Differential inhibition of sildenafil and macitentan on saxagliptin metabolism.

The development of diabetes mellitus (DM) is generally accompanied by erectile dysfunction (ED) and pulmonary arterial hypertension (PAH), which increases the use of combination drug therapy and the risk of drug-drug interactions. Saxagliptin for the treatment of DM, sildenafil for the treatment of ED and PAH, and macitentan for the treatment of PAH are all substrates of CYP3A4, which indicates their potential involvement in drug-drug interactions. Therefore, we investigated potential pharmacokinetic interactions between saxagliptin and sildenafil/macitentan. We investigated this speculation both in vitro and in vivo, and explored the underlying mechanism using in vitro hepatic metabolic models and molecular docking assays. The results showed that sildenafil substantially inhibited the metabolism of saxagliptin by occupying the catalytic site of CYP3A4 in a competitive manner, leading to the alterations in the pharmacokinetic properties of saxagliptin in terms of increased maximum plasma concentration (Cmax), area under the plasma concentration-time curve from time 0 to 24 h (AUC(0-t)), area under the plasma concentration-time curve from time 0 extrapolated to infinite time (AUC(0-∞)), decreased clearance rate (CLz/F), and prolonged terminal half-life (t1/2). In contrast, a slight inhibition was observed in saxagliptin metabolism when concomitantly used with macitentan, as no pharmacokinetic parameters were altered, except for CLz/F. Thus, dosage adjustment of saxagliptin may be required in combination with sildenafil to achieve safe therapeutic plasma concentrations and reduce the risk of potential toxicity, but it is not necessary for co-administration with macitentan.

Repeat-Enhancing Featured Ion-Guided Stoichiometry for Identification and Quantification of Direct Infusion Proteome.

The direct infusion-shotgun proteome analysis (DI-SPA) alongside data-independent acquisition mass spectrometry achieved fast proteome identification and quantification without chromatographic separation. However, robust peptide identification and quantification (label and label-free) for the DI-SPA data is still insufficient. We find that in the absence of chromatography, the identification of DI-SPA can be boosted by extending acquisition cycles repeatedly and maximizing the utilization of the featured repetition characteristics, combined with the machine learning-based automatic peptide scoring strategy. Here, we present the repeat-enhancing featured ion-guided stoichiometry (RE-FIGS), a complete and compact solution to (repeated) DI-SPA data. Using our strategy, the peptide identification can be improved above 30% with high reproducibility (70.0%). Notably, the label-free quantification of repeated DI-SPA can be successfully obtained with high accuracy (mean median error, 0.108) and high reproducibility (median error, 0.001). We believe our RE-FIGS method could boost the broad application of the (repeated) DI-SPA method and offer a new choice for proteomic analysis.

Physiological responses and altered halocarbon production in Phaeodactylum tricornutum after exposure to polystyrene microplastics.

Oceanic emissions are a major source of atmospheric, very short-lived, ozone-depleting, brominated substances. These substances can be produced by marine microalgae, estimates of their current and future emissions are imperfect, because the processes by which marine microalgae respond to environmental changes are rarely account for environmental pollutants. Here, concurrent measurements of the potential effects of polystyrene (PS) microplastics with concentrations of 25-100 mg/L on the growth of Phaeodactylum tricornutum and their volatile halocarbons (VHCs) production were made over a 20-day culture period. The maximum inhibition rates (IR) due to 0.1 µm and 0.5 µm PS microplastics on cell density were 40.11 % and 32.87 %, on Chl a content were 25.89 % and 20.73 %, and on Fv/Fm were 9.74 % and 9.00 %, respectively. All IR showed dose-dependent effects with maxima occurring in the logarithmic phase. However, in the stationary phase, P. tricornutum exposed to PS microplastics exhibited improved attributes. Enhanced biogenesis of VHCs was induced by the excess reactive oxygen species in algal cells due to microplastics exposure, and their production rates were higher in the logarithmic phase than stationary phase. This represents that oxidative stress to cells plays a dominant role in determining the release of CHBrCl2, CHBr2Cl, and CHBr3. Hence, we suggest that the widespread microplastics in the ocean may be partly responsible for the increase in the emission of VHCs by marine phytoplankton, thereby affecting the ozone layer recovery in the future.

