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SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to \"Long COVID\"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Neurological dysfunction after traumatic brain injury (TBI) is caused by both the primary injury and a secondary cascade of biochemical and metabolic events. Since TBI can be caused by a variety of mechanisms, numerous models have been developed to facilitate its study. The most prevalent models are controlled cortical impact and fluid percussion injury. Both typically use “sham” (craniotomy alone) animals as controls. However, the sham operation is objectively damaging, and we hypothesized that the craniotomy itself may cause a unique brain injury distinct from the impact injury. To test this hypothesis, 38 adult female rats were assigned to one of three groups: control (anesthesia only); craniotomy performed by manual trephine; or craniotomy performed by electric dental drill. The rats were then subjected to behavioral testing, imaging analysis, and quantification of cortical concentrations of cytokines. Both craniotomy methods generate visible MRI lesions that persist for 14 days. The initial lesion generated by the drill technique is significantly larger than that generated by the trephine. Behavioral data mirrored lesion volume. For example, drill rats have significantly impaired sensory and motor responses compared to trephine or naïve rats. Finally, of the seven tested cytokines, KC-GRO and IFN-γ showed significant increases in both craniotomy models compared to naïve rats. We conclude that the traditional sham operation as a control confers profound proinflammatory, morphological, and behavioral damage, which confounds interpretation of conventional experimental brain injury models. Any experimental design incorporating “sham” procedures should distinguish among sham, experimentally injured, and healthy/naïve animals, to help reduce confounding factors.

Background Analysis of somatic mutations from tumor whole exomes has fueled discovery of novel cancer driver genes. However, ~ 98% of the genome is non-coding and includes regulatory elements whose normal cellular functions can be disrupted by mutation. Whole genome sequencing (WGS), on the other hand, allows for identification of non-coding somatic variation and expanded estimation of background mutation rates, yet fewer computational tools exist for specific interrogation of this space. Results We present MutEnricher, a flexible toolset for investigating somatic mutation enrichment in both coding and non-coding genomic regions from WGS data. MutEnricher contains two distinct modules for these purposes that provide customizable options for calculating sample- and feature-specific background mutation rates. Additionally, both MutEnricher modules calculate feature-level and local, or “hotspot,” somatic mutation enrichment statistics. Conclusions MutEnricher is a flexible software package for investigating somatic mutation enrichment that is implemented in Python, is freely available, can be efficiently parallelized, and is highly configurable to researcher's specific needs. MutEnricher is available online at https://github.com/asoltis/MutEnricher .

In prostate cancer, emerging data highlight the role of DNA damage repair genes (DDRGs) in aggressive forms of the disease. However, DDRG mutations in African American men are not yet fully defined. Here, we profile germline mutations in all known DDRGs ( N  = 276) using whole genome sequences from blood DNA of a matched cohort of patients with primary prostate cancer comprising of 300 African American and 300 European Ancestry prostate cancer patients, to determine whether the mutation status can enhance patient stratification for specific targeted therapies. Here, we show that only 13 of the 46 DDRGs identified with pathogenic/likely pathogenic mutations are present in both African American and European ancestry patients. Importantly, RAD family genes ( RAD51, RAD54L, RAD54B ), which are potentially targetable, as well as PMS2 and BRCA1 , are among the most frequently mutated DDRGs in African American, but not in European Ancestry patients. DNA damage repair genes have been linked with increased aggressiveness of prostate cancer, however, the extent of mutation of these genes has not been analyzed within a cohort of African American patients. Here, the authors identify increased mutation rates in specific DNA repair genes, compared with prostate cancer patients with European Ancestry.

Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens. The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry. A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7. The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression.

