Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation.

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

Systemic proteome phenotypes reveal defective metabolic flexibility in Mecp2 mutants.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

Quantification and persistence of COVID-19 virus in recently deceased individuals before and after embalming.

The COVID-19 pandemic severely affected the medical education worldwide. The infection risk for medical students and healthcare personnel who work with COVID-19 positive cadavers or tissues remains unclear. Moreover, COVID-19 positive cadavers have been rejected by medical schools, adversely impacting the continuum of medical education. Herein, the viral genome abundance in tissues from four COVID-19 positive donors before and after embalming were compared. Tissue samples were collected from the lungs, liver, spleen, and brain both pre- and postembalming. The possible presence of infectious COVID-19 was determined by inoculating human tissue homogenates onto a monolayer of human A549-hACE2 cells and observing for cytopathic effects up to 72 h postinoculation. A real- time quantitative reverse transcription polymerase chain reaction was performed to quantify COVID-19 present in culture supernatants. Fully intact viral genome sequence was possible to obtain in samples with higher levels of virus, even several days postmortem. The embalming procedure described above substantially reduces the abundance of viable COVID-19 genomes in all tissues, sometimes even to undetectable levels. However, in some cases, COVID-19 RNA can still be detected, and a cytopathic effect can be seen both pre- and postembalmed tissues. This study suggests that embalmed COVID-19 positive cadavers might be used safely with appropriate precautions followed in gross anatomy laboratories and in clinical and scientific research. Deep lung tissue is the best specimen to test for the virus. If the tests on the lung tissues are negative, there is a very low likelihood that other tissues will show positive results.

Mitochondrial protein import clogging as a mechanism of disease.

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.

Inside our cells, compartments known as mitochondria generate the chemical energy required for life processes to unfold. Most of the proteins found within mitochondria are manufactured in another part of the cell (known as the cytosol) and then imported with the help of specialist machinery. For example, the TOM and TIM22 channels provide a route for the proteins to cross the two membrane barriers that separate the cytosol from the inside of a mitochondrion. ANT1 is a protein that is found inside mitochondria in humans, where it acts as a transport system for the cell's energy currency. Specific mutations in the gene encoding ANT1 have been linked to degenerative conditions that affect the muscles and the brain. However, it remains unclear how these mutations cause disease. To address this question, Coyne et al. recreated some of the mutations in the gene encoding the yeast equivalent of ANT1 (known as Aac2). Experiments in yeast cells carrying these mutations showed that the Aac2 protein accumulated in the TOM and TIM22 channels, creating a 'clog' that prevented other essential proteins from reaching the mitochondria. As a result, the yeast cells died. Mutant forms of the human ANT1 protein also clogged up the TOM and TIM22 channels of human cells in a similar way. Further experiments focused on mice genetically engineered to produce a "super-clogger" version of the mouse equivalent of ANT1. The animals soon developed muscle and neurological conditions similar to those observed in human diseases associated with ANT1. The findings of Coyne et al. suggest that certain genetic mutations in the gene encoding the ANT1 protein cause disease by blocking the transport of other proteins to the mitochondria, rather than by directly affecting ANT1's nucleotide trnsport role in the cell. This redefines our understanding of diseases associated with mitochondrial proteins, potentially altering how treatments for these conditions are designed.

Systemic Proteome Phenotypes Reveal Defective Metabolic Flexibility in Mecp2 Mutants.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

Increased prefrontal cortical cells positive for macrophage/microglial marker CD163 along blood vessels characterizes a neuropathology of neuroinflammatory schizophrenia.

