Retroperitoneal fibrosis (RPF) is a condition characterized by chronic inflammatory and fibrotic changes in the retroperitoneum that can lead to serious complications including kidney failure, mesenteric and limb ischemia, and deep venous thrombosis among others. Affected individuals may present with nonspecific symptomology that would require a high clinical index of suspicion for prompt diagnosis. We herein discuss a case of a young African-American man with recurrent deep venous thrombosis who presents with a 4-week history of constant aching pain of abdomen and back and kidney failure. Initial noncontrast computed tomogram (CT) only revealed mild bilateral hydroureteronephrosis with inflammatory changes but without obvious mass or lymphadenopathy. At the insistence of the renal consulting team to rule out RPF, a CT-urogram was performed which revealed an infiltrative mass encasing the aorta, inferior vena cava, and common iliac vessels. Laparoscopic biopsy revealed dense fibroadipose tissue, lymphocytic aggregates, focal scattered IgG4-positive plasma cells, and fibrin deposition. Patient underwent bilateral nephrostomy placement and empirical corticosteroid therapy with resolution of kidney failure. Our case illustrates a classic presentation of RPF with relatively benign findings on noncontrast CT that could have been missed if clinicians did not keep a high index of suspicion for the condition. Idiopathic retroperitoneal fibrosis (RPF) is a relatively uncommon condition characterized by chronic inflammatory and fibrotic changes in the retroperitoneum that can lead to diffuse encasement and compression of retroperitoneal structures and a myriad of associated complications. Clinical symptomology ranges from nonspecific constitutional symptoms, vague diffuse abdominal or back pain, abdominal distension, and lower extremity edema to more serious complications including kidney failure, mesenteric and limb ischemia, and deep venous thrombosis, among others –3. PrompIn patients who present with kidney failure from obstructive nephropathy, prompt diagnosis may be further hindered by the absence of significant hydronephrosis. It is plausible that compression of the kidneys, aorta, and renal arteries by inflammatory and fibrosing tissues leads to poor renal blood flow and low urine production, thus the appearance of relatively mild hydroureteronephrosis. Additionally, the level of RPF involvement may be more distal, which leads to hydroureters rather than overt hydronephrosis in early disease. Alternatively, kidney failure in patients with RPF may partly arise from renal ischemia due to the encasement of renal vasculature, in which case, hydronephrosis would not be observed. Finally, in the case of RPF associated with IgG4 related disease, kidney injury may be due to direct IgG4 related sclerosing interstitial nephritis independent of RPF-associated structural compromise [We herein discuss a case of a young African American male who presents with relatively acute kidney failure of unknown etiology whose initial kidney ultrasound and noncontrast computed tomogram (CT) of abdomen and pelvis only revealed mild bilateral hydroureteronephrosis.Clinical History. A 27-year-old African American male with a history of unprovoked recurrent right lower extremity deep vein thrombosis and bilateral testicular hydrocele presented with a 4-week history of bilateral flank and generalized nonradiating lower abdominal pain. The pain was described as constant ache, associated with early satiety, but without nausea/vomiting, diarrhea, melena, or hematochezia. He denied weight loss or any urinary symptoms. Social history was negative for tobacco smoking and alcohol or recreational drug use. Medications included diphenhydramine as needed for sleep and apixaban.Physical Exam. Vital vital signs revealed temperature 37.1°C, blood pressure 121/69 mmHg, pulse 96 beats/minute, and respiratory rate 16/minute. Lungs and heart exams were unremarkable. Lower half abdomen was mildly tender with palpation, but without mass, rebound, or guarding. There was mild right costovertebral angle tenderness and trace bilateral pedal edema. Genitalia were within normal limits without edema.Initial Laboratory Data. Routine chemistry revealed serum sodium 133 mEq/L, potassium 5.0 mEq/L, chloride 98 mEq/L, bicarbonate 25 mEq/L, blood urea nitrogen 57 mg/dL, creatinine 12.6 mg/dL (baseline of 1.0 mg/dL one month prior), estimated glomerular filtration rate 6 mL/min/1.73 m2, and calcium 8.6 mg/dL. Urinalysis revealed specific gravity 1.014, pH 8.0, no red or white blood cells, 100 mg/dL protein, and no glucose or blood. Urine protein to creatinine and albumin to creatinine ratios were 0.5 and 0.293 g/g creatinine respectively. Renal ultrasound and abdomen/pelvis CT without contrast revealed mild bilateral hydroureteronephrosis with nonspecific inflammatory changes  or obstructive uropathy. Differential diagnoses of obstructive uropathy relevant to current African American patient with unknown sickle cell history include recently passed papillary necrotic tissues or bladder stone, complicated medullary carcinoma (associated with sickle cell trait), diphenhydramine-induced neurogenic bladder, or retroperitoneal fibrosis (RPF). Rapidly progressive glomerulonephritis is thought to be less likely given the bland urinalysis.Additional Investigations. Given the mismatched relatively benign findings of both urinalyses and imaging studies and degree of kidney failure and associated symptomology suspicious for RPF, an abdomen and pelvis CT with intravenous contrast was obtained.Abdomen and pelvis computed tomogram with intravenous contrast revealed mild bilateral hydroureteronephrosis with point of obstruction at the level of distal abdominal aorta and inferior vena cava. There was an ill-defined, infiltrative soft tissue mass encasing both aorta, and inferior vena cava (approximately 7.2 cm). The mass appeared to infiltrate along the bilateral proximal common iliac vessels .Serologies to evaluate for autoimmune diseases and infectious etiologies including C-reactive-protein, human-immunodeficiency-virus, QuantiFERON gold, and antinuclear-antibody were negative. Serum lactate dehydrogenase obtained for possible lymphoproliferative disorder was mildly elevated at 248 IU (reference <192 IU). IgG4 level was 44.2 mg/dL (reference 4-86 mg/dL). Laparoscopic retroperitoneal mass biopsy revealed dense fibroadipose tissue with lymphocytic aggregates, focal scattered IgG4 positive plasma cells, and fibrin deposition without malignant cells.Diagnoses. The diagnosis of idiopathic RPF was made. Kidney failure was thought to be due to severe bilateral ureteral encasement by RPF.Clinical Follow-Up. Patient underwent bilateral nephrostomy placement with rapid improvement of serum creatinine. In addition, patient received a trial of prednisone 40 mg daily. At 4-month follow-up, kidney function normalized and CT revealed marked reduction in RPF size  and environmental exposures to tobacco smoking and asbestos are thought to be predisposing factors to the development of RPF [RPF may be associated with various autoimmune diseases including small-vessel vasculitis, rheumatoid arthritis, Sjogren's disease, and thyroiditis –3, 5 or t of RPF . The undt of RPF –12.Clinical manifestations reflect the underlying inflammatory process and dysfunction of affected structures, Histologically, RPF consists of inflammatory infiltrates, typically involving lymphocytes, plasma cells, and macrophages that are interspersed within collagen type I bundles or organized into nodular aggregates encircling small retroperitoneal vessels. RPF may be considered part of IgG4-related disease when plasma cells predominate inflammatory infiltrates, with IgG4+ to total IgG+ plasma cell ratio exceeding 40% –12.18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) has recently been suggested to be a beneficial tool in monitoring RPF activity and guiding ongoing therapy. 18F-FDG PET testing, however, is not specific for RPF and should not be used as a diagnostic tool [The diagnosis of RPF, particularly in the setting of kidney injury, relies on the clinicians' prompt recognition of its classic symptomology as initial low-quality imaging studies  may be unimpressive relative to the degree of kidney failure. Consideration for possible RPF should be given in cases of unexplained lower extremity edema or claudication, recurrent deep venous thrombosis, vague abdominal or flank pain, or even scrotal pain/edema, particularly in cases where a constellation of these symptoms are concurrently present. CT with intravenous contrast or magnetic resonance imaging may be used to diagnose RPF. RPF typically appears as a homogeneous mass encasing various retroperitoneal structures including the anterolateral sides of the abdominal aorta common iliac arteries, ureters, and inferior vena cava , 3, 7, 8tic tool . DefinitManagement involves symptomatic relief for affected organs such as relief of urinary obstruction via nephrostomy placement as needed for kidney failure and anticoagulation therapy for deep venous thrombosis. Whenever applicable, treatment of the underlying etiology or withdrawal of offending agents and exacerbating factors should be considered. Once infectious and malignant sources have been ruled out, a trial of immunosuppressive therapy may be initiated. Corticosteroid is currently the mainstay of therapy. There is no consensus on the optimal corticosteroid regimen, but empirical therapy has been suggested to be prednisone 0.5-1.0 mg/kg/day with gradual taper to 5.0-7.5 mg/day within 6-9 months , 15. Of In summary, RPF is presumably an uncommon and insidious disease that typically presents with nonspecific signs and symptoms. A strong clinical index of suspicion for RPF should be raised when patients present with an array of nonspecific signs and symptoms involving various structures from the abdomen and flank downward. Delayed diagnosis may lead to serious complications including kidney failure, deep venous thrombosis, bowel and extremity claudication, and even intestinal infarction. In patients with kidney involvement, the degree of kidney failure may be out of proportion to the presenting hydronephrosis. Awareness of this disease entity among clinicians is necessary to ensure prompt diagnosis and optimal outcomes. The mainstay of therapy involves the empirical use of corticosteroids but relapse rates are high with monotherapy. Close follow-up and addition of a second immunosuppressive agent may be required.


Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 μM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 μM, respectively. Among the tested xanthophyll-UGT pairs, β-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 . In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of β-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls.Despite the widespread use of the five major xanthophylls astaxanthin, β-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs). Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs  in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15) or recombinant supersomes (UGT2B15). We also predicted potential dietary supplement-drug interactions for β-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. β-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC Xanthophylls have been studied for their beneficial effects, including their powerful antioxidant activities and association with lower incidence of chronic diseases ,2,3,4. MOwing to the beneficial health effects of xanthophylls, their use as dietary supplements has been rapidly growing . The incDespite the widespread use of xanthophylls, there are few reports of interactions between drug-metabolizing enzymes and AS, βC, CA, LU and ZE. Gradelet et al.  reportedUGTs metabolize many endogenous substances  as well as numerous xenobiotics  ,26,27. TThe aim of this study was to investigate the inhibitory potential of five commonly used xanthophylls  on the seven major human hepatic UGT isozymes  in vitro and to quantitatively predict their potential for dietary supplement-drug interactions in vivo.50 values of the positive controls and five xanthophylls are shown in 50 values of the known potent inhibitors used in the inhibition studies were similar to previously reported values. The five tested xanthophylls showed only weak inhibitory effects on the six UGTs. βC mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40, and 34.9 ± 5.98 μM, respectively, and showed no apparent inhibition of UGT1A6, UGT1A9, and UGT2B7  were greater than 80%, except for UGT1A1 (61.2%) and UGT1A3 (64.2%) in the presence of ZE  for UGT1A1.The inhibitory effects of AS, βC, CA, LU, and ZE on the activity of six UGTs are illustrated in d UGT2B7 . CA weakce of ZE . Among t50 values of the positive control and five xanthophylls are listed in The inhibitory effects of AS, βC, CA, LU, and ZE on UGT2B15 activity in UGT2B15 supersomes are shown in i of 12.2 ± 0.985 μM. Nilotinib showed stronger competitive inhibition of UGT1A1, with a Ki of 0.811 ± 0.126 μM.To further characterize the inhibition of UGT1A1 by βC, enzyme kinetic experiments were performed in the presence of various concentrations of βC and β-estradiol. Samples containing the known potent UGT1A1 inhibitor nilotinib were included in the analysis. Representative Dixon plots showing UGT1A1 inhibition by βC and nilotinib in human liver microsomes are shown in S-oxazepam, a typical substrate of UGT2B15, and the corresponding glucuronide are not available, we used recombinant UGT2B15 supersomes and the nonspecific substrate 4-methylumbelliferone to evaluate UGT2B15 activity, as previously described in vivo value, and the fraction metabolized by the UGT1A1 of the co-administrated drug was supposed to be 1. A pharmacokinetic study found that, for a single-dose carotenoid supplement containing 1.3 mg of esterified βC in healthy subjects, the mean Cmax was approximately 0.15 μM [max value, the AUCi/AUC ratio was calculated as 1.012 for UGT1A1, indicating that the AUC of the co-administered drug may increase 1.2% when βC is co-administered with clinical drugs that undergo 100% UGT1A1-mediated elimination. Thus, our data strongly suggest that βC is unlikely to cause a clinically significant metabolic drug-drug interaction via inhibition of any of the seven major human hepatic UGTs involved in drug metabolism.As far as we know, for reversible inhibitors, the magnitude of the drug-drug interaction is affected by both the in vitro inhibition constant  were purchased from Sigma‒Aldrich Corporation . Efavirenz, nilotinib, propofol, propofol-O-glucuronide, serotonin-O-glucuronide, trifluoperazine-N-glucuronide, zidovudine-5′-glucuronide, and chenodeoxycholic acid 24-acyl-β-d-glucuronide were purchased from Toronto Research Chemicals . Deoxyschizandrin (purity: 97.7%) was kindly donated by Dr. Young-Won Chin . All solvents were of high-performance liquid chromatography (HPLC) grade and were obtained from Burdick & Jackson Company ; other chemicals were of the highest quality available.Pooled human liver microsomes from equal gender mix from 150 donors and a recombinant human UGT2B15 supersome were purchased from Corning Life Sciences . The human liver microsomes studies were performed in accordance with the Declaration of Helsinki and designated exempt from review by the Catholic University of Korea Institutional Review Board. Astaxanthin (AS), β-cryptoxanthin (βC), canthaxanthin (CA), lutein (LU), zeaxanthin (ZE), alamethicin, amitriptyline, β-estradiol, β-estradiol-3-glucuronide, chenodeoxycholic acid, diclofenac, 4-methylumbelliferone, 4-methylumbelliferyl glucuronide, trifluoperazine dihydrochloride, serotonin hydrochloride, zidovudine, hecogenin, niflumic acid, chlorpropamide, theophylline, MgCld-glucuronide, trifluoperazine-N-glucuronide, imipramine-N-β-d-glucuronide, serotonin-O-glucuronide, propofol-O-glucuronide, and zidovudine-5′-glucuronide were determined to probe the activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively, in human liver microsomes. The selected concentrations of UGT probe substrates were close to the reported Km values [The inhibitory effects of AS, βC, CA, LU and ZE on UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 were evaluated using pooled human liver microsomes, as previously described ,40. The m values ,41,42,432, the UGT-selective substrates, and five xanthophylls (0–50 μM), were pre-incubated for 30 min on ice to allow formation of alamethicin pores. Reac2345tions were initiated by the addition of 10 μL of UDPGA (5 mM) to a final reaction volume of 0.1 mL and incubated at 37 °C for 60 min (except 30 min for UGT1A9) in a shaking water bath. After incubation, each reaction was stopped by adding 50 μL of ice-cold acetonitrile containing chlorpropamide (300 ng/mL) or theophylline (30 μg/mL or 300 μg/mL) as an internal standard. The mixtures were then centrifuged  and 5-µL aliquots of the supernatants were injected into an LC-MS/MS system. Known potent inhibitors nilotinib (0–10 μM), deoxyschizandrin (0–100 μM), hecogenin (0–50 μM), diclofenac (0–500 μM), niflumic acid (0–5 μM) and efavirenz (0–200 μM) were included as positive controls for UGT1A1 [50 values). All incubations were performed in triplicate, and mean values were used for analysis. In brief, the 90-µL incubation mixture, including pooled human liver microsomes , 100 mM Tris buffer, 25 µg/mL alamethicin, 5 mM MgClr UGT1A1 , UGT1A3 r UGT1A1 , UGT1A4 r UGT1A1 , UGT1A6 r UGT1A1 , to evalv/v).All substrate and inhibitors were dissolved in methanol (except hecogenin and five xanthophylls which were dissolved in dimethylsulfoxide due to their low solubility) and serially diluted to the required concentrations. The final concentration of organic solvent in each incubation mixture including the control was 2.5% (0.5% of methanol and 2.0% of dimethylsulfoxide) , and five xanthophylls (0‒50 μM) were pre-incubated for 5 min at 37 °C. The reaction was initiated by addition of 10 µL 5 mM UDPGA  followed by incubation for 120 min at 37 °C in a shaking water bath. Reactions were stopped by addition of 50 µL of ice-cold acetonitrile containing chlorpropamide (300 ng/mL) as an internal standard, and they were chilled and centrifuged . An aliquot of 5 μL of supernatant was injected into an LC-MS/MS system. Additionally, identical parallel incubation samples containing a known UGT2B15 inhibitor amitriptyine (0–200 μM) was included as a positive control for UGT2B15. All incubations were performed in triplicate, and mean values were used for analysis.The inhibitory effects of AS, βC, CA, LU, and ZE on UGT2B15 were assessed in recombinant human UGT2B15 supersomes, as previously described ,32. Brie50 value, the inhibitor constant (Ki) values of the βC for UGT1A1 were determined. Briefly, β-estradiol, a specific substrate for UGT1A1, was incubated with βC or nilotinib, a well-known UGT1A1 inhibitor. For determination of Ki values, β-estradiol concentrations used were 5, 10 and 20 μM. The concentrations of nilotinib and βC were as follows: 0–2 μM and 0–50 μM, respectively. All incubations were performed in triplicate, and the mean values were used for the analysis. Other procedures were similar to those of the reversible inhibition studies.Based on the lowest ICd-glucuronide (UGT1A3), trifluoperazine-N-glucuronide (UGT1A4), propofol-O-glucuronide (UGT1A9), zidovudine-5′-glucuronide (UGT2B7), and 4-methylumbelliferyl glucuronide (UGT2B15); an Agilent Poroshell 120 EC-C18 column  was used for quantification of serotonin-O-glucuronide (UGT1A6). The isocratic mobile phase consisted of distilled water (A) and acetonitrile containing 0.1% formic acid (B) at a ratio (v/v) of 10:90 for chenodeoxycholic acid 24-acyl-β-d-glucuronide, 30:70 for trifluoperazine-N-glucuronide and zidovudine-5′-glucuronide, 50:50 for propofol-O-glucuronide and 4-methylumbelliferyl glucuronide, 65:35 for β-estradiol-3-glucuronide, and 90:10 for serotonin-O-glucuronide, at a flow rate of 0.5 mL/min. The total run time was 4.0 min per sample for each metabolite, with the exception of serotonin-O-glucuronide (6.0 min). Peak areas for all of the analytes were automatically integrated using Analyst software .The metabolites of the seven UGT-selective substrates were analyzed using an API 3200 triple quadrupole mass spectrometer  equipped with an Agilent 1260 HPLC system  operating in electrospray ionization interface mode, as described previously . ChromatThree-day validations were performed to confirm the effectiveness of the LC-MS/MS system for determination of each of the six UGT-selective substrate metabolites in microsomal incubation mixtures, and 4-methylumbelliferyl glucuronide in recombinant UGT2B15 supersomes. The calibration standards were prepared at six concentrations (0.01–50 µM for each metabolite) in blank microsomal or recombinant UGT2B15 incubation mixtures. We found that the precision (≤13.7%) and accuracy (87.1%–111.7%) values were within acceptable ranges; our method was thus both reliable and reproducible.50 values, the activities of each UGT isozyme in the presence of different concentrations of the five xanthophylls were compared with those of negative control incubations . The IC50 values were calculated via nonlinear least-squares regression analysis from plots of the logarithms of inhibitor concentration vs. the residual percentages of activities after inhibition, using WinNonlin . The apparent inhibitory constants  were estimated from fitted curves using the WinNonlin software. Inhibition data were fitted to different models of enzyme inhibition  using nonlinear least-squares regression analysis (WinNonlin software). The most appropriate inhibition model was selected based on goodness-of-fit criteria determined via visual inspection, by calculating correlations of determination , and using the corrected Akaike′s Information Criterion. For visual inspection, data were presented as Dixon plots.To determine IC


Context: Alzheimer’s disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions.Objective: This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta1-42 (Aβ1-42) induced cytotoxicity and IKKβ activity, respectively.Materials and methods: Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 μM) before treatment with Aβ1-42 (IC30 10 μM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKβ inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects.Results: Celastrol (1 μM) inhibited Aβ1-42 (10 μM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKβ as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol.Discussion and conclusion: The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKβ activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition. Alzheimer’s disease (AD) is the most common form of dementia affecting the elderly population. The extracellular accumulation of amyloid beta (Aβ) peptide deposits (neuritic plaques) and intracellular neurofibrillary tangles (NFT) are the characteristic microscopic lesions observed in AD brains  complex is the key enzyme in the activation of NF-κB. The IKK complex consists of three subunits: two catalytic subunits (IKKα and IKKβ) and a regulatory subunit IKKγ also termed as NEMO Gilmore . These pTripterygenium wilfordii), is a potent inhibitor of IKK-NF-κB signalling. Recent evidence has also shown celastrol as a promising therapeutics in models of AD, amyotrophic lateral sclerosis, Parkinson's disease and Huntington’s disease  and grown in DMEM  supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics  . IMR-32 cells were plated on 10 mm cell culture dishes coated with laminin . After 24 h of seeding, cells were differentiated with retinoic acid  to the final concentration of 10 μM for 7 days.4 cells/well in 200 μL of DMEM containing 10% FBS and incubated overnight. To study the effect of celastrol on Aβ1-42 induced cell damage, cells were pre-incubated with celastrol (1 μM)  for 12 h and then Aβ1-42 (10 μM)  was added to the medium for additional 24 h. A control (untreated) containing serum-free medium was also evaluated. Then plates were incubated with MTT solutions (0.5 mg/mL) for 3 h at 37 °C. The formazan was dissolved in 150 μL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 550 nm using microplate reader . Cell viability was expressed as a percentage of untreated cells.Differentiated IMR-32 cells were seeded in 96-well plates at a density of 