Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Extracellular vesicle RNAs reflect placenta dysfunction and are a biomarker source for preterm labour.

Preterm birth (PTB) can lead to lifelong complications and challenges. Identifying and monitoring molecular signals in easily accessible biological samples that can diagnose or predict the risk of preterm labour (PTL) in pregnant women will reduce or prevent PTBs. A number of studies identified putative biomarkers for PTL including protein, miRNA and hormones from various body fluids. However, biomarkers identified from these studies usually lack consistency and reproducibility. Extracellular vesicles (EVs) in circulation have gained significant interest in recent years as these vesicles may be involved in cell-cell communication. We have used an improved small RNA library construction protocol and a newly developed size exclusion chromatography (SEC)-based EV purification method to gain a comprehensive view of circulating RNA in plasma and its distribution by analysing RNAs in whole plasma and EV-associated and EV-depleted plasma. We identified a number of miRNAs in EVs that can be used as biomarkers for PTL, and these miRNAs may reflect the pathological changes of the placenta during the development of PTL. To our knowledge, this is the first study to report a comprehensive picture of circulating RNA, including RNA in whole plasma, EV and EV-depleted plasma, in PTL and reveal the usefulness of EV-associated RNAs in disease diagnosis.

Systems analysis identifies an essential role for SHANK-associated RH domain-interacting protein (SHARPIN) in macrophage Toll-like receptor 2 (TLR2) responses.

Precise control of the innate immune response is essential to ensure host defense against infection while avoiding inflammatory disease. Systems-level analyses of Toll-like receptor (TLR)-stimulated macrophages suggested that SHANK-associated RH domain-interacting protein (SHARPIN) might play a role in the TLR pathway. This hypothesis was supported by the observation that macrophages derived from chronic proliferative dermatitis mutation (cpdm) mice, which harbor a spontaneous null mutation in the Sharpin gene, exhibited impaired IL-12 production in response to TLR activation. Systems biology approaches were used to define the SHARPIN-regulated networks. Promoter analysis identified NF-κB and AP-1 as candidate transcription factors downstream of SHARPIN, and network analysis suggested selective attenuation of these pathways. We found that the effects of SHARPIN deficiency on the TLR2-induced transcriptome were strikingly correlated with the effects of the recently described hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO interact. We confirmed this interaction by co-immunoprecipitation analysis and furthermore found it to be abrogated by panr2. NEMO-dependent signaling was affected by SHARPIN deficiency in a manner similar to the panr2 mutation, including impaired p105 and ERK phosphorylation and p65 nuclear localization. Interestingly, SHARPIN deficiency had no effect on IκBα degradation and on p38 and JNK phosphorylation. Taken together, these results demonstrate that SHARPIN is an essential adaptor downstream of the branch point defined by the panr2 mutation in NEMO.

Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, it is developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of the coronavirus disease of 2019 (COVID-19). BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

Reinfection with SARS-CoV-2 and Waning Humoral Immunity: A Case Report.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a different strain harboring the spike variant D614G. This case of reinfection was one of the first cases of reinfection reported in 2020. With antibody, B cell and T cell analytics, we show correlates of adaptive immunity at reinfection, including a differential response in neutralizing antibodies to a D614G pseudovirus. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

Alterations in Plasma microRNA and Protein Levels in War Veterans with Chronic Mild Traumatic Brain Injury.

Blast-related mild traumatic brain injury (mTBI) is considered the "signature" injury of the wars in Iraq and Afghanistan. Identifying biomarkers that could aid in diagnosis and assessment of chronic mTBI are urgently needed, as little progress has been made toward identifying blood-based biomarkers of repetitive mTBI in the chronic state. Addressing this knowledge gap is especially important in the population of military veterans who are receiving assessment and care often years after their last exposure. Circulating microRNAs (miRNAs), especially those encapsulated in extracellular vesicles (EVs), have gained interest as a source of biomarkers for neurological conditions. To identify biomarkers for chronic mTBI, we used next generation sequencing (NGS) to analyze miRNAs in plasma and plasma-derived EVs from 27 Iraq and Afghanistan war veterans with blast-related chronic mTBI, 11 deployed veteran non-TBI controls, and 31 civilian controls. We identified 32 miRNAs in plasma and 45 miRNAs in EVs that significantly changed in the chronic mTBI cohort compared with control groups. These miRNAs were predominantly associated with pathways involved in neuronal function, vascular remodeling, blood-brain barrier integrity, and neuroinflammation. In addition, the plasma proteome was analyzed and showed that the concentrations of C-reactive protein (CRP) and membrane metalloendopeptidase (MME) were elevated in chronic mTBI samples. These plasma miRNAs and proteins could potentially be used as biomarkers and provide insights into the molecular processes associated with the long-term health outcomes associated with blast-related chronic mTBI.

