evaluation.

Candida auris is a globally emerging fungal pathogen that is associated with healthcare-related infections. The accurate and rapid detection of C. auris is crucial for effective infection prevention, control, and patient management. This study aimed to validate the analytical and diagnostic performance of the DiaSorin Molecular C. auris Detection Kit. The analytical specificity, sensitivity, and reproducibility of the assay were evaluated. The limit of detection (LOD) was determined to be 266 CFU/µL using the ZeptoMetrix Candida auris Z485 strain and standard calibration curves. The assay demonstrated high analytical specificity and showed no amplification against a diverse panel of bacteria and fungi. Clinical validation was conducted using deidentified residual axillary/groin surveillance culture specimens from C. auris culture-positive and culture-negative patients. The DiaSorin Molecular Detection Kit exhibited 100% agreement in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) when compared to cultures coupled with MALDI-TOF identification. Intra- and inter-reproducibility testing demonstrated consistent and reliable diagnostic performance. This validated assay offers rapid and accurate detection of C. auris, facilitating timely implementation of infection control measures and appropriate patient care. The DiaSorin Molecular C. auris Detection Kit has the potential to aid in controlling the outbreaks caused by this emerging fungal pathogen. Providing a reliable diagnostic tool can contribute to the effective management and containment of C. auris infections in healthcare settings and ultimately improve patient outcomes.

Molecular and immune signatures, and pathological trajectories of fatal COVID-19 lungs defined by in situ spatial single-cell transcriptome analysis.

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. In this study, we investigated the spatial single-cell molecular and cellular features of postmortem COVID-19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole-slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS-CoV-2. Compared with the non-COVID-19 control, COVID-19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-ß-SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single-cell atlas of cellular and molecular signatures of fatal COVID-19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

Marked elevations in lung and plasma ceramide in COVID-19 linked to microvascular injury.

The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide-rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFß activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFß kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Binding and Avidity Signatures of Polyclonal Sera From Individuals With Different Exposure Histories to Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Vaccination, and Omicron Breakthrough Infections.

BACKGROUND: The number of exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to vaccine antigens affect the magnitude and avidity of the polyclonal response. METHODS: We studied binding and avidity of different antibody isotypes to the spike, the receptor-binding domain (RBD), and the nucleoprotein (NP) of wild-type (WT) and BA.1 SARS-CoV-2 in convalescent, mRNA vaccinated and/or boosted, hybrid immune individuals and in individuals with breakthrough cases during the peak of the BA.1 wave. RESULTS: We found an increase in spike-binding antibodies and antibody avidity with increasing number of exposures to infection and/or vaccination. NP antibodies were detectible in convalescent individuals and a proportion of breakthrough cases, but they displayed low avidity. Omicron breakthrough infections elicited high levels of cross-reactive antibodies between WT and BA.1 antigens in vaccinated individuals without prior infection directed against the spike and RBD. The magnitude of the antibody response and avidity correlated with neutralizing activity against WT virus. CONCLUSIONS: The magnitude and quality of the antibody response increased with the number of antigenic exposures, including breakthrough infections. However, cross-reactivity of the antibody response after BA.1 breakthroughs, was affected by the number of prior exposures.

Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022.

Monkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Harnessing transcriptionally driven chromosomal instability adaptation to target therapy-refractory lethal prostate cancer.

Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

Applications of artificial intelligence in prostate cancer histopathology.

The diagnosis of prostate cancer (PCa) depends on the evaluation of core needle biopsies by trained pathologists. Artificial intelligence (AI) derived models have been created to address the challenges posed by pathologists' increasing workload, workforce shortages, and variability in histopathology assessment. These models with histopathological parameters integrated into sophisticated neural networks demonstrate remarkable ability to identify, grade, and predict outcomes for PCa. Though the fully autonomous diagnosis of PCa remains elusive, recently published data suggests that AI has begun to serve as an initial screening tool, an assistant in the form of a real-time interactive interface during histological analysis, and as a second read system to detect false negative diagnoses. Our article aims to describe recent advances and future opportunities for AI in PCa histopathology.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFb activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFb kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Evaluation and validation of an RT-PCR assay for specific detection of monkeypox virus (MPXV).

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with no clear epidemiological links to travel or animal exposure and with ever-increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real-time PCR-assay for MPXV diagnosis in humans and compare the performance of this novel assay against a Food & Drug Administration-cleared pan-Orthopox RT-PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT-PCR assay targeting the viral F3L-gene. In addition, we further evaluated MPXV-PCR-positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT-PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.

Development and validation of an AI-enabled digital breast cancer assay to predict early-stage breast cancer recurrence within 6 years.

BACKGROUND: Breast cancer (BC) grading plays a critical role in patient management despite the considerable inter- and intra-observer variability, highlighting the need for decision support tools to improve reproducibility and prognostic accuracy for use in clinical practice. The objective was to evaluate the ability of a digital artificial intelligence (AI) assay (PDxBr) to enrich BC grading and improve risk categorization for predicting recurrence. METHODS: In our population-based longitudinal clinical development and validation study, we enrolled 2075 patients from Mount Sinai Hospital with infiltrating ductal carcinoma of the breast. With 3:1 balanced training and validation cohorts, patients were retrospectively followed for a median of 6 years. The main outcome was to validate an automated BC phenotyping system combined with clinical features to produce a binomial risk score predicting BC recurrence at diagnosis. RESULTS: The PDxBr training model (n = 1559 patients) had a C-index of 0.78 (95% CI, 0.76-0.81) versus clinical 0.71 (95% CI, 0.67-0.74) and image feature models 0.72 (95% CI, 0.70-0.74). A risk score of 58 (scale 0-100) stratified patients as low or high risk, hazard ratio (HR) 5.5 (95% CI 4.19-7.2, p < 0.001), with a sensitivity 0.71, specificity 0.77, NPV 0.95, and PPV 0.32 for predicting BC recurrence within 6 years. In the validation cohort (n = 516), the C-index was 0.75 (95% CI, 0.72-0.79) versus clinical 0.71 (95% CI 0.66-0.75) versus image feature models 0.67 (95% CI, 0.63-071). The validation cohort had an HR of 4.4 (95% CI 2.7-7.1, p < 0.001), sensitivity of 0.60, specificity 0.77, NPV 0.94, and PPV 0.24 for predicting BC recurrence within 6 years. PDxBr also improved Oncotype Recurrence Score (RS) performance: RS 31 cutoff, C-index of 0.36 (95% CI 0.26-0.45), sensitivity 37%, specificity 48%, HR 0.48, p = 0.04 versus Oncotype RS plus AI-grade C-index 0.72 (95% CI 0.67-0.79), sensitivity 78%, specificity 49%, HR 4.6, p < 0.001 versus Oncotype RS plus PDxBr, C-index 0.76 (95% CI 0.70-0.82), sensitivity 67%, specificity 80%, HR 6.1, p < 0.001. CONCLUSIONS: PDxBr is a digital BC test combining automated AI-BC prognostic grade with clinical-pathologic features to predict the risk of early-stage BC recurrence. With future validation studies, we anticipate the PDxBr model will enrich current gene expression assays and enhance treatment decision-making.