Persistence and free chlorine disinfection of human coronaviruses and their surrogates in water.

The coronavirus disease 2019 pandemic illustrates the importance of understanding the behavior and control of human pathogenic viruses in the environment. Exposure via water (drinking, bathing, and recreation) is a known route of transmission of viruses to humans, but the literature is relatively void of studies on the persistence of many viruses, especially coronaviruses, in water and their susceptibility to chlorine disinfection. To fill that knowledge gap, we evaluated the persistence and free chlorine disinfection of human coronavirus OC43 (HCoV-OC43) and its surrogates, murine hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV), in drinking water and laboratory buffer using cell culture methods. The decay rate constants of human coronavirus and its surrogates in water varied, depending on virus and water matrix. In drinking water without disinfectant addition, MHV showed the largest decay rate constant (estimate ± standard error, 2.25 ± 0.09 day-1) followed by HCoV-OC43 (0.99 ± 0.12 day-1) and TGEV (0.65 ± 0.06 day-1), while in phosphate buffer without disinfectant addition, HCoV-OC43 (0.51 ± 0.10 day-1) had a larger decay rate constant than MHV (0.28 ± 0.03 day-1) and TGEV (0.24 ± 0.02 day-1). Upon free chlorine disinfection, the inactivation rates of coronaviruses were independent of free chlorine concentration and were not affected by water matrix, though they still varied between viruses. TGEV showed the highest susceptibility to free chlorine disinfection with the inactivation rate constant of 113.50 ± 7.50 mg-1 min-1 L, followed by MHV (81.33 ± 4.90 mg-1 min-1 L) and HCoV-OC43 (59.42 ± 4.41 mg-1 min-1 L). IMPORTANCE: This study addresses an important knowledge gap on enveloped virus persistence and disinfection in water. Results have immediate practical applications for shaping evidence-based water policies, particularly in the development of disinfection strategies for pathogenic virus control.

SIRPα Mediates IGF1 Receptor in Cardiomyopathy Induced by Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is characterized by increased myocardial mass despite near-normal blood pressure, suggesting the presence of a separate trigger. A potential driver is SIRPα (signal regulatory protein alpha)-a mediator impairing insulin signaling. The objective of this study is to assess the role of circulating SIRPα in CKD-induced adverse cardiac remodeling. METHODS: SIRPα expression was evaluated in mouse models and patients with CKD. Specifically, mutant, muscle-specific, or cardiac muscle-specific SIRPα KO (knockout) mice were examined after subtotal nephrectomy. Cardiac function was assessed by echocardiography. Metabolic responses were confirmed in cultured muscle cells or cardiomyocytes. RESULTS: We demonstrate that SIRPα regulates myocardial insulin/IGF1R (insulin growth factor-1 receptor) signaling in CKD. First, in the serum of both mice and patients, SIRPα was robustly secreted in response to CKD. Second, cardiac muscle upregulation of SIRPα was associated with impaired insulin/IGF1R signaling, myocardial dysfunction, and fibrosis. However, both global and cardiac muscle-specific SIRPα KO mice displayed improved cardiac function when compared with control mice with CKD. Third, both muscle-specific or cardiac muscle-specific SIRPα KO mice did not significantly activate fetal genes and maintained insulin/IGF1R signaling with suppressed fibrosis despite the presence of CKD. Importantly, SIRPα directly interacted with IGF1R. Next, rSIRPα (recombinant SIRPα) protein was introduced into muscle-specific SIRPα KO mice reestablishing the insulin/IGF1R signaling activity. Additionally, overexpression of SIRPα in myoblasts and cardiomyocytes impaired pAKT (phosphorylation of AKT) and insulin/IGF1R signaling. Furthermore, myotubes and cardiomyocytes, but not adipocytes treated with high glucose or cardiomyocytes treated with uremic toxins, stimulated secretion of SIRPα in culture media, suggesting these cells are the origin of circulating SIRPα in CKD. Both intracellular and extracellular SIRPα exert biologically synergistic effects impairing intracellular myocardial insulin/IGF1R signaling. CONCLUSIONS: Myokine SIRPα expression impairs insulin/IGF1R functions in cardiac muscle, affecting cardiometabolic signaling pathways. Circulating SIRPα constitutes an important readout of insulin resistance in CKD-induced cardiomyopathy.

