Low-frequency electroacupuncture exerts antinociceptive effects through activation of POMC neural circuit induced endorphinergic input to the periaqueductal gray from the arcuate nucleus.

It has been widely recognized that electroacupuncture (EA) inducing the release of ß-endorphin represents a crucial mechanism of EA analgesia. The arcuate nucleus (ARC) in the hypothalamus is a vital component of the endogenous opioid peptide system. Serving as an integration center, the periaqueductal gray (PAG) receives neural fiber projections from the frontal cortex, insular cortex, and ARC. However, the specific mechanisms how EA facilitates the release of ß-endorphin within the ARC, eliciting analgesic effects are yet to be elucidated. In this study, we conducted in vivo and in vitro experiments by transcriptomics, microdialysis, photogenetics, chemical genetics, and calcium imaging, combined with transgenic animals. Firstly, we detected 2 Hz EA at the Zusanli (ST36) increased the level of ß-endorphin and transcriptional level of proopiomelanocortin (POMC). Our transcriptomics profiling demonstrated that 2 Hz EA at the ST36 modulates the expression of c-Fos and Jun B in ARC brain nuclear cluster, and the transcriptional regulation of 2 Hz EA mainly occur in POMC neurons by Immunofluorescence staining verification. Meaning while, 2 Hz EA specifically activated the cAMP-PKA-CREB signaling pathway in ARC which mediating the c-Fos and Jun B transcription, and 2 Hz EA analgesia is dependent on the activation of cAMP-PKA-CREB signaling pathway in ARC. In order to investigate how the ß-endorphin produced in ARC transfer to integration center PAG, transneuronal tracing technology was used to observe the 2 Hz EA promoted the neural projection from ARC to PAG compared to 100 Hz EA and sham mice. Inhibited PAGGABA neurons, the transfer of ß-endorphin from the ARC nucleus to the PAG nucleus through the ARCPOMC-PAGGABA neural circuit. Furthermore, by manipulating the excitability of POMC neurons from ARCPOMC to PAGGABA using inhibitory chemogenetics and optogenetics, we found that this inhibition significantly reduced transfer of ß-endorphin from the ARC nucleus to the PAG nucleus and the effectiveness of 2 Hz EA analgesia in neurological POMC cyclization recombination enzyme (Cre) mice and C57BL/6J mice, which indicates that the transfer of ß-endorphin depends on the activation of POMC neurons prefect from ARCPOMC to PAGGABA. These findings contribute to our understanding of the neural circuitry underlying the EA pain-relieving effects and maybe provide valuable insights for optimizing EA stimulation parameters in clinical pain treatment using the in vivo dynamic visual investigating the central analgesic mechanism.

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis.

BACKGROUND: The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. METHODS: The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2-positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2-negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing. RESULTS: All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2-positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages. CONCLUSION: The developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Role of HLA class I and II alleles in susceptibility to ankylosing spondylitis in Chinese Han.

