The antigenic anatomy of SARS-CoV-2 receptor binding domain

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M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.     

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Neurological Soft Signs in Individuals with Pathological Gambling

Increased neurological soft signs (NSSs) have been found in a number of neuropsychiatric syndromes, including chemical addiction. The present study examined NSSs related to perceptual-motor and visuospatial processing in a behavioral addiction viz., pathological gambling (PG). As compared to mentally healthy individuals, pathological gamblers displayed significantly poorer ability to copy two- and three-dimensional figures, to recognize objects against a background noise, and to orient in space on a road-map test. Results indicated that PG is associated with subtle cerebral cortical abnormalities. Further prospective clinical research is needed to address the NSSs'' origin and chronology (e.g., predate or follow the development of PG) as well as their response to therapeutic interventions and/or their ability to predict such a response.     

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.     

Dynamic response characteristics of local muscle blood flow regulatory mechanisms in human forearm exercise.

We sought to understand the nature of control mechanisms involved in the adaptation of exercising muscle hyperemia. Seven subjects performed rhythmic dynamic forearm exercise under two exercise conditions: small step 1 [step increase from rest to 40% peak forearm vascular conductance (FVC), in ml.min(-1).100 mmHg-1] for 5 min followed by small step 2 (further increase to 80% peak FVC for 5 min), and large step (step increase from rest to 80% peak FVC for 5 min). FVC data were fit with a two- (small step 1) and three-component (small step 2, large step) exponential as appropriate. For the rapid phase I response, FVC dynamic response characteristics (time delay, time constant) were not affected by the magnitude of the work intensity increase when the transition began from rest, but were slower in the 40-80% transition. Rest-80% gain was greater than either rest-40% or 40-80% transitions but represented the same proportion of the phase I + phase II gain across all transitions (57 vs. 56 vs. 57%, respectively, P = 0.975). For the slower phase II response, dynamic response characteristics were not affected by the magnitude of the work intensity increase when initiated from rest. The time constant was not altered when the transition began from exercise vs. rest. We conclude that 1) dynamic response characteristics of exercise hyperemia control mechanisms are not affected by the magnitude of work rate increase when forearm exercise is initiated from rest, 2) phase I but not phase II dynamic response characteristics are sensitive to baseline exercise intensity, and 3) the mechanisms contributing to phase I result in the same relative response magnitude, regardless of the size of the step increase in exercise intensity or the baseline from which it is initiated.     

Predicting the Efficacy of Polycyclic Aromatic Hydrocarbon Bioremediation in Creosote-Contaminated Soil Using Bioavailability Assays

Nonexhaustive extraction (propanol, butanol, hydroxypropyl-β-cyclodextrin [HPCD]), persulfate oxidation and biodegradability assays were employed to determine the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil. After 16 weeks incubation, greater than 89% of three-ring compounds (acenaphthene, anthracene, fluorene, and phenanthrene) and 21% to 79% of four-ring compounds (benz[a]anthracene, chrysene, fluoranthene, and pyrene) were degraded by the indigenous microorganisms under biopile conditions. No significant decrease in five- (benzo[a]pyrene, benzo[b+k]fluoranthene) and six-ring compounds (benz[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) was observed. Desorption of PAHs using propanol or butanol could not predict PAH biodegradability: low-molecular-weight PAH biodegradability was underestimated whereas high-molecular-weight PAH biodegradability was overestimated. Persulfate oxidation and HPCD extraction of creosote-contaminated soil was able to predict three- and four-ring PAH biodegradability; however, the biodegradability of five-ring PAHs was overestimated. These results demonstrate that persulfate oxidation and HPCD extraction are good predictors of PAH biodegradability for compounds with octanol-water partitioning coefficients of < 6.     

Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice.

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.