Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification.

It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.

Acid and alkali-resistant fabric-based triboelectric nanogenerator for self-powered intelligent monitoring of protective clothing in highly corrosive environments.

The corrosion of materials severely limits the application scenarios of triboelectric nanogenerators (TENGs), especially in laboratories, chemical plants and other fields where leakage of chemically corrosive solutions is common. Here, we demonstrate a chemical-resistant triboelectric nanogenerator (CR-TENG) based on polysulfonamide (PSA) and polytetrafluoroethylene (PTFE) non-woven fabrics. The CR-TENG can stably harvest biological motion energy and perform intelligent safety protection monitoring in a strong corrosive environment. After treatment with strong acid and alkali solution for 7 days, the fabric morphology, diameter, tensile properties and output of CR-TENG are not affected, showing high reliability. CR-TENG integrated into protective equipment can detect the working status of protective equipment in real time, monitor whether it is damaged, and provide protection for wearers working in high-risk situations. In addition, the nonwoven-based CR-TENG has better wearing comfort and is promising for self-powered sensing in harsh environments.

Facile and foldable point-of-care biochip for nucleic acid based-colorimetric detection of murine norovirus in fecal samples using G-quadruplex and graphene oxide coated microbeads.

Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.

Differences in bio-incompatibility among four biocompatible dialyzer membranes using in maintenance hemodialysis patients.

BACKGROUND: Following the introduction of modified cellulosic and then synthetic membrane dialyzers, it was realized that the dialyzer bio-incompatibility depends on the membrane composition. We designed a prospective, randomized, cohort study of 6 months to determine several parameters of biocompatibility in maintenance hemodialysis (MHD) patients treated with four different membrane dialyzers. METHODS: There were 60 MHD patients enrolled in the study. In baseline, synthetic low-flux dialyzer, polysulfone (PS) membrane was used in all patients for at least 3 months. Then the patients were randomly divided into three groups according to different dialyzer membranes. Synthetic high-flux dialyzer group, polyethersulfone membrane, cellulose triacetate (CTA) high-flux membrane, and synthetic low-flux dialyzer, polymethylmethacrylate (PMMA) membrane were used in 6 months. A new dialyzer was used for each study treatment, and there was no dialyzer reuse. The biocompatibility markers and solutes removal markers were detected repeatedly at different time points. RESULTS: The blood levels of highly sensitive C reactive protein, interleukin (IL)-1ß, and interleukin (IL)-13 showed no difference among different groups at al time points. However, the blood complement levels and white blood cell counts were significantly different among three groups. When the dialyzers changed from PS to PMMA membrane, C3a levels and white blood cell counts changed significantly (p < 0.05). Moreover, the changes of C5a levels were significantly different between group CTA and group PMMA in month 3 (p < 0.05). CONCLUSION: There were much more differences on bio-incompatibility among different dialyzer membranes.

Electrospun Nanofibers Embedded with Copper Oxide Nanoparticles to Improve Antiviral Function.

Many studies on anti-bacterial/antiviral surfaces have been conducted to prevent epidemic spread worldwide. Several nanoparticles such as those composed of silver and copper are known to have antiviral properties. In this study, we developed copper oxide (CuO) nanoparticle-incorporated nanofibers to inactivate or remove viruses. The CuO nanoparticle-incorporated nanofiber was fabricated with a hydrophobic polymer-polyvinylpyrrolidone (PVP)-using electrospinning, and CuO nanoparticles were exposed from the PVP polymer surface by etching the nanofiber with oxygen plasma. The fabrication conditions of electrospinning and oxygen plasma etching were investigated by scanning electron microscopy (SEM), and field emission transmission electron microscopy (FETEM)/ energy dispersive spectrometry (EDS). H1N1 virus was utilized as the target sample and quantified by RT-qPCR. The antiviral efficacy of CuO nanoparticle-incorporated nanofibers was compared against bare CuO nanoparticles. Overall, 70% of the viruses were inactivated after CuO nanoparticle-incorporated nanofibers were incubated with 10² pfu/mL of H1N1 virus solution for 4 h. This indicates that the developed CuO nanoparticle-incorporated nanofibers have noticeable antiviral efficacy. As the developed CuO nanoparticle-incorporated nanofibers exerted promising antiviral effects against H1N1 virus, it is expected to benefit global health by preventing epidemic spread.

Discrimination and isolation of the virus from free RNA fragments for the highly sensitive measurement of SARS-CoV-2 abundance on surfaces using a graphene oxide nano surface.

