Population-based deep image prior for dynamic PET denoising: A data-driven approach to improve parametric quantification.

The high noise level of dynamic Positron Emission Tomography (PET) images degrades the quality of parametric images. In this study, we aim to improve the quality and quantitative accuracy of Ki images by utilizing deep learning techniques to reduce the noise in dynamic PET images. We propose a novel denoising technique, Population-based Deep Image Prior (PDIP), which integrates population-based prior information into the optimization process of Deep Image Prior (DIP). Specifically, the population-based prior image is generated from a supervised denoising model that is trained on a prompts-matched static PET dataset comprising 100 clinical studies. The 3D U-Net architecture is employed for both the supervised model and the following DIP optimization process. We evaluated the efficacy of PDIP for noise reduction in 25%-count and 100%-count dynamic PET images from 23 patients by comparing with two other baseline techniques: the Prompts-matched Supervised model (PS) and a conditional DIP (CDIP) model that employs the mean static PET image as the prior. Both the PS and CDIP models show effective noise reduction but result in smoothing and removal of small lesions. In addition, the utilization of a single static image as the prior in the CDIP model also introduces a similar tracer distribution to the denoised dynamic frames, leading to lower Ki in general as well as incorrect Ki in the descending aorta. By contrast, as the proposed PDIP model utilizes intrinsic image features from the dynamic dataset and a large clinical static dataset, it not only achieves comparable noise reduction as the supervised and CDIP models but also improves lesion Ki predictions.

Solving Missing Heritability in Patients With Familial Adenomatous Polyposis With DNA-RNA Paired Testing.

PURPOSE: Patients with germline pathogenic variants (PVs) in APC develop tens (attenuated familial adenomatous polyposis [AFAP]) to innumerable (classic FAP) adenomatous polyps in their colon and are at significantly increased lifetime risk of colorectal cancer. Up to 10% of FAP and up to 50% of patients with AFAP who have undergone DNA-only multigene panel testing (MGPT) do not have an identified PV in APC. We seek to demonstrate how the addition of RNA sequencing run concurrently with DNA can improve detection of germline PVs in individuals with a clinical presentation of AFAP/FAP. METHODS: We performed a retrospective query of individuals tested with paired DNA-RNA MGPT from 2021 to 2022 at a single laboratory and included those with a novel APC PV located in intronic regions infrequently covered by MGPT, a personal history of polyposis, and family medical history provided. All clinical data were deidentified in this institutional review board-exempt study. RESULTS: Three novel APC variants were identified in six families and were shown to cause aberrant splicing because of the creation of a deep intronic cryptic splice site that leads to an RNA transcript subject nonsense-mediated decay. Several carriers had previously undergone DNA-only genetic testing and had received a negative result. CONCLUSION: Here, we describe how paired DNA-RNA MGPT can be used to solve missing heritability in FAP families, which can have important implications in family planning and treatment decisions for patients and their families.

Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett's-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions.

BACKGROUND & AIMS: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression targeting SCJ LGR5+ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Circumventricular organ-hypothalamic circuit endoplasmic reticulum stress drives hepatic steatosis during obesity.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), characterized by excess liver triglyceride accumulation (hepatic steatosis), leads to an increased risk for cardiometabolic diseases and obesity-related mortality. Emerging evidence points to endoplasmic reticulum (ER) stress in the central nervous system as critical in NAFLD pathogenesis. Here, we tested the contribution of ER stress in a circumventricular organ-hypothalamic circuit in NAFLD development during obesity. METHODS: C57BL/6J male mice were fed a high-fat diet (HFD) or normal chow. A combination of histological, viral tracing, intersectional viral targeting, and in vivo integrative physiological approaches were used to examine the role of ER stress in subfornical organ to hypothalamic paraventricular nucleus projecting neurons (SFO➔PVN) in NAFLD during diet-induced obesity. RESULTS: Immunohistochemical analysis revealed marked unfolded protein response activation in the SFO, particularly in excitatory SFO➔PVN neurons of HFD-fed animals. Moreover, intersectional viral inhibition of ER stress in SFO➔PVN neurons resulted in a reduction in hepatomegaly, hepatic steatosis, and a blunted increase in body weight gain during diet-induced obesity, independent of changes in food intake, substrate partitioning, energy expenditure, and ambulatory activity. CONCLUSIONS: These results indicate that ER stress in an SFO➔PVN neural circuit contributes to hepatic steatosis during obesity.

