CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Rescuing Photoreceptors in RPE Dysfunction-Driven Retinal Degeneration: The Role of Small Extracellular Vesicles Secreted from Retinal Pigment Epithelium.

Dysfunction of the retinal pigment epithelium (RPE) is a common shared pathology in major degenerative retinal diseases despite variations in the primary etiologies of each disease. Due to their demanding and indispensable functional roles throughout the lifetime, RPE cells are vulnerable to genetic predisposition, external stress, and aging processes. Building upon recent advancements in stem cell technology for differentiating healthy RPE cells and recognizing the significant roles of small extracellular vesicles (sEV) in cellular paracrine and autocrine actions, we investigated the hypothesis that the RPE-secreted sEV alone can restore essential RPE functions and rescue photoreceptors in RPE dysfunction-driven retinal degeneration. Our findings support the rationale for developing intravitreal treatment of sEV. We demonstrate that intravitreally delivered sEV effectively penetrate the full thickness of the retina. Xenogenic intraocular administration of human-derived EVs did not induce acute immune reactions in rodents. sEV derived from human embryonic stem cell (hESC)-derived fully differentiated RPE cells, but not sEV-depleted conditioned cell culture media (CCM minus sEV), rescued photoreceptors and their function in a Royal College of Surgeons (RCS) rat model. This model is characterized by photoreceptor death and retinal degeneration resulting from a mutation in the MerTK gene in RPE cells. From the bulk RNA sequencing study, we identified 447 differently expressed genes in the retina after hESC-RPE-sEV treatment compared with the untreated control. Furthermore, 394 out of 447 genes (88%) showed a reversal in expression toward the healthy state in Long-Evans (LE) rats after treatment compared to the diseased state. Particularly, detrimental alterations in gene expression in RCS rats, including essential RPE functions such as phototransduction, vitamin A metabolism, and lipid metabolism were partially reversed. Defective photoreceptor outer segment engulfment due to intrinsic MerTK mutation was partially ameliorated. These findings suggest that RPE-secreted sEV may play a functional role similar to that of RPE cells. Our study justifies further exploration to fully unlock future therapeutic interventions with sEV in a broad array of degenerative retinal diseases.

An unbiased seed-based RNAi selection screen identifies small RNAs that inhibit androgen signaling and prostate cancer cell growth.

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3' UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa.

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

Autoantibodies identify primary Sjögren's syndrome in patients lacking serum IgG specific for Ro/SS-A and La/SS-B.

OBJECTIVE: Identify autoantibodies in anti-Ro/SS-A negative primary Sjögren's syndrome (SS). METHODS: This is a proof-of-concept, case-control study of SS, healthy (HC) and other disease (OD) controls. A discovery dataset of plasma samples (n=30 SS, n=15 HC) was tested on human proteome arrays containing 19 500 proteins. A validation dataset of plasma and stimulated parotid saliva from additional SS cases (n=46 anti-Ro+, n=50 anti-Ro-), HC (n=42) and OD (n=54) was tested on custom arrays containing 74 proteins. For each protein, the mean+3 SD of the HC value defined the positivity threshold. Differences from HC were determined by Fisher's exact test and random forest machine learning using 2/3 of the validation dataset for training and 1/3 for testing. Applicability of the results was explored in an independent rheumatology practice cohort (n=38 Ro+, n=36 Ro-, n=10 HC). Relationships among antigens were explored using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) interactome analysis. RESULTS: Ro+ SS parotid saliva contained autoantibodies binding to Ro60, Ro52, La/SS-B and muscarinic receptor 5. SS plasma contained 12 novel autoantibody specificities, 11 of which were detected in both the discovery and validation datasets. Binding to ≥1 of the novel antigens identified 54% of Ro- SS and 37% of Ro+ SS cases, with 100% specificity in both groups. Machine learning identified 30 novel specificities showing receiver operating characteristic area under the curve of 0.79 (95% CI 0.64 to 0.93) for identifying Ro- SS. Sera from Ro- cases of an independent cohort bound 17 of the non-canonical antigens. Antigenic targets in both Ro+ and Ro- SS were part of leukaemia cell, ubiquitin conjugation and antiviral defence pathways. CONCLUSION: We identified antigenic targets of the autoantibody response in SS that may be useful for identifying up to half of Ro seronegative SS cases.