It is highly important to sensitively measure the abundance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on various surfaces. Here, we present a nucleic acid-based detection method consisting of a new sample preparation protocol that isolates only viruses, not the free RNA fragments already present on the surfaces of indoor human-inhabited environments, using a graphene oxide-coated microbead filter. Wet wipes (100 cm2), not cotton swabs, were used to collect viruses from environmental surfaces with large areas, and viruses were concentrated and separated with a graphene oxide-coated microbead filter. Viral RNA from virus was recovered 88.10 ± 8.03% from the surface and free RNA fragment was removed by 99.75 ± 0.19% from the final eluted solution. When we tested the developed method under laboratory conditions, a 10-fold higher viral detection sensitivity (Detection limit: 1 pfu/100 cm2) than the current commercial protocol was observed. Using our new sample preparation protocol, we also confirmed that the virus was effectively removed from surfaces after chemical disinfection; we were unable to measure the disinfection efficiency using the current commercial protocol because it cannot distinguish between viral RNA and free RNA fragments. Finally, we investigated the presence of SARS-CoV-2 and bacteria in 12 individual negative pressure wards in which patients with SARS-CoV-2 infection had been hospitalized. Bacteria (based on 16 S DNA) were found in all samples collected from patient rooms; however, SARS-CoV-2 was mainly detected in rooms shared by two patients.

Effect of long intermittent hemodialysis on improving dialysis adequacy of maintenance hemodialysis patients.

BACKGROUND: With the increase in hemodialysis (HD) patients, the blood dialysis patient's quality of life (QoL) and long-term survival are still a challenge for clinicians. Recent studies have found that most of the HD patients have sleep disorders, which have a certain correlation with long-term survival and QoL. But there are few studies of Chinese in this field. This study aimed to investigate whether increasing the dialysis dose can improve sleep quality, so we treated HD patients on long intermittent hemodialysis (LIHD). METHODS: Forty patients who were treated by conventional HD at the Beijing Friendship Hospital Blood Purification Center were offered the option of LIHD. The patients' laboratory data, medication use, and questionnaire answers were analyzed. Conventional HD was delivered thrice weekly with 4 hours per treatment, and LIHD was delivered thrice weekly with 8 hours per treatment. The study lasted 6 months. Questionnaires included sleep quality survey and QoL SF-36; the former includes the Athens Insomnia Scale, Pittsburgh Sleep Quality Index (PSQI), and Epworth Sleepiness Scale (ESS). RESULTS: After conversion to LIHD the dialysis efficiency (Kt/V) significantly increased than before (P < 0.05) and clearance rate of urea nitrogen also increased from 67 to 78% (P < 0.01). After conversion, median values for Hb increased from 108.95 to 126.55 g/L (P < 0.01); albumin increased from 38.85 to 40.05 g/L (P < 0.01). Phosphorus decreased from 2.69 to 1.54 mmol/L (P < 0.01), but there was no alteration in blood calcium; phosphorus and calcium-phosphate product levels were under more control, but parathyroid hormone (iPTH) level did not change after conversion to LIHD. After conversion, blood pressure (BP) was better controlled than before and the mean number of antihypertensive drugs prescribed declined from 2.9 to 0.5 (P < 0.01). There was a significant reduction in the use of erythropoietin-stimulating agent of 5250 U/w (P < 0.01). Sleep quality significantly improved in the 2 months after conversion to LIHD, and the PSQI score decreased from 10.80 to 5.45 and the ESS score decreased from 12.05 to 5.30 (P < 0.01). However, sleep quality started to decline after 2 months on LIHD. QoL SF-36 score increased from 410.92 to 592.53 (P < 0.01). CONCLUSION: LIHD offers an effective improvement in dialysis adequacy for Chinese maintenance HD patients, but it improves sleep quality only briefly which may be related to loss of serum calcium and parathyroid dysfunction.

Improvement and influencing factors of blood pressure control by nephrologist referral in chronic kidney disease patients in China: a cohort study.

PURPOSE: Hypertension is an independent risk factor for mortality in chronic kidney disease (CKD) and is suboptimally controlled worldwide. Therefore, this study aimed to examine the rate of BP control and the main barriers to achieving target BP, according to K/DOQI guidelines, in China. METHODS: We performed a single-center, prospective cohort study. Two hundred and sixty CKD patients were referred by general physicians to nephrologists, and their BP was treated in accordance with K/DOQI guidelines for a 1-year follow-up. We evaluated improvement of BP target achievement and factors affecting BP control. We defined "not-at-goal" as persistence of systolic BP ≥ 130 mmHg and/or diastolic BP ≥ 80 mmHg after 1 year. RESULTS: The BP decreased from 138 ± 12/84 ± 7 mmHg at baseline to 124 ± 13/73 ± 7 mmHg after 1 year. The rate of achieving the BP goal (<130/80 mmHg) increased from 25.4 to 61.5 %. The decrease in BP was associated with a significant reduction of proteinuria (median, 0.14 vs 0.06 g/24 h; P < 0.05). Logistic regression analysis identified proteinuria levels ≥1.0 g/24 h (odds ratio [OR]: 5.21; 95 % confidence interval [CI]: 1.37-19.77) and high basal systolic BP (OR: 2.17; 95 % CI: 1.25-3.77) and diastolic BP (OR: 6.62; 95 % CI: 2.03-21.60) as independent predictors of not-at-goal BP. Higher educational level was independently associated with at-goal BP (OR: 0.21; 95 % CI: 0.06-0.78). CONCLUSIONS: In CKD patients, BP control is poor when managed by general physicians and may be improved after nephrologist referral. High basal BP and proteinuria levels ≥1.0 g/24 h are the main barriers that preclude the optimal control of BP.