Diagnostic Outcomes of Concurrent DNA and RNA Sequencing in Individuals Undergoing Hereditary Cancer Testing.

Importance: Personalized surveillance, prophylaxis, and cancer treatment options for individuals with hereditary cancer predisposition are informed by results of germline genetic testing. Improvements to genomic technology, such as the availability of RNA sequencing, may increase identification of individuals eligible for personalized interventions by improving the accuracy and yield of germline testing. Objective: To assess the cumulative association of paired DNA and RNA testing with detection of disease-causing germline genetic variants and resolution of variants of uncertain significance (VUS). Design, Setting, and Participants: Paired DNA and RNA sequencing was performed on individuals undergoing germline testing for hereditary cancer indication at a single diagnostic laboratory from March 2019 through April 2020. Demographic characteristics, clinical data, and test results were curated as samples were received, and changes to variant classification were assessed over time. Data analysis was performed from May 2020 to June 2023. Main Outcomes and Measures: Main outcomes were increase in diagnostic yield, decrease in VUS rate, the overall results by variant type, the association of RNA evidence with variant classification, and the corresponding predicted effect on cancer risk management. Results: A total of 43 524 individuals were included (median [range] age at testing, 54 [2-101] years; 37 373 female individuals [85.7%], 6224 male individuals [14.3%], and 2 individuals of unknown sex [<0.1%]), with 43 599 tests. A total of 2197 (5.0%) were Ashkenazi Jewish, 1539 (3.5%) were Asian, 3077 (7.1%) were Black, 2437 (5.6%) were Hispanic, 27 793 (63.7%) were White, and 2049 (4.7%) were other race, and for 4507 individuals (10.3%), race and ethnicity were unknown. Variant classification was impacted in 549 individuals (1.3%). Medically significant upgrades were made in 97 individuals, including 70 individuals who had a variant reclassified from VUS to pathogenic/likely pathogenic (P/LP) and 27 individuals who had a novel deep intronic P/LP variant that would not have been detected using DNA sequencing alone. A total of 93 of 545 P/LP splicing variants (17.1%) were dependent on RNA evidence for classification, and 312 of 439 existing splicing VUS (71.1%) were resolved by RNA evidence. Notably, the increase in positive rate (3.1%) and decrease in VUS rate (-3.9%) was higher in Asian, Black, and Hispanic individuals combined compared to White individuals (1.6%; P = .02; and -2.5%; P < .001). Conclusions and Relevance: Findings of this diagnostic study demonstrate that the ability to perform RNA sequencing concurrently with DNA sequencing represents an important advancement in germline genetic testing by improving detection of novel variants and classification of existing variants. This expands the identification of individuals with hereditary cancer predisposition and increases opportunities for personalization of therapeutics and surveillance.

A forebrain-hypothalamic ER stress driven circuit mediates hepatic steatosis during obesity.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 adults and contributes to advanced liver injury and cardiometabolic disease. While recent evidence points to involvement of the brain in NAFLD, the downstream neural circuits and neuronal molecular mechanisms involved in this response, remain unclear. Here, we investigated the role of a unique forebrain-hypothalamic circuit in NAFLD. METHODS: Chemogenetic activation and inhibition of circumventricular subfornical organ (SFO) neurons that project to the paraventricular nucleus of the hypothalamus (PVN; SFO→PVN) in mice were used to study the role of SFO→PVN signaling in NAFLD. Novel scanning electron microscopy techniques, histological approaches, molecular biology techniques, and viral methodologies were further used to delineate the role of endoplasmic reticulum (ER) stress within this circuit in driving NAFLD. RESULTS: In lean animals, acute chemogenetic activation of SFO→PVN neurons was sufficient to cause hepatic steatosis in a liver sympathetic nerve dependent manner. Conversely, inhibition of this forebrain-hypothalamic circuit rescued obesity-associated NAFLD. Furthermore, dietary NAFLD is associated with marked ER ultrastructural alterations and ER stress in the PVN, which was blunted following reductions in excitatory signaling from the SFO. Finally, selective inhibition of PVN ER stress reduced hepatic steatosis during obesity. CONCLUSIONS: Collectively, these findings characterize a previously unrecognized forebrain-hypothalamic-ER stress circuit that is involved in hepatic steatosis, which may point to future therapeutic strategies for NAFLD.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences.

