Dual thermal ecotypes coexist within a nearly genetically identical population of the unicellular marine cyanobacterium Synechococcus.

The extent and ecological significance of intraspecific functional diversity within marine microbial populations is still poorly understood, and it remains unclear if such strain-level microdiversity will affect fitness and persistence in a rapidly changing ocean environment. In this study, we cultured 11 sympatric strains of the ubiquitous marine picocyanobacterium Synechococcus isolated from a Narragansett Bay (RI) phytoplankton community thermal selection experiment. Thermal performance curves revealed selection at cool and warm temperatures had subdivided the initial population into thermotypes with pronounced differences in maximum growth temperatures. Curiously, the genomes of all 11 isolates were almost identical (average nucleotide identities of >99.99%, with >99% of the genome aligning) and no differences in gene content or single nucleotide variants were associated with either cool or warm temperature phenotypes. Despite a very high level of genomic similarity, sequenced epigenomes for two strains showed differences in methylation on genes associated with photosynthesis. These corresponded to measured differences in photophysiology, suggesting a potential pathway for future mechanistic research into thermal microdiversity. Our study demonstrates that present-day marine microbial populations can harbor cryptic but environmentally relevant thermotypes which may increase their resilience to future rising temperatures.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9.

BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

Epigallocatechin-3-gallate and cancer: focus on the role of microRNAs.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that affect gene expression. The role of miRNAs in different types of cancers has been published and it was shown that several miRNAs are inappropriately expressed in different cancers. Among the mechanisms that can cause this lack of proper expression are epigenetics, chromosomal changes, polymorphisms or defects in processing proteins. Recent research shows that phytochemicals, including epigallocatechin-3-gallate (EGCG), exert important epigenetic-based anticancer effects such as pro-apoptotic or anti proliferative through miRNA gene silencing. Given that EGCG is able to modulate a variety of cancer-related process i.e., angiogenesis, proliferation, metastasis and apoptosis via targeting various miRNAs such as let-7, miR-16, and miR-210. The discovery of new miRNAs and the differences observed in their expression when exposed to EGCG provides evidence that targeting these miRNAs may be beneficial as a form of treatment. In this review, we aim to provide an overview, based on current knowledge, on how phytochemicals, including epigallocatechin-3-gallate, can be considered as potential miRNAs modulator to improve efficacy of current cancer treatments.

A randomized controlled trial of iliopsoas plane block vs. femoral nerve block for hip arthroplasty.

BACKGROUND: Iliopsoas plane block (IPB) is a novel analgesic technique for hip surgery that retains quadriceps strength. However, evidence from randomized controlled trial is remains unavailable. We hypothesized that IPB, as a motor-sparing analgesic technique, could match the femoral nerve block (FNB) in pain management and morphine consumption, providing an advantage for earlier functional training in patients underwent hip arthroplasty. METHODS: We recruited ninety patients with femoral neck fracture, femoral head necrosis or hip osteoarthritis who were scheduled for unilateral primary hip arthroplasty were recruited and received either IPB or FNB. Primary outcome was the pain score during hip flexion at 4 h after surgery. Secondary outcomes included quadriceps strength and pain scores upon arrival at post anesthesia care unit (PACU) and at 2, 4, 6, 24, 48 h after surgery, the first time out of bed, total opioids consumption, patient satisfaction, and complications. RESULTS: There was no significant difference in terms of pain score during hip flexion at 4 h after surgery between the IPB group and FNB group. The quadriceps strength of patients receiving IPB was superior to those receiving FNB upon arrival at PACU and at 2, 4, 6 and 24 h after surgery. The IPB group showed a shorter first time out of bed compared to the FNB group. However, there were no significant differences in terms of pain scores within 48 h after surgery, total opioids consumption, patient satisfaction and complications between the two groups. CONCLUSION: IPB was not superior to FNB in terms of postoperative analgesia for hip arthroplasty. However, IPB could serve as an effective motor-sparing analgesic technique for hip arthroplasty, which would facilitate early recovery and rehabilitation. This makes IPB worth considering as an alternative to FNB. TRIAL REGISTRATION: The trial was registered prior to patient enrollment at the Chinese Clinical Trial Registry (ChiCTR2200055493; registration date: January 10, 2022; enrollment date: January 18, 2022; https://www.chictr.org.cn/searchprojEN.html ).

Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/ß-Catenin pathway.

BACKGROUND: Hypoxia naturally happens in embryogenesis and thus serves as an important environmental factor affecting embryo development. Hif-1α, an essential hypoxia response factor, was mostly considered to mediate or synergistically regulate the effect of hypoxia on stem cells. However, the function and relationship of hypoxia and Hif-1α in regulating mesendoderm differentiation remains controversial. RESULTS: We here discovered that hypoxia dramatically suppressed the mesendoderm differentiation and promoted the ectoderm differentiation of mouse embryonic stem cells (mESCs). However, hypoxia treatment after mesendoderm was established promoted the downstream differentiation of mesendoderm-derived lineages. These effects of hypoxia were mediated by the repression of the Wnt/ß-Catenin pathway and the Wnt/ß-Catenin pathway was at least partially regulated by the Akt/Gsk3ß axis. Blocking the Wnt/ß-Catenin pathway under normoxia using IWP2 mimicked the effects of hypoxia while activating the Wnt/ß-Catenin pathway with CHIR99021 fully rescued the mesendoderm differentiation suppression caused by hypoxia. Unexpectedly, Hif-1α overexpression, in contrast to hypoxia, promoted mesendoderm differentiation and suppressed ectoderm differentiation. Knockdown of Hif-1α under normoxia and hypoxia both inhibited the mesendoderm differentiation. Moreover, hypoxia even suppressed the mesendoderm differentiation of Hif-1α knockdown mESCs, further implying that the effects of hypoxia on the mesendoderm differentiation were Hif-1α independent. Consistently, the Wnt/ß-Catenin pathway was enhanced by Hif-1α overexpression and inhibited by Hif-1α knockdown. As shown by RNA-seq, unlike hypoxia, the effect of Hif-1α was relatively mild and selectively regulated part of hypoxia response genes, which fine-tuned the effect of hypoxia on mESC differentiation. CONCLUSIONS: This study revealed that hypoxia is fine-tuned by Hif-1α and regulates the mesendoderm and ectoderm differentiation by manipulating the Wnt/ß-Catenin pathway, which contributed to the understanding of hypoxia-mediated regulation of development.

A novel SLC8A1/LINC01913 intergenic region-ALK fusion identified by NGS and validated by IHC and FISH in a stage IIB lung adenocarcinoma patient who remains relapse-free during the treatment of crizotinib: a case report.

In resected non-small cell lung cancer (NSCLC), ALK rearrangements are associated with worse recurrence-free survival (RFS) than other driver genes. In addition, the micropapillary pattern of NSCLC is associated with a poor prognosis. In recent years, crizotinib tyrosine kinase inhibitors (TKIs) have been widely used to treat patients with advanced NSCLC with ALK fusion. Patient survival outcomes have become highly promising, reflecting the necessity of exploring the application of ALK-TKIs in resected, early stage NSCLC with ALK rearrangements. A 60-year-old Chinese man was diagnosed with stage IIB lung adenocarcinoma harboring a novel SLC8A1/LINC01913 intergenic region-ALK fusion identified by NGS and validated by immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH). Crizotinib (250 mg orally once daily) was administered to the patient following surgery. The patient remained relapse-free after four months and seven months. This report provided a valuable treatment plan for early lung adenocarcinoma patients with high risks to prevent a postoperative recurrence.

Number-dependent association of potentially inappropriate medications with clinical outcomes and expenditures among community-dwelling older adults: A population-based cohort study.