Mammalian Cell Genotoxicity of Potable Reuse and Conventional Drinking Waters.

Potable reuse water is increasingly part of the water supply portfolio for municipalities facing water shortages, and toxicity assays can be useful for evaluating potable reuse water quality. We examined the Chinese hamster ovary cell acute direct genotoxicity of potable reuse waters contributed by disinfection byproducts (DBPs) and anthropogenic contaminants and used the local conventional drinking waters as benchmarks for evaluating potable reuse water quality. Our results showed that treatment trains based on reverse osmosis (RO) were more effective than RO-free treatment trains for reducing the genotoxicity of influent wastewaters. RO-treated reuse waters were less genotoxic than the local tap water derived from surface water, whereas reuse waters not treated by RO were similarly genotoxic as the local drinking waters when frequent replacement of granular activated carbon limited contaminant breakthrough. The genotoxicity contributed by nonvolatile, uncharacterized DBPs and anthropogenic contaminants accounted for ≥73% of the total genotoxicity. The (semi)volatile DBPs of current research interest contributed 2-27% toward the total genotoxicity, with unregulated DBPs being more important genotoxicity drivers than regulated DBPs. Our results underscore the need to look beyond known, (semi)volatile DBPs and the importance of determining whole water toxicity when assessing the quality of disinfected waters.

Lysine and Arginine Reactivity and Transformation Products during Peptide Chlorination.

Chlorine reactions with peptide-bound amino acids form disinfection byproducts and contribute to pathogen inactivation by degrading protein structure and function. Peptide-bound lysine and arginine are two of the seven chlorine-reactive amino acids, but their reactions with chlorine are poorly characterized. Using N-acetylated lysine and arginine as models for peptide-bound amino acids and authentic small peptides, this study demonstrated conversion of the lysine side chain to mono- and dichloramines and the arginine side chain to mono-, di-, and trichloramines in ≤0.5 h. The lysine chloramines formed lysine nitrile and lysine aldehyde at ∼6% yield over ∼1 week. The arginine chloramines formed ornithine nitrile at ∼3% yield over ∼1 week but not the corresponding aldehyde. While researchers hypothesized that the protein aggregation observed during chlorination arises from covalent Schiff base cross-links between lysine aldehyde and lysine on different proteins, no evidence for Schiff base formation was observed. The rapid formation of chloramines and their slow decay indicate that they are more relevant than the aldehydes and nitriles to byproduct formation and pathogen inactivation over timescales relevant to drinking water distribution. Previous research has indicated that lysine chloramines are cytotoxic and genotoxic to human cells. The conversion of lysine and arginine cationic side chains to neutral chloramines should alter protein structure and function and enhance protein aggregation by hydrophobic interactions, contributing to pathogen inactivation.

Electrochemical Dechlorination of Municipal Wastewater Effluent.

The use of sodium bisulfite as an electron donor to quench chloramine disinfectant residuals in municipal wastewater effluents prior to discharge incurs the cost of purchasing and transporting bisulfite to the utility and increases the loading of salts to the receiving water. In this study, degradation of chloramine residuals within authentic municipal wastewater effluents was achieved within a 30 min timescale using a reductive electrochemical reactor, which supplied electrons via a stainless-steel cathode under galvanostatic conditions without an ion exchange membrane separating the cathode and anode. Application of a 0.26 mA/cm2 cathodic current density reduced chloramines to ammonia and avoided oxidation at the IrO2-coated titanium anode of chloride to chlorine or chlorate and of ammonia to nitrite or nitrate. Net chloramine production was observed at a higher current density (2 mA/cm2). Chloramine degradation rates and Coulombic efficiencies were highest and electrical energy per order (EEO) values were lowest for the 304-grade stainless-steel cathode, which contains the highest nickel content, and for a stainless-steel cathode with a high surface area. Differences in ionic strength and pH were less important. For chloraminated municipal wastewater samples, the highest Coulombic efficiency was 4.1% and the lowest EEO value was 0.08 kWh/m3. An initial comparison indicated that the electricity cost associated with this EEO value would be comparable to the cost of sodium bisulfite for areas with low electricity costs.