Transcript levels of cytokines and SERPINA3 have been used to define a substantial subset (40%) of individuals with schizophrenia with elevated inflammation and worse neuropathology in the dorsolateral prefrontal cortex (DLPFC). In this study, we tested if inflammatory proteins are likewise related to high and low inflammatory states in the human DLFPC in people with schizophrenia and controls. Levels of inflammatory cytokines (IL6, IL1ß, IL18, IL8) and a macrophage marker (CD163 protein) were measured in brains obtained from the National Institute of Mental Health (NIMH) (N = 92). First, we tested for diagnostic differences in protein levels overall, then we determined the percentage of individuals that could be defined as "high" inflammation using protein levels. IL-18 was the only cytokine to show increased expression in schizophrenia compared to controls overall. Interestingly, two-step recursive clustering analysis showed that IL6, IL18, and CD163 protein levels could be used as predictors of "high and low" inflammatory subgroups. By this model, a significantly greater proportion of schizophrenia cases (18/32; 56.25%; SCZ) were identified as belonging to the high inflammatory (HI) subgroup compared to control cases (18/60; 30%; CTRL) [χ2(1) = 6.038, p = 0.014]. When comparing across inflammatory subgroups, IL6, IL1ß, IL18, IL8, and CD163 protein levels were elevated in both SCZ-HI and CTRL-HI compared to both low inflammatory subgroups (all p < 0.05). Surprisingly, TNFα levels were significantly decreased (-32.2%) in schizophrenia compared to controls (p < 0.001), and were most diminished in the SCZ-HI subgroup compared to both CTRL-LI and CTRL-HI subgroups (p < 0.05). Next, we asked if the anatomical distribution and density of CD163+ macrophages differed in those with schizophrenia and high inflammation status. Macrophages were localized to perivascular sites and found surrounding small, medium and large blood vessels in both gray matter and white matter, with macrophage density highest at the pial surface in all schizophrenia cases examined. A higher density of CD163+ macrophages, that were also larger and more darkly stained, was found in the SCZ-HI subgroup (+154% p < 0.05). We also confirmed the rare existence of parenchymal CD163+ macrophages in both high inflammation subgroups (schizophrenia and controls). Brain CD163+ cell density around blood vessels positively correlated with CD163 protein levels. In conclusion, we find a link between elevated interleukin cytokine protein levels, decreased TNFα protein levels, and elevated CD163+ macrophage densities especially along small blood vessels in those with neuroinflammatory schizophrenia.

Changes in cytokine and cytokine receptor levels during postnatal development of the human dorsolateral prefrontal cortex.

In addition to their traditional roles in immune cell communication, cytokines regulate brain development. Cytokines are known to influence neural cell generation, differentiation, maturation, and survival. However, most work on the role of cytokines in brain development investigates rodents or focuses on prenatal events. Here, we investigate how mRNA and protein levels of key cytokines and cytokine receptors change during postnatal development of the human prefrontal cortex. We find that most cytokine transcripts investigated (IL1B, IL18, IL6, TNF, IL13) are lowest at birth and increase between 1.5 and 5 years old. After 5 years old, transcriptional patterns proceeded in one of two directions: decreased expression in teens and young adults (IL1B, p = 0.002; and IL18, p = 0.004) or increased mean expression with maturation, particularly in teenagers (IL6, p = 0.004; TNF, p = 0.002; IL13, p < 0.001). In contrast, cytokine proteins tended to remain elevated after peaking significantly around 3 years of age (IL1B, p = 0.012; IL18, p = 0.026; IL6, p = 0.039; TNF, p < 0.001), with TNF protein being highest in teenagers. An mRNA-only analysis of cytokine receptor transcripts found that early developmental increases in cytokines were paralleled by increases in their ligand-binding receptor subunits, such as IL1R1 (p = 0.033) and IL6R (p < 0.001) transcripts. In contrast, cytokine receptor-associated signaling subunits, IL1RAP and IL6ST, did not change significantly between age groups. Of the two TNF receptors, the 'pro-death' TNFRSF1A and 'pro-survival' TNFRSF1B, only TNFRSF1B was significantly changed (p = 0.028), increasing first in toddlers and again in young adults. Finally, the cytokine inhibitor, IL13, was elevated first in toddlers (p = 0.006) and again in young adults (p = 0.053). While the mean expression of interleukin-1 receptor antagonist (IL1RN) was highest in toddlers, this increase was not statistically significant. The fluctuations in cytokine expression reported here support a role for increases in specific cytokines at two different stages of human cortical development. The first is during the toddler/preschool period (IL1B, IL18, and IL13), and the other occurs at adolescence/young adult maturation (IL6, TNF and IL13).