AIMS: The aim of this study was to investigate the prevalence of potentially inappropriate medication (PIM) prescribing and its number-dependent association (PIM = 1, 2, ≥3) with all-cause hospitalizations, emergency department (ED) visits, and medication expenditures in Beijing, China. METHODS: A retrospective cohort analysis was conducted to analyse PIM prescribing in community-dwelling older adults aged ≥65 years within the Beijing Municipal Medical Insurance Database (data from July to September 2016). The prevalence of PIMs was estimated based on the 2015 Beers Criteria. Logistic models were utilized to investigate the associations between PIM use and all-cause hospitalizations and ED visits. Generalized linear models with the logic link and gamma distribution were used to analyse associations between PIM use and medication expenditures. RESULTS: Among the 506 214 older adults, the prevalence of PIM was 38.07%. After adjusting for covariables, prescribing two and three or more PIMs was associated with increased risks of hospitalizations (PIM = 2: odds ratio [OR] 1.34, 95% confidence interval [CI]: 1.22-1.47; PIM ≥ 3: OR = 1.47, 95% CI: 1.32-1.63) and ED visits (PIM = 2: OR = 1.29, 95% CI 1.12-1.48; PIM ≥ 3: OR = 1.23, 95% CI: 1.04-1.44). Exposures to two and three or more PIMs were associated with higher medication expenditures for inpatient visits (PIM = 2: incidence rate ratio [IRR] = 1.08, 95% CI 1.01-1.16; PIM ≥ 3: IRR = 1.18, 95% CI: 1.08-1.28). Vasodilators were the most frequent PIM prescribing group among patients who were hospitalized or had to visit the ED. CONCLUSIONS: PIMs were prescribed at a high rate among community-dwelling older adults in Beijing. Two or more PIMs were associated with increased risks of hospitalizations, ED visits, and increased inpatient medication expenditures. Effective interventions are needed to target unnecessary and inappropriate medications in older adults.

Salivary IgA antibody to malondialdehyde-acetaldehyde associates with mild periodontal pocket depth.

OBJECTIVE: Oxidized epitopes such as malondialdehyde-acetaldehyde (MAA) play a crucial role in the progression of atherosclerosis through activation of the humoral immune response. The exact mechanism of the association between atherosclerosis and periodontal diseases is not fully understood. The aim of the current study is to evaluate the association of oral humoral immune response to oxidized epitopes with parameters of periodontal disease. MATERIALS AND METHODS: The Parogene cohort consist of patients who have undergone coronary angiography due to cardiac symptoms. In this study, 423 patients were randomly selected for an extensive oral examination. Salivary Immunoglobulin A to oxidized epitopes and bacterial antigens was determined by chemiluminescence immunoassay. RESULTS: In a binary logistic regression model adjusted with periodontal disease confounders, periodontal pocket depth (PPD) 4-5 mm associated with salivary IgA antibodies to MAA-LDL (p = 0.034), heat shock protein 60 of Aggregatibacter actinomycetemcomitans (p = 0.045), Porphyromonas gingivalis (p = 0.045), A. actinomycetemcomitans (p = 0.005), P. intermedia (p = 0.020), and total IgA (p = 0.003). CONCLUSIONS: The current study shows the association of salivary IgA to MAA-LDL with PPD 4-5 mm in a cohort of patients with chronic coronary artery disease. Humoral immune cross-reactivation to oxidized epitopes such MAA-LDL could partly explain the link of periodontitis with systemic diseases.

The effect of continuous adductor canal block combined with distal interspace between the popliteal artery and capsule of the posterior knee block for total knee arthroplasty: a randomized, double-blind, controlled trial.