Malaria affects ∼ » billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

An Enigmatic Case of Skeletal Fluorosis: Inhalant-Induced Toxicity.

ABSTRACT: Skeletal fluorosis is more common in the developing world, but is occasionally seen in the United States. We present radiographic, scintigraphic, CT, and clinical images of a 26-year-old woman with rapidly progressive, debilitating, polyostotic periostitis, and diffuse osteosclerosis typical of skeletal fluorosis. Laboratory analyses supported this diagnosis. The source of excess fluoride intake was elusive until a concurrent mental health workup revealed the patient's proclivity for inhaling air-duster cans containing difluoroethane. Difluoroethane inhalant abuse is an increasingly reported cause of skeletal fluorosis that astute clinicians should recognize. Discontinuation and sobriety from this toxic agent are essential for recovery.

C57BL/6J mice exposed to perfluorooctanoic acid demonstrate altered immune responses and increased seizures after Theiler's murine encephalomyelitis virus infection.

Introduction: Neurological diseases can stem from environmental influences such as antecedent viral infections or exposure to potential toxicants, some of which can trigger immune responses leading to neurological symptoms. Theiler's murine encephalomyelitis virus (TMEV) is used to model human neurological conditions associated with prior viral infections, with outcomes partly attributable to improper induction and regulation of the immune response. Perfluorooctanoic acid (PFOA) can alter pathologies known to influence neurological disease such as inflammatory responses, cytokine expression, and glial activation. Co-exposure to TMEV and PFOA was used to test the hypothesis that early life exposure to the potential immunotoxicant PFOA would affect immune responses so as to render TMEV-resistant C57BL/6J (B6) mice susceptible to viral-induced neurological disease. Methods: Neonate B6 mice were exposed to different treatments: non-injected, sham-infected with PBS, and TMEV-infected, with the drinking water of each group including either 70 ppt PFOA or filtered water. The effects of PFOA were evaluated by comparing neurological symptoms and changes in immune-related cytokine and chemokine production induced by viral infection. Immune responses of 23 cytokines and chemokines were measured before and after infection to determine the effects of PFOA exposure on immune response. Results: Prior to infection, an imbalance between Th1, Th2, and Treg cytokines was observed in PFOA-exposed mice, suppressing IL-4 and IL-13 production. However, the balance was restored and characterized by an increase in pro-inflammatory cytokines in the non-infected group, and a decrease in IL-10 in the PFOA + TMEV group. Furthermore, the PFOA + TMEV group experienced an increase in seizure frequency and severity. Discussion: Overall, these findings provide insight into the complex roles of immune responses in the pathogenesis of virus-associated neurological diseases influenced by co-exposures to viruses and immunotoxic compounds.

Large-field-of-view scanning electron microscopy of the paraventricular nucleus of the hypothalamus during diet-induced obesity.

Dysregulation in the paraventricular nucleus of the hypothalamus (PVN) is associated with a variety of diseases including those related to obesity. Although most investigations have focused on molecular changes, structural alterations in PVN neurons can reveal underlying functional disruptions. Although electron microscopy (EM) can provide nanometer resolution of brain structures, an inherent limitation of traditional transmission EM is the single field of view nature of data collection. To overcome this, we used large-field-of-view high-resolution backscatter scanning electron microscopy (bSEM) of the PVN. By stitching high-resolution bSEM images, taken from normal chow and high-fat diet mice, we achieved interactive, zoomable maps that allow for low-magnification screening of the entire PVN and high-resolution analyses of ultrastructure at the level of the smallest cellular organelle. Using this approach, quantitative analysis across the PVN revealed marked electron-dense regions within neuronal nucleoplasm following high-fat diet feeding, with an increase in kurtosis, indicative of a shift away from a normal distribution. Furthermore, measures of skewness indicated a shift toward darker clustered electron-dense regions, potentially indicative of heterochromatin clusters. We further demonstrate the utility to map out healthy and altered neurons throughout the PVN and the ability to remotely perform bSEM imaging in situations that require social distancing, such as the COVID-19 pandemic. Collectively, these findings present an approach that allows for the precise placement of PVN cells within an overall structural and functional map of the PVN. Moreover, they suggest that obesity may disrupt PVN neuronal chromatin structure.NEW & NOTEWORTHY Paraventricular nucleus of the hypothalamus (PVN) alterations are linked to obesity-related conditions, but limited knowledge exists about neuroanatomical changes in this region. A large-field-of-view backscatter scanning electron microscopy (bSEM) method was used, which allowed the identification of up to 40 PVN neurons in individual samples. During obesity in mice, bSEM revealed changes in PVN neuronal nucleoplasm, possibly indicating chromatin clustering. This microscopy advancement offers valuable insights into neuroanatomy in both healthy and disease conditions.