OBJECTIVE: The objective of the study was to clarify the associations of HLA class I and II alleles with ankylosing spondylitis (AS) among Chinese Han. METHODS: We performed HLA genotyping and Sanger sequencing for 68 HLA-B*27(-), 62 HLA-B*27(+) AS patients, and 70 controls. Case-control analyses and separate analyses of HLA-B*27(-) patients were performed. One-way ANOVA and Kruskal-Wallis multiple comparisons test were used to analyze the effects of HLA-A\B\C\DRB1\DQB1 alleles on clinical characteristics of HLA-B*27(-) and HLA-B*27(+) patients. RESULTS: In the HLA-B*27(+) group, positive associations were seen with A*11:02, B*27:04, B*27:05, C*02:02, C*12:02, and DRB1*04:01 and negative associations were seen with A*33:03, B*07:02, B*57:01, and C*07:02. The age at onset was greater in HLA-B*27(-) patients than in HLA-B*27(+) patients (30.03 ± 15.15 vs. 23.08 ± 7.79 years). In the HLA-B*27(-) group, those with A*01:01, B*13:01, B*13:02, C*01:02, C*04:01, DQB1*02:01, DQB1*06:01, and DRB1*03:01 had an earlier onset than those without these alleles, while patients carrying B*40:02, C*07:02, C*12:02, C*15:02, DQB1*05:02, and DQB1*05:03 had a delayed onset. In the HLA-B*27(-) group, A*32:01(+), C*08:01(+), and DRB1*04:05(-) women were likely to develop AS. In the HLA-B*27(+) group, DQB1*03:02(+) women may be more likely to develop AS. DRB1*12:02 and HLA-B*27 interacted with the distribution of AS-affected sites. In the HLA-B*27(+) group, DRB1*12:02(+) patients were likely to have peripheral joint involvement. CONCLUSION: HLA class I and II alleles other than HLA-B*27 contribute to AS predisposition and characteristics among Chinese Han patients.

A novel method for early detection of colorectal cancer based on detection of methylation of two fragments of syndecan-2 (SDC2) in stool DNA.

BACKGROUND: Methylated SDC2 has been proved as a diagnostic marker for human colorectal cancer (CRC), noninvasive stool DNA-based methylation testing also emerges as a novel approach for detecting CRC. The aim of this study was to evaluate the clinical performance of stool DNA-based SDC2 methylation test by a new qPCR detection reagent for early detection of CRC. METHODS: A new qPCR detection reagent contained two differentially methylated regions in SDC2 CpG islands for the detection of CRC was used in this study. Performance of the SDC2 methylation detection reagent was evaluated by analyzing limit of detection, precision, and specificity. The effect of interfering substances on assay performance was also tested. 339 subjects (102 CRC patients, 50 patients with advanced adenomas, 39 patients with non-advanced adenomas, 18 colitis patients and 130 normal individuals) from the China-Japan Friendship Hospital were evaluated. Approximately 2.5 g of stool sample was collected from each participant. Stool DNA was extracted and bisulfite-converted, followed by qPCR assay, which contained two pairs of primers for the methylation detection of two fragments of the SDC2 gene (named SDC2-A and SDC2-B). The diagnostic value of this test in CRC was evaluated by calculating receiver operating characteristic (ROC) curve, and value of the area under the curve (AUC). RESULTS: The test kit was able to detect methylated SDC2 in stool DNA samples with concentrations as low as 90 copies/µL in 100% of replicates. The sensitivity for detecting CRC by methylated SDC2-A alone was 85.29% (95% CI 77.03-91.00%) with a specificity of 96.15% (95% CI 91.08-98.58%). The sensitivity by methylated SDC2-B alone was 83.33% (95% CI 74.82-89.42%) with a specificity of 97.69% (95% CI 93.14-99.51%). However, when methylated SDC2-A and methylated SDC2-B were combined, the sensitivity for CRC detection improved to 87.25% (95% CI 79.27-92.53%) with a specificity of 94.62% (95% CI 89.11-97.56%). Further, the detection reagent achieved ROC-AUC 0.874 (95% CI 0.822-0.927) for SDC2-A, 0.906 (95% CI 0.859-0.952) for SDC2-B, and 0.939 (95% CI 0.902-0.977) for SDC2-Combine A&B. CONCLUSIONS: This study validated the capability of stool DNA-based SDC2 methylation test for early screening of CRC, and combined detection of two fragments of SDC2 gene could improve detection sensitivity.

Rapid detection of the irinotecan-related UGT1A1*28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe.

BACKGROUND: Determination of UGT1A1 (TA)n polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA)n polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1*1 [(TA)6 ] and *28 [(TA)7 ] genotypes. METHODS: After protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1*1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China-Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra- and inter-run precision of the melting temperatures (Tm s) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity. RESULTS: All genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra- and inter-run coefficients of variation for the Tm s were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA. CONCLUSION: The developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low-cost detection of UGT1A1 (TA)n polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Effects of Compound 511 on BDNF-TrkB Signaling in the Mice Ventral Tegmental Area in Morphine-Induced Conditioned Place Preference.