Purified Chlorine Dioxide as an Alternative to Chlorine Disinfection to Minimize Chlorate Formation During Postharvest Produce Washing.

The washwater used to wash produce within postharvest washing facilities frequently contains high chlorine concentrations to prevent pathogen cross-contamination. To address concerns regarding the formation and uptake of chlorate (ClO3-) into produce, this study evaluated whether switching to chlorine dioxide (ClO2) could reduce chlorate concentrations within the produce. Because ClO2 exhibits lower disinfectant demand than chlorine, substantially lower concentrations can be applied. However, ClO3- can form through several pathways, particularly by reactions between ClO2 and the chlorine used to generate ClO2 via reaction with chlorite (ClO2-) or chlorine that forms when ClO2 reacts with produce. This study demonstrates that purging ClO2 from the chlorine and ClO2- mixture used for its generation through a trap containing ClO2- can scavenge chlorine, substantially reducing ClO3- concentrations in ClO2 stock solutions. Addition of low concentrations of ammonia to the produce washwater further reduced ClO3- formation by binding the chlorine produced by ClO2 reactions with produce as inactive chloramines without scavenging ClO2. While chlorate concentrations in lettuce, kale, and broccoli exceeded regulatory guidelines during treatment with chlorine, ClO3- concentrations were below regulatory guidelines for each of these vegetables when treated with ClO2 together with these two purification measures. Switching to purified ClO2 also reduced the concentrations of lipid-bound oleic acid chlorohydrins and protein-bound chlorotyrosines, which are exemplars of halogenated byproducts formed from disinfectant reactions with biomolecules within produce.

Cytotoxicity Comparison between Drinking Water Treated by Chlorination with Postchloramination versus Granular Activated Carbon (GAC) with Postchlorination.

Granular activated carbon treatment with postchlorination (GAC/Cl2) and chlorination followed by chloramination (Cl2/NH2Cl) represent two options for utilities to reduce DBP formation in drinking water. To compare the total cytotoxicity of waters treated by a pilot-scale GAC treatment system with postchlorination (and in some instances with prechlorination upstream of GAC (i.e., (Cl2)/GAC/Cl2)) and chlorination/chloramination (Cl2/NH2Cl) at ambient and elevated Br- and I- levels and at three different GAC ages, we applied the Chinese hamster ovary (CHO) cell cytotoxicity assay to whole-water extracts in conjunction with calculations of the cytotoxicity contributed by the 33 (semi)volatile DBPs lost during extractions. At both ambient and elevated Br- and I- levels, GAC/Cl2 and Cl2/NH2Cl achieved comparable reductions in the formation of regulated trihalomethanes (THMs) and haloacetic acids (HAAs). Nonetheless, GAC/Cl2 always resulted in lower total cytotoxicity than Cl2/NH2Cl, even at up to 65% total organic carbon breakthrough. Prechlorination formed (semi)volatile DBPs that were removed by the GAC, yet there was no substantial difference in total cytotoxicity between Cl2/GAC/Cl2 and GAC/Cl2. The poorly characterized fraction of DBPs captured by the bioassay dominated the total cytotoxicity when the source water contained ambient levels of Br- and I-. When the water was spiked with Br- and I-, the known, unregulated (semi)volatile DBPs and the uncharacterized fraction of DBPs were comparable contributors to total cytotoxicity; the contributions of regulated THMs and HAAs were comparatively minor.