Left ventricular mass index and aortic arch calcification score are independent mortality predictors of maintenance hemodialysis patients.

To analyze predictive factors for all-cause mortality, cardiovascular (CV) mortality, nonfatal CV events (CVE) in maintenance hemodialysis (MHD) patients, and to compare the effects of standard hemodialysis (HD) and online hemodiafiltration (HDF) on these factors and outcomes. A total of 333 MHD patients were prospectively followed up for 50 ± 15 months and all-cause death, CV death and CVE were registered. At the baseline, demographic, clinical, and laboratory data of the whole population were recorded. Then, patients were stratified into two groups according to the dialysis modalities, HD (n = 268) and HDF (n = 65). At the end of 6th month, clinical and laboratory data were recorded again. The predictive factors at baseline for all-cause mortality, CV mortality, and CVE were analyzed by Cox regression. The effects of HD and HDF on these factors at the 6th month and long-term outcomes were compared by t-test and Kaplan-Meier method, respectively. Age, gender, left ventricular mass index (LVMI), aortic arch calcification score (AoACS), hemoglobin (Hb) <10 g/dL, and ferritin >500 ng/mL maintained independent associations with all-cause mortality. C-reactive protein (CRP), LVMI, AoACS, and Hb <10 g/dL were associated with CV mortality. Prior cardiovascular disease (CVD), AoACS and LVMI were independent predictors of nonfatal CVE. Higher body mass index (BMI), body weight, total serum cholesterol, Hb concentration, and lower CRP level, LVMI, and AoACS were found in patients on HDF at the end of the 6th month. Improved outcomes with longer survival time for all-cause mortality, CV mortality, and CVE were found in HDF group. Age, gender, LVMI, AoACS, Hb, and ferritin were predictors of all-cause mortality in MHD patients. CRP, LVMI, AoACS, and Hb were associated with CV mortality. Prior CVD, AoACS, and LVMI were independent predictors of nonfatal CVE. HDF could improve BMI, body weight, total serum cholesterol, Hb, CRP, LVMI, AoACS, and long-term outcomes, including all-cause mortality, CV mortality, and CVE.

Characterization of a small molecule inhibitor of tumor necrosis factor-alpha production.

BACKGROUND: Numerous studies have shown that reducing the level of tumor necrosis factor-alpha (TNFα) through the use of anti-TNF antibodies or soluble TNF receptor is a safe and efficacious treatment to inflammatory diseases such as rheumatoid arthritis. Therefore, novel approaches to achieve this outcome are desired. The aim of this study was to investigate the characterization of a small molecule inhibitor, Y316, which blocks TNF mRNA upregulation and TNF production by lipopolysaccharides (LPS) stimulated monocytes. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy volunteers were plated in 24-well plates and stimulated with LPS (1 µg/ml), phorbol-12-myristate-13-acetate (PMA) (100 ng/ml), zymosan (10 µg/ml) and Tsst (100 ng/ml). Supernatants were collected after 4-hour culture at 37°C, and quantitative determination of TNFα, interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10 and IL-2 production in the supernatants was performed by colorimetric enzyme-linked immunosorbent assay (ELISA). Total RNA of PBMC was isolated and cytokine mRNA quantitation was performed by using a RNA level measuring kit (R & D Systems). PBMC were pretreated with Y316 (10 µmol/L, 1 µmol/L, 0.1 µmol/L, 0.01 µol/L and 0.001 µmol/L) or dimethyl sulfoxide at 37°C for 10 minutes, and then stimulated with LPS or PMA, protein concentrations of p44.42, IKBα, P38 and Jun NH2-terminal kinase were determined by Western blotting. Cyclic adenosine-3',5'-monophosphate (cAMP) of PBMC was measured by enzyme immunoassay kit (Amersham Pharmacia Biotech). RESULTS: Y316 blocked TNF production and inhibited the upregulation of TNF mRNA levels in response to LPS, and also prevented the production of IL-1 and IL-6. In contrast, Y316 augmented the production of IL-10 in LPS-stimulated monocytes. Y316 failed to prevent the production of IL-2 and TNF in antigen-stimulated T cells, suggesting that its effects may be cell-type specific. Y316 prevented the phosphorylation and activation of the MAPK, ERK, and therefore appeared to mediate its effects on TNF by acting at an early point in the signaling cascade induced in response to LPS. There was no effect of Y316 on cAMP levels either alone or in the presence of LPS. CONCLUSIONS: Y316 appears to be a small molecule inhibiting TNF production, which may act via a novel mechanism. Identification of the target of Y316 may lead to the development of alternative strategies for achieving selective cytokine inhibition.