Safer at school early alert: an observational study of wastewater and surface monitoring to detect COVID-19 in elementary schools.

Background: Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods: The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 to March 2021. Findings: In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88%-98%); 67% were associated with a positive wastewater sample (95% CI: 57%-77%), and 40% were associated with a positive surface sample (95% CI: 29%-52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation: Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy. Funding: County of San Diego, Health and Human Services Agency, National Institutes of Health, National Science Foundation, Centers for Disease Control.

Wastewater and surface monitoring to detect COVID-19 in elementary school settings: The Safer at School Early Alert project.

Background: Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods: The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 - March 2021. Findings: In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88% - 98%); 67% were associated with a positive wastewater sample (95% CI: 57% - 77%), and 40% were associated with a positive surface sample (95% CI: 29% - 52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation: Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy. Funding: County of San Diego, Health and Human Services Agency, National Institutes of Health, National Science Foundation, Centers for Disease Control.

Central Feminization of Obese Male Mice Reduces Metabolic Syndrome.

Metabolic syndrome encompasses a spectrum of conditions that increases the risk for cardiovascular and metabolic diseases. It is widely accepted that the sex hormone estrogen plays a protective metabolic role in premenopausal women, in part through central nervous system (CNS) mechanisms. However, most work to date has focused on the loss of estrogen in females (e.g., menopause). Interestingly, transgender individuals receiving feminizing gender affirming therapy (i.e., estrogen) are relatively protected from metabolic syndrome conditions, pointing to a role for CNS estrogen in the development of metabolic syndrome in men. Here, we show that estrogen signaling in the brain protects males from metabolic syndrome and obesity related complications. First, short-term CNS specific supplementation of low-dose 17-ß-estradiol in diet-induced obese male mice resulted in a significant reduction in body weight in parallel with a decrease in food intake without alterations in energy expenditure. In conjunction, central supplementation of estrogen reduced visceral adiposity, including epididymal and abdominal regions, with slighter decreases in subcutaneous inguinal and thermogenic brown adipose tissue. Furthermore, central estrogen administration reduced the liver manifestation of metabolic syndrome including hepatomegaly and hepatic steatosis. Collectively, these findings indicate that a lack of estrogen action in the brain may predispose males to metabolic syndrome pathogenesis.

Viral Clearance and Neuroinflammation in Acute TMEV Infection Vary by Host Genetic Background.

A wide range of viruses cause neurological manifestations in their hosts. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the brain, depending in part on host genetic background. The interaction between host genetic background, neurological response to viral infection, and subsequent clinical manifestations remains poorly understood. In the present study, we used the genetically diverse Collaborative Cross (CC) mouse resource to better understand how differences in genetic background drive clinical signs and neuropathological manifestations of acute Theiler's murine encephalomyelitis virus (TMEV) infection. For the first time, we characterized variations of TMEV viral tropism and load based on host genetic background, and correlated viral load with microglial/macrophage activation. For five CC strains (CC002, CC023, CC027, CC057, and CC078) infected with TMEV, we compared clinical signs, lesion distribution, microglial/macrophage response, expression, and distribution of TMEV mRNA, and identified genetic loci relevant to the early acute (4 days post-infection [dpi]) and late acute (14 dpi) timepoints. We examined brain pathology to determine possible causes of strain-specific differences in clinical signs, and found that fields CA1 and CA2 of the hippocampal formation were especially targeted by TMEV across all strains. Using Iba-1 immunolabeling, we identified and characterized strain- and timepoint-specific variation in microglial/macrophage reactivity in the hippocampal formation. Because viral clearance can influence disease outcome, we used RNA in situ hybridization to quantify viral load and TMEV mRNA distribution at both timepoints. TMEV mRNA expression was broadly distributed in the hippocampal formation at 4 dpi in all strains but varied between radiating and clustered distribution depending on the CC strain. We found a positive correlation between microglial/macrophage reactivity and TMEV mRNA expression at 4 dpi. At 14 dpi, we observed a dramatic reduction in TMEV mRNA expression, and localization to the medial portion of field CA1 and field CA2. To better understand how host genetic background can influence pathological outcomes, we identified quantitative trait loci associated with frequency of lesions in a particular brain region and with microglial/macrophage reactivity. These QTL were located near several loci of interest: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1), and transmembrane protein 106 B (Tmem106b). Together, these results provide a novel understanding about the influences of genetic variation on the acute neuropathological and immunopathological environment and viral load, which collectively lead to variable disease outcomes. Our findings reveal possible avenues for future investigation which may lead to more effective intervention strategies and treatment regimens.

