Early pandemic in-hospital outcomes and mortality risk factors in COVID-19 solid organ transplant patients.

Background: Solid organ transplant (SOT) recipients with COVID-19 have a higher risk of mortality than those without COVID-19. However, it is unclear how SOT patient outcomes compare to the general population without SOT who contract COVID-19. Methods: We used the National Inpatient Sample from January to December 2020 to investigate inpatient outcomes seen in SOT recipients after contracting COVID-19 compared to nontransplant patients. We identified our study sample using ICD-10 CM and excluded those <18 years of age and those with dual organ transplants. Inpatient outcomes were compared in SOT and non-SOT COVID cohorts, and we further evaluated predictors of mortality in the SOT with COVID population. Results: Out of the 1,416,445 COVID-19 admissions included in the study, 8315 (0.59%) were single SOT recipients. Our analysis that adjusted for multiple baseline characteristics and comorbidities demonstrated that COVID-19 in SOT patients was associated with higher rates of acute kidney injury (adjusted odds ratio [aOR] 2.34, 95% confidence interval [CI] 1.81-3.02, P < 0.01), lower rates of acute respiratory distress syndrome (aOR 0.68, 95% CI 0.54-0.85, P < 0.01), and similar rates of cardiac arrest, pulmonary embolism, circulatory shock, cerebrovascular events, and in-hospital mortality. Age >65 was associated with mortality in SOT patients. Conclusion: In this nationally representative sample, SOT patients presenting with COVID-19 experienced similar rates of mortality compared to those without SOT. SOT patients were more likely to develop acute kidney injury. Further research is needed to understand the complex relationship between transplant patient outcomes and COVID-19.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ~0.9 in as little as six-weeks with a mean firing rate of ~13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of sporadic Alzheimer's disease by mimicking blood-brain barrier breakdown using a human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

The reverse transcriptase inhibitor 3TC protects against age-related cognitive dysfunction.

Aging is the primary risk factor for most neurodegenerative diseases, including Alzheimer's disease. Major hallmarks of brain aging include neuroinflammation/immune activation and reduced neuronal health/function. These processes contribute to cognitive dysfunction (a key risk factor for Alzheimer's disease), but their upstream causes are incompletely understood. Age-related increases in transposable element (TE) transcripts might contribute to reduced cognitive function with brain aging, as the reverse transcriptase inhibitor 3TC reduces inflammation in peripheral tissues and TE transcripts have been linked with tau pathology in Alzheimer's disease. However, the effects of 3TC on cognitive function with aging have not been investigated. Here, in support of a role for TE transcripts in brain aging/cognitive decline, we show that 3TC: (a) improves cognitive function and reduces neuroinflammation in old wild-type mice; (b) preserves neuronal health with aging in mice and Caenorhabditis elegans; and (c) enhances cognitive function in a mouse model of tauopathy. We also provide insight on potential underlying mechanisms, as well as evidence of translational relevance for these observations by showing that TE transcripts accumulate with brain aging in humans, and that these age-related increases intersect with those observed in Alzheimer's disease. Collectively, our results suggest that TE transcript accumulation during aging may contribute to cognitive decline and neurodegeneration, and that targeting these events with reverse transcriptase inhibitors like 3TC could be a viable therapeutic strategy.

TDP-43 knockdown in mouse model of ALS leads to dsRNA deposition, gliosis, and neurodegeneration in the spinal cord.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

A long-term obesogenic high-fat diet in mice partially dampens the anti-frailty benefits of late-life intermittent fasting.

The global obesity pandemic coupled with ever-growing life expectancies equates to hundreds of millions of individuals with potentially longer but not healthier lives. Aging is one of the risk factors for numerous maladies such as metabolic disorder and frailty, which are exacerbated under obesity. Thus, therapeutic approaches that address obesity to ultimately improve affected individuals' quality of life and extend their lifespan are needed. We previously reported that the every other day (EOD) fasting initiated late-life improved metabolic, musculoskeletal, and cognitive endpoints in standard rodent diet-fed mice. In the present study, using the same dietary intervention methodology, we tested if 2.5 months of EOD fasting could improve metabolic, physiological, and cognitive endpoints in mice after an 18 month obesogenic high-fat diet (HFD). The positive effects of EOD fasting were generally consistent across the endpoints; EOD fasting decreased total body mass, maintained more %lean mass, improved glucose tolerance and utilization, and improved neuromuscular function. In contrast to our previous study, grip strength, hippocampal-dependent memory, and renal hydrogen sulfide (H2S) production were not improved by the HFD EOD fasting. Thus, efficacy for late-life initiated intermittent fasting to improve specific frailty markers may be partially dependent on nutritional compositions of the diet.

