SARS-CoV-2 raw wastewater surveillance from student residences on an urban university campus.

The COVID-19 pandemic brought about an urgent need to monitor the community prevalence of infection and detect the presence of SARS-CoV-2. Testing individual people is the most reliable method to measure the spread of the virus in any given community, but it is also the most expensive and time-consuming. Wastewater-based epidemiology (WBE) has been used since the 1960s when scientists implemented monitoring to measure the effectiveness of the Polio vaccine. Since then, WBE has been used to monitor populations for various pathogens, drugs, and pollutants. In August 2020, the University of Tennessee-Knoxville implemented a SARS-CoV-2 surveillance program that began with raw wastewater surveillance of the student residence buildings on campus, the results of which were shared with another lab group on campus that oversaw the pooled saliva testing of students. Sample collection began at 8 am, and the final RT-qPCR results were obtained by midnight. The previous day's results were presented to the campus administrators and the Student Health Center at 8 am the following morning. The buildings surveyed included all campus dormitories, fraternities, and sororities, 46 buildings in all representing an on-campus community of over 8,000 students. The WBE surveillance relied upon early morning "grab" samples and 24-h composite sampling. Because we only had three Hach AS950 Portable Peristaltic Sampler units, we reserved 24-h composite sampling for the dormitories with the highest population of students. Samples were pasteurized, and heavy sediment was centrifuged and filtered out, followed by a virus concentration step before RNA extraction. Each sample was tested by RT-qPCR for the presence of SARS-CoV-2, using the CDC primers for N Capsid targets N1 and N3. The subsequent pooled saliva tests from sections of each building allowed lower costs and minimized the total number of individual verification tests that needed to be analyzed by the Student Health Center. Our WBE results matched the trend of the on-campus cases reported by the student health center. The highest concentration of genomic copies detected in one sample was 5.06 × 107 copies/L. Raw wastewater-based epidemiology is an efficient, economical, fast, and non-invasive method to monitor a large community for a single pathogen or multiple pathogen targets.

Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories.

Introduction: Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater. Methods: The decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4°C and 20°C. Results: Temperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day-1 at 4°C and 0.261 day-1 at 20°C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day-1, respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions. Discussion: The first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.

Coding-Complete Genome Sequence of a SARS-CoV-2 Variant Obtained from Raw Sewage at the University of Tennessee-Knoxville Campus.

Reported here is a coding-complete genome sequence of a SARS-CoV-2 variant obtained from raw wastewater samples at the University of Tennessee-Knoxville campus. This sequence provides insight into SARS-CoV-2 variants that circulate on large college campuses but remain mostly undetected.

Detection of Chlamydiaceae in ocular swabs from Australian pre-export feedlot sheep.

Infectious Ovine Keratoconjunctivitis (IOK) is a contagious ocular disease of sheep. A range of organisms have been observed as the aetiological agents of IOK. In this study, the presence of chlamydial pathogens (C. pecorum, C. abortus, C. psittaci) in conjunctival swabs was tested for. The swabs were collected from sheep with varying grades of IOK in an Australian pre-export feedlot. The sheep had been rejected from a shipment because of the eye disease. The relative contribution of chlamydial pathogens to IOK and the rejection of animals was evaluated. In total, 149 conjunctival swabs were taken from rejected sheep (IOK Grades 1 to 6; n = 126) as well as those with healthy eyes (Grade 0; n = 23). Screening for chlamydial pathogens was done using species-specific qPCR assays. Chlamydial DNA was detected in 35.6% (53/149) of conjunctival samples. C. pecorum was the most predominant species with an overall prevalence of 28.9% (43/149). C. psittaci prevalence was 6.7% (10/149). Both organisms were detected in healthy as well as IOK-affected eyes. All swabs tested negative for C. abortus. The results from this study demonstrate that Chlamydia spp can be readily detected in sheep presenting with IOK. The zoonotic C. abortus was not detected in any of the samples in this study, providing further evidence to the suggestion that this pathogen remains absent from Australia. Although the exact contribution of Chlamydia spp in the IOK pathogenesis is unclear, such studies are anticipated to be of benefit to Australian domestic and live export production systems.

The Scottish enhanced Staphylococcus aureus bacteraemia surveillance programme: the first 18 months of data in children.