Study on Defective T Junction-Mediated Strand Displacement Amplification and Its Application in Microchip Electrophoretic Detection of Longer Bacterial 16S rDNA.

Current strand displacement amplification (SDA)-based nucleic acid sensing methods generally rely on a ssDNA template that involves complementary bases to the endonuclease recognition sequence, which has the limitation of detecting only short nucleic acids. Herein, a new SDA method in which the defective T junction structure is first used to support SDA (dT-SDA) was proposed and applied in longer DNA detection. In dT-SDA, an auxiliary probe and a primer were designed to specifically identify the target gene, following the formation of a stable defective T junction structure through proximity hybridization, and the formation of defective T junctions could further trigger cascade SDA cycling to produce numerous ssDNA products. The quantity of these ssDNA products was detected through microchip electrophoresis (MCE) and could be transformed to the concentration of the target gene. Moreover, the applicability of this developed strategy in detecting long genomic DNA was verified by detecting bacterial 16S rDNA. This proposed dT-SDA strategy consumes less time and has satisfactory sensitivity, which has great potential for effective bacterial screening and infection diagnosis.

Clinical characteristics of children with airway malacia complicated by pneumonia.

BACKGROUND: Airway malacia is an important cause of noisy breathing, recurrent wheezing and respiratory infections, chronic coughing, and episodes of respiratory distress in young children. As the clinical manifestations of airway malacia are not common, many clinicians have insufficient understanding of this disease. So the purpose of this study is to summarize the pathogenic bacteria and clinical manifestations of airway softening complicated with pneumonia in children. METHODS: Children hospitalized with airway malacia complicated by pneumonia were eligible for enrollment from January 1, 2013 to December 31, 2019. Medical records of patients were reviewed for etiology, clinical characteristics, and laboratory examination results. RESULTS: A total of 164 pneumonia patients with airway malacia were admitted. The male-to-female ratio was 3:1. The age of patients ranged from 1 month to 4 years old. The median age was 6 (3-10) months. The most commonly detected pathogen were Mycoplasma pneumoniae (25/164, 15.24%), Streptococcus pneumoniae (18/164, 10.98%), and respiratory syncytial virus (16/164, 9.76%). Common signs among the 164 patients with confirmed airway malacia included cough (98.78%), wheezing (67.07%), fever (35.37%), intercostal retractions (23.17%), dyspnea (10.98%), cyanosis (11.11%), and crackles (50%). Compared with those without airway malacia, the incidence of premature delivery and mechanical ventilation was higher, and the duration of symptoms before admission (median, 13.5 d) and hospital stay (median 10.0 d) were longer. Of the children with pneumonia, 11.59% of those with airway malacia required supplemental oxygen compared with 4.88% of those without airway malacia (p < 0.05). CONCLUSION: The median age of children with airway malacia was 6 months. The most common pathogen in patients with airway malacia complicated by pneumonia was Mycoplasma pneumoniae. Patients with airway malacia complicated by pneumonia often presented with a longer disease course, more severe symptoms, and had delayed recovery.

Simultaneous electrochemical determination of nuc and mecA genes for identification of methicillin-resistant Staphylococcus aureus using N-doped porous carbon and DNA-modified MOF.