Differences in Cancer Phenotypes Among Frequent CHEK2 Variants and Implications for Clinical Care-Checking CHEK2.

Importance: Germline CHEK2 pathogenic variants (PVs) are frequently detected by multigene cancer panel testing (MGPT), but our understanding of PVs beyond c.1100del has been limited. Objective: To compare cancer phenotypes of frequent CHEK2 PVs individually and collectively by variant type. Design, Setting, and Participants: This retrospective cohort study was carried out in a single diagnostic testing laboratory from 2012 to 2019. Overall, 3783 participants with CHEK2 PVs identified via MGPT were included. Medical histories of cancer in participants with frequent PVs, negative MGPT (wild type), loss-of-function (LOF), and missense were compared. Main Outcomes and Measures: Participants were stratified by CHEK2 PV type. Descriptive statistics were summarized including median (IQR) for continuous variables and proportions for categorical characteristics. Differences in age and proportions were assessed with Wilcoxon rank sum and Fisher exact tests, respectively. Frequencies, odds ratios (ORs), 95% confidence intervals were calculated, and P values were corrected for multiple comparisons where appropriate. Results: Of the 3783 participants with CHEK2 PVs, 3473 (92%) were female and most reported White race. Breast cancer was less frequent in participants with p.I157T (OR, 0.66; 95% CI, 0.56-0.78; P<.001), p.S428F (OR, 0.59; 95% CI. 0.46-0.76; P<.001), and p.T476M (OR, 0.74; 95% CI, 0.56-0.98; P = .04) PVs compared with other PVs and an association with nonbreast cancers was not found. Following the exclusion of p.I157T, p.S428F, and p.T476M, participants with monoallelic CHEK2 PV had a younger age at first cancer diagnosis (P < .001) and were more likely to have breast (OR, 1.83; 95% CI, 1.66-2.02; P < .001), thyroid (OR, 1.63; 95% CI, 1.26-2.08; P < .001), and kidney cancer (OR, 2.57; 95% CI, 1.75-3.68; P < .001) than the wild-type cohort. Participants with a CHEK2 PV were less likely to have a diagnosis of colorectal cancer (OR, 0.62; 95% CI, 0.51-0.76; P < .001) compared with those in the wild-type cohort. There were no significant differences between frequent CHEK2 PVs and c.1100del and no differences between CHEK2 missense and LOF PVs. Conclusions and Relevance: CHEK2 PVs, with few exceptions (p.I157T, p.S428F, and p.T476M), were associated with similar cancer phenotypes irrespective of variant type. CHEK2 PVs were not associated with colorectal cancer, but were associated with breast, kidney, and thyroid cancers. Compared with other CHEK2 PVs, the frequent p.I157T, p.S428F, and p.T476M alleles have an attenuated association with breast cancer and were not associated with nonbreast cancers. These data may inform the genetic counseling and care of individuals with CHEK2 PVs.

Effective Functional Immunogenicity of a DNA Vaccine Combination Delivered via In Vivo Electroporation Targeting Malaria Infection and Transmission.