Clinical Features of Genetic Resilience in Chronic Obstructive Pulmonary Disease.

Introduction: In the personalized risk quantification of chronic obstructive pulmonary disease (COPD), genome-wide association studies and polygenic risk scores (PRS) complement traditional risk factors, such as age and cigarette smoking. However, despite being at considerable levels of risk, some individuals do not develop COPD. Research on COPD resilience remains largely unexplored. Methods: We applied the previously published COPD PRS to whole genome sequencing data from non-Hispanic white and African American individuals in the COPDGene study. We defined genetic resilience as individuals unaffected by COPD with a polygenic risk score above the 90 th percentile. We defined risk-matched case individuals as those with COPD (i.e., FEV 1 /FVC < 0.70) and a PRS above the 90 th percentile. We defined low risk individuals without COPD (i.e., FEV 1 /FVC > 0.70) as a polygenic risk score below the 10 th percentile. We compared genetically resilient individuals to risk-matched individuals with COPD and low risk individuals by demographics, lung function, respiratory symptoms, co-morbidities, and chest CT scan measurements. We also performed survival analyses, differential expression analysis, and matching for sensitivity analyses. Results: We identified 211 resilient individuals without COPD, 605 genetic risk-matched individuals with COPD, and 527 low-risk individuals without COPD. Resilient individuals had higher FEV 1 % predicted and lower percent emphysema. In contrast, resilient individuals had higher airway wall thickness compared to low-risk unaffected individuals. While there was no difference in survival between low-risk and resilient individuals, resilient individuals had higher survival compared to risk matched cases. We also identified two genes that were differentially expressed between low-risk unaffected individuals and resilient individuals. Conclusion: Genetically resilient individuals had a reduced burden of COPD disease-related measures compared to risk-matched cases but had subtly increased measures compared to low-risk unaffected individuals. Further genetic studies will be needed to illuminate the underlying pathobiology of our observations.

The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

Infection with Borrelia burgdorferi Increases the Replication and Dissemination of Coinfecting Powassan Virus in Ixodes scapularis Ticks.

Powassan virus (POWV) is a tick-borne neuroinvasive flavivirus endemic to North America. It is generally transmitted by the tick, Ixodes scapularis. This species also transmits Borrelia burgdorferi, the causative agent of Lyme disease. Infection with B. burgdorferi can result in arthritis, carditis, and neuroborreliosis. These pathogens experience sylvatic overlap. To determine the risk of human exposure to coinfected ticks, the interactions between POWV and B. burgdorferi are assessed in laboratory-infected I. scapularis. Adult male and female I. scapularis ticks are orally inoculated with either both pathogens, POWV only, B. burgdorferi only, or uninfected media. After twenty-one days, the ticks are dissected, and RNA is extracted from their midguts and salivary glands. In infected midguts, the quantity of POWV in coinfected ticks was elevated compared to those with only POWV. In addition, the salivary glands of ticks with infected midguts had increased POWV dissemination to those with only POWV. RNA sequencing is performed to identify the potential mechanism for this pattern, which varies between the organs. Ixodes scapularis ticks are found to be capable of harboring both POWV and B. burgdorferi with a benefit to POWV replication and dissemination.

Distinct Phenotypes of Inflammation Associated Macrophages and Microglia in the Prefrontal Cortex Schizophrenia Compared to Controls.