1 × 101-42, cells were lysed by the addition of cold RIPA buffer  and fresh protease inhibitor cocktail, and cell lysate was centrifuged at 14,000 rpm at 4 °C for 20 min. The supernatant was harvested and analyzed for protein content using BCA method . Protein was denatured in sample buffer, then separated on 12% SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked for 2 h at room temperature with Tris-buffered saline  containing 5% non-fat milk. The blots were washed three times with TBST  and incubated with p-IκBα and p-p65 primary antibodies  (1:1000 dilutions) at 4 °C overnight. The blots were incubated for 1 h at room temperature with secondary antibodies (1:5000 dilutions), and detected by ECL detection reagent. To ensure that equal amounts of sample protein were applied for electrophoresis, GAPDH was used as an internal control. Densitometry analysis was done using Image lab™  software.The cells were seeded in 10 mm Petri plates in 1.5 mL of DMEM containing 10% FBS overnight. After treatment with celastrol and Aβhttp://www.acdlabs.com, Advanced Chemistry Development, Inc. Toronto, Ontario, Canada). These 2D structures were converted into three dimensional (3D) structures format by importing into Accelrys Discovery Studio 3.5 visualizer (DS 3.5)  and saved in .mol format for each compound. In addition, these molecules were optimized using the Conjugate Gradients method . This protein structure was determined by X-ray crystallography with 2.83 Å resolution   was used to identify pharmacologically active molecules from celastrol dataset. Also, range of acquired drug-like properties of these compounds was compared with those 95% of the known drugs. The physiochemical and pharmacokinetic properties predicted in this study included serum protein binding (QPlogKhsa), logP for octanol/water (QPlogpo/w), blood brain barrier level (QPlogBB), and human oral absorption in gastrointestinal tract (QP %) . The QikProp module encoded in Schrodinger program  Harvey . Further) Harvey .A computational program called Prediction of Activity Spectra for Substances (PASS) was used for predicting possible biological activity . Differences between the groups were assessed by Student’s In vitro studies have shown that Aβ directly activates microglia and provokes the secretion of pro-inflammatory cytokines, including IL-1β, IL-6, TNFα (Lucin & Wyss-Coray Alzheimer’s disease (AD) is characterized by an extracellular amyloid-β (Aβ) deposition, which activates microglia, triggers neuroinflammation and involves neuronal death and cognitive deficits  . The co-crystallized known inhibitor KSA was utilized as control ligand and its binding region (ATP binding pocket located at the hinge loop) with IKKβ was considered as favourable region for inhibition.The analogues chosen for this study were modified either at C-20 carboxylic acid or at A/B rings  of celastrol. Although A/B ring modification offered more active analogues, the intact quinone methide moiety in the A/B ring was reported to be important for both cytotoxicity and neuroprotection , the central ubiquitin-like domain, ULD (310–404), and the C-terminal dimerization domain, SDD (408–664). This hinge region (residues 95–100) of KD included three active site residues: Glu97, Tyr98 and Cys99 and these residues were critical for hydrogen-bonding interactions with inhibitors. Celastrol formed two hydrogen bonds with Glu97 and one with Met96, gatekeeper residue which controls the access of inhibitor to binding pocket. An additional hydrogen bond with Asn150 and multiple hydrophobic interactions with Gly loop (residues 20-30) and activation loop (residues 166-194) greatly stabilizes the celastrol IKKβ interaction with the binding score of −10.56 kcal/mol . The ana28 formed one hydrogen bond with Tyr98. Compound 15 formed two hydrogen bonds (Glu97 and Cys99 each) with highest binding energy of −10.79 kcal/mol. The bindings were also stabilized with multiple nonbonding interaction with the activation loop residues. These compounds were further analyzed for drug like properties and PASS prediction.Among the selected analogues, seventeen were A/B ring modified while eight were C-20 modified. Thus, analogues were interacted with IKKβ irrespective of intact or modified quinone methide moiety. Twenty-two analogues specifically formed hydrogen bonding with Cys99 residue and compound Despite celastrol has good pharmacological activities, poor solubility and cytotoxicity, it is of major concern to be used as a drug. Hence, in this study, we examined the drug likeness of selected celastrol analogues, implementing computational predictive methods, which inferred to possess anti-inflammatory property and also possible candidates for treating dementia.2 and 34. Eight analogues showed one violation but they can be considered for the drug development as they are derived from natural products (Ganesan Supplementary Tables S2 and S3.Qikprop module from Schrodinger program was utilized to determine the drug-like behaviours of celastrol compounds. The results were evaluated based on their pharmacokinetic properties such as serum protein binding, octanol/water partition coefficient, CNS activity, blood brain barrier level and human oral absorption in gastrointestinal tract. Among these, BBB and CNS, are the most important descriptors in Alzheimer’s drug discovery (Hou et al.  Ganesan . The det2 and 34 showed high Pa value for dementia treatment. The results were shown in Supplementary Table S4. Collectively, compound 15 has the highest binding affinity, CNS activity and more drug likeness than parent celastrol. Although this study is an initial screening using in silico approach, further studies are needed to confirm the potency of compound 15 and other celastrol analogues for IKKβ inhibition and neuroprotection.Recently, several computational tools were used to identify potential targets from natural product molecules (Lagunin et al. Overall, IKKβ is an attractive anti-inflammatory target in neurodegenerative diseases. Celastrol, a known NF-κB inhibitor modulates NF-κB signalling through inhibition of IKKβ. The present study revealed the binding pattern of celastrol and its analogues within the active site of IKKβ. Molecular-docking analysis displayed 25 celastrol molecules located well within the ATP-binding site. Each compound formed at least one hydrogen-bond interaction with active site residues Glu97, Tyr98 and Cys99. Non-bonded interactions were also seen with activation loop residues stabilizing the ligand and IKKβ interaction. Analysis of drug-like properties demonstrated few compounds were capable of crossing BBB, non-toxic and obeyed softened Lipinski rule of 5. The biological property prediction revealed that celastrol anti-neuroinflammatory and neuroprotective properties have been retained in its analogues. Further experimental studies are needed to validate the IKK inhibition and neuroprotection of selected celastrol analogues.