The detection of Staphylococcus aureus specific gene in combination with the mecA gene is vitally important for accurate identification of methicillin-resistant Staphylococcus aureus (MRSA). A homogeneous electrochemical DNA sensor was fabricated for simultaneous detection of mecA and nuc gene in MRSA. Metal-organic framework (type UiO-66-NH2) was applied as nanocarrier. Two electroactive dyes, methylene blue (MB) and epirubicin (EP), were encapsulated in UiO-66-NH2, respectively, and were locked by the hybrid double-stranded DNA. Based on the target-response electroactive dye release strategy, once target DNA exists, it completely hybridizes with displacement DNA (DEP and DMB). So DEP and DMB is displaced from the MOF surface, causing the release of electroactive dyes. Co-Zn bimetallic zeolitic imidazolate framework-derived N-doped porous carbon serves for electrode modification to improve electrocatalytic performance and sensitivity. The differential pulse voltammetry peak currents of MB and EP were accurately detected at - 0.14 V and - 0.53 V versus the Ag/AgCl reference electrode, respectively. Under the optimal conditions, the detection limits of mecA gene and nuc gene were 3.7 fM and 1.6 fM, respectively. Combining the effective application of MOFs and the homogeneous detection strategy, the sensor exhibited satisfactory performance for MRSA identification in real samples. The recovery was 92.6-103%, and the relative standard deviation was less than 5%. Besides, MRSA and SA can also be distinguished. This sensor has great potential in practical applications.

An electrochemical sensor for bacterial lipopolysaccharide detection based on dual functional Cu2+-modified metal-organic framework nanoparticles.

An electrochemical sensor based on dual functional Cu2+-modified metal-organic framework nanoparticles (Cu2+-NMOFs) for sensitive detection of bacterial lipopolysaccharide (LPS) is reported. Cu2+-NMOFs were prepared and characterized by SEM, EDS, XRD, and XPS. In this LPS sensor, LPS firstly immobilized in gold nanoparticles/reduced graphene oxide by C18 alkane thiol chains, since the LPS can interact with the C18 alkyl chains by strong intermolecular interactions. Then the Cu2+-NMOFs were captured by the anionic groups of the carbohydrate portions of LPS molecules and played a vital role of recognition unit. More importantly, the Cu2+-NMOFs can catalyze dopamine oxidation to generate aminochrome, resulting in a strong electrochemical oxidation signal. The electrochemical sensor based on dual functional Cu2+-NMOFs was investigated by differential pulse voltammetry, and the stripping peak currents of dopamine oxidized to aminochrome were used to monitor the level of LPS. The developed method demonstrated a wide linear range from 0.0015 to 750 ng/mL with a limit of detection of 6.1 × 10-4 ng/mL. The fabricated sensor was applied to detect LPS in mouse blood serum and satisfactory results were achieved. Compared to other detection schemes by using the LPS-binding proteins, peptides, and aptamer, the proposed LPS determination based on the catalytic peroxidase-mimicking NMOFs has some advantages such as good reproducibility, low detection limit, and excellent specificity. Graphical abstract An electrochemical sensor based on dual functional Cu2+-modified metal-organic framework was developed for detection of bacterial lipopolysaccharide. This sensor combined a metal ion-based target recognition and electrocatalytic detection, and provided a high sensitive strategy for detection of lipopolysaccharide.

Selective detection of cytochrome C by microchip electrophoresis based on an aptamer strategy.

The release of cytochrome C (Cyt C) plays an important role in apoptosis. In this study, selective and sensitive detection of Cyt C based on an aptamer strategy coupled with MCE was developed. Following the binding of a specific aptamer to Cyt C, the aptamer exhibited an irregular state, reducing the binding affinity of a fluorescent probe, and thus preventing the aptamer-Cyt C complexes from detection within the MCE. The height of the detection peak of the residual aptamer linearly decreased, and therefore, the difference in peak height of residual aptamer compared to that of the initial aptamer was used to quantify the captured protein concentration. Experimental conditions such as incubation time, pH, temperature, and ionic strength were optimized. A measurement of Cyt C concentration by MCE was achieved within 135 s, with a limit of detection as low as 0.4 nM. The proposed method has high selectivity and good stability for the detection of Cyt C. The experimental results demonstrate that this method is quick, consumes only a small quantity of sample, is highly selectivity and exhibits high sensitivity.

Electrochemical determination of Salmonella typhimurium by using aptamer-loaded gold nanoparticles and a composite prepared from a metal-organic framework (type UiO-67) and graphene.