BACKGROUND: The optimal analgesia for total knee arthroplasty (TKA) requires excellent analgesia while preserving muscle strength. This study aimed to determine the hypothesis that continuous adductor canal block (CACB) combined with the distal interspace between the popliteal artery and the posterior capsule of the knee (IPACK) block could effectively alleviate the pain of the posterior knee, decrease opioids consumption, and promote early recovery and discharge. METHODS: Patients undergoing unilateral, primary TKA were allocated into group CACB+SHAM (receiving CACB plus sham block) or group CACB+IPACK (receiving CACB plus IPACK block). The primary outcome was cumulative opioid consumption. Secondary outcomes included the incidence of postoperative pain originated from the posterior knee, visual analogue scale (VAS) score, range of motion, ambulation distance, and satisfaction for pain management. RESULTS: The incidence of moderate-severe pain of the posterior knee was lower in group CACB+IPACK than that of the group CACB+SHAM at 4 hours (17.1% vs. 42.8%; p = 0.019), 8 hours (11.4% vs. 45.7%; p = 0.001), and 24 hours (11.4% vs. 34.3%; p = 0.046) after TKA. The VAS scores of the posterior knee were lower in group CACB+IPACK than that of the group CACB+SHAM at 4 hours [2 (2) vs. 3 (2-4); p = 0.000], 8 hours [1 (1, 2) vs. 3 (2-4); p = 0.001], and 24 hours [1(0-2) vs. 2 (1-4); p = 0.002] after TKA. The overall VAS scores were lower in group CACB+IPACK than that of the group CACB+SHAM at 4 hours [3 (2, 3) vs. 3 (3, 4); p = 0.013] and 8 hours [2 (2, 3) vs. 3 (2-4); p = 0.032] at rest and 4 hours [3 (3, 4) vs. 4 (4, 5); p = 0.001], 8 hours [3 (2-4) vs. 4 (3-5); p = 0.000], 24 hours [2 (2, 3) vs. 3 (2-4); p = 0.001] during active flexion after TKA. The range of motion (59.11 ± 3.90 vs. 53.83 ± 5.86; p = 0.000) and ambulation distance (44.60 ± 4.87 vs. 40.83 ± 6.65; p = 0.009) were superior in group CACB+IPACK than that of the group CACB+SHAM in postoperative day 1. The satisfaction for pain management was higher in group CACB+IPACK than that of the group CACB+SHAM [9 (8, 9) vs. 8 (7-9); p = 0.024]. There was no difference in term of cumulative opioids consumption between group CACB+IPACK and group CACB+SHAM [120(84-135) vs. 120(75-135); p = 0.835]. CONCLUSION: The combination of CACB and distal IPACK block could decrease the incidences of moderate-severe posterior knee pain, improve the postoperative pain over the first 24 hours after TKA, as well as promoting recovery of motor function. However, the opioids consumption was not decreased by adding distal IPACK to CACB. TRIAL REGISTRATION: This study was registered at Chinese Clinical Trial Registry ( ChiCTR2200059139 ; registration date: 26/04/2022; enrollment date: 16/11/2020; http://www.chictr.org.cn ).

Recipient C7 rs9292795 genotype and the risk of hepatocellular carcinoma recurrence after orthotopic liver transplantation in a Han Chinese population.

BACKGROUND: Complement component(C7) gene has been shown to influence the prognosis in Hepatocellular carcinoma (HCC) patients. The association between C7 and HCC recurrence after orthotopic liver transplantation (OLT), however, is still unknown. The purpose of this study was to evaluate whether the donor and recipient C7 gene polymorphisms are related to HCC recurrence after OLT in the Han Chinese population. METHODS: A total of 73 consecutive patients with HCC who had undergone OLT, both donors and recipients, were involved in this research. A single nucleotide polymorphism of C7, rs9292795, was genotyped using Sequenom MassARRAY in the cohort. The expression of C7 and the association between C7 gene polymorphisms and HCC recurrence following OLT were analyzed by bioinformatics and statistical analysis, respectively. RESULTS: As shown in database, the expression of C7 was higher in HCC tissues than that in normal tissues, and represented a worse prognosis. We also found that recipient C7 rs9292795 polymorphism, rather than the donor, was significantly associated with HCC recurrence after OLT. Multivariate logistic regression analysis confirmed that TNM stage (P = 0.001), Milan criteria (P = 0.000) and recipient rs9292795 genotype (TT vs AA/AT, P = 0.008) were independent risk factors for HCC recurrence. Furthermore, the recipient carrying AA/AT showed higher recurrence-free survival (RFS) and overall survival (OS) than that carrying TT (P < 0.05). In Cox proportional hazards model, TNM stage, recipient rs9292795 genotype, and Milan criteria were identified as independent factors for RFS and OS (P < 0.05) as well as pre-OLT serum alpha fetoprotein (AFP) level was associated with OS (P < 0.05). CONCLUSIONS: Recipient C7 rs9292795 gene polymorphism is related to the recurrence of HCC after OLT, which may be a helpful prognostic marker for HCC patients who receive OLT.

Perineural Dexmedetomidine Reduces the Median Effective Concentration of Ropivacaine for Adductor Canal Block.