Compound 511 (511) is specially developed for opioid addiction treatment based on the Ancient Chinese drug rehabilitation literature, and its composition has profound effects in the treatment of drug addiction in various clinical trials and animal experiments. The effect of 511 on the rewarding properties of morphine and craving responses and its potential mechanisms remain unclear. Here, we have applied a conditioned place preference (CPP) paradigm in mice to measure morphine-induced rewarding effects under the treatment of 511. Then we used the RNA sequencing strategy to screen its potential mechanisms. In our research, firstly, we found 511 could decrease CPP score, locomotor activity, self-administration, jumping behavior, weight loss, wet-dog shakes, and stereotyped behavior. Then the brain VTA region tissues were performed mRNA sequencing to detect potential mechanisms. We found the brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) were downregulated in morphine-induced CPP, whereas the decreased BDNF and TrkB were reversed after 511 treatment. We retested the levels of BDNF and TrkB using qRT-PCR and Western blot and found the similar results to mRNA sequencing. It has been widely reported that BDNF-TrkB signaling in the VTA is involved in multiple facets of addiction, including reward and motivation, so we focused on the BDNF-TrkB signaling to investigate the anti-addiction mechanisms of 511 in morphine addiction mice. We studied the downstream pathway of BDNF-TrkB and the soma size of dopaminergic neurons. The results showed 511 could increase the phosphorylation levels of PI3K and AKT, which were decreased in morphine-induced CPP. Simultaneously, 511 could decrease the level of PLCγ1 and the phosphorylation levels of ERK and S6K, which were increased in morphine-induced CPP. In addition, 511 also enlarged the soma size of VTA dopaminergic neurons, which was reduced in morphine-induced CPP. Hence, our research indicated 511 maybe mediate the BDNF-TrkB signaling in VTA to improve morphine addiction behavior.

Genetically elevated circulating homocysteine concentrations increase the risk of diabetic kidney disease in Chinese diabetic patients.

Diabetic kidney disease (DKD) is a devastating and frequent complication of diabetes mellitus. Here, we first adopted methylenetetrahytrofolate reductase (MTHFR) gene C677T polymorphism as an instrument to infer the possible causal relevance between circulating homocysteine and DKD risk in a Chinese population and next attempted to build a risk prediction model for DKD. This is a hospital-based case-control association study. Total 1107 study participants were diagnosed with type 2 diabetes mellitus, including 547 patients with newly diagnosed and histologically confirmed DKD. MTHFR gene C677T polymorphism was determined using the TaqMan method. Carriers of 677TT genotype (14.55 µmol/L) had significantly higher homocysteine concentrations than carriers of 677CT genotype (12.88 µmol/L) (P < 0.001). Carriers of 677TT genotype had a 1.57-fold increased risk of DKD (odds ratio: 1.57, 95% CI: 1.21-2.05, P = 0.001) relative to carriers of 677CT genotype after adjusting for confounders. Mendelian randomization analysis revealed that the odds ratio for DKD relative to diabetes mellitus per 5 µmol/L increment of circulating homocysteine concentrations was 3.86 (95% confidence interval: 1.21-2.05, P < 0.001). In the Logistic regression analysis, hypertension, homocysteine and triglyceride were significantly associated with an increased risk of DKD and they constituted a risk prediction model with good test performance and discriminatory capacity. Taken together, our findings provide evidence that elevated circulating homocysteine concentrations were causally associated with an increased risk of DKD in Chinese diabetic patients.

Interaction of MTHFR C677T polymorphism with smoking in susceptibility to diabetic nephropathy in Chinese men with type 2 diabetes.