An aptamer based assay is described for the determination of Salmonella typhimurium (S.typhimurium). A metal-organic framework-graphene composite of type UiO-67/GR is used as the substrate, and an aptamer-gold nanoparticles-horseradish peroxidase (Apt-AuNP-HRP) conjugate the signal amplification probe. A phosphate-terminal and partially complementary DNA (cDNA) of the aptamer is covalently bound to UiO-67/GR via the chemical complexation between phosphate and Zr-OH groups of UiO-67, and then S. typhimurium and cDNA will compete for the binding sites. The binding of Apt-AuNP-HRP to S.typhimurium leads to the formation of strong conjugates. The unbound signal probes then attach to the surface of a glassy carbon electrode via hybridization with cDNA. This generates a large current response (best measured at a potential as low as -0.02 V vs. saturated calomel electrode) under the catalytic action of HRP on the H2O2-hydroquinone system. Under the optimal conditions, the differential pulse voltammetric signal decreases linearly in the 2 × 101 - 2 × 108 cfu·mL-1 S.typhimurium concentration range, with a lower detection limit of 5 cfu·mL-1 (based on S/N = 3). The method was successfully applied to the detection of S. typhimurium in spiked milk samples. Graphical abstract Schematic presentation of electrochemical determination of Salmonella typhimurium (S.typhimurium). A metal-organic framework (type UiO-67) and graphene (GR) composite were used as substrate, and gold nanoparticles carrying horseradish peroxidase (HRP) for signal amplification. HQ: hydroquinone; cDNA: complementary DNA of aptamer.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Pharmacokinetics of Anti-Rheumatic drugs Methotrexate and Tofacitinib with its metabolite M9 in Rats by UPLC-MS/MS.

BACKGROUND: Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. METHODS: Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. RESULTS: The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. CONCLUSION: The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

A magnetic graphene oxide and UiO-66 based homogeneous dual recognition electrochemical aptasensor for accurate and sensitive detection of aflatoxin B1.

Aflatoxin (AFs) contamination is one of the serious food safety issues. Aflatoxin B1 (AFB1) is the most common and toxic aflatoxin, which has been classified as a class 1 carcinogen by the International Agency for Research on Cancer (IARC). It is extremely destructive to liver tissue. Developing a convenient and sensitive detection technique is essential. In this paper, we developed a homogeneous dual recognition strategy based electrochemical aptasensor for accurate and sensitive detection of aflatoxin B1 (AFB1) based on the magnetic graphene oxide (MGO) and UiO-66. The MGO was synthesized for the recognition and magnetic separation of AFB1 from complex samples. UiO-66/ferrocenecarboxylic acid (Fc)/aptamer composites were constructed as both recognition and signal probes. The probes would specifically capture AFB1 enriched by MGO, which enables dual recognition in homogeneous solution, thus further improving the accuracy of AFB1 detection. The electrochemical aptasensor for AFB1 had a linear range from 0.005 to 500 ng mL-1. Additionally, the limit of detection was 1 pg mL-1. It shows a favorable potential for both sensitive and accurate detection of AFB1 in real samples.

Rapid detection of foodborne pathogens in diverse foodstuffs by universal electrochemical aptasensor based on UiO-66 and methylene blue composites.

Rapid and sensitive detection of foodborne pathogens in complex environments is essential for food protection. A universal electrochemical aptasensor was fabricated for the detection of three common foodborne pathogens, including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Salmonella typhimurium (S. typhimurium). The aptasensor was developed based on the homogeneous and membrane filtration strategy. Zirconium-based metal-organic framework (UiO-66)/methylene blue (MB)/aptamer composite was designed as a signal amplification and recognition probe. Bacteria were quantitatively detected by the current changes of MB. By simply changing the aptamer, different bacteria could be detected. The detection limits of E. coli, S. aureus and S. typhimurium were 5, 4 and 3 CFU·mL-1, respectively. In humidity and salt environments, the stability of the aptasensor was satisfactory. The aptasensor exhibited satisfactory detection performance in different real samples. This aptasensor has excellent potential for rapid detection of foodborne pathogens in complex environments.

