scholarly journals Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration

2021 ◽  
Vol 4 (2) ◽  
pp. 32
Author(s):  
Brienna L. Anderson-Coughlin ◽  
Adrienne E. H. Shearer ◽  
Alexis N. Omar ◽  
K. Eric Wommack ◽  
Kalmia E. Kniel
Keyword(s):  
Enteric Virus ◽  
Clinical Testing ◽  
Human Feces ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Waste ◽  
Safety Research ◽  
New Castle County ◽  
Wastewater Systems ◽  
High Concentrations ◽  
Centrifugal Ultrafiltration

The COVID-19 pandemic is a global crisis and continues to impact communities as the disease spreads. Clinical testing alone provides a snapshot of infected individuals but is costly and difficult to perform logistically across whole populations. The virus which causes COVID-19, SARS-CoV-2, is shed in human feces and urine and can be detected in human waste. SARS-CoV-2 can be shed in high concentrations (>107 genomic copies/mL) due to its ability to replicate in the gastrointestinal tract of humans through attachment to the angiotensin-converting enzyme 2 (ACE-2) receptors there. Monitoring wastewater for SARS-CoV-2, alongside clinical testing, can more accurately represent the spread of disease within a community. This protocol describes a reliable and efficacious method to recover SARS-CoV-2 in wastewater, quantify genomic RNA levels, and evaluate concentration fluctuations over time. Using this protocol, viral levels as low as 10 genomic copies/mL were successfully detected from 30 mL of wastewater in more than seven-hundred samples collected between August 2020 and March 2021. Through the adaptation of traditional enteric virus methods used in food safety research, targets have been reliably detected with no inhibition of detection (RT-qPCR) observed in any sample processed. This protocol is currently used for surveillance of wastewater systems across New Castle County, Delaware.

Changes in the Binding of Radioactive Conjugated Bile Salts to Serum Proteins in Cholestatic Jaundice

1983 ◽  
Vol 65 (1) ◽  
pp. 77-84 ◽  
Author(s):  
A. Chitranukroh ◽  
B. H. Billing
Keyword(s):  
Fatty Acids ◽  
Liver Disease ◽  
Bile Salts ◽  
Serum Proteins ◽  
Cholestatic Liver Disease ◽  
Normal Individuals ◽  
High Concentrations ◽  
Conjugated Bile Salts ◽  
Centrifugal Ultrafiltration

1. A micro-partition centrifugal ultrafiltration technique has been used to monitor the percentage of[14C]glycocholate,[3H]glycochenodeoxycholate and[3H]glycochenodepxycholate-3-sulphate bound to serum proteins of patients with cholestatic liver disease. 2. in comparison with normal individuals the percentage of binding of [14C]glycocholate and, to a lesser extent, of [3H]glycochenodeoxycholate and [3H]glycochenodeoxycholate-3-sulphate was reduced. 3. The binding of [14C]glycocholate was inversely related to the serum bile salt and bilirubin concentrations. in contrast, the binding of [3H]glycochenodeoxycholate and [3H]glycochenodeoxycholate-3-sulphate were not altered by the severity of the cholestasis. 4. Studies in vitro indicated that the reduction in the binding of [14C]glycocholate in cholestatic liver disease was not due to high concentrations of bile salts, unconjugated bilirubin or fatty acids.

Breakdown of psyllium hydrocolloid by strains of Bacteroides ovatus from the human intestinal tract

1978 ◽  
Vol 24 (3) ◽  
pp. 336-338 ◽  
Author(s):  
Abigail A. Salyers ◽  
Charlotte J. Harris ◽  
Tracy D. Wilkins
Keyword(s):  
Intestinal Tract ◽  
Extracellular Enzymes ◽  
Human Feces ◽  
Bacteroides Ovatus ◽  
High Concentrations ◽  
Human Intestinal Tract

Strains of Bacteroides ovatus from human feces produced extracellular enzymes which degraded psyllium hydrocolloid. Other species of saccharolytic bacteria which are found in high concentrations in human feces did not degrade psyllium.

Effects of Water Quality on Microporous Filter Methods for Enteric Virus Concentration

1985 ◽  
Vol 17 (4-5) ◽  
pp. 665-679 ◽  
Author(s):  
M. D. Sobsey ◽  
T. Cromeans ◽  
A. R. Hickey ◽  
J. S. Glass
Keyword(s):  
Fulvic Acid ◽  
Bentonite Clay ◽  
Enteric Virus ◽  
Fulvic Acids ◽  
Virus Concentration ◽  
Humic And Fulvic Acids ◽  
Virus Adsorption ◽  
Filter Methods ◽  
High Concentrations ◽  
Virus Recovery