Treatment with ultra-therapeutic doses of HI or glargine (p < 0.05) increased the expression of Ki-67 in thyroid cells. The results demonstrated that therapeutic doses of glargine have a longer-lasting hypoglycemic control than HI. Based on the results, HI or glargine did not stimulate thyroid cell proliferation at therapeutic doses, but high doses did.Our aim was to investigate whether human insulin (HI) or insulin glargine treatment could promote the proliferation of thyroid cells and determine the association between type 2 diabetes and thyroid disease. Rats were treated with different doses of HI and insulin glargine. Plasma glucose and the phosphorylation levels of the insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (ERK1/2) were measured. A total of 105 rats were randomly assigned to three groups as follows: control group, HI group, and glargine group. Both drugs promoted the phosphorylation of IR, Akt, and ERK1/2 in a dose-dependent manner ( Over the past few decades, it has emerged that diabetes increases the risk of many types of cancer, including thyroid cancer, colon cancer, pancreatic cancer, breast cancer, bladder cancer, and non-Hodgkin lymphoma . Among tin vitro experiments  elevated levels of insulin, insulin resistance, insulin injections, or the use of sulfonylurea drugs; (2) increased BMI; (3) increased thyroid-stimulating hormone levels; 4) chronically elevated blood sugar and triglyceride levels; or 5) vitamin D deficiency  were housed in temperature-controlled environments and fed with standard chow n = 35 animals per group). Each treatment group was sub-divided into seven sub-groups, according to the time points; 0, 15, 30, 45, 60, 90, and 120 min (n = 5–10 per sub-group). Refer to Emily Jane Gallagher for a description of the grouping method (n = 5–10). Two weeks before testing, all animals are acclimated to the feeding, injection and fasting protocol for 2 h before injection, but the water was injected in lieu of drug. Injectable drugs were prepared immediately before administration. HI  was diluted with saline, while glargine  was diluted with PBS (pH = 4). Drugs were diluted in 100 μl 0.9% saline or PBS according to the following formula: weight (kg) of rat × experimental dose  × 10 μl (glargine or human insulin) = mass of drug.Rats were randomly assigned to one of three treatment groups after weighing: control group, HI group, or glargine group . The tissue was cut using ophthalmic scissors and homogenized with a tissue homogenizer on ice at 1,500 r/min. After mixing for 30 min, tissue homogenates were centrifuged at 12,000 r/min for 15 min at 4°C. The supernatant was collected.The protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto polyvinylidene fluoride membranes . All specific antibodies were purchased from Cell Signaling Technology. After blocking in Tris buffered saline and Tween 20 (TBST) containing 5% non-fat dried milk for 2 h, the membranes were incubated with specific antibodies in the following order: IR , pIGF-1R , IGF-1R , pErk1/2 , pAkt , Akt , β-actin antibodies . After 3 washes, the blots were incubated with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 2 h, and visualized with ECL Plus chemiluminescence reagent kit  by being exposed to an autoradiographic film . Protein expression levels were quantified using Quantity One software.Samples were incubated with magnetic beads under rotation for 2 h at 4°C, after which they were washed with ice-cold RIPA buffer. Antigens were eluted using loading buffer, and then boiled at 96°C for 5 min. The supernatant was collected after centrifugation.Antigen retrieval was performed with high temperature and high pressure using citrate buffer . Harris hematoxylin was used to stain the nuclei, and ethanol was used for dehydration. The horseradish peroxidase (HRP)-positive cells appeared yellow, with brown granules in the cytoplasm and cell membrane. Image-Pro Plus 6.0 software was used to select a region that acted as a uniform standard to evaluate the positive photos.P < 0.05 were considered significant.Data were analyzed using one-way ANOVA, as appropriate. All statistics and data analysis were performed using SPSS21.0 software. Data are presented as the mean ± SD, and p < 0.05). The difference between HI and glargine group was significantly different at 15 min and 45 min time-points (p < 0.01). During the experiment, rats displayed normal levels of activity and no rats died after drug or saline injection.Wistar rats were fasted for 2 h before treatment. Blood sugar levels after three treatments were comparable to the levels prior to drug treatment: 5.92 ± 0.25, 5.85, and 5.97 ± 0.23 mmol/L . SubcutaWistar rats were fasted for 2 h, and then were injected with 1 U/kg of HI or glargine. Previous studies by Hansen et al.  and TennThe phosphorylation level of IR in thyroid cells was slightly altered after treatment. In HI group, phosphorylation level peaked at 2.26 ± 0.57-fold change at 15 min, compared to the level of onset, and then decreased rapidly. Phosphorylation levels were close to the limit of detection at 60 min post-injection . Phospho*p < 0.05, **p < 0.01, ***p < 0.001). After 30 min, Akt phosphorylation levels were 1.2–2.3 higher in glargine group than in HI group.Rats were fasted for 2 h, after which they were injected with 1 U/kg of HI or glargine. The phosphorylation level of Akt was measured within 120 min. Fifteen min after injection of HI, phosphorylation of Akt reached a peak level, then fell to 19% of the maximum . In glar*p < 0.05, **p < 0.01). At 30 min post-injection, ERK1/2 phosphorylation levels were about 7.6–6.2 times higher in glargine group than in HI group.The rats were fasted for 2 h, then injected with 1 U/kg of HI or glargine, and the phosphorylation levels of ERK1/2 were measured. At 15 min post-injection of HI, phosphorylation of ERK1/2 reached a maximum value of 6.39 ± 1.97-fold (which was similar to Akt), then fell and stabilized at lower levels after 30 min, and eventually dropped to 4.63 ± 0.78-folds  after 12After 2 h of fasting, Wistar rats were injected with 1, 12.5, 50, or 200 U/kg of HI or glargine. At 60 min after injection, the average blood sugar level was 1.8 mmol/L, and there were no obvious hypoglycemic effects, with all rats surviving.***p < 0.001) in terms of IR and IGF-1R phosphorylation. In addition, there was a dose-dependent effect of both insulin treatments on the phosphorylation level of IGF-1Rβ/IRβ (p*** < 0.001). At each dose, protein phosphorylation was higher in glargine group than in HI group . Increasing doses of insulin did not have any saturating effect on IGF-1Rβ/IRβ receptors' phosphorylation; even at the highest dose of 200 U/kg, phosphorylation was significantly higher than the other doses, especially in glargine group.At each dose, the effects of HI or glargine treatments were significantly different from the control group . Rats injected with high doses of glargine (50 and 200 U/kg) had a significantly higher phosphorylation compared to the control group . IR phosphorylation level increased with the drug dose. At the same doses, there were significant differences between HI and glargine groups . The phosphorylation level of IR was remarkably (1.71- to 2.3-fold) higher in HI group compared to glargine group at doses above 12.5 U/kg. These findings demonstrate that HI acts on the insulin receptor in thyroid tissue.The IR phosphorylation levels measured at each dose are shown in *P < 0.05, **P < 0.01, ***P < 0.001). The phosphorylation levels of IGF-1R increased with insulin treatment dose, with the exception of the highest HI dose, which reduced the phosphorylation level relative to lower doses. At the same dose, HI and glargine produced significantly different effects on IGF-1R phosphorylation (###P < 0.001). At lower doses (1 and 12.5 U/kg) HI produced a higher phosphorylation than glargine. At higher doses (50 and 200 U/kg) glargine caused higher IGF-1R phosphorylation than HI (~1.48- and 1.92-fold higher) or blank control (7.02- to 7.12-fold higher). We conclude that high doses of glargine increase the phosphorylation of insulin receptors in thyroid tissue.According to WB analysis, high doses of glargine caused higher increases in IGF-1Rβ/IRβ phosphorylation than HI. No obvious dose-dependent changes in the phosphorylation level of IR were found in the precipitation analysis. Next, we measured the dose-dependent effects of the drugs on IGF-1R phosphorylation . The IGF*p < 0.05, **p < 0.01, ***p < 0.001), and the effects followed a dose-dependent pattern. However, it is interesting to note that Akt phosphorylation was similar after treatment with 200 U/kg dose and 50 U/kg dose of HI (2.80 ± 0.10 and 0.10 ± 0.12), indicating the saturation of Akt phosphorylation. At 12.5 and 200 U/kg doses, pAkt levels were higher after glargine treatment compared to HI treatment . At the highest dosage (200 U/kg), Akt phosphorylation was 3.8 times higher in glargine than in the control group.Having detected changes in the phosphorylation of IR and IGF-1R, we explored changes in phosphorylation of the downstream signaling proteins Akt and ERK1/2. As shown in **p < 0.05, **p < 0.01, ***p < 0.001). At all dosages, glargine caused