We investigated the interaction of MTHFR C677T polymorphism (rs1801133) with smoking in susceptibility to diabetic nephropathy (DN) in Chinese men with type 2 diabetes mellitus (T2DM). We studied 655 Chinese men with T2DM, who were divided into two groups (321 with DN and 334 without DN). The genotype of MTHFR C677T polymorphism was detected by real-time polymerase chain reaction. MTHFR TT genotype carried a higher risk of DN compared with the CC genotype (OR = 2.05; P = 0.002). The T allele showed marked association with DN development in patients who smoked, using additive, recessive, and dominant models (OR = 1.60, 1.83, and 1.88, respectively; P = 0.006, 0.002, and 0.04, respectively), which was not observed in the nonsmoking group. Patients with TT and CT genotypes, who smoked had a higher risk of DN compared with the control group (non-smoking with CC genotype; OR = 3.73 and 2.28, respectively; P < 0.001 and P = 0.004, respectively), whereas the other groups were not observed. In conclusion, the T allele of rs1801133 may be a risk factor for DN in Chinese men with T2DM, and synergy appears to exist between the MTHFR rs1801133 and smoking in susceptibility to DN.

Transplantation of human fetal pancreatic progenitor cells ameliorates renal injury in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a severe complication of diabetes mellitus (DM). Pancreas or islet transplantation has been reported to prevent the development of DN lesions and ameliorate or reverse existing glomerular lesions in animal models. Shortage of pancreas donor is a severe problem. Islets derived from stem cells may offer a potential solution to this problem. OBJECTIVE: To evaluate the effect of stem cell-derived islet transplantation on DN in a rat model of streptozotocin-induced DM. METHODS: Pancreatic progenitor cells were isolated from aborted fetuses of 8 weeks of gestation. And islets were prepared by suspension culture after a differentiation of progenitor cells in medium containing glucagon-like peptide-1 (Glp-1) and nicotinamide. Then islets were transplanted into the liver of diabetic rats via portal vein. Blood glucose, urinary volume, 24 h urinary protein and urinary albumin were measured once biweekly for 16 weeks. Graft survival was evaluated by monitoring human C-peptide level in rat sera and by immunohistochemical staining for human mitochondrial antigen and human C-peptide in liver tissue. The effect of progenitor-derived islets on filtration membrane was examined by electron microscopy and real-time polymerase chain reaction (PCR). Immunohistochemical staining, real-time PCR and western blot were employed for detecting fibronectin, protein kinase C beta (PKCß), protein kinase A (PKA), inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD). RESULTS: Islet-like clusters derived from 8th gestational-week human fetal pancreatic progenitors survived in rat liver. And elevated serum level of human C-peptide was detected. Blood glucose, 24 h urinary protein and urinary albumin were lower in progenitor cell group than those in DN or insulin treatment group. Glomerular basement membrane thickness and fibronectin accumulation decreased significantly while podocytes improved morphologically in progenitor cell group. Furthermore, receptor of advanced glycation end products and PKCß became down-regulated whereas PKA up-regulated by progenitor cell-derived islets. And iNOS rose while SOD declined. CONCLUSIONS: DN may be reversed by transplantation of human fetal pancreatic progenitor cell-derived islets. And fetal pancreatic progenitor cells offer potential resources for cell replacement therapy.

Detection the Frequency and Characteristics of FLT3 Internal Tandem Duplication Mutations by Capillary Electrophoresis Assay and Next-Generation Sequencing in.