Approximately 40% of people with schizophrenia are classified as having "high inflammation." This subgroup has worse neuropathology than patients with "low inflammation." Thus, one would expect the resident microglia and possibly monocyte-derived macrophages infiltrating from the periphery to be "activated" in those with schizophrenia with elevated neuroinflammation. To test whether microglia and/or macrophages are associated with increased inflammatory signaling in schizophrenia, we measured microglia- and macrophage-associated transcripts in the postmortem dorsolateral prefrontal cortex of 69 controls and 72 people with schizophrenia. Both groups were stratified by neuroinflammatory status based on cortical mRNA levels of cytokines and SERPINA3. We found microglial mRNAs levels were either unchanged (IBA1 and Hexb, p > 0.20) or decreased (CD11c, <62% p < 0.001) in high inflammation schizophrenia compared to controls. Conversely, macrophage CD163 mRNA levels were increased in patients, substantially so in the high inflammation schizophrenia subgroup compared to low inflammation subgroup (>250%, p < 0.0001). In contrast, high inflammation controls did not have elevated CD163 mRNA compared to low inflammation controls (p > 0.05). The pro-inflammatory macrophage marker (CD64 mRNA) was elevated (>160%, all p < 0.05) and more related to CD163 mRNA in the high inflammation schizophrenia subgroup compared to high inflammation controls, while anti-inflammatory macrophage and cytokine markers (CD206 and IL-10 mRNAs) were either unchanged or decreased in schizophrenia. Finally, macrophage recruitment chemokine CCL2 mRNA was increased in schizophrenia (>200%, p < 0.0001) and CCL2 mRNA levels positively correlated with CD163 mRNA (r = 0.46, p < 0.0001). Collectively, our findings support the co-existence of quiescent microglia and increased pro-inflammatory macrophages in the cortex of people with schizophrenia.

Inflammation-related transcripts define "high" and "low" subgroups of individuals with schizophrenia and bipolar disorder in the midbrain.

Dopamine dysregulation in schizophrenia may be associated with midbrain inflammation. Previously, we found elevated levels of pro-inflammatory cytokine mRNAs in the post-mortem midbrain of people with schizophrenia (46%) but not from unaffected controls (0%) using a brain cohort from Sydney, Australia. Here, we measured cytokine mRNAs and proteins in the midbrain in the Stanley Medical Research Institute (SMRI) array cohort (N = 105). We tested if the proportions of individuals with schizophrenia and with high inflammation can be replicated, and if individuals with bipolar disorder with elevated midbrain cytokines can be identified. mRNA levels of 7 immune transcripts from post-mortem midbrain tissue were measured via RT-PCR and two-step recursive clustering analysis was performed using 4 immune transcripts to define "high and low" inflammatory subgroups. The clustering predictors used were identical to our earlier midbrain study, and included: IL1B, IL6, TNF, and SERPINA3 mRNA levels. 46% of schizophrenia cases (16/35 SCZ), 6% of controls (2/33 CTRL), and 29% of bipolar disorder cases (10/35 BPD) were identified as belonging to the high inflammation (HI) subgroups [χ2 (2) = 13.54, p < 0.001]. When comparing inflammatory subgroups, all four mRNAs were significantly increased in SCZ-HI and BPD-HI compared to low inflammation controls (CTRL-LI) (p < 0.05). Additionally, protein levels of IL-1ß, IL-6, and IL-18 were elevated in SCZ-HI and BPD-HI compared to all other low inflammatory subgroups (all p < 0.05). Surprisingly, TNF-α protein levels were unchanged according to subgroups. In conclusion, we determined that almost half of the individuals with schizophrenia were defined as having high inflammation in the midbrain, replicating our previous findings. Further, we detected close to one-third of those with bipolar disorder to be classified as having high inflammation. Elevations in some pro-inflammatory cytokine mRNAs (IL-1ß and IL-6) were also found at the protein level, whereas TNF mRNA and protein levels were not concordant.

High Sensitivity and Specificity of Dormitory-Level Wastewater Surveillance for COVID-19 during Fall Semester 2020 at Syracuse University, New York.