Muscle Atrophy in CKD: A Historical Perspective of Advancements in Its Understanding.

OBJECTIVE: This perspective reviews the seminal clinical and experimental observations that led to today's current mechanistic model of muscle protein loss (wasting) in patients with chronic kidney disease (CKD). RESULTS AND CONCLUSION: Early International Society of Renal Nutrition and Metabolism (ISRNM) meetings facilitated discussions and hypotheses about the causes of muscle wasting in CKD. It became widely recognized that wasting is common and correlated with increased risks of mortality and morbidity. Although anorexia and dietary restrictions contribute to muscle loss, several features of CKD-associated wasting cannot be explained by malnutrition alone. The protein catabolism-inducing actions of metabolic acidosis, inflammation, insulin resistance, endocrine disorders and uremic toxins were progressively identified. Continued research to understand the interactions of inflammation, anabolic resistance, mitochondrial dysfunction, exercise, and nutrition on muscle protein turnover in patients with CKD will hopefully accelerate discoveries and treatments to ameliorate muscle wasting as well as the progression of CKD.

Electrochemical Reduction of Halogenated Alkanes and Alkenes Using Activated Carbon-Based Cathodes.

Granular activated carbon (GAC) is used to sorb a broad range of halogenated contaminant classes, but spent GAC disposal is costly. Taking advantage of GAC's conductivity, this study evaluated the conversion of the GAC to cathodes for electrochemical reductive dehalogenation of 15 halogenated alkanes and alkenes exhibiting a diversity of structures (type of halogen, number of halogens, functional groups) and including contaminants of practical importance (e.g., trichloroethylene). Alkane degradation rates increased with the number of halogens and in the order: chlorine < bromine < iodine. Quantitative structure-activity relationships (QSARs) correlating experimental first-order degradation rate constants for alkanes with molecular descriptors associated with an outer-sphere one-electron transfer calculated using density functional theory indicated that correlations with molecular descriptors improved in the order: aqueous phase reduction potentials (E0,aq) < energy of the substrate's lowest unoccupied molecular orbital (ELUMO) < Marcus theory activation free energies (ΔG‡) ∼ gas-phase standard reduction free energies (ΔG0,gas). Chlorinated alkene degradation rates increased with decreasing number of chlorines, and QSAR correlations were opposite those of alkanes, indicating a different reaction mechanism. Degradation timescales ranged from 1 min to 3 h with halides as predominant products. These results suggest that the electrochemical reduction of halogenated alkanes and alkenes can be used to regenerate spent GAC.

Formation of Oleic Acid Chlorohydrins in Vegetables during Postharvest Chlorine Disinfection.

High chlorine doses (50-200 mg/L) are used in postharvest washing facilities to control foodborne pathogen outbreaks. However, chlorine can react with biopolymers (e.g., lipids) within the produce to form chlorinated byproducts that remain in the food. During chlorination of micelles of oleic acid, an 18-carbon alkene fatty acid, chlorine added rapidly across the double bond to form the two 9,10-chlorohydrin isomers at a 100% yield. The molar conversion of lipid-bound oleic acid to 9,10-chlorohydrins in chlorine-treated glyceryl trioleate and produce was much lower, reflecting the restricted access of chlorine to lipids. Yields from spinach treated with 100 mg/L chlorine at 7.5 °C for 2 min increased from 0.05% (0.9 nmol/g-spinach) for whole leaf spinach to 0.11% (2 nmol/g) when shredding increased chlorine access. Increasing temperature (21 °C) and chlorine contact time (15 min) increased yields from shredded spinach to 0.83% (22 nmol/g) at 100 mg/L chlorine and to 1.8% (53 nmol/g) for 200 mg/L chlorine. Oleic acid 9,10-chlorohydrin concentrations were 2.4-2.7 nmol/g for chlorine-treated (100 mg/L chlorine at 7.5 °C for 2 min) broccoli, carrots, and butterhead lettuce, but 0.5-1 nmol/g for cabbage, kale, and red leaf lettuce. Protein-bound chlorotyrosine formation was higher in the same vegetables (5-32 nmol/g). The Chinese hamster ovary cell chronic cytotoxicity LC50 value for oleic acid 9,10-chlorohydrins was 0.106 mM. The cytotoxicity associated with the chlorohydrins and chlorotyrosines in low masses (9-52 g) of chlorine-washed vegetables would be comparable to that associated with trihalomethanes and haloacetic acids at levels of regulatory concern in drinking water.