Identifying an Optimal Neuroinflammation Treatment Using a Nanoligomer Discovery Engine.

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leading to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and it is causally linked to a range of neurodegenerative diseases such as Alzheimer's diseases (AD), Multiple Sclerosis (MS), Parkinson's disease (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier, making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer Discovery Engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1ß or IL-1ß, tumor necrosis factor-alpha or TNF-α, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-κß) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and artificial intelligence-based ranking method to design sequence-specific peptide molecules to up- or downregulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-κß (SB.201.17D.8_NF-κß1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low dose (5 mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

Incidence of Post-Heart Transplant Chronic Thyroiditis and Its Association With Pretransplant Amiodarone Use.

BACKGROUND: Chronic thyroiditis (CT) is a common cause of thyroid dysfunction and could therefore adversely affect outcomes in patients undergoing heart transplant (HT). The incidence of post-HT CT and whether amiodarone, a commonly used anti-arrhythmic drug in patients with heart failure during pre-HT period, is associated with the development of post-HT CT are unknown. METHODS: A retrospective review of HT recipients from February 2, 2010 to October 16, 2018 was performed. Patients who lacked relevant pre-/post-HT records, underwent thyroidectomy, had pre-HT thyroid dysfunction or thyroiditis within 15 days post-HT, and those on amiodarone during the post-HT period were excluded, yielding a final cohort of 75 patients. RESULTS: Patients had a mean age of 63.3 ± 1.4 years and were predominantly male (90.7%) and white (80%). The incidence of post-HT CT was 32% with the majority (83.3%) manifesting as hypothyroidism. Median time to diagnosis of CT after transplant was 10.2 months (interquartile range, 4-27.4). Additionally, the CT group had higher pre-HT use of amiodarone (non-CT vs CT: 21.6% vs 50%, P = .01), higher prevalence of atrial fibrillation (non-CT vs CT: 23.5% vs 45.8%; P = .05), and more stage IV/V chronic kidney disease (non-CT vs CT: 2% vs 16.7%, P = .02). On multivariate analysis, pre-HT amiodarone use was associated with the development of post-HT CT after adjustment for age, sex, and chronic kidney disease (odds ratio, 3.65; 95% CI, 1.17-11.44; P = .03). CONCLUSION: The incidence of post-HT CT is high and is strongly associated with pre-HT amiodarone use underpinning the importance of closely following the post-HT thyroid profile in these patients.

Amyloid beta acts synergistically as a pro-inflammatory cytokine.

The amyloid beta (Aß) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aß are incompletely understood. Here, we report that nanomolar concentrations of Aß act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aß can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aß have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aß at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aß often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aß and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

Peripubertal Bisphenol A Exposure Imparts Detrimental Age-Related Changes in Body Composition, Cognition, and Hydrogen Sulfide Production Capacities.

Aims: Peripubertal endocrine disruption has immediate and lifelong consequences on health, cognition, and lifespan. Disruption comes from dietary, environmental, and pharmaceutical sources. The plasticizer Bisphenol A (BPA) is one such endocrine disrupting chemical. However, it is unclear whether peripubertal BPA exposure incites long-lasting physiological, neuro-cognitive, and/or longevity-related metabolic impairments. Catabolism of cysteine via transsulfuration enzymes produces hydrogen sulfide (H2S), a redox-modulating gasotransmitter causative to endocrine and metabolic homeostasis and improved cognitive function with age. As thyroid hormone (TH) regulates hepatic H2S production and BPA is a TH receptor antagonist, we hypothesized that BPA exposure during peripubertal development impairs metabolic and neuro-cognitive/behavioral endpoints in aged mice, in part, due to altered peripheral and/or localized H2S production and redox status. Results: To test this, male C57BL/6J mice at 5 weeks of age were orally exposed daily for 5 weeks to 250 µg BPA/kg, defined as low dose group (LD BPA), or 250 mg BPA/kg, defined as high dose group (HD BPA). Both LD and HD BPA exposure decreased lean mass and increased fat mass accompanied by decreased serum total TH at advanced ages. In addition, LD BPA had an anxiogenic effect whereas HD BPA caused cognitive deficits. Notably, HD BPA disrupted tissue-specific H2S production capacities and/or protein persulfidation, with the former negatively correlated with memory deficits and oxidative stress. Innovation and Conclusion: These findings provide a potential mechanism of action for acute and long-term health impacts of BPA-induced peripubertal endocrine disruption and bolster the need for improved monitoring and limitation of adolescent BPA exposure.

Is There a Brain Microbiome?