Reinfection with SARS-CoV-2 and Failure of Humoral Immunity: a case report.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a new strain harboring the spike variant D614G. With antibody and B cell analytics, we show correlates of adaptive immunity, including a differential response to D614G. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

Chronic elevation of plasma vascular endothelial growth factor-A (VEGF-A) is associated with a history of blast exposure.

Mounting evidence points to the significance of neurovascular-related dysfunction in veterans with blast-related mTBI, which is also associated with reduced [18F]-fluorodeoxyglucose (FDG) uptake. The goal of this study was to determine whether plasma VEGF-A is altered in veterans with blast-related mTBI and address whether VEGF-A levels correlate with FDG uptake in the cerebellum, a brain region that is vulnerable to blast-related injury 72 veterans with blast-related mTBI (mTBI) and 24 deployed control (DC) veterans with no lifetime history of TBI were studied. Plasma VEGF-A was significantly elevated in mTBIs compared to DCs. Plasma VEGF-A levels in mTBIs were significantly negatively correlated with FDG uptake in cerebellum. In addition, performance on a Stroop color/word interference task was inversely correlated with plasma VEGF-A levels in blast mTBI veterans. Finally, we observed aberrant perivascular VEGF-A immunoreactivity in postmortem cerebellar tissue and not cortical or hippocampal tissues from blast mTBI veterans. These findings add to the limited number of plasma proteins that are chronically elevated in veterans with a history of blast exposure associated with mTBI. It is likely the elevated VEGF-A levels are from peripheral sources. Nonetheless, increasing plasma VEGF-A concentrations correlated with chronically decreased cerebellar glucose metabolism and poorer performance on tasks involving cognitive inhibition and set shifting. These results strengthen an emerging view that cognitive complaints and functional brain deficits caused by blast exposure are associated with chronic blood-brain barrier injury and prolonged recovery in affected regions.

Wnt signaling promotes proliferation and stemness regulation of spermatogonial stem/progenitor cells.

Spermatogonial stem cells (SSCs) self-renew throughout life to produce progenitor cells that are able to differentiate into spermatozoa. However, the mechanisms underlying the cell fate determination between self-renewal and differentiation have not yet been delineated. Culture conditions and growth factors essential for self-renewal and proliferation of mouse SSCs have been investigated, but no information is available related to growth factors that affect fate determination of human spermatogonia. Wnts form a large family of secreted glycoproteins, the members of which are involved in cell proliferation, differentiation, organogenesis, and cell migration. Here, we show that Wnts and their receptors Fzs are expressed in mouse spermatogonia and in the C18-4 SSC line. We demonstrate that WNT3A induces cell proliferation, morphological changes, and cell migration in C18-4 cells. Furthermore, we show that beta-catenin is activated during testis development in 21-day-old mice. In addition, our study demonstrates that WNT3A sustained adult human embryonic stem (ES)-like cells derived from human germ cells in an undifferentiated stage, expressing essential human ES cell transcription factors. These results demonstrate for the first time that Wnt/beta-catenin pathways, especially WNT3A, may play an important role in the regulation of mouse and human spermatogonia.

The Impact of Moyamoya Disease and RNF213 Mutations on the Spectrum of Plasma Protein and MicroRNA.

