Functional analysis of extracellular polymeric substances (EPS) during the granulation of aerobic sludge: Relationship among EPS, granulation and nutrients removal.

Extracellular polymeric substances (EPS) with high molecular weights, secreted from microorganisms, play a critical functional role in the aerobic granular sludge (AGS). To investigate the level and function of EPS during the granulation of aerobic sludge and in the mature AGS, a sequencing batch reactor (SBR) was operated for 70 days. Aerobic granules with an average diameter of 0.25 mm were obtained with reducing settling time of sludge. Simultaneous removals of COD, nitrogen and phosphorus by the mature AGS exceeded 90, 95 and 95%, respectively. The EPS content increased significantly to above 333 mg/g MLVSS during the initial stage, and after that, it stabilized at about 240 mg/g MLVSS as the mature AGS formed, higher than that of the seed sludge (212 mg/g MLVSS). The increased EPS contents showed a negative correlation with SVI values, while a strong positive relationship with the formation of the AGS. The protein/polysaccharide (PN/PS) ratio in the EPS increased from 1.42 to 4.17, and TP/MLSS increased to about 6%, with the formation of AGS. The proportion of extracellular-P increased with the increase of EPS, and then maintained stable at about 20%, indicating EPS promoted the removal of phosphorus. Furthermore, the results from the Standards, Measurements and Testing (SMT) and X-Ray Diffraction (XRD) showed that phosphorus in the AGS mainly existed in the form of inorganic phosphorus (IP) and the proportion of Ca5(PO4)3(OH) in IP was up to 92%. This investigation demonstrated that EPS had a positive relationship with the sludge granulation and nutrients removal.

Factors Influencing the Sample Adequacy of Ultrasound-Guided Fine-Needle Aspiration from Solid Thyroid Nodules for Liquid-Based Cytology: A Demographic, Sonographic, and Technical Perspective.

Background and Objectives: To identify factors that influence the sample adequacy of solid thyroid nodules based on ultrasound-guided fine-needle aspiration (FNA) with subsequent liquid-based cytology. Materials and Methods: We retrospectively reviewed 855 patients who underwent ultrasound-guided FNA at our hospital between July 2019 and July 2020. The final analysis included 801 solid thyroid nodules in 801 patients. After reviewing the demographic data, ultrasonic features, and FNA technique-related factors, we defined 14 potential variables. For cytological results, the Bethesda categories II−VI were defined as adequate sample results. Univariate and multivariate analyses were performed to identify factors that influenced sample adequacy. Results: The adequate sample rate was 87.1%. The univariate analysis showed that four factors were related to adequate sampling in patients with thyroid FNA. These factors included age (p < 0.001), nodule orientation (p = 0.0232), calcification (p = 0.0034), and operator experience (p = 0.0286). After the multivariate analysis, five independent factors were identified to improve the diagnostic results of FNA for solid thyroid nodules: (1) the presence of Hashimoto's thyroiditis (odds ratio (OR) = 1.810; 95% confidence interval (CI): 1.076−3.045; p = 0.0254), (2) a taller-than-wide orientation (OR = 2.038; 95% CI: 1.260−3.296; p = 0.0037), (3) the presence of calcification (OR = 1.767; 95% CI: 1.115−2.799; p = 0.0153), (4) four needle passes to obtain material (OR = 1.750; 95% CI: 1.094−2.799; p = 0.0196), and (5) an experienced operator (OR = 0.561; 95% CI: 0.319−0.987; p = 0.0451). Conclusions: A taller-than-wide orientation, the presence of calcification, and the presence of Hashimoto's thyroiditis were found to affect the sample adequacy of ultrasound-guided FNA with liquid-based cytology. The sample adequacy could be improved when FNA is performed with four needle passes by experienced doctors.

A facile signal-on electrochemical DNA sensing platform for ultrasensitive detection of pathogenic bacteria based on Exo III-assisted autonomous multiple-cycle amplification.