Model enteric viruses were concentrated from seeded samples of raw, finished and granular activated carbon (GAC)-treated waters and from GAC-treated waters supplemented with humic or fulvic acid or bentonite clay particles by adsorption to electropositive (Virosorb 1MDS) filters at pH 7.5 or electronegative (Filterite) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Natural particulates in raw and finished waters had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared to GAC-treated water, but the extent of interference varied with virus type and adsorption conditions. Humic and fulvic acids caused appreciable reductions in poliovirus adsorption and recovery efficiencies with both types of filters. Fulvic acid caused greater reductions in virus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with virus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced virus recoveries by both types of filters, its greatest effect was on virus elution and recovery from Filterite filters. Bentonite clay enhanced poliovirus retention by both filter types as much as 8-fold. However, the presence of clay often interfered with elution of retained viruses from both filter types. Under some conditions, overall virus recoveries were considerably lower from water with clay than from clay-free control water. The results of this study suggest that high concentrations of soluble organic matter such as humic and fulvic acids and suspended solids such as bentonite clay may interfere somewhat with virus recovery by filter adsorption-elution methods.

Simulated Human Feces for Testing Human Waste Processing Technologies in Space Systems

2006 ◽  
Author(s):  
Kanapathipillai Wignarajah ◽  
Eric Litwiller ◽  
John W. Fisher ◽  
John Hogan
Keyword(s):  
Waste Processing ◽  
Space Systems ◽  
Human Feces ◽  
Processing Technologies ◽  
Human Waste

scholarly journals SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

2021 ◽  
Author(s):  
Bojan F. Hörnich ◽  
Anna K. Großkopf ◽  
Sarah Schlagowski ◽  
Matthias Tenbusch ◽  
Hannah Kleine-Weber ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Inhibitory Activity ◽  
Cleavage Site ◽  
Receptor Expression ◽  
Proteolytic Activation ◽  
Angiotensin Converting Enzyme 2 ◽  
High Concentrations ◽  
Dose Dependent ◽  
Initial Target ◽  
Cell Cell

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2’ site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCE Cell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2’ cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

Freeze-fracture, freeze-etch complementary pairs of virus-infected cells reveal value of etching even when relatively high concentrations of cryoprotectants are used

1978 ◽  
Vol 36 (2) ◽  
pp. 136-137
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Plant Tissue ◽  
Phosphate Buffer ◽  
Infected Plant ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Virus Infected Plant ◽  
Infected Cells ◽  
High Concentrations

It has been assumed by many involved in freeze-etch or freeze-fracture studies that it would be useless to etch specimens which were cryoprotected by more than 15% glycerol. We presumed that the amount of cryoprotective material exposed at the surface would serve as a contaminating layer and prevent the visualization of fine details. Recent unexpected freeze-etch results indicated that it would be useful to compare complementary replicas in which one-half of the frozen-fractured specimen would be shadowed and replicated immediately after fracturing whereas the complement would be etched at -98°C for 1 to 10 minutes before being shadowed and replicated.Standard complementary replica holders (Steere, 1973) with hinges removed were used for this study. Specimens consisting of unfixed virus-infected plant tissue infiltrated with 0.05 M phosphate buffer or distilled water were used without cryoprotectant. Some were permitted to settle through gradients to the desired concentrations of different cryoprotectants.

Effect of verapamil on cytoplasmic distribution of granules and microfilaments in amphibian urinary bladder

1988 ◽  
Vol 46 ◽  
pp. 324-325
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Morphological Changes ◽  
Granular Cell ◽  
Cytosolic Calcium ◽  
Calcium Storage ◽  
Amphibian Urinary Bladder ◽  
Vesicular Membrane ◽  
High Concentrations ◽  
Cytoskeletal System

The amphibian urinary bladder has been used as a ‘model’ system for studies of the mechanism of action of antidiuretic hormone (ADH) in stimulating transepithelial water flow. The increase in water permeability is accompanied by morphological changes that include the stimulation of apical microvilli, mobilization of microtubules and microfilaments and vesicular membrane fusion events . It has been shown that alterations in the cytosolic calcium concentrations can inhibit ADH transmembrane water flow and induce alterations in the epithelial cell cytomorphology, including the cytoskeletal system . Recently, the subapical granules of the granular cell in the amphibian urinary bladder have been shown to contain high concentrations of calcium, and it was suggested that these cytoplasmic constituents may act as calcium storage sites for intracellular calcium homeostasis. The present study utilizes the calcium antagonist, verapamil, to examine the effect of calcium deprivation on the cytomorphological features of epithelial cells from amphibian urinary bladder, with particular emphasis on subapical granule and microfilament distribution.

