Tactile estimation of hedonic and sensory properties during active touch: An electroencephalography study.

Perceptual judgements about our physical environment are informed by somatosensory information. In real-world exploration, this often involves dynamic hand movements to contact surfaces, termed active touch. The current study investigated cortical oscillatory changes during active exploration to inform the estimation of surface properties and hedonic preferences of two textured stimuli: smooth silk and rough hessian. A purpose-built touch sensor quantified active touch, and oscillatory brain activity was recorded from 129-channel electroencephalography. By fusing these data streams at a single trial level, oscillatory changes within the brain were examined while controlling for objective touch parameters (i.e., friction). Time-frequency analysis was used to quantify changes in cortical oscillatory activity in alpha (8-12 Hz) and beta (16-24 Hz) frequency bands. Results reproduce findings from our lab, whereby active exploration of rough textures increased alpha-band event-related desynchronisation in contralateral sensorimotor areas. Hedonic processing of less preferred textures resulted in an increase in temporoparietal beta-band and frontal alpha-band event-related desynchronisation relative to most preferred textures, suggesting that higher order brain regions are involved in the hedonic processing of texture. Overall, the current study provides novel insight into the neural mechanisms underlying texture perception during active touch and how this process is influenced by cognitive tasks.

Epigenetics may characterize asymptomatic COVID-19 infection.

RT-PCR is the foremost clinical test for diagnosis of COVID-19. Unfortunately, PCR-based testing has limitations and may not result in a positive test early in the course of infection before symptoms develop. Enveloped RNA viruses, such as coronaviruses, alter peripheral blood methylation and DNA methylation signatures may characterize asymptomatic versus symptomatic infection. We used Illumina's Infinium MethylationEPIC BeadChip array to profile peripheral blood samples from 164 patients who tested positive for SARS-CoV-2 by RT-PCR, of whom 8 had no symptoms. Epigenome-wide association analysis identified 10 methylation sites associated with infection and a quantile-quantile plot showed little inflation. These preliminary results suggest that differences in methylation patterns may distinguish asymptomatic from symptomatic infection.

Acute Illness Associated With Cannabis Use, by Route of Exposure: An Observational Study.

Background: Little is known about the relative harms of edible and inhalable cannabis products. Objective: To describe and compare adult emergency department (ED) visits related to edible and inhaled cannabis exposure. Design: Chart review of ED visits between 1 January 2012 and 31 December 2016. Setting: A large urban academic hospital in Colorado. Participants: Adults with ED visits with a cannabis-related International Classification of Diseases, Ninth or 10th Revision, Clinical Modification (ICD-9-CM or ICD-10-CM), code. Measurements: Patient demographic characteristics, route of exposure, dose, symptoms, length of stay, disposition, discharge diagnoses, and attribution of visit to cannabis. Results: There were 9973 visits with an ICD-9-CM or ICD-10-CM code for cannabis use. Of these, 2567 (25.7%) visits were at least partially attributable to cannabis, and 238 of those (9.3%) were related to edible cannabis. Visits attributable to inhaled cannabis were more likely to be for cannabinoid hyperemesis syndrome (18.0% vs. 8.4%), and visits attributable to edible cannabis were more likely to be due to acute psychiatric symptoms (18.0% vs. 10.9%), intoxication (48% vs. 28%), and cardiovascular symptoms (8.0% vs. 3.1%). Edible products accounted for 10.7% of cannabis-attributable visits between 2014 and 2016 but represented only 0.32% of total cannabis sales in Colorado (in kilograms of tetrahydrocannabinol) during that period. Limitation: Retrospective study design, single academic center, self-reported exposure data, and limited availability of dose data. Conclusion: Visits attributable to inhaled cannabis are more frequent than those attributable to edible cannabis, although the latter is associated with more acute psychiatric visits and more ED visits than expected. Primary Funding Source: Colorado Department of Public Health and Environment.