A facile signal-on electrochemical DNA biosensor has been developed for ultrasensitive detection of pathogenic bacteria using an Exo III-assisted autonomous multiple-cycle amplification strategy. The strategy relies on pathogens and aptamer binding-initiated release of a trigger, which combines with the 3'-protruding terminus of the hairpin probe 1, leading to the formation of double-stranded DNA with a blunt 3' terminus which starts the Exo III-assisted multiple signal amplification reaction. In addition, hairpin probe 2 labeled with an electroactive reporter at the middle of the loop region is ingeniously designed to contain a short hairpin-embedded segment, which can fold into a hairpin structure via an Exo III-assisted cleavage reaction, thus bringing the redox molecule in proximity to the electrode surface for "signal-on" sensing. Under optimal conditions, this biosensor exhibits a very low detection limit as low as 8 cfu mL-1 and a wide linear range from 1.0 × 101 to 1.0 × 107 cfu mL-1 of target pathogenic bacteria. As far as we know, this is the first time that the Exo III-assisted autonomous multiple-cycle amplification strategy has been used for signal-on electrochemical sensing of pathogenic bacteria. In addition, the proposed sensor can also be used for highly sensitive detection of other targets by changing the aptamer sequence, such as nucleic acids, proteins and small molecules. Therefore, the proposed signal-on electrochemical sensing strategy might provide a simple and practical new platform for detection of pathogenic bacteria and related biological analysis, food safety inspection and environmental monitoring.

Circular exponential amplification of photoinduced electron transfer using hairpin probes, G-quadruplex DNAzyme and silver nanocluster-labeled DNA for ultrasensitive fluorometric determination of pathogenic bacteria.

The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650 nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8 cfu·mL-1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis. Graphical abstract The reaction system includes three hairpin probes (H1, H2 and H3), primer probe (P), Phi 29 DNA ploymerase (Phi 29) and nicking endonuclease Nt.AlwI (Nt.AlwI). Phi 29 and Nt.AlwI -assisted signal amplification leads to the recycling of target and produces numerous single stranded-DNAs (S). Strand displacement amplification leads to photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA/AgNCs. HAP-based circular exponential amplification of PET results in an ultrasensitive fluorometric assay.

Anti-factor Xa level monitoring of low-molecular-weight heparin for prevention of venous thromboembolism in critically ill patients (AXaLPE): protocol of a randomised, open-label controlled clinical trial.

INTRODUCTION: Whether and when to monitor the amount of anti-factor Xa (aFXa) activity in critically ill patients with complex diseases to prevent venous thromboembolism (VTE) remain unclear. This study is a randomised controlled trial to investigate the effect of aFXa level monitoring on reducing VTE and to establish a new method for accurately preventing VTE in critically ill patients with low-molecular-weight heparin (LMWH). METHODS AND ANALYSIS: A randomised controlled trial is planned in two centres with a planned sample size of 858 participants. Participants will be randomly assigned to three groups receiving LMWH prophylaxis at a 1:1:1 ratio: in group A, peak aFXa levels will serve as the guide for the LMWH dose; in group B, the trough aFXa levels will serve as the guide for the LMWH dose; and in group C, participants serving as the control group will receive a fixed dose of LMWH. The peak and trough aFXa levels will be monitored after LMWH (enoxaparin, 40 mg, once daily) reaches a steady state for at least 3 days. The monitoring range for group A's aFXa peak value will be 0.3-0.5 IU/mL, between 0.1 and 0.2 IU/mL is the target range for group B's aFXa trough value. In order to reach the peak or trough aFXa levels, groups A and B will be modified in accordance with the monitoring peak and trough aFXa level. The incidence of VTE will serve as the study's primary outcome indicator. An analysis using the intention-to-treat and per-protocol criterion will serve as the main outcome measurement. ETHICS AND DISSEMINATION: The Xuanwu Hospital Ethics Committee of Capital Medical University and Peking University First Hospital Ethics Committee have approved this investigation. It will be released in all available worldwide, open-access, peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT05382481.

Two-stage nicking enzyme signal amplification (NESA)-based biosensing platform for the ultrasensitive electrochemical detection of pathogenic bacteria.

The sensitive and selective detection of pathogenic bacteria represents an essential approach in food safety analysis and clinical diagnostics. We report the development of a simple, rapid, and low-cost electrochemical biosensing strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. The biosensor relies on the target and aptamer binding-triggered two-stage nicking enzyme signal amplification (NESA) and three-way junction probe-mediated electrochemical signal transduction. In the presence of the target S. typhimurium, the specific binding of S. typhimurium and aptamer results in the release of a primer, which hybridizes with HAP1 and initiates an extension reaction with the aid of polymerase and dNTPs. A specific recognition site for Nt.BsmaI is generated in the DNA duplex; thus, the produced DNA is nicked and the secondary primer is released (named recycle I). Subsequently, the reaction solution supplemented with a helper DNA is dropped on the electrode surface, and a three-way junction probe containing a specific recognition site for Nt.BsmaI is thus formed. The MB-labeled probe is nicked with the help of Nt.BsmaI and the dissociated primer-helper DNA duplex combines with another HAP2 (named recycle II). Thus, a remarkably decreased electrochemical signal is generated because the electroactive MB is far away from the electrode surface. As far as we know, this work is the first time that NESA and three-way junction probe-mediated electrochemical signal transduction has been used for pathogenic bacteria detection. Under optimal conditions, the results reveal that the calibration plot obtained for S. typhimurium is approximately linear from 9.6 to 9.6 × 105 cfu mL-1 with the limit of detection of 8 cfu mL-1. Additionally, the proposed strategy has been successfully applied to the quantitative assay of S. typhimurium in the real samples. Therefore, the NESA-based biosensing strategy might create a useful and practical platform for pathogenic bacteria identification, and the related food safety analysis and clinical diagnosis.