significantly larger increases in pERK than HI (###p < 0.001). From lowest to highest dose, glargine treatment increased pERK by 1.95-, 2.39-, 1.34-, and 1.22-fold more than HI.In general, pERK increased with the dose of insulin treatment . ERK1/2 n = 10 animals per dose). The highest dose (200 U/kg) had lethal effects; and 40% of rats in this group died. We performed an immunohistochemical examination of Ki-67, Akt, and ERK1/2 in thyroid tissue at 4 weeks after treatment. The results are shown in To explore the effect of high doses of HI and glargine on thyroid cell proliferation, we divided female Wistar rats into HI and glargine treatment groups, and tested the effect of four different doses of the drugs  4 weeks after injection  is named after its histidine substituted for aspartic acid in the No. 10 B chain. Insulin B10 aspart has higher affinity for IR and IGF-1R than human insulin, and the duration of binding to IR is longer. In addition, insulin B10 aspart has stronger proliferation-promoting effects on mammalian cells than human insulin. In 1992, Drejer  tested teriments –24. In ceriments , 27. Theeriments .In vitro experiments have shown that HI can promote the proliferation of thyroid follicular cells and human endometrial cells, and can increase malignant transformation of human endometrial cells (It is worth noting, however, that high doses of HI increase thyroid cell proliferation. al cells , 30. Resal cells . IR-A exal cells . Pandinial cells  found thal cells  postulatal cells  showed tin vivo study on the effect of HI treatment for 4 weeks on thyroid tissue revealed a significant increase in pERK1/2, but no effect on pAkt level. Thus, Akt phosphorylation may not be the primary signaling pathway that mediates thyroid cell proliferation after HI administration, and pERK1/2 may play a greater role, however further studies are needed to clarify this hypothesis.As mentioned above, HI and its analogs activate the PI3K/Akt and MAPK signaling pathways. PI3K/Akt signaling increases the number of cells by inhibiting cell apoptosis, which plays a key role in modulating the effects of insulin and IGF-1 on metabolism and mitosis . MAPK siin vivo and in vitro. It also suggests that clinicians should consider the possibility that insulin may stimulate thyroid cell proliferation during the treatment of diabetes.HI and glargine treatment produced a higher phosphorylation of ERK1/2 than control control, with glargine having slightly stronger effects compared to HI. According to insulin cell-signaling pathways, this could be due to the higher affinity between glargine and IGF-1R relative to IR , becausein vivo experiments show that therapeutic doses of glargine have a longer-lasting hypoglycemic effect than HI, and promote the phosphorylation of IR, Akt, and ERK1/2. High doses of HI and glargine can promote thyroid cell proliferation, while therapeutic doses of HI and glargine do not cause thyroid cell proliferation. HI may be associated with IR phosphorylation, while glargine may be associated with IGF-1 phosphorylation. Both receptors can activate PI3K-Akt and MAPK signaling pathways to promote proliferation.Our All datasets generated for this study are included in the manuscript and/or the supplementary files.This study was carried out in accordance with the recommendations of Zhejiang Chinese Medical University of guidelines of committee. The protocol was approved by the Zhejiang Chinese Medical University of committee.XS, KY, AS, ST, and DY substantially contributed to the conception and design of this study, drafted the work, gave final approval of the version to be published, and agreed to be accountable for all aspects of this work and ensured that issues pertaining to the accuracy or integrity of any part of this work are appropriately investigated and resolved. XZ and TC critically revised the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to test the efficacy of the ST as a hand-cleaning product. Two sets of laboratory tests, with 16 volunteers in each, were conducted to test the efficacy of different prototypes of the ST. Volunteers pre-contaminated their hands with nonpathogenic Escherichia coli. Comparisons were made between hand cleaning with the ST and handwashing with the reference soap, using a crossover design. Participants also completed a questionnaire about product perceptions. Three of the prototypes of the ST were more efficacious at removing E. coli from pre-contaminated hands than handwashing with soap . The ST prototypes used less water than handwashing with soap, were well accepted, and were considered preferable in communal settings. The ST has the potential to be a suitable complementary hand-cleaning product for humanitarian emergencies.Handwashing with soap reduces the transmission of diarrheal pathogens, but access to hand-washing facilities, water, and soap in humanitarian emergencies is limited. The SuperTowel Fecal–oral pathogens can cause diarrheal diseases, some respiratory infections, and many outbreak-related diseases . These diseases are a leading cause of preventable illness and death across all types of humanitarian crises.2 Handwashing with soap is one of the most effective preventative strategies for the control of fecal–oral pathogens. It has the potential to reduce diarrhea by up to 48%3 and reduce acute respiratory infections by up to 27%.4In the wake of an emergency, populations often find themselves displaced and living in crowded conditions, with poor sanitation and hygiene facilities and a limited amount of water. These conditions create an ideal environment for the spread of communicable diseases.6 This is partly because when water and soap are scarce, they are prioritized for tasks other than handwashing.9 Second, it is often difficult for NGOs to meet the demand of beneficiaries—particularly in protracted crises. The Sphere Standards10 recommend that in an emergency, one person should have 250 g of bathing soap per month and at least 15 L of water per day. In 2016, 65.6 million people were displaced11 from their homes because of conflict, persecution, and crises, and therefore, meeting the needs of all these people was a mammoth logistical task—particularly for a product like soap that is heavy (when ordered in bulk) and requires regular replacement.Despite this, access to hand-washing facilities, water, and soap in humanitarian emergencies is often limited. Those affected by emergencies typically rely on nongovernment organizations (NGOs) to distribute soap and water. However, this approach alone has not been found to increase hand-washing rates.12 Consider this typical example of a camp setting. Most camps have shared latrines, and alongside these, there are shared hand-washing facilities . Invariably, the soap that NGOs provide is rarely placed at these shared facilities5 because crisis-affected populations worry about it being stolen and used by others. Instead, if individuals wanted to wash their hands with soap after using the toilet, they would be required to walk to the toilet carrying their own bar of soap. On reaching the shared toilet, there is nowhere for these individuals to store the soap hygienically—perhaps it would be placed on the unclean floor. If they were then to wash their hands with soap after using the toilet, then this would mean that they would have to walk back to their shelter with the slimy bar of soap, meaning that their hands would still be covered with soap at the end of the process. Alternatively, people may try to wash their hands within their shelters—bringing contamination into the home environment. In practice, both options are inconvenient and impractical, so hands are normally just rinsed with water or not washed at all.5In many camp settings, hand-washing and toilet facilities are not designed in ways that make it easy for crisis-affected populations to always wash their hands with soap.bc,13 and there are records of it being used widely across history and cultures. Although it is an ideal hand-washing product in the vast majority of settings, the particular dynamics of humanitarian crises reduce its practicality and usability.Soap is thought to have been in existence since 2,800 www.realreliefway.com). It is a durable fabric with permanent antimicrobial bonding. The treated fabric must be dipped in water and then rubbed against the hands so that pathogens will be transferred to the fabric, where they will be killed. The antimicrobial technology does not involve toxic chemicals. Instead, it is achieved by long chains of carbon atoms attached to positively charged nitrogen atoms bonded to a silica layer of the fabric. The positively charged layer attracts negatively charged microbes , causing membrane disruption of the microbes. A fabric with the same antimicrobial bonding has already been tested and used as part of hospital linen and as part of reusable menstrual pads. Tests associated with these products have shown that the antimicrobial treatment was able to reduce 99.9% of Escherichia coli, Staphylococcus, Aspergillus brasiliensis, Aspergillus niger, Pseudomonas aeruginosa, Clostridium sporogenes, and Klebsiella pneumoniae and 99.3% of Candida albicans.14 These tests were based on