Absence of Either Ripk3 or Mlkl Reduces Incidence of Hepatocellular Carcinoma Independent of Liver Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. Because necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis-induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet-induced HCC. Male and female wild-type (WT) mice and mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed either a control diet, choline-deficient low-fat diet or choline-deficient high-fat diet. Blocking necroptosis reduced markers of inflammation [proinflammatory cytokines (TNFα, IL6, and IL1ß), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation-associated oncogenic pathways (JNK, PD-L1/PD-1, ß-catenin), and HCC in male mice. We demonstrate that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. Whereas in female mice, blocking necroptosis reduced HCC independent of inflammation. Our data show a sex-specific difference in the development of inflammation, fibrosis, and HCC in WT mice. However, blocking necroptosis reduced HCC in both males and females without altering liver fibrosis. Thus, our study suggests that necroptosis is a valid therapeutic target for NAFLD-mediated HCC. IMPLICATIONS: Necroptosis is a major contributor to hepatic inflammation that drives the progression of NAFLD to HCC and therefore represents a valid target for NAFLD-mediated HCC.

The Isolation and In Vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions.

The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and the suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

Dietary protection against the visual and motor deficits induced by experimental autoimmune encephalomyelitis.

Introduction: Five to eight percent of the world population currently suffers from at least one autoimmune disorder. Despite multiple immune modulatory therapies for autoimmune demyelinating diseases of the central nervous system, these treatments can be limiting for subsets of patients due to adverse effects and expense. To circumvent these barriers, we investigated a nutritional intervention in mice undergoing experimental autoimmune encephalomyelitis (EAE), a model of autoimmune-mediated demyelination that induces visual and motor pathologies similar to those experienced by people with multiple sclerosis (MS). Methods: EAE was induced in female and male mice and the impact of limiting dietary carbohydrates by feeding a ketogenic diet (KD) enriched in medium chain triglycerides (MCTs), alpha-linolenic acid (an omega-3 fatty acid), and fiber was evaluated in both a preventive regimen (prior to immunization with MOG antigen) and an interventional regimen (following the onset of symptoms). Motor scores were assigned daily and visual acuity was measured using optokinetic tracking. Immunohistochemical analyses of optic nerves were done to assess inflammatory infiltrates and myelination status. Fatty acid and cytokine profiling from blood were performed to evaluate systemic inflammatory status. Results: The KD was efficacious when fed as a preventive regimen as well as when initiated as an interventional regimen following symptom onset. The KD minimally impacted body weight during the experimental time course, increased circulating ketones, prevented motor and ocular deficits, preserved myelination of the optic nerve, and reduced infiltration of immune cells to optic nerves. The KD also increased anti-inflammatory-associated omega-3 fatty acids in the plasma and reduced select cytokines in the circulation associated with EAE-mediated pathological inflammation. Discussion: In light of ongoing clinical trials using dietary strategies to treat people with MS, these findings support that a KD enriched in MCTs, omega-3 fatty acids, and fiber promotes a systemic anti-inflammatory milieu and ameliorates autoimmune-induced demyelinating visual and motor deficits.

Lipid hydroperoxides and oxylipins are mediators of denervation induced muscle atrophy.

Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis.

The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

A novel sulindac derivative protects against oxidative damage by a cyclooxygenase-independent mechanism.

Oxidative damage is believed to play a major role in the etiology of many age-related diseases and the normal aging process. We previously reported that sulindac, a cyclooxygenase (COX) inhibitor and FDA approved anti-inflammatory drug, has chemoprotective activity in cells and intact organs by initiating a pharmacological preconditioning response, similar to ischemic preconditioning (IPC). The mechanism is independent of its COX inhibitory activity as suggested by studies on the protection of the heart against oxidative damage from ischemia/reperfusion and retinal pigmented endothelial (RPE) cells against chemical oxidative and UV damage . Unfortunately, sulindac is not recommended for long-term use due to toxicities resulting from its COX inhibitory activity. To develop a safer and more efficacious derivative of sulindac, we screened a library of indenes and identified a lead compound, MCI-100, that lacked significant COX inhibitory activity but displayed greater potency than sulindac to protect RPE cells against oxidative damage. MCI-100 also protected the intact rat heart against ischemia/reperfusion damage following oral administration. The chemoprotective activity of MCI-100 involves a preconditioning response similar to sulindac, which is supported by RNA sequencing data showing common genes that are induced or repressed by sulindac or MCI-100 treatment. Both sulindac and MCI-100 protection against oxidative damage may involve modulation of Wnt/ß-catenin signaling resulting in proliferation while inhibiting TGFb signaling leading to apoptosis. In summary MCI-100, is more active than sulindac in protecting cells against oxidative damage, but without significant NSAID activity, and could have therapeutic potential in treatment of diseases that involve oxidative damage. Significance Statement In this study, we describe a novel sulindac derivative, MCI-100, that lacks significant COX inhibitory activity, but is appreciably more potent than sulindac in protecting retinal pigmented epithelial (RPE) cells against oxidative damage. Oral administration of MCI-100 markedly protected the rat heart against ischemia/reperfusion damage. MCI-100 has potential therapeutic value as a drug candidate for age-related diseases by protecting cells against oxidative damage and preventing organ failure.