Low-background and visual detection of antibiotic based on target-activated colorimetric split peroxidase DNAzyme coupled with dual nicking enzyme signal amplification.

Herein, we have reported the development of a simple, rapid, and low cost colorimetric method for the detection of antibiotic based on target-activated split peroxidase DNAzyme coupled with dual nicking enzyme signal amplification (NESA). To lower background signal in G-quadruplex DNAzyme-based detection, the two split G-rich parts are caged into two different hairpin probes, respectively, preventing the two parts from assembling into the G-quadruplex structure. By the combination of restriction endonuclease-assisted cleavage reaction with the spilt G-quadruplex probes, target-modulated release of the two split G-rich parts is achieved, affording high specificity of antibiotic detection. Our strategy features with several aspects. First, the less background signal produced by the self-assembly of G-quadruplex in the absence of target is effectively eliminated owing to the pre-blocking of the two split G-rich parts. Second, dual NESA coupled G-quadruplex DNAzyme amplification strategy is integrated with colorimetric assay of antibiotic, which significantly improves the detection sensitivity. Third, peroxidase-mimicking DNAzyme is used as biocatalyst in our reaction system, which can catalyze the oxidation of 2,2' - azino - bis (3 - ethylbenzothiozoline - 6 - sulfonic acid) (ABTS2-) mediated by H2O2 to generate the colored radical anion (ABTS•-), allowing to low cost and visual detection of antibiotic by the naked eye. Under optimized conditions, the results revealed the proposed biosensor exhibits excellent specificity and sensitivity toward kanamycin with a detection limit as low as 14.7 pM. Hence, the target-activated split G-quadruplex DNAzyme and dual NESA-based strategy provides a useful and practical platform for antibiotic residues determination and other analytes detection in bio-analysis.

CKII-SIRT1-SM22α loop evokes a self-limited inflammatory response in vascular smooth muscle cells.

AIMS: Sirtuin 1 (SIRT1) inhibits nuclear factor kappa B (NF-κB) activity in response to the inflammatory cytokine tumour necrosis factor alpha (TNF-α). Smooth muscle (SM) 22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signalling cascades in vascular smooth muscle cells (VSMCs). Sm22α knockout results in increased expression of pro-inflammatory genes in the aortas which are controlled by NF-κB. This study aimed to investigate the relationship between SM22α and SIRT1 in the control of vascular inflammation. METHODS AND RESULTS: The ligation injury model of Sirt1-Tg/Sm22α-/- mice displayed an increased level of the inflammatory molecules in the carotid arteries compared with Sirt1-Tg mice, accompanied with aggravating neointimal hyperplasia. In the in vitro study, on the one hand, we showed that TNF-α induced the epigenetic silencing of SM22α transcription via EZH2-mediated H3K27 methylation in the SM22α promoter region, contributing to inflammatory response. On the other hand, TNF-α simultaneously induced SIRT1 phosphorylation via CKII and thereby protected against inflammation. Phosphorylated SIRT1 interacted with and deacetylated EZH2 and, subsequently, promoted SM22α transcription by inhibiting EZH2 activity. Increased SM22α in turn facilitated the phosphorylation and activation of SIRT1 via recruitment of CKII to SIRT1, which amplified the anti-inflammatory effect of SIRT1. CONCLUSION: Our findings demonstrate that, in response to TNF-α stimulation, CKII-SIRT1-SM22α acts in a loop to reinforce the expression of SM22α, which limits the inflammatory response in VSMCs in vivo and in vitro. The anti-inflammatory effect of SIRT1 may be dependent on SM22α to some extent. Our data point to targeted activation of SIRT1 in VSMCs as a promising therapeutic avenue in preventing cardiovascular diseases.