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae isolates among children in Suzhou, China.

Background: Streptococcus pneumoniae (SP) is responsible for pneumococcal diseases with severe morbidity and mortality. High rates of drug resistance constitute serious public health concerns. Vaccination has proven to be an effective means of reducing disease burden. Epidemiological information of antibiotic susceptibilities and serotype distribution will be of great help to the management of pneumococcal infections. This study reported the serotype distribution and antibiotic resistance pattern of SP in hospitalized children in Suzhou during the years 2017-2018. The aim is to reduce pneumococcal resistance and guide vaccination. Methods: The clinical data of hospitalized children with SP were collected and analyzed. A total of 2,446 strains of SP were isolated from these patients. Serotypes were determined using the Quellung reaction. Antibiotic resistance was tested using the E-test diffusion method. Results: The non-susceptible rates of the isolates to penicillin, amoxicillin, and cefotaxime were 9.5%, 27.7%, and 27.2%, respectively. And 97.6% of SP isolates showed multidrug-resistant (MDR). The most common resistance pattern of non-invasive isolates was macrolides + sulfamethoxazole + clindamycin + tetracycline. The major serotypes of this resistance pattern were 6A, 23F, 6B, 19F, 15B. The most extensive resistance pattern of invasive isolates was macrolides + ß-lactams + sulfamethoxazole + clindamycin + tetracycline. The most common serotypes of the pattern were 19F, 19A, 6B, 23F, 6A. The most common serotypes were 19F (28.6%), 6B (11.9), 23F (11.2%), 6A (10.6%), and 19A (9.1%). In the isolates with MDR, the first five most common serotypes were 19F, non-vaccine serotype (NVT), 6B, 6A and 23F. Strains belonging to different serotypes exhibited distinct antimicrobial resistance patterns and were found to be associated with different diseases. The coverage rates of pneumococcal conjugate vaccine (PCV)7 and PCV13 in all isolates reached 60.4% (310/513) and 80.9% (415/513), respectively. Conclusions: The main serotypes of SP in Suzhou were 19F, 6B, 23F, 6A, and 19A. The use of PCV13 is beneficial to children in Suzhou.

Selective, direct activation of high-conductance, calcium-activated potassium channels causes smooth muscle relaxation.

High-conductance calcium-activated potassium (Maxi-K) channels are present in smooth muscle where they regulate tone. Activation of Maxi-K channels causes smooth muscle hyperpolarization and shortening of action-potential duration, which would limit calcium entry through voltage-dependent calcium channels leading to relaxation. Although Maxi-K channels appear to indirectly mediate the relaxant effects of a number of agents, activators that bind directly to the channel with appropriate potency and pharmacological properties useful for proof-of-concept studies are not available. Most agents identified to date display significant polypharmacy that severely compromises interpretation of experimental data. In the present study, a high-throughput, functional, cell-based assay for identifying Maxi-K channel agonists was established and used to screen a large sample collection (>1.6 million compounds). On the basis of potency and selectivity, a family of tetrahydroquinolines was further characterized. Medicinal chemistry efforts afforded identification of compound X, from which its two enantiomers, Y and Z, were resolved. In in vitro assays, Z is more potent than Y as a channel activator. The same profile is observed in tissues where the ability of either agent to relax precontracted smooth muscles, via a potassium channel-dependent mechanism, is demonstrated. These data, taken together, suggest that direct activation of Maxi-K channels represents a mechanism to be explored for the potential treatment of a number of diseases associated with smooth muscle hyperexcitability.

Characterization of the substituted N-triazole oxindole TROX-1, a small-molecule, state-dependent inhibitor of Ca(V)2 calcium channels.