Plasmodium falciparum circumsporozoite protein (PfCSP) and Pfs25 are leading candidates for the development of pre-erythrocytic and transmission-blocking vaccines (TBV), respectively. Although considerable progress has been made in developing PfCSP- and Pfs25-based vaccines, neither have elicited complete protection or transmission blocking in clinical trials. The combination of antigens targeting various life stages is an alternative strategy to develop a more efficacious malaria vaccine. In this study, female and male mice were immunized with DNA plasmids encoding PfCSP and Pfs25, administered alone or in combination via intramuscular in vivo electroporation (EP). Antigen-specific antibodies were analyzed for antibody titers, avidity and isotype by ELISA. Immune protection against sporozoite challenge, using transgenic P. berghei expressing PfCSP and a GFP-luciferase fusion protein (PbPfCSP-GFP/Luc), was assessed by in vivo bioluminescence imaging and blood-stage parasite growth. Transmission reducing activity (TRA) was evaluated in standard membrane feeding assays (SMFA). High levels of PfCSP- and Pfs25-specific antibodies were induced in mice immunized with either DNA vaccine alone or in combination. No difference in antibody titer and avidity was observed for both PfCSP and Pfs25 between the single DNA and combined DNA immunization groups. When challenged by PbPfCSP-GFP/Luc sporozoites, mice immunized with PfCSP alone or combined with Pfs25 revealed significantly reduced liver-stage parasite loads as compared to mice immunized with Pfs25, used as a control. Furthermore, parasite liver loads were negatively correlated with PfCSP-specific antibody levels. When evaluating TRA, we found that immunization with Pfs25 alone or in combination with PfCSP elicited comparable significant transmission reduction. Our studies reveal that the combination of PfCSP and Pfs25 DNAs into a vaccine delivered by in vivo EP in mice does not compromise immunogenicity, infection protection and transmission reduction when compared to each DNA vaccine individually, and provide support for further evaluation of this DNA combination vaccine approach in larger animals and clinical trials.

Serum Cytokines Predict Neurological Damage in Genetically Diverse Mouse Models.

Viral infections contribute to neurological and immunological dysfunction driven by complex genetic networks. Theiler's murine encephalomyelitis virus (TMEV) causes neurological dysfunction in mice and can model human outcomes to viral infections. Here, we used genetically distinct mice from five Collaborative Cross mouse strains and C57BL/6J to demonstrate how TMEV-induced immune responses in serum may predict neurological outcomes in acute infection. To test the hypothesis that serum cytokine levels can provide biomarkers for phenotypic outcomes of acute disease, we compared cytokine levels at pre-injection, 4 days post-injection (d.p.i.), and 14 d.p.i. Each strain produced unique baseline cytokine levels and had distinct immune responses to the injection procedure itself. Thus, we eliminated the baseline responses to the injection procedure itself and identified cytokines and chemokines induced specifically by TMEV infection. Then, we identified strain-specific longitudinal cytokine profiles in serum during acute disease. Using stepwise regression analysis, we identified serum immune markers predictive for TMEV-induced neurological phenotypes of the acute phase, e.g., IL-9 for limb paralysis; and TNF-α, IL-1ß, and MIP-1ß for limb weakness. These findings indicate how temporal differences in immune responses are influenced by host genetic background and demonstrate the potential of serum biomarkers to track the neurological effects of viral infection.

An Evaluation of Phylogenetic Workflows in Viral Molecular Epidemiology.

The use of viral sequence data to inform public health intervention has become increasingly common in the realm of epidemiology. Such methods typically utilize multiple sequence alignments and phylogenies estimated from the sequence data. Like all estimation techniques, they are error prone, yet the impacts of such imperfections on downstream epidemiological inferences are poorly understood. To address this, we executed multiple commonly used viral phylogenetic analysis workflows on simulated viral sequence data, modeling Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), and Ebolavirus, and we computed multiple methods of accuracy, motivated by transmission-clustering techniques. For multiple sequence alignment, MAFFT consistently outperformed MUSCLE and Clustal Omega, in both accuracy and runtime. For phylogenetic inference, FastTree 2, IQ-TREE, RAxML-NG, and PhyML had similar topological accuracies, but branch lengths and pairwise distances were consistently most accurate in phylogenies inferred by RAxML-NG. However, FastTree 2 was the fastest, by orders of magnitude, and when the other tools were used to optimize branch lengths along a fixed FastTree 2 topology, the resulting phylogenies had accuracies that were indistinguishable from their original counterparts, but with a fraction of the runtime.

RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.