Cultivation and characterisation of the surface markers and carbohydrate profile of human corneal endothelial cells.

BACKGROUND: The study aims to characterise human corneal endothelial cell (HCEnC) cultures generated by the peel-and-digest method based on their surface protein/carbohydrate expression pattern. METHODS: Quantitative polymerase chain reaction was used to compare expression of vimentin, CD90, Cytokeratin-19, ZO-1 and Claudin 14 in cultured HCEnC and cell line B4G12 versus stromal cells. Fluorescence-activated cell sorting was used to assess surface protein distribution of cultured and uncultured HCEnC. Distribution of surface proteins/carbohydrates was visualised by immunofluorescent and lectin staining. RESULTS: Human corneal endothelial cell and B4G12 showed lower expression level for vimentin, CD90, Cytokeratin-19 compared with stromal cells; while ZO-1 was expressed in endothelial cells, Claudin 14 was detected in B4G12 only. Fluorescence-activated cell sorting analyses revealed CD166, CD47, CD44, CD54, CD73, CD90, CD105, CD106, CD112, CD146 and CD325 to be present, with CD34 to be absent from cultured HCEnC. Freshly isolated, non-cultivated HCEnCs were CD90, CD73, CD146 and CD325 positive. Carbohydrates were detected by lectins LCA, PHA E, PHA L, PSA, sWGA, Con A, RCA 120 and WGA, but cultured HCEnC showed negative for GSL I, SBA, DBA, PNA and UEA I. CONCLUSION: Cultures established by the peel-and-digest method are probably not prone to stromal contamination, but the cells are likely to undergo endothelial-to mesenchymal transition as suggested by apparent morphological changes.

Concise review: evidence for CD34 as a common marker for diverse progenitors.

CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34-positive (CD34(+) ) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34(+) cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34(+) cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34(+) cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

BACKGROUND AIMS: The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. METHODS: Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS: All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. CONCLUSIONS: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.

Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium.

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies.

BACKGROUND: The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. METHODS: Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at -80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. RESULTS: The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 µm2 in LEC to 392,887 µm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/µl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 µl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 µm2 to 130,0000 µm2. RNA concentration of these samples ranged from 10.88 ng/12 µl to 25.8 ng/12 µl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. CONCLUSIONS: The optimised protocol for sample collection and laser microdissection improved the RNA yield of the in situ ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.

An interpretable framework for sleep posture change detection and postural inactivity segmentation using wrist kinematics.

Sleep posture and movements offer insights into neurophysiological health and correlate with overall well-being and quality of life. Clinical practices utilise polysomnography for sleep assessment, which is intrusive, performed in unfamiliar environments, and requires trained personnel. While sensor technologies such as actigraphy are less invasive alternatives, concerns about their reliability and precision in clinical practice persist. Moreover, the field lacks a universally accepted algorithm, with methods ranging from raw signal thresholding to data-intensive classification models that may be unfamiliar to medical staff. This paper proposes a comprehensive framework for objectively detecting sleep posture changes and temporally segmenting postural inactivity using clinically relevant joint kinematics, measured by a custom-made wearable sensor. The framework was evaluated on wrist kinematic data from five healthy participants during simulated sleep. Intuitive three-dimensional visualisations of kinematic time series were achieved through dimension reduction-based preprocessing, providing an out-of-the-box framework explainability that may be useful for clinical monitoring and diagnosis. The proposed framework achieved up to 99.2% F1-score and 0.96 Pearson's correlation coefficient for posture detection and inactivity segmentation respectively. This work paves the way for reliable home-based sleep movement analysis, serving patient-centred longitudinal care.

Neural correlates of perceptual texture change during active touch.