Recovery of Clean Water and Ammonia from Domestic Wastewater: Impacts on Embodied Energy and Greenhouse Gas Emissions.

Treatment of domestic wastewater can recover valuable resources, including clean water, energy, and ammonia. Important metrics for these systems are greenhouse gas (GHG) emissions and embodied energy, both of which are location- and technology-dependent. Here, we determine the embodied energy and GHG emissions resulting from a conventional process train, and we compare them to a nonconventional process train. The conventional train assumes freshwater conveyance from a pristine source that requires energy for pumping (US average of 0.29 kWh/m3), aerobic secondary treatment with N removal as N2, and Haber-Bosch synthesis of ammonia. Overall, we find that this process train has an embodied energy of 1.02 kWh/m3 and a GHG emission of 0.77 kg-CO2eq/m3. We compare these metrics to those of a nonconventional process train that features anaerobic secondary treatment technology followed by further purification of the effluent by reverse osmosis and air stripping for ammonia recovery. This "short-cut" process train reduces embodied energy to 0.88 kWh/m3 and GHG emissions to 0.42 kg-CO2eq/m3, while offsetting demand for ammonia from the Haber-Bosch process and decreasing reliance upon water transported over long distances. Finally, to assess the potential impacts of nonconventional nitrogen removal technology, we compared the embodied energy and GHG emissions resulting from partial nitritation/anammox coupled to anaerobic secondary treatment. The resulting process train enabled a lower embodied energy but increased GHG emissions, largely due to emissions of N2O, a potent greenhouse gas.

Sunlight-Driven Chlorate Formation during Produce Irrigation with Chlorine- or Chloramine-Disinfected Water.

The increasing use of chlorine- or chloramine-containing irrigation waters to minimize foodborne pathogens is raising concerns about the formation and uptake of disinfection byproducts into irrigated produce. Chlorate has received particular attention in the European Union. While previous research demonstrated the formation of chlorate from dark disproportionation reactions of free chlorine and uptake of chlorate into produce from roots, this study evaluated chlorate formation from solar irradiation of chlorine- and chloramine-containing irrigation droplets and uptake through produce surfaces. Sunlight photolysis of 50 µM (3.6 mg/L as Cl2) chlorine significantly enhanced the formation of chlorate, with a 7.2% molar yield relative to chlorine. Chlorate formation was much less significant in sunlit chloramine solutions. In chlorinated solutions containing 270 µg/L bromide, sunlight also induced the conversion of bromide to 280 µg/L bromate. Droplet evaporation and the resulting increase in chlorine concentrations approximately doubled sunlight-induced chlorate formation relative to that in the bulk solutions in which evaporation is negligible. When vegetables (broccoli, cabbage, chicory, lettuce, and spinach) were sprayed with chlorine-containing irrigation water in a sunlit field, sunlight promoted chlorate formation and uptake through vegetable surfaces to concentrations above maximum residue levels in the European Union. Spraying with chloramine-containing waters in the dark minimized chlorate formation and uptake into the vegetables.

Disinfection Byproduct Recovery during Extraction and Concentration in Preparation for Chemical Analyses or Toxicity Assays.