the fabric directly being spiked with these pathogens. Tests also assessed whether the efficacy of the antimicrobial treatment declined with use, but no such decrease was observed.To overcome some of the limitations of soap, we wanted to develop and test the efficacy of an alternative soap product—the ST. The ST is a product developed by Real Relief  whether the ST could effectively remove microbes from hands and transfer them to the towel and 2) whether hand cleaning with the ST could result in an average log15 In selecting the materials for the second round of testing, we considered the water absorption and feel of the fabric (based on the assumption that the ST would have to feel nice when rubbed on the hands to be used) and explored different textures that could facilitate cleaning under fingernails (because nails are known to harbor pathogens).18Two rounds of laboratory testing were conducted on the ST. For the first round, we applied the antimicrobial bonding to a soft satin polyester/cotton fabric, similar to what had been used in hospitals for bed linen. We realized that the satin polyester/cotton fabric was likely to be poor at removing pathogens from hands and was not pleasant to rub on the skin. This led us to explore microfiber fabrics. Some microfiber fabrics have been found to remove 99% of bacteria from surfaces.19 which is designed to evaluate the ability of hand-wash agents to eliminate transient pathogens from volunteers’ hands without regard to resident microorganisms. This procedure is based on the “post-contamination treatment” of hands and involves the placement of the test organism (E. coli [ATCC 11229]) on the hands of test subjects, followed by exposure of the test product.To test the efficacy of the ST, we used an adapted protocol of the European Committee for Standardization (EN 1499)The studies were performed in the Department of Microbiology, Kelkar Education Trust’s (KET’s) Scientific Research Centre, Mumbai (India). The first test was conducted in December 2017 and the second set of tests in April 2018. The studies were approved by the London School of Hygiene and Tropical Medicine Ethics Committee and the KET’s Institutional Scientific & Ethics Committee.21 so removal was necessary so as not to affect the recovery pre- and post-values of the tests.Sixteen adult volunteers were selected for each of the laboratory test rounds. Written consent was obtained from all of them. Volunteers were students at nearby universities and were recruited by using poster advertisements that were placed within these nearby institutions. Eligible volunteers had to be male, older than 18 years; must have short fingernails with no artificial nails; must have no cuts or wounds on their hands; must have no history of drug allergies; and must not have taken any antibiotics in the last 2 weeks. All volunteers were physically examined before their involvement in the studies to ensure they were healthy and had healthy skin . Participants were asked to remove all forms of jewelry from their hands before handwashing. Rings are known to retain bacteria,2. In the first set of experiments, we tested the original ST (ST0) product. We used a crossover design in which the 16 volunteers were allotted randomly to two groups of the same size. One group applied the new test formulation (ST0) and the other a reference hand-washing product (Johnson & Johnson odor-free liquid soap) using a standardized procedure. In a consecutive run, the two groups reversed roles (crossover design). At the end of the whole series of runs, every subject had used each hand-washing product once.The STs used in the tests were produced by Real Relief and measured 25 cmThree new prototypes were created and tested against the same reference soap during the second round of tests. SuperTowel prototype 1 (ST1) was a cloth made out of terry towel fabric. The cloth is a microfiber, composed of 80% polyester and 20% polyamide. SuperTowel prototype 2 (ST2) was a cloth made of thin pique woven material. The cloth’s fiber composition is 80% polyester and 20% polyamide. SuperTowel prototype 3 (ST3) was made from the same material as ST1 but had one corner that was made from a scourer-like material. This was designed to act as a nail scrubbing pad. All the prototypes were treated to have the antimicrobial bonding. In the second set of tests, we used a Latin square design where 16 different sequences of the four treatments  were created beforehand. The sequences were allotted to individual volunteers by means of a number draw. This process meant that each volunteer used all of the hand-washing products once and did so in the order prescribed by the randomly selected sequence.E. coli (ATCC 11229) 8.3 × 108 cfu/mL. Excess of fluid was drained off, and the hands were air-dried for 3 minutes.The hands of each volunteer were washed with a non-medicated soap, dried (thoroughly with paper towels), and immersed up to the mid-metacarpals for 5 seconds with fingers spread apart in a contamination fluid containing nonpathogenic 22Immediately after drying, the fingertips and thumbs of the left and right hands were rubbed in separate Petri dishes containing 10 mL of tryptone soya broth (TSB)  for 60 seconds to assess the release of the test organism before treatment of the hands . The pre-value was estimated using the standard serial dilution method.23 (a diagram of the steps was given to them). Afterward, volunteers were asked to rinse their hands under running tap water for an additional 15 seconds and then air-dry them for 3 minutes (without the use of a machine).When testing the reference soap, volunteers were asked to use 5 mL of non-medicated liquid soap and 1 mL of water. They were asked to wash their pre-contaminated hands for 60 seconds by following the “WHO guidelines for handwashing when hands are visibly soiled”23 as a guide for cleaning their hands with the ST (this excluded the steps related to soap and water). In the case of ST3, the nail-cleaning procedure illustrated in picture 7 of the WHO guideline was performed by rubbing the fingertips on the nail scrubbing pad. Participants were not given guidance on how long they should scrub their nails within the 60 seconds. At the end of the 60 seconds, the volunteers’ hands were allowed to air-dry for 3 minutes (without a machine).The ST procedure was the same irrespective of the prototype being used. The process involved soaking the ST in water by submersing it completely in a bucket filled with tap water. The amount of water absorbed by the ST was recorded by means of weighing the towel before and after soaking. Participants rubbed their pre-contaminated hands with the wet ST for 60 seconds. They were asked to use steps 2–7 of the “WHO guidelines for how to wash hands when visibly soiled”A subsample of participants were video-recorded while completing the tests, and one of the authors (R. K.) was present for all of the tests to maintain standardization and monitor quality.22 After the procedure, the volunteers were given medicated soap to wash their hands.When participants’ hands were dry, the fingertips and thumbs of the left and right hands were rubbed in separate Petri dishes containing 10 mL of TSB  for 60 seconds to assess the release of the test organism after treatment of the hands . Post-values were determined using the dilution method.At the end of the second study, each volunteer was invited to complete an administered questionnaire, aimed at collecting information about the perceived feasibility of using the ST at different critical times for handwashing and identifying user preferences between the prototypes. The questionnaire was developed by the authors and was designed to provide initial insights into the product’s acceptability. The questions were not related to humanitarian contexts as none of the participants had been personally affected by crises. Instead, they explored the potential use of the ST within the home and in public settings.10 counts from the left and right hands of each subject were averaged separately, for both pre- and post-values. The arithmetic means of all individual log10 reduction values were calculated. The statistical analysis was performed with the statistical package STATA version 13.0 . The distribution of the data was assessed using kurtosis and skewness tests. Given that the data were not normally distributed, nonparametric tests were used. In the first set of experiments, Wilcoxon matched-pair signed-rank test was used to test for the difference between the first ST product and the reference soap. In the second set of experiments, as more than one product was compared, we used the Kruskal–Wallis test, and if evidence of a significant effect was observed, then we used post hoc pair-wise tests (Wilcoxon signed-rank tests) to assess the differences between specific pairs. The new product was considered to have the same efficacy as the reference product (soap) if the mean log10 reduction factor was not significantly smaller for the former than for the latter. Because of the confirmative nature of the test on this application, the level of significance is set at P = 0.05 and the test used is two-sided. The discrimination efficiency of