Numerous studies have identified microbial sequences or epitopes in pathological and non-pathological human brain samples. It has not been resolved if these observations are artifactual, or truly represent population of the brain by microbes. Given the tempting speculation that resident microbes could play a role in the many neuropsychiatric and neurodegenerative diseases that currently lack clear etiologies, there is a strong motivation to determine the "ground truth" of microbial existence in living brains. Here I argue that the evidence for the presence of microbes in diseased brains is quite strong, but a compelling demonstration of resident microbes in the healthy human brain remains to be done. Dedicated animal models studies may be required to determine if there is indeed a "brain microbiome."

Application of a bioinformatic pipeline to RNA-seq data identifies novel virus-like sequence in human blood.

Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data and in a reconstruction experiment using synthetic data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq datasets currently being generated.

Late-life intermittent fasting decreases aging-related frailty and increases renal hydrogen sulfide production in a sexually dimorphic manner.

Global average life expectancy continues to rise. As aging increases the likelihood of frailty, which encompasses metabolic, musculoskeletal, and cognitive deficits, there is a need for effective anti-aging treatments. It is well established in model organisms that dietary restriction (DR), such as caloric restriction or protein restriction, enhances health and lifespan. However, DR is not widely implemented in the clinic due to patient compliance and its lack of mechanistic underpinnings. Thus, the present study tested the effects of a somewhat more clinically applicable and adoptable DR regimen, every-other-day (EOD) intermittent fasting, on frailty in 20-month-old male and female C57BL/6 mice. Frailty was determined by a series of metabolic, musculoskeletal, and cognitive tasks performed prior to and toward the end of the 2.5-month dietary intervention. Late-life EOD fasting attenuated overall energy intake, hypothalamic inflammatory gene expression, and frailty in males. However, it failed to reduce overall caloric intake and had a little positive effect in females. Given that the selected benefits of DR are dependent on augmented production of the gasotransmitter hydrogen sulfide (H2S) and that renal H2S production declines with age, we tested the effects of EOD fasting on renal H2S production capacity and its connection to frailty in males. EOD fasting boosted renal H2S production, which positively correlated with improvements in multiple components of frailty tasks. Therefore, late-life initiated EOD fasting is sufficient to reduce aging-related frailty, at least in males, and suggests that renal H2S production capacity may modulate the effects of late-life EOD fasting on frailty.

Dietary restriction transforms the mammalian protein persulfidome in a tissue-specific and cystathionine γ-lyase-dependent manner.

Hydrogen sulfide (H2S) is a cytoprotective redox-active metabolite that signals through protein persulfidation (R-SSnH). Despite the known importance of persulfidation, tissue-specific persulfidome profiles and their associated functions are not well characterized, specifically under conditions and interventions known to modulate H2S production. We hypothesize that dietary restriction (DR), which increases lifespan and can boost H2S production, expands tissue-specific persulfidomes. Here, we find protein persulfidation enriched in liver, kidney, muscle, and brain but decreased in heart of young and aged male mice under two forms of DR, with DR promoting persulfidation in numerous metabolic and aging-related pathways. Mice lacking cystathionine γ-lyase (CGL) have overall decreased tissue protein persulfidation numbers and fail to functionally augment persulfidomes in response to DR, predominantly in kidney, muscle, and brain. Here, we define tissue- and CGL-dependent persulfidomes and how diet transforms their makeup, underscoring the breadth for DR and H2S to impact biological processes and organismal health.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

The gut microbiome-derived metabolite trimethylamine N-oxide modulates neuroinflammation and cognitive function with aging.

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r2 = 0.07, p < 0.05). Study 2: In mice, TMAO concentrations in plasma and the brain increased in parallel with aging (r2 = 0.60), suggesting TMAO crosses the blood-brain barrier. The greater TMAO concentrations in old mice (27 months) were associated with higher brain pro-inflammatory cytokines and markers of astrocyte activation vs. young adult mice (6 months). Study 3: To determine if TMAO independently induces an "aging-like" decline in cognitive function, young mice (6 months) were supplemented with TMAO in chow for 6 months. Compared with controls, TMAO-supplemented mice performed worse on the novel object recognition test, indicating impaired memory and learning, and had increased neuroinflammation and markers of astrocyte activation. Study 4: Human astrocytes cultured with TMAO vs. control media exhibited changes in cellular morphology and protein markers consistent with astrocyte activation, indicating TMAO directly acts on these cells. Our results provide translational insight into a novel pathway that modulates neuroinflammation and cognitive function with aging, and suggest that TMAO might be a promising target for prevention of neuroinflammation and cognitive decline with aging.

IL-2-Agonist-Induced IFN-γ Exacerbates Systemic Anaphylaxis in Food Allergen-Sensitized Mice.