BACKGROUND: FLT3-internal tandem duplication mutations (ITDs) are found in approximately 30% of patients with acute myeloid leukemia (AML) and are markers of poor prognosis. However, the characteristics of FLT3/ ITDs in Chinese AML patients have rarely been reported. The aim of this study was to analyze the frequency and characteristics of FLT3/ITDs in Chinese AML patients. METHODS: In the selected 152 cases of Chinese AML patients, capillary electrophoresis (CE) was used to analyze the frequency and characteristics of FLT3/ITDs. Next-generation sequencing was used to analyze the sequences of FLT3/ITDs positive patients. The differences of clinical features between FLT3/ITD positive group and FLT3/ITD negative group were estimated by statistical analysis. RESULTS: 42 cases (27.6%) were FLT3/ITDs-positive, in which 34 cases (81%) had a single duplication, and the remaining 8 cases (19%) had 2 or more ITDs. Median ITD size was 42 bp and median ITD allelic ratio was 0.25. Using next-generation sequencing, ITDs integrating in non-juxtamembrane (JM) domains were detected in 12 ITDs (24%) and in JM domains were detected in 38 ITDs (76%). Furthermore, duplication of at least one residue between Y591 and Y599 was detected in 48 (96%) of all 50 ITDs, and the insertion site was strongly correlated with ITD size: more C-terminal located inserted fragments were significantly longer. CONCLUSIONS: Our data provide further evidence of the heterogeneity of FLT3/ITDs among different subgroups in Chinese AML patients. ITDs varied widely, but hotspots were concentrated. These results also suggest that nextgeneration sequencing is a useful method for detection of FLT3/ITDs sequences.

C-peptide ameliorates renal injury in type 2 diabetic rats through protein kinase A-mediated inhibition of fibronectin synthesis.

Type 1 diabetes mellitus (T1DM) is characterized by the deficiencies of insulin and C-peptide. Mounting evidences have proved the beneficial effects of C-peptide on the renal function in T1DM. However, it is still controversial about the roles of C-peptide in T2DM nephropathy since the level of C-peptide fluctuates greatly at different stages of T2DM. In the present study, we found that the serum C-peptide concentration was much lower in GK rats with diabetic nephropathy than that in normal counterparts. A sustained supplementation of C-peptide at a physiological level could ameliorate urinary albumin, independent of blood glucose control. C-peptide treatment improved glomerulosclerosis and podocyte morphology and reduced the thickness of glomerular basement membrane as compared with saline treatment control. Moreover, it decreased fibronectin synthesis in diabetic glomeruli and in cultured rat mesangial cells accompanied by a down-regulation of RAGE and an up-regulation of PKA. Interestingly, H-89, a PKA inhibitor, could reverse the inhibition effect of C-peptide on fibronectin production in cultured mesangial cells. These findings suggest that C-peptide level is low in T2DM rats with nephropathy and a treatment with a physiological dose of C-peptide can prevent renal injury in diabetic GK rats.

H2O2 oxidation - CaO precipitation pretreatment combining forward osmosis for electroless nickel spent tank liquid (ENSTL) reduction.

The electroless nickel spent tank liquid (ENSTL), as a typical hazardous waste, contains a variety of refractory organic substances as well as heavy metals and inorganic salts. Generally, ENSTL is delegated for disposing by qualified hazardous waste disposal departments in China. However, the temporary storage, transportation, and higher entrusted disposal expenses increase the burden on enterprises producing the hazardous ENSTL. This paper explored an oxidation/precipitation pretreatment and forward osmosis (FO) combined process for ENSTL reduction. 400 mmol/L Hydrogen peroxide and 5.0 wt% calcium oxide were selected as the optimal pretreatment in order to minimize the osmotic pressure of ENSTL, by which the conductivity was significantly reduced from 50.8 mS/cm to 26.8 mS/cm. As a result, the concentrating factor (N) could be dramatically increased from 2.45 by the direct FO to 8.71 by the combined system. Accordingly, the average water flux during the 24 h concentrating cycle increased from 2.47 L/(m2·h) to 4.56 L/(m2·h). TOC rejection rate decreased from 90.23% to 84.39% due to the transformation of organic matter forms by the chemical oxidation during the pretreatment. Meanwhile, TP, Ni and NH4+ rejection rates decreased to a certain extent, which may related to the mitigation of membrane fouling by the pretreatment.