Introduction: Texture changes occur frequently during real-world haptic explorations, but the neural processes that encode perceptual texture change remain relatively unknown. The present study examines cortical oscillatory changes during transitions between different surface textures during active touch. Methods: Participants explored two differing textures whilst oscillatory brain activity and finger position data were recorded using 129-channel electroencephalography and a purpose-built touch sensor. These data streams were fused to calculate epochs relative to the time when the moving finger crossed the textural boundary on a 3D-printed sample. Changes in oscillatory band power in alpha (8-12 Hz), beta (16-24 Hz) and theta (4-7 Hz) frequency bands were investigated. Results: Alpha-band power reduced over bilateral sensorimotor areas during the transition period relative to ongoing texture processing, indicating that alpha-band activity is modulated by perceptual texture change during complex ongoing tactile exploration. Further, reduced beta-band power was observed in central sensorimotor areas when participants transitioned from rough to smooth relative to transitioning from smooth to rough textures, supporting previous research that beta-band activity is mediated by high-frequency vibrotactile cues. Discussion: The present findings suggest that perceptual texture change is encoded in the brain in alpha-band oscillatory activity whilst completing continuous naturalistic movements across textures.

Perception of fluids with diverse rheology applied to the underarm versus forearm skin.

Little is known about the tactile-perceptual structure of fluids. Therefore, ten fluids with diverse, characterized rheologies were rated by 16 females, on 27 sensory attributes (e.g., "slippery") and 14 emotional attributes (e.g., "enjoyable") via five-point categorical scales. Fluids were assessed against the volar forearm and underarm, sites that commonly experience contact with fluids during the use of personal care products. Application of fluids was either by the participant to their own body ("self-applied") or by the experimenter to the participant's body ("experimenter-applied"). Separate factor analyses of the sensory and emotional attributes for different body sites and modes of touch suggested approximately the same factorial structure in each case. Four general sensory factors emerged, labeled Lubricating, Textured, Silken, and Viscous, and two emotional factors, Comfortable and Arousing. These factors resembled those from equivalent work that used solid materials as stimuli, emphasizing that despite the differences in perceptual structure between fluid-coated and dry, solid surfaces, different body sites, and modes of touch influence the perception of fluid and dry stimuli similarly. As expected, fluids varied widely in how they scored on the factors. Site-wise differences were found, whereby stimuli assessed against the forearm were rated as more Lubricating, less Textured, more Silken, and more Comfortable than they were against the underarm. Self-applied touch was less Comfortable than experimenter-applied. The physical and perceptual were linked insofar as greater measured viscosity at low shear rates was associated with perceptions of cold and wet, whereas at high shear rates, greater viscosity was associated with greater perceived thickness.

Signalling pathways involved in ribonuclease-7 expression.

Antimicrobial peptides are host defence molecules that play a potential role in preventing infection at the epithelial surfaces. Ribonuclease (RNase)-7 has been shown to possess a broad spectrum of microbicidal activity against various pathogens. Here, we demonstrate that RNase-7 protein is localised to the superficial layers of ocular surface cells and increased in response to interleukin (IL)-1ß, suggesting an active role during inflammation related to ocular surface infection. Signal transduction pathways involved in RNase-7 expression are unknown. Involvement of transforming growth factor ß-activated kinase-1 (TAK-1) activated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway molecules [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] were studied because of their importance in infection and inflammation. Blocking the MAPKs resulted in inhibition of RNase-7 expression in response to IL-1ß. However, RNase-7 induction by IL-1ß was not affected by inhibiting the NF-κB signalling pathway. In conclusion, our results indicate that RNase-7 expression is specifically mediated via MAPKs but not NF-κB signalling pathways.

Immune mechanisms associated with sex-based differences in severe COVID-19 clinical outcomes.

BACKGROUND: Although biological males and females are equally likely to become infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), evidence has mounted that males experience higher severity and fatality compared to females. MAIN: The objective of this review is to examine the existing literature on biological mechanisms underlying sex-based differences that could contribute to SARS-CoV-2 infection clinical outcomes. Sex-based differences in immunologic response and hormonal expression help explain the differences in coronavirus disease 2019 (COVID-19) outcomes observed in biological males and females. X inactivation facilitates a robust immune response to COVID-19 in females, who demonstrate a more profound antibody response and faster recovery when compared to males. Low testosterone levels also help explain the dysregulated inflammatory response and poor outcomes observed in some males with COVID-19. Gender differences in health expression and behaviors further compound these observed differences. CONCLUSION: Understanding the biology of sex-based differences in COVID-19 severity and mortality could help inform preventative measures, treatment decisions, and development of personalized, sex-specific therapies.