Proteomic Insight into the Role of Exosomes in Proliferative Vitreoretinopathy Development.

Purpose: To characterize vitreous humor (VH) exosomes and to explore their role in the development of proliferative vitreoretinopathy (PVR) using mass spectrometry-based proteome profiling. Methods: Exosomes were isolated from undiluted VH from patients with retinal detachment (RD) with various stages of PVR (n = 9), macular hole (MH; n = 5), or epiretinal membrane (ERM; n = 5) using differential ultracentrifugation. The exosomal size, morphology, and exosome markers were analyzed using a nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and an exosome detection antibody array. The tryptic fragment sequencing of exosome-contained proteins was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a Thermo Lumos Fusion Tribrid Orbitrap mass spectrometer. The pathway analysis of the MS data was performed. Results: The number of exosome particles were significantly increased only in the RD with severe PVR group compared with the control groups and the RD without PVR or with mild PVR groups. Of 724 exosome proteins identified, 382 were differentially expressed (DE) and 176 were uniquely present in PVR. Both DE proteins and exosome proteins that were only present in PVR were enriched in proteins associated with previously known key pathways related to PVR development, including reactive retinal gliosis, pathologic cellular proliferation, inflammation, growth of connective tissues, and epithelial mesenchymal transition (EMT). The SPP1, CLU, VCAN, COL2A1, and SEMA7A that are significantly upregulated in PVR were related to the tissue remodeling. Conclusions: Exosomes may play a key role in mediating tissue remodeling along with a complex set of pathways involved in PVR development.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Multi-tissue DNA methylation microarray signature is predictive of gene function.

Background Transcriptional correlation networks derived from publicly available gene expression microarrays have been previously shown to be predictive of known gene functions, but less is known about the predictive capacity of correlated DNA methylation at CpG sites. Guilt-by-association co-expression methods can adapted for use with DNA methylation when a representative methylation value is created for each gene. We examine how methylation compares to expression in predicting Gene Ontology terms using both co-methylation and traditional machine learning approaches across different types of representative methylation values per gene. Methods We perform guilt-by-association gene function prediction with a suite of models called Methylation Array Network Analysis, using a network of correlated methylation values derived from over 24,000 samples. In generating the correlation matrix, the performance of different methods of collapsing probe-level data effect on the resulting gene function predictions was compared, along with the use of different regions surrounding the gene of interest. Results Using mean comethylation of a given gene to its annotated term had an overall highest prediction macro-AUC of 0.60 using mean gene body methylation, across all Gene Ontology terms. This was increased using the logistic regression approach with the highest macro-AUC of 0.82 using mean gene body methylation, compared to the naive predictor of 0.72. Conclusion Genes correlated in their methylation state are functionally related. Genes clustered in co-methylation space were enriched for chromatin state, PRC2, immune response, and development-related terms.

Pharmacologic treatment with OKN-007 reduces alpha-motor neuron loss in spinal cord of aging mice.

