Implementing wastewater surveillance for SARS-CoV-2 on a university campus: Lessons learned.

Wastewater surveillance, also known as wastewater-based epidemiology (WBE), has been successfully used to detect SARS-CoV-2 and other viruses in sewage in many locations in the United States and globally. This includes implementation of the surveillance on college and university campuses. A two-phase study was conducted during the 2020-2021 academic year to test the feasibility of a WBE system on campus and to supplement the clinical COVID-19 testing performed for the student, staff, and faculty body. The primary objective during the Fall 2020 semester was to monitor a large portion of the on-campus population and to obtain an understanding of the spreading of the SARS-CoV-2 virus. The Spring 2021 objective was focused on selected residence halls and groups of residents on campus, as this was more efficient and relevant for an effective follow-up response. Logistical problems and planning oversights initially occurred but were corrected with improved communication and experience. Many lessons were learned, including effective mapping, site planning, communication, personnel organization, and equipment management, and obtained along the way, thereby paving an opportune guide for future planning efforts. PRACTITIONER POINTS: WBE was successful in the detection of many SARS-CoV-2 variants incl. Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron. Careful planning and contingencies were essential for a successful implementation of a SARS-CoV-2 monitoring program. A surveillance program may be important for detection and monitoring of other public health relevant targets in wastewater incl. bacteria, viruses, fungi and viruses. Diverse lessons were learned incl. effective mapping, site planning, communication, personnel organization, and equipment management, thereby providing a guide for future planning efforts.

Modulation of factors affecting optic nerve head astrocyte migration.

PURPOSE: The authors investigated the role of myosin light chain kinase (MYLK) and transforming growth factor beta (TGFbeta) receptor pathways in optic nerve head (ONH) astrocyte migration. They further investigated how the expression of these genes is altered by elevated hydrostatic pressure (HP). METHODS: PCR was used to determine the isoforms of MYLK expressed in ONH astrocytes. siRNAs against MYLK (all isoforms) and TGFbeta receptor 2 (TGFBR2) were prepared and tested for effects on the migration of cultured ONH astrocytes. Finally, the effects of elevated HP (24-96 hours) on the expression of MYLK isoforms and selected TGFbeta pathway components were measured. RESULTS: Multiple isoforms of MYLK are present in ONH astrocytes from Caucasian (CA) and African American (AA) donors. Both populations express the short form (MYLK-130) and the long form (MYLK-210) of MYLK and a splicing variant within MYLK-210. MYLK-directed siRNA decreased MYLK expression and cell migration compared with control siRNA. siRNA directed against TGFbeta receptor 2 also decreased cell migration compared with control and decreased extracellular matrix genes regulated by TGFbeta signaling. Elevated HP increased the expression of MYLK-130 and MYLK-210 in both populations of astrocytes. However, TGFbeta2 was uniquely upregulated by exposure to elevated HP in CA compared with AA astrocytes. CONCLUSIONS: Differential expression of TGFbeta pathway genes and MYLK isoforms observed in populations of glaucomatous astrocytes applies to the elevated HP model system. MYLK may be a new target for intervention in glaucoma to alter reactive astrocyte migration in the ONH.

Susceptibility to glaucoma: differential comparison of the astrocyte transcriptome from glaucomatous African American and Caucasian American donors.

BACKGROUND: Epidemiological and genetic studies indicate that ethnic/genetic background plays an important role in susceptibility to primary open angle glaucoma (POAG). POAG is more prevalent among the African-descent population compared to the Caucasian population. Damage in POAG occurs at the level of the optic nerve head (ONH) and is mediated by astrocytes. Here we investigated differences in gene expression in primary cultures of ONH astrocytes obtained from age-matched normal and glaucomatous donors of Caucasian American (CA) and African American (AA) populations using oligonucleotide microarrays. RESULTS: Gene expression data were obtained from cultured astrocytes representing 12 normal CA and 12 normal AA eyes, 6 AA eyes with POAG and 8 CA eyes with POAG. Data were normalized and significant differential gene expression levels detected by using empirical Bayesian shrinkage moderated t-statistics. Gene Ontology analysis and networks of interacting proteins were constructed using the BioGRID database. Network maps included regulation of myosin, actin, and protein trafficking. Real-time RT-PCR, western blots, ELISA, and functional assays validated genes in the networks. CONCLUSION: Cultured AA and CA glaucomatous astrocytes retain differential expression of genes that promote cell motility and migration, regulate cell adhesion, and are associated with structural tissue changes that collectively contribute to neural degeneration. Key upregulated genes include those encoding myosin light chain kinase (MYLK), transforming growth factor-beta receptor 2 (TGFBR2), rho-family GTPase-2 (RAC2), and versican (VCAN). These genes along with other differentially expressed components of integrated networks may reflect functional susceptibility to chronic elevated intraocular pressure that is enhanced in the optic nerve head of African Americans.

Gene expression and functional studies of the optic nerve head astrocyte transcriptome from normal African Americans and Caucasian Americans donors.

PURPOSE: To determine whether optic nerve head (ONH) astrocytes, a key cellular component of glaucomatous neuropathy, exhibit differential gene expression in primary cultures of astrocytes from normal African American (AA) donors compared to astrocytes from normal Caucasian American (CA) donors. METHODS: We used oligonucleotide Affymetrix microarray (HG U133A & HG U133A 2.0 chips) to compare gene expression levels in cultured ONH astrocytes from twelve CA and twelve AA normal age matched donor eyes. Chips were normalized with Robust Microarray Analysis (RMA) in R using Bioconductor. Significant differential gene expression levels were detected using mixed effects modeling and Statistical Analysis of Microarray (SAM). Functional analysis and Gene Ontology were used to classify differentially expressed genes. Differential gene expression was validated by quantitative real time RT-PCR. Protein levels were detected by Western blots and ELISA. Cell adhesion and migration assays tested physiological responses. Glutathione (GSH) assay detected levels of intracellular GSH. RESULTS: Multiple analyses selected 87 genes differentially expressed between normal AA and CA (P<0.01). The most relevant genes expressed in AA were categorized by function, including: signal transduction, response to stress, ECM genes, migration and cell adhesion. CONCLUSIONS: These data show that normal astrocytes from AA and CA normal donors display distinct expression profiles that impact astrocyte functions in the ONH. Our data suggests that differences in gene expression in ONH astrocytes may be specific to the development and/or progression of glaucoma in AA.