BACKGROUND: National enhanced surveillance of Staphylococcus aureus bacteraemia (SAB) commenced on 1st October 2014 to gain a more in-depth understanding of the epidemiology of SAB in Scotland. Children under 16 years of age were analysed separately from adults because previous studies had demonstrated epidemiological differences. AIM: To identify risk factors and patient populations at greatest risk to enable the development of focused improvement plans. METHODS: All National Health Service (NHS) boards within NHS Scotland take part in the mandatory enhanced surveillance, with data collected by trained data collectors using nationally agreed definitions. FINDINGS: Analysis of the first 18 months of data showed that hospital-acquired SAB was mostly associated with neonates with device risk factors, whereas community-associated SAB was found in older children who had few, if any, risk factors and most presented with a bone or joint infection. CONCLUSION: The enhanced SAB data highlighted the difference in risk factors and entry points for the acquisition of SAB within the paediatric population.

The Scottish enhanced Staphylococcus aureus bacteraemia surveillance programme: the first 18 months of data in adults.

BACKGROUND: Staphylococcus aureus bacteraemia (SAB) is the second most common source of positive blood cultures, after Escherichia coli, reported within NHS Scotland. Laboratory surveillance has been mandatory in Scotland for SAB since 2001. AIM: To gain an understanding of the epidemiology of SAB cases and associated risk factors for healthcare and true community onset. Identification of these factors and the patient populations at greatest risk enables the development of focused improvement plans. METHODS: All NHS boards within NHS Scotland take part in the mandatory enhanced surveillance, with data collected by trained data collectors using nationally agreed definitions. FINDINGS: Between 1st October 2014 and 31st March 2016, 2256 episodes of SAB in adults were identified. The blood cultures were taken in 58 hospitals and across all 15 Scottish health boards. The data demonstrated that approximately one-third of all SAB cases are true community cases. Vascular access devices continue to be the most reported entry point (25.7%) in individuals who receive health care, whereas skin and soft tissue risk factors are present in all origins. A significant risk factor unique to community cases is illicit drug injection. CONCLUSION: Improvement plans for reduction of SAB should be targeted more widely than hospital care settings alone.

Effects of protein kinase C activation on human platelet cyclic AMP metabolism.

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).

The role of ligand in estrogen receptor regulation of gene expression.

We believe that steroid binding is not required for receptor binding to DNA, but instead induces a conformational change in the receptor domains involved in the protein-protein interactions proposed above. Data from Hansen and Gorski (1986), and more recent studies (M. Fritsch and J. Gorski, unpublished results) strongly suggest that the steroid binding domain when bound to estrogens undergoes a dramatic change in conformation characterized by a loss of hydrophobic surface. This marked change in the steroid binding domain probably affects the so-called dimerization region located in this domain and thus the interaction of receptor with nuclear proteins in vivo. In our working model, ER is bound to specific DNA sequences or response elements of a variety of genes with or without estrogen. Ligand binding induces conformational changes in the steroid binding and perhaps other domains of the receptor that in turn change receptor interaction with the transcriptional machinery. The nature of this change is not at all clear at present, and the possibility of enzymatic modification of receptor or associated transcription factors should not be excluded. Whatever the mechanism of receptor action on transcription, we expect it kinetically will be closely related to the occupancy of the receptor with estrogen. Finally, any model of ER interactions with target genes also needs to account for the drastic ligand effect on the extractability of all ER from the nucleus.

Primary peripheral T cell lymphoma of the endometrium.

A case of a primary peripheral T cell lymphoma arising in the endometrium is presented. Primary lymphomas of the female genital tract are rare, with endometrial lymphomas and those of T cell type being rarer still. Extensive investigations revealed no other sites of disease and the patient was treated by hysterectomy and chemotherapy. She remains well 33 months later. We believe that this case is exceptionally unusual.

Intraperitoneal medetomidine: a novel analgesic strategy for postoperative pain management in pregnant sheep.