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca(V)2.2) as therapeutic targets for chronic pain. A state-dependent Ca(V)2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca(V)2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca(V)2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca(V)2.2 channels with an IC(50) of 0.11 µM. The voltage dependence of Ca(V)2.2 inhibition was examined using automated electrophysiology. TROX-1 IC(50) values were 4.2, 0.90, and 0.36 µM at -110, -90, and -70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca(V)2.2 with a 10-fold IC(50) separation between first (27 µM) and last (2.7 µM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca(V)2.2 channels with an IC(50) of 9.5 µM under hyperpolarized conditions and 0.69 µM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC(50) values were 0.29, 0.19, and 0.28 µM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 µM by calcium influx for Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non-subtype-selective Ca(V)2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca(V)2.2-selective inhibitor ziconotide in preclinical models of chronic pain.

Biosensing bacterial 16S rDNA by microchip electrophoresis combined with a CRISPR system based on real-time crRNA/Cas12a formation.

The accurate, simple and sensitive detection of bacterial infections at the early stage is highly valuable in preventing the spread of disease. Recently, CRISPR-Cas12a enzyme-derived nucleic acid detection methods have emerged along with the discovery of the indiscriminate single-stranded DNA (ssDNA) cleavage activity of Cas12a. These nucleic acid detection methods are made effective and sensitive by combining them with isothermal amplification technologies. However, most of the proposed CRISPR-Cas12a strategies involve Cas-crRNA complexes in the preassembled mode, which result in inevitable nonspecific background signals. Besides, the signal ssDNA used in these strategies needs tedious pre-labeling of the signal molecules. Herein, a post-assembly CRISPR-Cas12a method has been proposed based on target-induced transcription amplification and real-time crRNA generation for bacterial 16S rDNA biosensing. This strategy is label-free through the combination of microchip electrophoresis (MCE) detection. In addition, this method eliminates the need for a protospacer adjacent motif (PAM) on the target sequences, and has the potential to be an effective and simple method for nucleic acid detection and infectious disease diagnosis.

Clinical characteristics and etiology of children with bronchiolitis before and during the COVID-19 pandemic in Suzhou, China.

Objective: We sought to compare the clinical characteristics and etiology of children with bronchiolitis in Suzhou before the pandemic of coronavirus disease 2019 (COVID-19) with those during the pandemic. Methods: Children who were hospitalized with bronchiolitis in the Department of Respiratory Disease, Children's Hospital of Soochow University were retrospectively enrolled over 3 consecutive years (2019, 2020, and 2021) from February 1 to January 31. Medical records were reviewed for etiology, clinical manifestations, and laboratory examination results. Results: The pathogen detection rate and the positive respiratory syncytial virus (RSV) detection rate were lowest in 2020 and highest in 2021. The rate of human rhinovirus detection in 2021 was higher than that in 2019 but similar to that in 2020. The RSV-positive rate differences among the 3 years varied by age group. Regarding the monthly distribution of RSV-positive cases over the 3-year study, all age groups showed a significant increase in the number of cases during the winter of 2021, and this increase started as early as October. With regard to clinical manifestations, the proportion of children presenting with stuffy nose rhinorrhea in 2021 [73.33% (165/225)] was greater than that in 2019 [48.61% (122/251)] and 2020 [57.06% (97/170)], while the proportion of children with gastrointestinal symptoms in 2021 [11.56% (26/225)] was smaller than that in 2019 [25.50% (64/251)] but similar to that in 2020 [17.06% (29/170)]. Conclusions: After the implementation of COVID-19 pandemic-related interventions, significantly lower pathogen detection and RSV-positive rates were observed in children with bronchiolitis in 2020. An upward trend in these rates was observed in 2021, coinciding with the relaxation of COVID-19 prevention measures. Strengthening infection control and surveillance systems is extremely important for future work.

Comparison of the clinical features of human bocavirus and metapneumovirus lower respiratory tract infections in hospitalized children in Suzhou, China.