A residential building's wastewater presents a potential non-invasive method of surveilling numerous infectious diseases, including SARS-CoV-2. We analyzed wastewater from 16 different residential locations at Syracuse University (Syracuse, NY, USA) during fall semester 2020, testing for SARS-CoV-2 RNA twice weekly and compared the presence of clinical COVID-19 cases to detection of the viral RNA in wastewater. The sensitivity of wastewater surveillance to correctly identify dormitories with a case of COVID-19 ranged from 95% (95% confidence interval [CI] = 76-100%) on the same day as the case was diagnosed to 73% (95% CI = 53-92%), with 7 days lead time of wastewater. The positive predictive value ranged from 20% (95% CI = 13-30%) on the same day as the case was diagnosed to 50% (95% CI = 40-60%) with 7 days lead time. The specificity of wastewater surveillance to correctly identify dormitories without a case of COVID-19 ranged from 60% (95% CI = 52-67%) on the day of the wastewater sample to 67% (95% CI = 58-74%) with 7 days lead time. The negative predictive value ranged from 99% (95% CI = 95-100%) on the day of the wastewater sample to 84% (95% CI = 77-91%) with 7 days lead time. Wastewater surveillance for SARS-CoV-2 at the building level is highly accurate in determining if residents have a COVID-19 infection. Particular benefit is derived from negative wastewater results that can confirm a building is COVID-19 free.

Development and Validation of Two RT-qPCR Diagnostic Assays for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 Genomic Targets across Two Specimen Types.

Following the outbreak and subsequent pandemic of coronavirus disease 2019 (COVID-19), clinical diagnostic laboratories worldwide sought accurate and reliable testing methodologies. However, many laboratories were and still are hindered by a number of factors, including an unprecedented demand for testing, reagent and laboratory supply shortages and availability of qualified staff. To respond to these concerns, two separate laboratory-developed tests were validated for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using two different specimen types. In addition, these assays target different genomic regions of SARS-CoV-2, allowing for viral detection and mitigating genetic variation. Lower limit of detection and clinical evaluation studies showed detection of SARS-CoV-2 at 500 cp/mL with nasopharyngeal and saliva samples. These multiplexed RT-qPCR assays, although based on modified CDC, New York State Department of Health, and World Health Organization Emergency Use Authorization tests, allow for higher throughput and rapid turnaround time, benefiting patients, clinicians, and communities as a whole. These cost-effective tests also use readily obtainable reagents, circumventing commercial assay supply chain issues. The laboratory-developed tests described here have improved patient care and are highly adaptable should the need arise at other clinical diagnostic laboratories. Furthermore, the foundation and design of these assays may be modified in the future for detection of COVID-19 variants or other RNA-based viral detection tests.

Cytosolic adaptation to mitochondria-induced proteostatic stress causes progressive muscle wasting.

Mitochondrial dysfunction causes muscle wasting in many diseases and probably also during aging. The underlying mechanism is poorly understood. We generated transgenic mice with unbalanced mitochondrial protein loading and import, by moderately overexpressing the nuclear-encoded adenine nucleotide translocase, Ant1. We found that these mice progressively lose skeletal muscle. Ant1-overloading reduces mitochondrial respiration. Interestingly, it also induces small heat shock proteins and aggresome-like structures in the cytosol, suggesting increased proteostatic burden due to accumulation of unimported mitochondrial preproteins. The transcriptome of Ant1-transgenic muscles is drastically remodeled to counteract proteostatic stress, by repressing protein synthesis and promoting proteasomal function, autophagy, and lysosomal amplification. These proteostatic adaptations collectively reduce protein content thereby reducing myofiber size and muscle mass. Thus, muscle wasting can occur as a trade-off of adaptation to mitochondria-induced proteostatic stress. This finding could have implications for understanding the mechanism of muscle wasting, especially in diseases associated with Ant1 overexpression, including facioscapulohumeral dystrophy.

Coupling freedom from disease principles and early warning from wastewater surveillance to improve health security.