Background Cardiac glycosides such as digoxin, digitoxin and ouabain are still used around the world to treat patients with chronic heart failure with reduced ejection fraction (HFrEF) and/or atrial fibrillation (AF). However, in the US, only digoxin is licensed for treating these illnesses, and the use of digoxin for this group of patients is increasingly being replaced in the US by a new standard of care with groups of more expensive drugs. However, ouabain and digitoxin, and less potently digoxin, have also recently been reported to inhibit SARS-CoV-2 virus penetration into human lung cells, thus blocking COVID-19 infection. COVID-19 is known to be a more aggressive disease in patients with cardiac comorbidities, including heart failure. Objective We therefore considered the possibility that digoxin might provide at least a measure of relief from COVID-19 in digoxin-treated heart failure patients. To this end, we hypothesized that treatment with digoxin rather than standard of care might equivalently protect heart failure patients with regard to diagnosis of COVID-19, hospitalization and death. Methods To test this hypothesis, we conducted a cross-sectional study by using the US Military Health System (MHS) Data Repository to identify all MHS TRICARE Prime and Plus beneficiaries aged 18–64 years with a heart failure (HF) diagnosis during the period April 2020 to August 2021. In the MHS, all patients receive equal, optimal care without regard to rank or ethnicity. Analyses included descriptive statistics on patient demographics and clinical characteristics, and logistic regressions to determine likelihood of digoxin use. Results We identified 14,044 beneficiaries with heart failure in the MHS during the study period. Of these, 496 were treated with digoxin. However, we found that both digoxin-treated and standard-of-care groups were equivalently protected from COVID-19. We also noted that younger active duty service members and their dependents with HF were less likely to receive digoxin compared with older, retired beneficiaries with more comorbidities. Conclusion The hypothesis of equivalent protection by digoxin treatment of HF patients in terms of susceptibility to COVID-19 infection appears to be supported by the data.

Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Aβ). We have hypothesized that the intrinsic Aβ calcium channel activity of the oligomeric Aβ polymer may be responsible for the neurotoxic properties of Aβ, and that Aβ channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Aβ channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Aβ channel and protect neurons from Aβ toxicity. Both block the Aβ channel with similar potency (≈500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.

Proteomics for blast traumatic brain injury (bTBI) research represents an exciting new approach that can greatly help to address the complex pathology of this condition. Antibody-based platforms, antibody microarrays (AbMA), and reverse capture protein microarrays (RCPM) can complement the classical methods based on 2D gel electrophoresis and mass spectrometry (2DGE/MS). These new technologies can address problematic issues, such as sample complexity, sensitivity, quantitation, reproducibility, and analysis time, which are typically associated with 2DGE/MS. Combined with bioinformatics analysis and interpretation of primary microarray data, these methods will generate a new level of understanding about bTBI at the level of systems biology. As biological and clinical knowledge and the availability of these systems become more widely established, we expect that AbMA and RCPM will be used routinely in clinical diagnostics, and also for following therapeutic progress. At the technical level, we anticipate that these platforms will evolve to accommodate comprehensive, high-speed, label-free analysis on a human proteome-wide scale.

Cerebral inflammatory responses may initiate secondary cascades following traumatic brain injury (TBI). Changes in the expression of both cytokines and chemokines may activate, regulate, and recruit innate and adaptive immune cells associated with secondary degeneration, as well as alter a host of other cellular processes. In this study, we quantified the temporal expression of a large set of inflammatory mediators in rat cortical tissue after brain injury. Following a controlled cortical impact (CCI) on young adult male rats, cortical and hippocampal tissue of the injured hemisphere and matching contralateral material was harvested at early (4, 12, and 24 hours) and extended (3 and 7 days) time points post-procedure. Naïve rats that received only anesthesia were used as controls. Processed brain homogenates were assayed for chemokine and cytokine levels utilizing an electrochemiluminescence-based multiplex ELISA platform. The temporal profile of cortical tissue samples revealed a multi-phasic injury response following brain injury. CXCL1, IFN-γ, TNF-α levels significantly peaked at four hours post-injury compared to levels found in naïve or contralateral tissue. CXCL1, IFN-γ, and TNF-α levels were then observed to decrease at least 3-fold by 12 hours post-injury. IL-1β, IL-4, and IL-13 levels were also significantly elevated at four hours post-injury although their expression did not decrease more than 3-fold for up to 24 hours post-injury. Additionally, IL-1β and IL-4 levels displayed a biphasic temporal profile in response to injury, which may suggest their involvement in adaptive immune responses. Interestingly, peak levels of CCL2 and CCL20 were not observed until after four hours post-injury. CCL2 levels in injured cortical tissue were significantly higher than peak levels of any other inflammatory mediator measured, thus suggesting a possible use as a biomarker. Fully elucidating chemokine and cytokine signaling properties after brain injury may provide increased insight into a number of secondary cascade events that are initiated or regulated by inflammatory responses.