Objective: We compared the clinical data of hospitalized children with lower respiratory tract infections caused by human bocavirus (HBoV) and human metapneumovirus (hMPV). Methods: In total, 8,430 children admitted to the Department of Respiration, Children's Hospital of Soochow University for lower respiratory tract infections from January 2017 to October 2021 were enrolled. Seven common respiratory viruses, including respiratory syncytial virus, influenza virus A, influenza virus B, parainfluenza virus (PIV) I, PIV II, PIV III, and adenovirus, were detected by direct immunofluorescence assay, whereas human rhinovirus and hMPV were detected by reverse transcription-polymerase chain reaction. Mycoplasma pneumoniae (MP) and HBoV were detected by real-time fluorescence quantitative polymerase chain reaction. Bacteria was detected in blood, nasopharyngeal secretion, bronchoalveolar lavage specimen or pleural fluid by culture. In parallel, MP was detected by enzyme-linked immunosorbent assay. In addition, we performed metagenomic testing of alveolar lavage fluid from some of the patients in our study. Results: The detection rate of HBoV was 6.62% (558/8430), whereas that of hMPV was 2.24% (189/ 8430). The detection rate of HBoV was significantly higher in children aged 1 to <3 years than in other age groups, but there were no significant differences in positivity rates for hMPV by age. Before 2020, the incidence of HBoV infection peaked in summer and autumn, whereas that of hMPV peaked in spring. The epidemiology of both HBoV and hMPV has changed because of the impact of the novel coronavirus. Among the positive cases, the HBoV mixed infection rate was 51.6%, which was similar to that for hMPV mixed infection (44.4%). Comparing clinical characteristics between HBoV and hMPV single infection, the median age of children was 17 months in the HBoV group and 11 months in the hMPV group. In the HBoV single infection group, 31 patients (11.5%) had pulse oxygen saturation of less than 92% on admission, 47 (17.4%) had shortness of breath, and 26 (9.6%) presented with dyspnea. Meanwhile, four patients (3.8%) in the hMPV single infection group had pulse oxygen saturation of less than 92% on admission, eight (7.6%) displayed shortness of breath, and three (2.9%) had dyspnea. The proportion of patients requiring mechanical ventilation and the rate of PICU admission were higher in the HBoV group than in the hMPV group. Conclusion: The prevalence of HBoV infection is higher than that of hMPV infection in children with lower respiratory tract infection in Suzhou, and HBoV is more likely to cause severe infection than hMPV. Public health interventions for COVID-19 outbreaks have affected the prevalence of HBoV and hMPV.

Construction of Pd Single Site Anchored on Nitrogen-Doped Porous Carbon and Its Application for Total Antioxidant Level Detection.

Natural enzymes have excellent catalytic activity. However, due to their unstable nature and high cost, current research has turned to the synthesis and development of enzyme-like nanomaterials and single-atomic nanozymes. In this study, a single-atomic palladium-loaded nitrogen-doped porous carbon catalyst (SA-Pd/NPC) was prepared and used as a mimetic peroxidase to catalyze the substrates oxidation. The catalytic capability of the SA-Pd/NPC was tested by the TMB-H2O2 system, and it expressed a superior catalytic capability owing to the plentiful catalytic centers of the single-atom Pd, its high porosity, the large specific surface area, and the strong electron transfer capability of the NPC. For the color reaction of TMB, thiol antioxidants (e.g., glutathione, GSH) and non-thiol antioxidants (e.g., ascorbic acid, AA) are suitable for different inhibition mechanisms. GSH and AA are typical substances of these two main antioxidant types, respectively. Here, we demonstrate that this prepared catalyst could be used to simultaneously determine a variety of major known physiologically relevant thiol-containing and thiol-free antioxidants, accompanied by a blue color gradient change with UV-Vis spectra at 652 nm through the SA-Pd/NPC-catalyzed TMB-H2O2 system. Linear responses to GSH and AA could be obtained in the concentration ranges of 0.01-0.10 mM and 1-13 µM (both R2 values were greater than 0.970), respectively, while the limits of detection were 3 µM and 0.3 µM, respectively. The ability of the nanozyme to detect overall antioxidant levels (TAL) was also confirmed in subsequent tests on artificial saliva and biological samples.