Aging is associated with molecular and functional declines in multiple physiologic systems. We have previously reported age-related changes in spinal cord that included a decline in α-motor neuron numbers, axonal loss, and demyelination associated with increased inflammation and blood-spinal cord barrier (BSCB) permeability. These changes may influence other pathologies associated with aging, in particular loss of muscle mass and function (sarcopenia), which we and others have shown is accompanied by neuromuscular junction disruption and loss of innervation. Interventions to protect and maintain motor neuron viability and function in aging are currently lacking and could have a significant impact on improving healthspan. Here we tested a promising compound, OKN-007, that has known antioxidant, anti-inflammatory and neuroprotective properties, as a potential intervention in age-related changes in the spinal cord. OKN-007 is a low molecular weight disulfonyl derivative of (N-tert Butyl-α-phenylnitrone) (PBN) that can easily cross the blood-brain barrier. We treated middle age (16 month) wild-type male mice with OKN-007 in drinking water at a dose of 150 mg/kg/day until 25 months of age. OKN-007 treatment exerted a number of beneficial effects in the aging spinal cord, including a 35% increase in the number of lumbar α-motor neurons in OKN-treated old mice compared to age-matched controls. Brain spinal cord barrier permeability, which is increased in aging spinal cord, was also blunted by OKN-007 treatment. Age-related changes in microglia proliferation and activation are blunted by OKN-007, while we found no effect on astrocyte proliferation. Transcriptome analysis identified expression changes in a number of genes that are involved in neuronal structure and function and revealed a subset of genes whose changes in response to aging are reversed by OKN-007 treatment. Overall, our findings suggest that OKN-007 exerts neuroprotective and anti-inflammatory effects on the aging spinal cord and support OKN-007 as a potential therapeutic to improve α-motor neuron health.

Detecting anomalous referencing patterns in PubMed papers suggestive of author-centric reference list manipulation.

Although citations are used as a quantifiable, objective metric of academic influence, references could be added to a paper solely to inflate the perceived influence of a body of research. This reference list manipulation (RLM) could take place during the peer-review process, or prior to it. Surveys have estimated how many people may have been affected by coercive RLM at one time or another, but it is not known how many authors engage in RLM, nor to what degree. By examining a subset of active, highly published authors (n = 20,803) in PubMed, we find the frequency of non-self-citations (NSC) to one author coming from a single paper approximates Zipf's law. Author-centric deviations from it are approximately normally distributed, permitting deviations to be quantified statistically. Framed as an anomaly detection problem, statistical confidence increases when an author is an outlier by multiple metrics. Anomalies are not proof of RLM, but authors engaged in RLM will almost unavoidably create anomalies. We find the NSC Gini Index correlates highly with anomalous patterns across multiple "red flags", each suggestive of RLM. Between 81 (0.4%, FDR < 0.05) and 231 (1.1%, FDR < 0.10) authors are outliers on the curve, suggestive of chronic, repeated RLM. Approximately 16% of all authors may have engaged in RLM to some degree. Authors who use 18% or more of their references for self-citation are significantly more likely to have NSC Gini distortions, suggesting a potential willingness to coerce others to cite them.

The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation.

Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air-liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation.

Many chronological aging clocks can be found throughout the epigenome: Implications for quantifying biological aging.

Epigenetic alterations are a hallmark of aging and age-related diseases. Computational models using DNA methylation data can create "epigenetic clocks" which are proposed to reflect "biological" aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under-represented in canonical regions of epigenetic regulation. There was only a weak association between "accelerated" epigenetic aging and disease. We also compare tissue-specific and pan-tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate "epigenetic aging" in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti-aging interventions. Purpose-built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose-built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context.

Insulin/IGF-1 signaling and heat stress differentially regulate HSF1 activities in germline development.

Germline development is sensitive to nutrient availability and environmental perturbation. Heat shock transcription factor 1 (HSF1), a key transcription factor driving the cellular heat shock response (HSR), is also involved in gametogenesis. The precise function of HSF1 (HSF-1 in C. elegans) and its regulation in germline development are poorly understood. Using the auxin-inducible degron system in C. elegans, we uncovered a role of HSF-1 in progenitor cell proliferation and early meiosis and identified a compact but important transcriptional program of HSF-1 in germline development. Interestingly, heat stress only induces the canonical HSR in a subset of germ cells but impairs HSF-1 binding at its developmental targets. Conversely, insulin/insulin growth factor 1 (IGF-1) signaling dictates the requirement for HSF-1 in germline development and functions through repressing FOXO/DAF-16 in the soma to activate HSF-1 in germ cells. We propose that this non-cell-autonomous mechanism couples nutrient-sensing insulin/IGF-1 signaling to HSF-1 activation to support homeostasis in rapid germline growth.

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.