Neural correlates of texture perception during active touch.

Previous studies have shown attenuation of cortical oscillations over bilateral sensorimotor cortex areas during passive perception of smooth textures applied to the skin. However, humans typically explore surfaces using dynamic hand movements. As movements may both modulate texture-related cortical activity and induce movement-related cortical activation, data from passive texture perception cannot be extrapolated to active texture perception. In the present study, we used electroencephalography to investigate cortical oscillatory changes during texture perception throughout active touch exploration. Three natural textured stimuli were selected: smooth silk, soft brushed cotton, and rough hessian. Texture samples were mounted on a purpose-built touch sensor which measured the load and position of the index finger, whilst electroencephalography from 129 channels recorded oscillatory brain activity. The data were fused to investigate oscillatory changes relating to active touch. Changes in oscillatory band power, event-related desynchronisation/synchronisation (ERD/ERS), were investigated in alpha (8-12 Hz) and beta (16-24 Hz) frequency bands. Active texture exploration revealed bilateral activation patterns over sensorimotor cortical areas. Beta-band ERD increased over contralateral sensorimotor regions for soft and smooth textures, and over ipsilateral sensorimotor areas for the smoothest texture. Analysis of covariance revealed that individual differences in perception of softness and smoothness were related to variations in cortical oscillatory activity. Differences may be due to increased high frequency vibrations for smooth and soft textures compared to rough. For the first time, active touch was quantified and fused with electroencephalography data streams, contributing to the understanding of the neural correlates of texture perception during active touch.

Comparison of choroidal and retinal endothelial cells: characteristics and response to VEGF isoforms and anti-VEGF treatments.

Neovascular eye diseases such as wet age-related macular degeneration and proliferative diabetic retinopathy are two of the most common causes of irreversible visual loss. Although mediated by vascular endothelial growth factor (VEGF), the mechanisms of these diseases are not fully understood. Molecular inhibitors of VEGF including pegaptanib, ranibizumab and bevacizumab are used as treatments for these diseases. However, there have been very few direct comparisons between these agents, and as dose and treatment regimes differ their relative efficacies are hard to determine. In vitro comparisons tend to use cells from different sites or species, which show heterogeneity in their responses. The aim of this study was to compare the characteristics of primary cultures of isolated human choroidal endothelial cells (hCEC) and retinal endothelial cells (hREC), and their proliferation responses to stimulation with VEGF 121 and 165, and to compare the anti-proliferative effects of these three drugs. hCEC and hREC were positive for the cell markers VEGFR1, VEGFR2, CD31, CD34 and von Willebrand's factor (vWF), with greater expression of CD34 on the hREC compared to hCEC. Contrary to previous assumptions VEGF isoforms 121 and 165 were found to be equally potent in stimulating endothelial cell proliferation. However, hREC exhibited higher proliferation with either VEGF isoform compared to hCEC. The anti-VEGF treatments ranibizumab and bevacizumab were effective in decreasing proliferation of hCEC induced by the two VEGF isoforms, individually and in combination, with ranibizumab being moderately more effective, particularly in hREC. Pegaptanib was effective in controlling the proliferation of hCEC stimulated by VEGF 165, but was ineffective against the stimulatory effect of VEGF 121. There were found to be significant differences in microvascular endothelial cells from the retina and choroid, both in the expression of cell markers and their behaviour in response to growth factors and currently available anti-VEGF agents, highlighting the importance of targeting treatments to specific intraocular vascular beds and/or diseases.