Hair cell changes after cationic stimulation of corti's organ

1989 ◽  
Vol 47 ◽  
pp. 798-799
Author(s):  
Cesar D. Fermin ◽  
Hans-Peter Zenner
Keyword(s):  
Organ Of Corti ◽  
Cross Sectional ◽  
Surface Areas ◽  
High K ◽  
Anatomical Arrangement ◽  
Cell Cytosol ◽  
High Concentrations ◽  
K Concentration ◽  
Cell Changes

Contraction of outer and inner hair cells (OHC&IHC) in the Organ of Corti (OC) of the inner ear is necessary for sound transduction. Getting at HC in vivo preparations is difficult. Thus, isolated HCs have been used to study OHC properties. Even though viability has been shown in isolated (iOHC) preparations by good responses to current and cationic stimulation, the contribution of adjoining cells can not be explained with iOHC preparations. This study was undertaken to examine changes in the OHC after expossure of the OHC to high concentrations of potassium (K) and sodium (Na), by carefully immersing the OC in either artifical endolymph or perilymph. After K and Na exposure, OCs were fixed with 3% glutaraldehyde, post-fixed in osmium, separated into base, middle and apex and embedded in Araldite™. One μm thick sections were prepared for analysis with the light and E.M. Cross sectional areas were measured with Bioquant™ software.Potassium and sodium both cause isolated guinea pig OHC to contract. In vivo high K concentration may cause uncontrolled and sustained contractions that could contribute to Meniere's disease. The behavior of OHC in the vivo setting might be very different from that of iOHC. We show here changes of the cell cytosol and cisterns caused by K and Na to OHC in situs. The table below shows results from cross sectional area measurements of OHC from OC that were exposed to either K or Na. As one would expect, from the anatomical arrangement of the OC, OHC#l that are supported by rigid tissue would probably be displaced (move) less than those OHC located away from the pillar. Surprisingly, cells in the middle turn of the cochlea changed their surface areas more than those at either end of the cochlea. Moreover, changes in surface area do not seem to differ between K and Na treated OCs.

ACE2 as therapeutic agent

2020 ◽  
Vol 134 (19) ◽  
pp. 2581-2595
Author(s):  
Qiuhong Li ◽  
Maria B. Grant ◽  
Elaine M. Richards ◽  
Mohan K. Raizada
Keyword(s):  
Therapeutic Agent ◽  
Renin Angiotensin System ◽  
Peptide Hormone ◽  
Experimental Models ◽  
Electrolyte Balance ◽  
Ang Ii ◽  
Angiotensin Converting Enzyme 2 ◽  
Beneficial Effects ◽  
Overwhelming Evidence ◽  
Future Challenges

Abstract The angiotensin-converting enzyme 2 (ACE2) has emerged as a critical regulator of the renin–angiotensin system (RAS), which plays important roles in cardiovascular homeostasis by regulating vascular tone, fluid and electrolyte balance. ACE2 functions as a carboxymonopeptidase hydrolyzing the cleavage of a single C-terminal residue from Angiotensin-II (Ang-II), the key peptide hormone of RAS, to form Angiotensin-(1-7) (Ang-(1-7)), which binds to the G-protein–coupled Mas receptor and activates signaling pathways that counteract the pathways activated by Ang-II. ACE2 is expressed in a variety of tissues and overwhelming evidence substantiates the beneficial effects of enhancing ACE2/Ang-(1-7)/Mas axis under many pathological conditions in these tissues in experimental models. This review will provide a succinct overview on current strategies to enhance ACE2 as therapeutic agent, and discuss limitations and future challenges. ACE2 also has other functions, such as acting as a co-factor for amino acid transport and being exploited by the severe acute respiratory syndrome coronaviruses (SARS-CoVs) as cellular entry receptor, the implications of these functions in development of ACE2-based therapeutics will also be discussed.

Spine Impairment Evaluation: Sixth Edition Approaches

2009 ◽  
Vol 14 (1) ◽  
pp. 1-5
Author(s):  
Craig Uejo ◽  
Marjorie Eskay-Auerbach ◽  
Christopher R. Brigham
Keyword(s):  
Functional Status ◽  
Spinal Injury ◽  
Current Medical ◽  
Clinical Testing ◽  
Medical Evidence ◽  
Sixth Edition ◽  
Whole Person ◽  
Rating Process ◽  
Impairment Ratings ◽  
Class Assignment

Abstract Evaluators who use the AMA Guides to the Evaluation of Permanent Impairment (AMA Guides), Sixth Edition, should understand the significant changes that have occurred (as well as the Clarifications and Corrections) in impairment ratings for disorders of the cervical spine, thoracic spine, lumbar spine, and pelvis. The new methodology is an expansion of the Diagnosis-related estimates (DRE) method used in the fifth edition, but the criteria for defining impairment are revised, and the impairment value within a class is refined by information related to functional status, physical examination findings, and the results of clinical testing. Because current medical evidence does not support range-of-motion (ROM) measurements of the spine as a reliable indicator of specific pathology or permanent functional status, ROM is no longer used as a basis for defining impairment. The DRE method should standardize and simplify the rating process, improve validity, and provide a more uniform methodology. Table 1 shows examples of spinal injury impairment rating (according to region of the spine and category, with comments about the diagnosis and the resulting class assignment); Table 2 shows examples of spine impairment by region of the spine, class, diagnosis, and associated whole person impairment ratings form the sixth and fifth editions of the AMA Guides.

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