Over 700 disinfection byproducts (DBPs) have been identified, but they account for only ∼30% of total organic halogen (TOX). Extracting disinfected water is necessary to assess the overall toxicity of both known and unknown DBPs. Commonly used DBP extraction methods include liquid-liquid extraction (LLE) and solid-phase extraction (SPE), which may use either XAD resins or other polymeric sorbents. With few exceptions, DBP recoveries have not been quantified. We compared recoveries by LLE, XAD resins, and a mixture of Phenomenex Sepra SPE sorbents (hereafter SPE) for (semi-)volatile DBPs and nonvolatile model compounds at the 1-L scale. We scaled up the three methods to extract DBPs in 10 L of chlorinated creek waters. For (semi-)volatile DBPs, XAD resulted in lower recoveries than LLE and SPE at both 1- and 10-L scales. At the 10-L scale, recovery of certain trihalomethanes and trihalogenated haloacetic acids by XAD was negligible, while recovery of other (semi-)volatile DBPs extracted by XAD (<30%) was lower than by SPE or LLE (30-60%). TOX recovery at the 10-L scale was generally similar by the three extraction methods. The low TOX recovery (<30%) indicates that the toxicity assessed by bioassays predominantly reflects the contribution of the nonvolatile, hydrophobic fraction of DBPs.

Evaluation of Histidine Reactivity and Byproduct Formation during Peptide Chlorination.

The covalent modifications resulting from chlorine reactions with peptide-bound amino acids contribute to pathogen inactivation and disinfection byproduct (DBP) formation. Previous research suggested that histidine is the third most reactive of the seven chlorine-reactive amino acids, leading to the formation of 2-chlorohistidine, 2-oxohistidine, or low-molecular-weight byproducts such as trihalomethanes. This study demonstrates that histidine is less reactive toward formation of chlorine transformation products (transformation time scale of hours to days) than five of the seven chlorine-reactive amino acids, including tyrosine (transformation time scale of minutes). Chlorine targeted tyrosine in preference to histidine within peptides, indicating that chlorine reactions with tyrosine and other more reactive amino acids could contribute more to the structural modifications to proteins over the short time scales relevant to pathogen inactivation. Over the longer time scales relevant to disinfection byproduct formation in treatment plants or distribution systems, this study identified ß-cyanoalanine as the dominant transformation product of chlorine reactions with peptide-bound histidine, with molar yields of ∼50% after 1 day. While a chlorinated histidine intermediate was observed at lower yields (maximum ∼5%), the cumulative concentration of the conventional low-molecular-weight DBPs (e.g., trihalomethanes) was ≤7%. These findings support the need to identify the high-yield initial transformation products of chlorine reactions with important precursor structures to facilitate the identification of unknown DBPs.

Mechanisms Regulating Muscle Protein Synthesis in CKD.

BACKGROUND: CKD induces loss of muscle proteins partly by suppressing muscle protein synthesis. Muscles of mice with CKD have increased expression of nucleolar protein 66 (NO66), as do muscle biopsy specimens from patients with CKD or those undergoing hemodialysis. Inflammation stimulates NO66 expression and changes in NF-κB mediate the response. METHODS: Subtotal nephrectomy created a mouse model of CKD with BUN >80 mg/dl. Crossing NO66flox/flox with MCK-Cre mice bred muscle-specific NO66 (MCK-NO66) knockout mice. Experiments assessed the effect of removing NO66. RESULTS: Muscle-specific NO66 knockout in mice blocks CKD-induced loss of muscle mass and improves protein synthesis. NO66 suppression of ribosomal biogenesis via demethylase activity is the mechanism behind these responses. In muscle cells, expression of NO66, but not of demethylase-dead mutant NO66, decreased H3K4me3 and H3K36me3 and suppressed pre-rRNA expression. Knocking out NO66 increased the enrichment of H3K4me3 and H3K36me3 on ribosomal DNA. In primary muscle cells and in muscles of mice without NO66, ribosomal RNA, pre-rRNA, and protein synthesis all increased. CONCLUSIONS: CKD suppresses muscle protein synthesis via epigenetic mechanisms that NO66 mediates. Blocking NO66 could suggest strategies that counter CKD-induced abnormal muscle protein catabolism.