Characteristics and Comparative Analysis of Six Mitogenomes of Genus Kiefferulus Goetghebuer, 1922 (Diptera: Chironomidae).

Chironomidae is a cosmopolitan and species-rich family of insects, with many species serving as useful indicators of aquatic ecosystem health. In this study, we newly sequenced six species of Kiefferulus Goetghebuer, 1922 (Chironomidae: Chironominae) by high-throughput sequencing technology. We analyzed characters of the mitochondrial genome, including the sequence length, nucleotide composition, and evolutionary rates of this genus. The size of the newly obtained sequences ranged from 15,588 to 15,767 bp, and all of them included 22 tRNAs, 13 PCGs, 2 rRNAs, and 1 CR. The CR showed the highest AT content relative to the PCGs, rRNAs, and tRNAs. Relative synonymous codon usage analysis showed that UUA, UUU, and AUU are the preferred codons. The ratio of nonsynonymous (Ka) to synonymous (Ks) substitution rates showed that all Ka/Ks of PCGs were lower than 1, with ATP8 having the highest evolution rate, while COX1 exhibited the lowest evolution rate. We reconstructed the phylogenetic relationship of the genus Kiefferulus based on eight species (six ingroups and two outgroups), using five matrices and employing Maximum likelihood and Bayesian inference approaches. Phylogenetic analysis of the Kiefferulus showed that six species within this genus were classified into a monophyletic clade.

Optimizing Flexible Microelectrode Designs for Enhanced Efficacy in Electrical Stimulation Therapy.

To investigate the impact of electrode structure on Electrical Stimulation Therapy (EST) for chronic wound healing, this study designed three variants of flexible microelectrodes (FMs) with Ag-Cu coverings (ACCs), each exhibiting distinct geometrical configurations: hexagonal, cross-shaped, and serpentine. These were integrated with PPY/PDA/PANI (3/6) (full name: polypyrrole/polydopamine/polyaniline 3/6). Hydrogel dressing comprehensive animal studies, coupled with detailed electrical and mechanical modeling and simulations, were conducted to assess their performance. Results indicated that the serpentine-shaped FM outperformed its counterparts in terms of flexibility and safety, exhibiting minimal thermal effects and a reduced risk of burns. Notably, FMs with metal coverings under 3% demonstrated promising potential for optoelectronic self-powering capabilities. Additionally, simulation data highlighted the significant influence of hydrogel non-uniformity on the distribution of electrical properties across the skin surface, providing critical insights for optimizing EST protocols when employing hydrogel dressings.

Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus.

Objective: A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods: Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results: By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 µmol/L and 0.2 µmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 µmol/L; the final concentrations of FluA probe and primer were 0.2 µmol/L and 0.3 µmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 µmol/L and 0.25 µmol/L, respectively. Conclusion: A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.

Tangshen formula improves diabetic nephropathy in STZ-induced diabetes rats fed with hyper-methionine by regulating the methylation status of kidney.

BACKGROUND: The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250-300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. RESULTS: Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. CONCLUSION: While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

Synthesis and Antibacterial Evaluation of Novel Vancomycin Derivatives Containing Quaternary Ammonium Moieties.

A series of novel vancomycin analogues with quaternary ammonium moieties have been designed and synthesized for fighting with clinically isolated drug-resistant bacteria. Partial target molecules exhibited potent activity against the tested strains. Among all of the compounds, a triazole quaternary ammonium vancomycin (QAV) derivative QAV-a1 exerted the best antibacterial activities. QAV-a1 was found to be 4- to 32-fold more efficacious than vancomycin against MRSA. Meanwhile, QAV-a1 showed a good pharmacokinetic profile with a half-life of 5.19 ± 0.10 h, which is longer than that of vancomycin (4.3 ± 1.9 h). These results provided guidance for the further exploitation of vancomycin derivatives against drug-resistant bacteria.