Food allergies are common, costly and potentially life-threatening disorders. They are driven by Th2, but inhibited by Th1 reactions. There is also evidence indicating that IL-2 agonist treatment inhibits allergic sensitization through expansion of regulatory T cells. Here, we tested the impact of an IL-2 agonist in a novel model for food allergy to hen´s egg in mice sensitized without artificial adjuvants. Prophylactic IL-2 agonist treatment expanded Treg populations and inhibited allergen-specific sensitization. However, IL-2 agonist treatment of already sensitized mice increased mast cell responses and allergic anaphylaxis upon allergen re-challenge. These effects depended on allergen-specific IgE and were mediated through IFN-γ, as shown by IgE transfer and blockade of IFN-γ with monoclonal antibodies. These results suggest that although shifting the allergic reaction toward a Treg/Th1 response inhibits allergic sensitization, the prototypic Th1 cytokine IFN-γ promotes mast cell activation and allergen-induced anaphylaxis in individuals that are already IgE-sensitized. Hence, while a Th1 response can prevent the development of food allergy, IFN-γ has the ability to exacerbate already established food allergy.

Bone Marrow Plasma Cells Modulate Local Myeloid-Lineage Differentiation via IL-10.

Bone marrow plasma cells have been reported to represent a major source of IL-10; however, the impact of plasma cell derived IL-10 in that tissue remains poorly understood. We confirm in this study that even in the absence of acute immune reactions, mature plasma cells represent the dominant IL-10+ cell population in the bone marrow, and identify myeloid-lineage cells as a main local target for plasma cell derived IL-10. Using Vert-X IL-10 transcriptional reporter mice, we found that more than 50% of all IL-10+ cells in bone marrow were CD138+ plasma cells, while other IL-10+ B lineage cells were nearly absent in this organ. Accordingly, IL-10 was found in the supernatants of short-term cultures of FACS-sorted bone marrow plasma cells, confirming IL-10 secretion from these cells. IL-10+ bone marrow plasma cells showed a B220-/CD19-/MHCII low phenotype suggesting that these cells represent a mature differentiation stage. Approximately 5% of bone marrow leucocytes expressed the IL-10 receptor (IL-10R), most of them being CD115+/Ly6C+/CD11c- monocytes. Compared to littermate controls, young B lineage specific IL-10 KO mice showed increased numbers of CD115+ cells but normal populations of other myeloid cell types in bone marrow. However, at 7 months of age B lineage specific IL-10 KO mice exhibited increased populations of CD115+ myeloid and CD11c+ dendritic cells (DCs), and showed reduced F4/80 expression in this tissue; hence, indicating that bone marrow plasma cells modulate the differentiation of local myeloid lineage cells via IL-10, and that this effect increases with age. The effects of B cell/plasma cell derived IL-10 on the differentiation of CD115+, CD11c+, and F4/80+ myeloid cells were confirmed in co-culture experiments. Together, these data support the idea that IL-10 production is not limited to early plasma cell stages in peripheral tissues but is also an important feature of mature plasma cells in the bone marrow. Moreover, we provide evidence that already under homeostatic conditions in the absence of acute immune reactions, bone marrow plasma cells represent a non-redundant source for IL-10 that modulates local myeloid lineage differentiation. This is particularly relevant in older individuals.

TDP-43 knockdown causes innate immune activation via protein kinase R in astrocytes.

TAR-DNA binding protein 43 (TDP-43) is a multifunctional RNA binding protein directly implicated in the etiology of amyotrophic lateral sclerosis (ALS). Previous studies have demonstrated that loss of TDP-43 function leads to intracellular accumulation of non-coding repetitive element transcripts and double-stranded RNA (dsRNA). These events could cause immune activation and contribute to the neuroinflammation observed in ALS, but this possibility has not been investigated. Here, we knock down TDP-43 in primary rat astrocytes via siRNA, and we use RNA-seq, immunofluorescence, and immunoblotting to show that this results in: 1) accumulation of repetitive element transcripts and dsRNA; and 2) pro-inflammatory gene and protein expression consistent with innate immune signaling and astrocyte activation. We also show that both chemical inhibition and siRNA knockdown of protein kinase R (PKR), a dsRNA-activated kinase implicated in the innate immune response, block the expression of all activation markers assayed. Based on these findings, we suggest that intracellular accumulation of endogenous dsRNA may be a novel and important mechanism underlying the pathogenesis of ALS (and perhaps other neurodegenerative diseases), and that PKR inhibitors may have the potential to prevent reactive astrocytosis in ALS.

Heat shock in C. elegans induces downstream of gene transcription and accumulation of double-stranded RNA.

We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.