Designing a Nanoscale Three-phase Electrochemical Pathway to Promote Pt-catalyzed Formaldehyde Oxidation.

Gas-phase heterogeneous catalysis is a process spatially constrained on the two-dimensional surface of a solid catalyst. Here, we introduce a new toolkit to open up the third dimension. We discovered that the activity of a solid catalyst can be dramatically promoted by covering its surface with a nanoscale-thin layer of liquid electrolyte while maintaining efficient delivery of gas reactants, a strategy we call three-phase catalysis. Introducing the liquid electrolyte converts the original surface catalytic reaction into an electrochemical pathway with mass transfer facilitated by free ions in a three-dimensional space. We chose the oxidation of formaldehyde as a model reaction and observed a 25000-times enhancement in the turnover frequency of Pt in three-phase catalysis as compared to conventional heterogeneous catalysis. We envision three-phase catalysis as a new dimension for catalyst design and anticipate its applications in more chemical reactions from pollution control to the petrochemical industry.

Pharmacokinetics and pharmacodynamics of TTI-101, a STAT3 inhibitor that blocks muscle proteolysis in rats with chronic kidney disease.

Loss of muscle proteins increases the morbidity and mortality of patients with chronic kidney disease (CKD), and there are no reliable preventive treatments. We uncovered a STAT3/CCAAT-enhancer-binding protein-δ to myostatin signaling pathway that activates muscle protein degradation in mice with CKD or cancer; we also identified a small-molecule inhibitor of STAT3 (TTI-101) that blocks this pathway. To evaluate TTI-101 as a treatment for CKD-induced cachexia, we measured TTI-101 pharmacokinetics and pharmacodynamics in control and CKD rats that were orally administered TTI-101or its diluent. The following two groups of gavage-fed rats were studied: sham-operated control rats and CKD rats. Plasma was collected serially (0, 0.25, 0.5, 1, 2, 4, 8, and 24 h) following TTI-101 administration (at oral doses of 0, 10, 30, or 100 mg/kg). Plasma levels of TTI-101 were measured by LC-MS/MS, and pharmacokinetic results were analyzed with the PKSolver program. Plasma TTI-101 levels increased linearly with doses; the maximum plasma concentrations and time to maximal plasma levels (~1 h) were similar in sham-operated control rats and CKD rats. Notably, gavage treatment of TTI-101 for 3 days produced TTI-101 muscle levels in sham control rats and CKD rats that were not significantly different. CKD rats that received TTI-101 for 7 days had suppression of activated STAT3 and improved muscle grip strength; there also was a trend for increasing body and muscle weights. TTI-101 was tolerated at doses of 100 mg·kg-1·day-1 for 7 days. These results with TTI-101 in rats warrant its development as a treatment for cachexia in humans.

Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40.

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.

Effects of Sodium Bicarbonate in CKD Stages 3 and 4: A Randomized, Placebo-Controlled, Multicenter Clinical Trial.