The absorption of medetomidine released by continuous infusion from an osmotic pump in the abdominal cavity was studied in pregnant sheep during the 24 h postoperative period. Additionally pain and sedation was assessed. Eleven sheep were studied: six were treated with a medetomidine loaded osmotic pump delivering 10 µL/h (3 µg/kg/h medetomidine); and five with a saline loaded osmotic pump (control). Serial blood samples were taken and analysed to determine plasma medetomidine levels. Medetomidine was absorbed from the peritoneal cavity and a steady plasma concentration was achieved within 10 h, mean (SD) peak concentration was 2.87 (0.22) ng/mL. Sheep receiving medetomidine analgesia had significantly lower pain scores at 10 h than controls. Four control sheep required rescue analgesia, compared with 0 in the treatment group. Delivery of 3 µg/kg/h medetomidine by an intraperitoneal osmotic pump to pregnant sheep in the 24 h postoperative period provides adequate plasma concentrations of medetomidine for analgesia without sedation.

Glutamine promotes colony formation in bone marrow and HL-60 cells; accelerates myeloid differentiation in induced HL-60 cells.

Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis.

Benign mixed tumor of the vagina: case report with expanded immunohistochemical profile.

We present a case of benign mixed tumor of the vagina with its typical clinicopathologic and expanded immunohistochemical features. The strong coexpression of the mesenchymal spindle cell component with epithelial markers such as CK7, together with strong CD10, Bcl2, and estrogen and progesterone receptors favors a mullerian derived tumor. The tumor is best labeled as benign mixed mullerian tumor as originally designated.

Marked effects of salt on estrogen receptor binding to DNA: biologically relevant discrimination between DNA sequences.

Avidin-biotin complexed with DNA (ABCD) assays were employed to determine the binding affinity of estrogen receptor (ER) to DNA under various salt conditions. Type and concentration of salt in the reaction buffer dramatically affected the ability of the ER to discriminate between DNA sequences. Under appropriate salt conditions, ER was able to bind to the estrogen response element from the Xenopus vitellogenin A2 gene with at least 3 orders of magnitude greater affinity than a two base pair mutant sequence, and 5 orders of magnitude greater affinity than plasmid DNA. In these studies, the best discrimination was observed under conditions of salt type and concentration that more closely approximated intracellular conditions, i.e., 100-150 mM potassium salts. Analysis of the binding affinities for ER to all three types of DNA over a range of KCl concentrations indicated that the ionic interactions upon ER binding were the same for the three DNA molecules tested. Therefore, the additional stability of ER binding to target DNA sequences was contributed by nonionic interactions.

High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen.

We have developed an antibody-based DNA binding assay to study the effects of the absence or presence of an estrogen receptor agonist and two antagonists on the thermodynamic binding parameters for estrogen receptor binding to a consensus estrogen response element in vitro. We first demonstrate that antibodies raised against a region of the estrogen receptor directly adjacent to the DNA-binding domain do not interfere with the receptor's ability to preferentially bind to the vitellogenin estrogen response element in plasmid DNA. Exploiting this property to develop a quantitative DNA binding assay, we demonstrate that estrogen is not required for high affinity binding of the receptor for an oligonucleotide containing this element, nor do antiestrogens alter this interaction. In addition, we find that 1 mol of estrogen receptor is complexed with high affinity to 1 mol of vitellogenin estrogen response element, instead of two estrogen receptors/response element as would be predicted if estrogen receptor homodimer formation was required for high affinity DNA binding. Our data best fit a model in which the active estrogen receptor form is a monomer or heterodimer, but not a homodimer, and the regulatory role of estrogen on the estrogen receptor is the induction of protein-protein, but not protein-DNA, interactions.

Identification of a unique liganded estrogen receptor complex released from the nucleus by decavanadate.

Unoccupied estrogen receptor (ER) can be extracted from tissues by homogenization with a hypotonic buffer, whereas hormone-occupied ER becomes tightly bound to the nuclear pellet and must be extracted with high-salt-containing buffers. The molecular basis for estrogen-induced tight nuclear binding of ER remains an important puzzle. The different subcellular fractionation behaviors of the occupied and unoccupied ER are presumed to be due to a difference in their ability to interact with nuclear components, such as DNA and proteins. The proteins that are the targets for interaction with the hormone-occupied ER may be important for transcriptional regulation. However, the salt-extracted ER is recovered as a homodimer, and associated proteins are presumably lost due to the high-salt conditions. We have discovered an alternate method of releasing the occupied ER from the nucleus. Inclusion of 2 mM orthovanadate, polymerized primarily to decavanadate, in a hypotonic buffer efficiently releases over 90% of estrogen-bound ER from the nuclear pellet. The recovered ER complex is fully functional in terms of estrogen and DNA binding and is full-length by western blot analysis. Our data suggest that the mechanism of ER release is by decavanadate competition with nuclear DNA, rather than by inhibition of a phosphotyrosine phosphatase. Of particular interest, the decavanadate released occupied ER complex shows distinct behavior by sucrose density gradient sedimentation analysis. It is larger than the salt-extracted transformed ER, suggesting that an occupied ER in complex with nuclear proteins may be released from the nucleus by decavanadate.

Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol.

Gel shift assays were employed to distinguish between the contribution of 17 beta-estradiol (E2) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis gave a dissociation constant of 390 +/- 40 pM for the E2-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperatively was not observed. A dissociation constant of 450 +/- 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-fold lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.

Isolation and characterization of subgingival staphylococci from periodontitis patients and controls.

OBJECTIVES: To isolate and characterize subgingival staphylococci from patients with periodontal disease and from periodontally healthy controls, to evaluate the periodontal environment as a potential source for systemic staphylococcal infections. METHODS: Periopaper strips were used to isolate subgingival staphylococci from 28 patients with chronic periodontitis and 28 periodontally healthy age and sex-matched controls. Staphylococci were identified by microbiological methods and antibiotic resistance profiles determined. RESULTS: Staphylococci were isolated from 54% diseased subgingival and 43% healthy subgingival sites in over 50% periodontitis patients and from 29% healthy subgingival sites in 54% controls. No significant differences in the frequency of isolation or numbers of staphylococci isolated from diseased and healthy sites were noted. Staphylococcus epidermidis was the predominant oral species. Seventy per cent (115 of 165) of all isolates were penicillin-resistant. CONCLUSIONS: Subgingival staphylococci are present in both periodontitis patients and controls. In periodontitis there is an increased risk of bacteraemia because of the increased dentogingival surface area. The dental and periodontal health of patients at risk from haematogenous infections should therefore be maintained at a high level. Antibiotic resistance profiles of the oral staphylococcal isolates suggest that amoxicillin may no longer be a suitable antibiotic for prophylaxis against systemic infections such as prosthetic valve endocarditis.

DNA allosterically modulates the steroid binding domain of the estrogen receptor.

The ability of DNA to allosterically alter the conformation of the estrogen receptor's (ER) steroid binding domain was investigated. Using dissociation kinetics we observed that when DNA was bound to the DNA binding domain of the rat uterine ER the rate of estrogen dissociation from the steroid binding domain increased almost 2-fold. This change in the rate of estrogen dissociation depended on the concentration of DNA used and correlated with the thermodynamic binding affinities (Kd) of the ER for two different DNA sequences. We were unable to detect a DNA-induced change in the trypsin cleavage pattern of the amino terminal end of the ER. Using a whole cell dissociation kinetic assay with MCF-7 breast cancer cells we observed a 7-fold slower rate of estrogen dissociation from the ER within the cell than from the ER in vitro. This suggests that additional factors, other than DNA binding, may modify the steroid binding domain within the cell. We conclude that DNA can allosterically modulate the structure of the steroid binding domain of the ER, and we hypothesize that this conformational change may be necessary for the full transcriptional activity of the ER.

Estrogen receptor binding to DNA: affinity for nonpalindromic elements from the rat prolactin gene.

The estrogen receptor (ER) binds with high affinity to the nonclassical estrogen response elements (ERE) found in the rat prolactin gene. There are two putative EREs in this gene; at -1582 and -1722 upstream of the transcriptional start site. We used DNA binding assays based on the immunoprecipitation of receptor with bound DNA to quantitate the binding of ER to these two elements. ER bound each element with significantly higher affinity than it bound to nonspecific DNA, but with 10-100-fold less affinity than that for the classical ERE sequence derived from the vitellogenin A2 gene. These comparisons rank the prolactin gene sequences as weak EREs. We also observed a 1:1 ratio of ER to ERE in the bound complexes, indicating that these high-affinity interactions were not dependent upon an ER homodimer. These data support the role of these sequences in mediating estrogen regulation of the prolactin gene.