Host methylation predicts SARS-CoV-2 infection and clinical outcome.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, most clinical testing has focused on RT-PCR1. Host epigenome manipulation post coronavirus infection2-4 suggests that DNA methylation signatures may differentiate patients with SARS-CoV-2 infection from uninfected individuals, and help predict COVID-19 disease severity, even at initial presentation. METHODS: We customized Illumina's Infinium MethylationEPIC array to enhance immune response detection and profiled peripheral blood samples from 164 COVID-19 patients with longitudinal measurements of disease severity and 296 patient controls. RESULTS: Epigenome-wide association analysis revealed 13,033 genome-wide significant methylation sites for case-vs-control status. Genes and pathways involved in interferon signaling and viral response were significantly enriched among differentially methylated sites. We observe highly significant associations at genes previously reported in genetic association studies (e.g. IRF7, OAS1). Using machine learning techniques, models built using sparse regression yielded highly predictive findings: cross-validated best fit AUC was 93.6% for case-vs-control status, and 79.1%, 80.8%, and 84.4% for hospitalization, ICU admission, and progression to death, respectively. CONCLUSIONS: In summary, the strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to infection status, disease severity, and clinical deterioration provides insights useful for diagnosis and prognosis of patients with viral infections.

Host methylation predicts SARS-CoV-2 infection and clinical outcome.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, most clinical testing has focused on RT-PCR1. Host epigenome manipulation post coronavirus infection2-4 suggests that DNA methylation signatures may differentiate patients with SARS-CoV-2 infection from uninfected individuals, and help predict COVID-19 disease severity, even at initial presentation. METHODS: We customized Illumina's Infinium MethylationEPIC array to enhance immune response detection and profiled peripheral blood samples from 164 COVID-19 patients with longitudinal measurements of disease severity and 296 patient controls. RESULTS: Epigenome-wide association analysis revealed 13,033 genome-wide significant methylation sites for case-vs-control status. Genes and pathways involved in interferon signaling and viral response were significantly enriched among differentially methylated sites. We observe highly significant associations at genes previously reported in genetic association studies (e.g. IRF7, OAS1). Using machine learning techniques, models built using sparse regression yielded highly predictive findings: cross-validated best fit AUC was 93.6% for case-vs-control status, and 79.1%, 80.8%, and 84.4% for hospitalization, ICU admission, and progression to death, respectively. CONCLUSIONS: In summary, the strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to infection status, disease severity, and clinical deterioration provides insights useful for diagnosis and prognosis of patients with viral infections.

Viral infections affect the body in many ways, including via changes to the epigenome, the sum of chemical modifications to an individual's collection of genes that affect gene activity. Here, we analyzed the epigenome in blood samples from people with and without COVID-19 to determine whether we could find changes consistent with SARS-CoV-2 infection. Using a combination of statistical and machine learning techniques, we identify markers of SARS-CoV-2 infection as well as of severity and progression of COVID-19 disease. These signals of disease progression were present from the initial blood draw when first walking into the hospital. Together, these approaches demonstrate the potential of measuring the epigenome for monitoring SARS-CoV-2 status and severity.

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics.

BACKGROUND: The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye. Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC). RESULTS: LEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence. CONCLUSIONS: Our transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.

Preparation of Dried Amniotic Membrane for Corneal Repair.

Amniotic membrane transplantation is an established therapeutic and biological adjunct for several clinical situations, including treatment of diabetic foot ulcers and ocular surface disease. However, poorly standardized and validated clinical preparation and storage procedures can render the final product highly variable and an unpredictable biomaterial. We have therefore developed a novel, standardized method for processing and dry-preserving amniotic membrane, minimizing biochemical, compositional, and structure damage to produce a potentially superior membrane suitable for clinical use. The intellectual property associated with this methodology was patented by the University of Nottingham and licensed to NuVision® Biotherapies which formed the basis of the Tereo® manufacturing process which is used to manufacture Omnigen®.