RATIONALE & OBJECTIVE: Metabolic acidosis associated with chronic kidney disease (CKD) may contribute to muscle dysfunction and bone disease. We aimed to test whether treatment with sodium bicarbonate improves muscle and bone outcomes. STUDY DESIGN: Multicenter, randomized, placebo-controlled, clinical trial. SETTING & PARTICIPANTS: 149 patients with CKD stages 3 and 4 between July 2011 and April 2016 at 3 centers in Cleveland, OH, and the Bronx, NY. INTERVENTION: Sodium bicarbonate (0.4 mEq per kg of ideal body weight per day) (n=74) or identical-appearing placebo (n=75). OUTCOMES: Dual primary outcomes were muscle function assessed using sit-to-stand test and bone mineral density. Muscle biopsies were performed at baseline and 2 months. Participants were seen at baseline and 2, 6, 12, and 24 months. RESULTS: Mean baseline serum bicarbonate level was 24.0±2.2 (SD) mEq/L and mean baseline estimated glomerular filtration rate was 36.3±11.2mL/min/1.73m2. Baseline characteristics did not differ between groups. Mean serum bicarbonate levels in the intervention arm during follow-up were 26.4±2.2, 25.5±2.3, 25.6±2.6, and 24.4±2.8 mEq/L (at 2, 6, 12, and 24 months). These were significantly higher than in the placebo group (P<0.001). Compared to the placebo group, participants randomly assigned to sodium bicarbonate treatment had no significant differences in sit-to-stand time (5 repetitions: P=0.1; and 10 repetitions P=0.07) or bone mineral density (P=0.3). Sodium bicarbonate treatment caused a decrease in serum potassium levels that was of borderline statistical significance (P=0.05). There were no significant differences in estimated glomerular filtration rates, blood pressure, weight, serious adverse events, or levels of muscle gene expression between the randomly assigned groups. LIMITATIONS: Initial mean serum bicarbonate level was in the normal range. CONCLUSIONS: Sodium bicarbonate therapy in patients with CKD stages 3 and 4 significantly increases serum bicarbonate and decreases potassium levels. No differences were found in muscle function or bone mineral density between the randomly assigned groups. Larger trials are required to evaluate effects on kidney function. FUNDING: National Institutes of Health grant. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT01452412.

A Tale of Two Treatments: The Multiple Barrier Approach to Removing Chemical Contaminants During Potable Water Reuse.

In response to water scarcity and an increased recognition of the risks associated with the presence of chemical contaminants, environmental engineers have developed advanced water treatment systems that are capable of converting municipal wastewater effluent into drinking water. This practice, which is referred to as potable water reuse, typically relies upon reverse osmosis (RO) treatment followed by exposure to ultraviolet (UV) light and addition of hydrogen peroxide (H2O2). These two treatment processes individually are capable of controlling many of the chemical and microbial contaminants in wastewater; however, a few chemicals may still be present after treatment at concentrations that affect water quality. Low-molecular weight (<200 Da), uncharged compounds represent the greatest challenge for RO treatment. For potable water reuse systems, compounds of greatest concern include oxidation products formed during treatment (e.g., N-nitrosodimethylamine, halogenated disinfection byproducts) and compounds present in wastewater effluent (e.g., odorous compounds, organic solvents). Although the concentrations of most of these compounds decrease to levels where they no longer compromise water quality after they encounter the second treatment barrier (i.e., UV/H2O2), low-molecular weight compounds that are resistant to direct photolysis and exhibit low reactivity with hydroxyl radical (·OH) may persist. While attempts to identify the compounds that pass through both barriers have accounted for approximately half of the dissolved organic carbon remaining after treatment, it is unlikely that a significant fraction of the remaining unknowns will ever be identified with current analytical techniques. Nonetheless, the toxicity-weighted concentration of certain known compounds (e.g., disinfection byproducts) is typically lower in RO-UV/H2O2 treated water than conventional drinking water. To avoid the expense associated with managing the concentrate produced by RO, environmental engineers have begun to employ alternative treatment barriers. The use of alternatives such as nanofiltration, ozonation followed by biological filtration, or activated carbon filtration avoids the problems associated with the production and disposal of RO concentrate, but they may allow a larger number of chemical contaminants to pass through the treatment process. In addition to the transformation products and solvents that pose risks in the RO-UV/H2O2 system, these alternative barriers are challenged by larger, polar compounds that are not amenable to oxidation, such as perfluoroalkyl acids and phosphate-containing flame retardants. To fully protect consumers who rely upon potable water reuse systems, new policies are needed to prevent chemicals that are difficult to remove during advanced treatment from entering the sewer system. By using knowledge about the composition of municipal wastewater and the mechanisms through which contaminants are removed during treatment, it should be possible to safely reuse municipal wastewater effluent as a drinking water source.