Infectious disease surveillance is vitally important to maintaining health security, but these efforts are challenged by the pace at which new pathogens emerge. Wastewater surveillance can rapidly obtain population-level estimates of disease transmission, and we leverage freedom from disease principles to make use of nondetection of SARS-CoV-2 in wastewater to estimate the probability that a community is free from SARS-CoV-2 transmission. From wastewater surveillance of 24 treatment plants across upstate New York from May through December of 2020, trends in the intensity of SARS-CoV-2 in wastewater correlate with trends in COVID-19 incidence and test positivity (⍴ > 0.5), with the greatest correlation observed for active cases and a 3-day lead time between wastewater sample date and clinical test date. No COVID-19 cases were reported 35% of the time the week of a nondetection of SARS-CoV-2 in wastewater. Compared to the United States Centers for Disease Control and Prevention levels of transmission risk, transmission risk was low (no community spared) 50% of the time following nondetection, and transmission risk was moderate or lower (low community spread) 92% of the time following nondetection. Wastewater surveillance can demonstrate the geographic extent of the transmission of emerging pathogens, confirming that transmission risk is either absent or low and alerting of an increase in transmission. If a statewide wastewater surveillance platform had been in place prior to the onset of the COVID-19 pandemic, policymakers would have been able to complement the representative nature of wastewater samples to individual testing, likely resulting in more precise public health interventions and policies.

Longitudinal stability of salivary microRNA biomarkers in children and adolescents with autism spectrum disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a complex neurological condition with increasing prevalence. Few tools accurately predict the developmental trajectory of children with ASD. Such tools would allow clinicians to provide accurate prognoses and track the efficacy of therapeutic interventions. Salivary RNAs that reflect the genetic-environmental interactions underlying ASD may provide objective measures of symptom severity and developmental outcomes. This study investigated whether salivary RNAs previously identified in childhood ASD remain perturbed in older children. We also explored whether RNA candidates changed with therapeutic intervention. METHOD: A case-control design was used to characterize levels of 78 saliva RNA candidates among 96 children (48 ASD, 48 non-ASD, mean age: 11 years). Thirty-one children (22 ASD, 9 non-ASD developmental delay, mean age: 4 years) were followed longitudinally to explore changes of RNA candidates during early intervention. Saliva RNA and standardized behavioral assessments were collected for each participant. Associations between candidate RNAs and behavioral scores were determined in both groups via Spearman Correlation. Changes in candidate RNAs across two time-points were assessed in the younger cohort via Wilcoxon rank-sum test. RESULTS: Seven RNAs were associated with VABS-II and BASC scores in the older group ([R] >0.25, FDR< 0.15). Within the younger cohort, 12 RNAs displayed significant changes over time (FDR< 0.05). Three microRNAs were associated with behavioral scores and changed over time (miR-182-5p, miR-146b-5p, miR-374a-5p). CONCLUSION: Several salivary RNAs are strongly associated with autistic behaviors in older individuals with ASD and change as early as three months after therapy initiation in younger children. These molecules could be used to track treatment effectiveness and provide prognoses. Further validation is necessary.

Saliva RNA biomarkers predict concussion duration and detect symptom recovery: a comparison with balance and cognitive testing.

OBJECTIVE: The goals of this study were to assess the ability of salivary non-coding RNA (ncRNA) levels to predict post-concussion symptoms lasting ≥ 21 days, and to examine the ability of ncRNAs to identify recovery compared to cognition and balance. METHODS: RNA sequencing was performed on 505 saliva samples obtained longitudinally from 112 individuals (8-24-years-old) with mild traumatic brain injury (mTBI). Initial samples were obtained ≤ 14 days post-injury, and follow-up samples were obtained ≥ 21 days post-injury. Computerized balance and cognitive test performance were assessed at initial and follow-up time-points. Machine learning was used to define: (1) a model employing initial ncRNA levels to predict persistent post-concussion symptoms (PPCS) ≥ 21 days post-injury; and (2) a model employing follow-up ncRNA levels to identify symptom recovery. Performance of the models was compared against a validated clinical prediction rule, and balance/cognitive test performance, respectively. RESULTS: An algorithm using age and 16 ncRNAs predicted PPCS with greater accuracy than the validated clinical tool and demonstrated additive combined utility (area under the curve (AUC) 0.86; 95% CI 0.84-0.88). Initial balance and cognitive test performance did not differ between PPCS and non-PPCS groups (p > 0.05). Follow-up balance and cognitive test performance identified symptom recovery with similar accuracy to a model using 11 ncRNAs and age. A combined model (ncRNAs, balance, cognition) most accurately identified recovery (AUC 0.86; 95% CI 0.83-0.89). CONCLUSIONS: ncRNA biomarkers show promise for tracking recovery from mTBI, and for predicting who will have prolonged symptoms. They could provide accurate expectations for recovery, stratify need for intervention, and guide safe return-to-activities.