the test procedure described has been set to detect a difference between the two mean log10 reduction factors of approximately 0.6 log10 at a power of 95%. This results in a sample size of N = 16 for each set of experiments.For both the reference soap and the ST products, logA total of 32 people participated in the tests: 16 in the first set of experiments and 16 in the second one. All were male, lived in an urban area, and ranged in age from 18 to 43 years (average age 21 years).10 pre-values was 7.6, and the maximum detectable log10 reduction observed in these experiments was 4.95. In the first set of experiments, we observed that use of the ST0 prototype resulted in a mean log10 reduction of 2.20 ± 0.57. For ST0, the reduction observed was significantly less effective than that with the reference soap . In the second set of tests, we observed differences in log10 reduction with the different ST products . A mean log10 reduction of 4.11 ± 0.47 for ST1, 3.84 ± 0.61 for ST2, and 3.71 ± 0.67 for ST3 was detected, which was significantly higher than that observed with the reference soap of 3.01 ± 0.63 . SuperTowel prototype 1 was more efficient in reducing bacteria than ST3 . The bacterial reductions from all tests are shown in The process of hand rinsing for 15 seconds after handwashing with the reference soap consumed 1.2 L of water. The ST prototypes absorbed on average 36 ± 9 mL (ST0), 318 ± 14 mL (ST1), 80 ± 4 mL (ST2), and 171 ± 15 mL (ST3) of water.In the questionnaire, the 32 participants were asked to compare the reference soap with the STs. These results are summarized in The first ST prototype (ST0) showed less efficacy in reducing bacteria from artificially contaminated hands than soap. Three new microfiber prototypes of ST were designed, and all showed better efficacy in removing bacteria than soap. SuperTowel Prototype 1 was the most efficacious.24Through our design workshop, we identified that the smoothness of the ST0 fabric (made from a satin cotton/polyester blend) probably prevented pathogens from being transferred from the hands to the towel. This led us to explore and include microfiber fabrics in the second round of testing. The second set of laboratory tests found that all three of the microfiber ST prototypes were more efficacious than handwashing with soap. Microfibers are produced by combining many very thin polyester and polyamide fibers into a single fiber during production. However, unlike conventional cloths, they have thousands of randomly arranged sharp-edged microfiber strands, which improve cleaning efficacy.25 This could explain why three of the ST prototypes were able to remove more bacteria from volunteers’ hands than soap. The added advantage of the ST in comparison with other hand-wiping materials is that the antimicrobial bonding technology ensures pathogens landing on the fabric are destroyed because of membrane disruption.Different studies have found that mechanical friction applied during handwashing plays a role in removing microorganisms adhering to the hands.The most effective prototype was ST1, which was made from a relatively thick and soft terry towel fabric. SuperTowel prototype 3 was made from the same fabric but included a corner made from scourer-like fabric, designed for cleaning nails. Although ST3 was found to be more efficacious than handwashing with soap, it was the least effective of the three microfiber prototypes. By re-watching the video recordings, we identified that when using ST3, participants prioritized scrubbing their nails on the pad and did not allocate as much of the 60 seconds to wiping the cloth over the rest of their hands. This likely explains the lower rates of bacteria removal. Although participants preferred using the ST3 prototype, these observed patterns of use and its reduced efficacy suggest that ST1 is a more reliable product.All four versions of the ST used much less water than handwashing with soap, indicating its potential usefulness in water-scarce settings. The questionnaire responses indicated that the ST would be preferable to soap in public environments or when outside the home. This supports the hypothesized use of this product during or in the wake of humanitarian crises because facilities are commonly shared or not always available. It also suggests that the product could be appealing in noncrisis settings, whenever handwashing with soap is not desirable or convenient.Moringa oleifera plant powder as a hand-cleaning product for potential use in domestic settings.26 We also do not know what size of bacterial log10 reduction would have a meaningful public health impact in domestic settings.28 A further limitation of this European Committee Standard is that it requires 60 seconds of hand cleaning with the reference soap and each of the test products. Commonly, in real-world settings, people wash their hands for just 5–10 seconds.29There were several limitations of our study. To be comparable with other studies, the tests followed an adapted version of the European Committee Standard for evaluating hand antiseptic agents. However, these standards are principally designed for assessing the efficacy of new hand-wash or hand-rub agents to be used in health-care settings. At present, there is a lack of clarity about whether this study protocol is also appropriate for domestic-focused efficacy studies like this one. The same protocol has previously been used to test the efficacy of The studies described here provide a useful proof of concept, under laboratory conditions, but further testing could be carried out to explore the comparable efficacy of the ST when used for a more realistic hand-washing duration or on hands that were naturally contaminated. It is likely that future users of the ST would have to carry it around on their person. Therefore, there would be value in assessing whether the product can achieve comparable efficacy if used when moist rather than soaking wet. Third, it would be interesting to assess whether the ST can achieve equivalent efficacy if a person’s hands are visibly dirty  or oily. Given the process by which the antimicrobial treatment kills pathogens, it is biologically plausible that the ST could be used with water of any quality . However, it would be useful to verify this through laboratory-based efficacy tests with a range of water qualities and field-based acceptability studies to determine whether people would feel comfortable with gray water being used for hand cleaning in this way.31 For studies to do with hygiene, this is important because of gendered differences in real-world hand-washing practices (women tend to wash their hands more frequently and more thoroughly than men)39 and because in the Indian context, women are primarily responsible for preparing food and may also have different attitudes toward handwashing after defecation. In this study, it would have been particularly useful to get the opinions of women as part of the questionnaires.For practical reasons, we excluded women from our study. Indian regulations require that if any new product is to be tested on women, then they must undergo pregnancy screening. We did not want to subject female participants to this and, therefore, decided to have male participants only. The lack of female participants is unlikely to have had an impact on the primary results, given that hand cleaning was performed in a prescribed and standardized way during these studies. However, scientific research should always aim to include women equally.Clostridium) from hands and transfer them to the ST without killing them . This inability to kill nonencapsulated viruses is not unique to the ST. For example, norovirus, rotavirus, and Clostridium difficile have been found to be resistant to typical infection control measures  and persist in the environment and on hands and surfaces for a long time.42The antimicrobial treatment used on the ST can effectively kill all bacteria, fungi, and protozoa; however, it only kills viruses that are encapsulated. It is likely that the microfiber material used in the ST will remove diarrhea-causing nonencapsulated viruses (such as norovirus and rotavirus) and bacterial spores  where handwashing with soap is not a feasible, convenient, or desirable option. Our findings indicate that the ST is appropriate for use in contexts where soap and water availability is scarce or irregular, and where sanitation and hygiene facilities are not available or are shared and, therefore, considered less desirable to use. Given that the ST is more lightweight than soap and longer lasting, we envisage that the ST would also be beneficial to humanitarian actors working in areas where logistical and security issues make regular hygiene product distribution challenging. We recommend that further testing should be carried out to assess the efficacy of the ST under conditions that more closely mirror real-world hand-washing practices. Further development of the ST should be carried out in consultation with crisis-affected populations to ensure the product meets their needs.These studies have demonstrated that under controlled conditions, the microfiber prototypes of the ST were more efficacious at removing nonpathogenic