A pilot-scale SNAD-MBBR process for treating anaerobic digester liquor of swine wastewater: performance and microbial community.

In this pilot-scale study, simultaneous partial nitrification, anammox, and denitrification (SNAD) process was achieved successfully in a moving bed biofilm reactor (MBBR) for treating anaerobic digester liquor of swine wastewater. After 95 days of operation, when the total nitrogen loading rate of SNAD-MBBR process was 1.09 kg TN/m3/day, the total nitrogen removal rate could reach 0.87 kg TN/m3/day, and the removal efficiencies of ammonium and total nitrogen were 92.0% and 79.7%, respectively. The optimum pH and temperature for SNAD-MBBR process were 8.5 and 35 °C, respectively, and the optimum dissolved oxygen for SNAD1 and SNAD2 were 0.30 and 0.07 mg/L, respectively. The 16S rRNA sequencing suggested that Candidatus Kuenenia, Candidatus Brocadia, Nitrosomonas, and Denitratisoma were the dominant nitrogen removal bacteria. Some of the co-existing bacteria (Truepera, Limnobacter, and Anaerolineaceae uncultured) promoted ammonium oxidation and guaranteed the growth of the anammox bacteria under adverse environmental conditions. Overall, this study demonstrated that the SNAD-MBBR process would be an energy-saving and cost-effective method for the removal of nitrogen from swine wastewater and provided important process parameters for stable operation of the full-scale SNAD process.

Immunosuppression by opioids: Mechanisms of action on innate and adaptive immunity.

Opioids are excellent analgesics for the clinical treatment of various types of acute and chronic pain, particularly cancer-related pain. Nevertheless, it is well known that opioids have some nasty side effects, including immunosuppression, which is commonly overlooked. As a result, the incidence of opportunistic bacterial and viral infections increases in patients with long-term opioid use. Nowadays, there are no effective medications to alleviate opioid-induced immunosuppression. Understanding the underlying molecular mechanism of opioids in immunosuppression can enable researchers to devise effective therapeutic interventions. This review comprehensively summarized the exogenous opioids-induced immunosuppressive effects and their underlying mechanisms, the regulatory roles of endogenous opioids on the immune system, the potential link between opioid immunosuppressive effect and the function of the central nervous system (CNS), and the future perspectives in this field.

Fast and Sensitive Detection of SARS-CoV-2 Nucleic Acid Using a Rapid Detection System Free of RNA Extraction.

Objectives: To establish and evaluate the analytical and clinical performance of the Flash20 SARS-CoV-2 nucleic acid rapid detection system free of RNA extraction. Methods: The limit of detection (LoD) was determined using a negative nasopharyngeal swab matrix spiked with different concentrations of SARS-CoV-2 virus; a total of 734,337 reference sequences of viral genomes from GenBank were used for the in-silico analysis to assess the inclusivity of the assay. The specificity of the system was evaluated by testing 27 medically relevant organisms. A total of 115 clinical specimens were collected and tested on the Flash20 SARS-CoV-2 detection system and with an FDA-approved comparator test to assess the clinical performance of the system. Results: The LoD of the Flash20 SARS-CoV-2 detection system is 250 copies/mL with a positive rate ≥90% (n = 20); alignments results showed that over 99% identity of the primer and probe of the Flash20 SARS-CoV-2 nucleic acid rapid detection system to the available SARS-CoV-2 sequences; the omicron samples tested 100% positive. None of the 27 organisms showed cross-reactivity with the Flash20 SARS-CoV-2 nucleic acid rapid detection system. Among all the 215 clinical samples, the Flash20 SARS-CoV-2 nucleic acid rapid detection system exhibits a high sensitivity of 99.24% (131/132) and 100% (83/83) specificity. Conclusion: The nucleic acid rapid detection system provides sensitive and accurate detection of SARS-CoV-2 free of RNA extraction. The high sensitivity and short time to results of approximately 35 minutes may impact earlier infection control and disease management.