Co-quantification of crAssphage increases confidence in wastewater-based epidemiology for SARS-CoV-2 in low prevalence areas.

Wastewater surveillance of SARS-CoV-2 RNA is increasingly being incorporated into public health efforts to respond to the COVID-19 pandemic. In order to obtain the maximum benefit from these efforts, approaches to wastewater monitoring need to be rapid, sensitive, and relatable to relevant epidemiological parameters. In this study, we present an ultracentrifugation-based method for the concentration of SARS-CoV-2 wastewater RNA and use crAssphage, a bacteriophage specific to the human gut, to help account for RNA loss during transit in the wastewater system and sample processing. With these methods, we were able to detect, and sometimes quantify, SARS-CoV-2 RNA from 20 mL wastewater samples within as little as 4.5 hours. Using known concentrations of bovine coronavirus RNA and deactivated SARS-CoV-2, we estimate recovery rates of approximately 7-12% of viral RNA using our method. Results from 24 sewersheds across Upstate New York during the spring and summer of 2020 suggested that stronger signals of SARS-CoV-2 RNA from wastewater may be indicative of greater COVID-19 incidence in the represented service area approximately one week in advance. SARS-CoV-2 wastewater RNA was quantifiable in some service areas with daily positives tests of less than 1 per 10,000 people or when weekly positive test rates within a sewershed were as low as 1.7%. crAssphage DNA concentrations were significantly lower during periods of high flow in almost all areas studied. After accounting for flow rate and population served, crAssphage levels per capita were estimated to be about 1.35 × 1011 and 2.42 × 108 genome copies per day for DNA and RNA, respectively. A negative relationship between per capita crAssphage RNA and service area size was also observed likely reflecting degradation of RNA over long transit times. Our results reinforce the potential for wastewater surveillance to be used as a tool to supplement understanding of infectious disease transmission obtained by traditional testing and highlight the potential for crAssphage co-detection to improve interpretations of wastewater surveillance data.

Evidence-based use of scalable biomarkers to increase diagnostic efficiency and decrease the lifetime costs of autism.

Challenges associated with the current screening and diagnostic process for autism spectrum disorder (ASD) in the US cause a significant delay in the initiation of evidence-based interventions at an early age when treatments are most effective. The present study shows how implementing a second-order diagnostic measure to high risk cases initially flagged positive from screening tools can further inform clinical judgment and substantially improve early identification. We use two example measures for the purposes of this demonstration; a saliva test and eye-tracking technology, both scalable and easy-to-implement biomarkers recently introduced in ASD research. Results of the current cost-savings analysis indicate that lifetime societal cost savings in special education, medical and residential care are estimated to be nearly $580,000 per ASD child, with annual cost savings in education exceeding $13.3 billion, and annual cost savings in medical and residential care exceeding $23.8 billion (of these, nearly $11.2 billion are attributable to Medicaid). These savings total more than $37 billion/year in societal savings in the US. Initiating appropriate interventions faster and reducing the number of unnecessary diagnostic evaluations can decrease the lifetime costs of ASD to society. We demonstrate the value of implementing a scalable highly accurate diagnostic in terms of cost savings to the US. LAY SUMMARY: This paper demonstrates how biomarkers with high accuracy for detecting autism spectrum disorder (ASD) could be used to increase the efficiency of early diagnosis. Results also show that, if more children with ASD are identified early and referred for early intervention services, the system would realize substantial costs savings across the lifespan.