Moyamoya disease (MMD) is a rare cerebrovascular disorder characterized by occlusion of bilateral internal carotid and intracerebral arteries with the compensatory growth of fragile small vessels. MMD patients develop recurrent infarctions in the basal ganglia and subcortical regions. Symptoms include transient ischemic attack or stroke, seizures, and headaches, which may occur suddenly or in a stepwise progression. Mutations in Ring Finger Protein 213 (RNF213), a Zinc ring finger protein, have been identified in some MMD patients but the etiology of MMD is still largely unknown. To gain insight into the pathophysiology of MMD, we characterized the impact of the RNF213 mutations on plasma protein and RNA profiles. Isobaric tags for relative and absolute quantitation and proximity extension assay were used to characterize the plasma proteome. Next generation sequencing-based small RNAseq was used to analyze the cell-free small RNAs in whole plasma and RNA encapsulated in extracellular vesicles. The changes of miRNAs and proteins identified are associated with signaling processes including angiogenesis and immune activities which may reflect the pathology and progression of MMD.

Wnt10b induces chemotaxis of osteosarcoma and correlates with reduced survival.

BACKGROUND: Osteosarcoma (OS) is a primary malignant tumor of the bone that typically presents in the second decade of life and has a poor prognosis, especially in metastatic cases. Wnt signaling contributes to the pathogenesis of tumors such as colon cancer and malignant melanoma. Wnt signaling controls normal bone formation during embryogenesis and homeostasis in adult organisms, thus we evaluated Wnt signaling in OS. PROCEDURE: We surveyed the expression of Wnts, their receptors, Frizzleds and LRPs, and soluble Wnt inhibitors (sFRPs) in four OS cell lines by RT-PCR. We also tested biological response of OS cell lines to exogenous Wnts by measuring beta-catenin stabilization, Dvl phosphorylation, TOPFLASH activity and chemotaxis. Human OS tumor microarrays were evaluated for expression of Wnt10b by immunohistochemistry. RESULTS: All cell lines tested showed expression of at least three Wnts and one Frizzled. Exogenous Wnt3a and Wnt10b treatment induced Dvl phosphorylation, beta-catenin stabilization and TCF4 transcriptional activity in both metastatic and non-metastatic murine OS cell lines. Metastatic OS cell lines showed better chemotaxis response to Wnts than the non-metastatic OS cell lines. Immunohistochemistry studies of 44 human OS samples demonstrated that Wnt10b expression correlated with decreased overall survival. CONCLUSIONS: These results further supports a possible autocrine or paracrine Wnt pathway in metastatic potential of OS.

Activation of the canonical Wnt pathway during genital keratinocyte transformation: a model for cervical cancer progression.

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic beta-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from beta-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of beta-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of beta-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.

PDZD7-MYO7A complex identified in enriched stereocilia membranes.

While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.

Effects of a fluorescent Myosin light chain phosphatase inhibitor on prostate cancer cells.

Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/(32)P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI(50) values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. ß-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype.

Beta-catenin accelerates human papilloma virus type-16 mediated cervical carcinogenesis in transgenic mice.

Human papilloma virus (HPV) is the principal etiological agent of cervical cancer in women, and its DNA is present in virtually all of these tumors. However, exposure to the high-risk HPV types alone is insufficient for tumor development. Identifying specific collaborating factors that will lead to cervical cancer remains an unanswered question, especially because millions of women are exposed to HPV. Our earlier work using an in vitro model indicated that activation of the canonical Wnt pathway in HPV-positive epithelial cells was sufficient to induce anchorage independent growth. We therefore hypothesized that constitutive activation of this pathway might function as the "second hit." To address this possibility, we developed two double-transgenic (DT) mouse models, K14-E7/ΔN87ßcat and K14-HPV16/ΔN87ßcat that express either the proteins encoded by the E7 oncogene or the HPV16 early region along with constitutively active ß-catenin, which was expressed by linking it to the keratin-14 (K14) promoter. We initiated tumor formation by treating all groups with estrogen for six months. Invasive cervical cancer was observed in 11% of the K14-ΔN87ßcat mice, expressing activated ß-catenin and in 50% of the animals expressing the HPV16 E7 oncogene. In double-transgenic mice, coexpression of ß-catenin and HPV16 E7 induced invasive cervical cancer at about 7 months in 94% of the cases. We did not observe cervical cancer in any group unless the mice were treated with estrogen. In the second model, K14-HPV16 mice suffered cervical dysplasias, but this phenotype was not augmented in HPV16/ΔN87ßcat mice. In summary, the phenotypes of the K14-E7/ΔN87ßcat mice support the hypothesis that activation of the Wnt/ß-catenin pathway in HPV-associated premalignant lesions plays a functional role in accelerating cervical carcinogenesis.