BACKGROUND Multimodal analgesic regimens are well known as the best option for total knee arthroplasty. They include the adductor canal block, combined with local infiltration analgesia and a block of the interspace between the popliteal artery and the capsule of the posterior knee. However, these analgesic techniques all require a large amount of local anesthetics. In this study, we explored whether the quantity of local anesthetics could be decreased by using dexmedetomidine for the adductor canal block. MATERIAL AND METHODS Fifty-four patients scheduled for unilateral, primary total knee arthroplasty were allocated into 2 groups: the ropivacaine group (group R) and the dexmedetomidine group (group RD). Ropivacaine 0.5% was chosen as the initial concentration, and the concentration was decreased or increased according to the response of the previous participant. Based on Dixon's up-and-down method, the median effective concentration was calculated. RESULTS The quadriceps strength was similar between the 2 groups, both at 30 min after adductor canal block and during recovery from general anesthesia in the Postanesthesia Care Unit. None of the patients in this study exhibited bradycardia or hypotension. The median effective concentration of ropivacaine for adductor canal block was 0.29% (95% confidence interval [CI], 0.28-0.31%) in group RD, which was lower than that in group R (0.38% [95% CI, 0.36-0.41%]). CONCLUSIONS This study found perineural dexmedetomidine 1 µg/kg could reduce the median effective concentration of ropivacaine for the adductor canal block.

Application of 3-Dimensional Printing in Pediatric Living Donor Liver Transplantation: A Single-Center Experience.

Three-dimensional (3D) printing has been used to support organ transplantations. However, whether it helps remains unclear. This study aimed to present and assess the application of 3D-printed liver models in pediatric living donor liver transplantation (LDLT). The 3D images were printed to touchable liver models with transparent liver parenchyma, specifically colored hepatic vessels, and biliary structures. A total of 30 consecutive recipients were enrolled in the study: 10 were operated on with the support of 3D printing (3D-printing group) and 20 (control group) were operated on without it. Detailed photographs and data of the cases in the 3D-printing group were presented. One patient underwent auxiliary partial orthotopic liver transplantation using the left lobe graft, in which the abdominal cavity model was also printed to test whether the planned graft fit the recipient's abdominal cavity. The 3D-printed models facilitated surgical planning and procedures, particularly in the management of hepatic veins and in the prevention of large-for-size syndrome. The operative time of donors in the 3D-printing group was significantly shorter compared with the control group (2.3 ± 0.4 versus 3.0 ± 0.4 hours; P < 0.001). Inpatient costs for donors in the 3D-printing group were 17.1% lower than those in the control group (34.6 ± 6.6 versus 41.7 ± 10.4 thousand ¥; P = 0.03). In conclusion, in small infants and complicated pediatric LDLT patients, 3D-printed models can help minimize the risk of large-for-size syndrome and graft reduction. The 3D-printed models may be conducive to liver graft procurement and intraoperative assistance in pediatric LDLT.

Diagnostic value of near-infrared or fluorescent indocyanine green guided sentinel lymph node mapping in gastric cancer: A systematic review and meta-analysis.

BACKGROUND: This meta-analysis was conducted to evaluate the diagnostic value of near-infrared (NIR) or fluorescent indocyanine green (ICG) guided sentinel lymph node (SLN) mapping in gastric cancer (GC). METHODS: This meta-analysis was registered at the PROSPERO. Clinical studies were retrieved from the electronic database Pubmed, Embase, Medline, Web of science, and the Cochrane Library. Quality assessment was conducted by an adapted checklist of QUADAS-2. A bivariate mixed-effects model was used to pool the data. Evaluation of articles quality, analysis for publication bias, summary receiver operator characteristic (SROC) curves, and meta-regression were also performed. Subgroup analysis was used to explain the heterogeneities. RESULTS: A total of 13 clinical studies (971 patients) were included. The NIR or fluorescent imaging (FI) involved infrared ray electronic endoscopy (IREE), infrared ray laparoscopic system (IRLS), and FI system. Significant evidence of heterogeneity was found for sensitivity and specificity (I 2 = 91.1% and I 2 = 98.2%), respectively. The pooled SLN sensitivity (Sen), specificity (Spe), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were 0.94(95%CI: 0.80-0.99), 1.00(95%CI: 0.60-1.00), 34.0(95%CI: 9.25-125.29), 0.06(95%CI:0.02-0.22), and 252.50(95%CI: 94.93-671.61), respectively. Area under curve (AUC) of SROC curve was 1.00 (95%CI: 0.99-1.00), and the summary operating point (cut-off value) was SENS = 0.94(95%CI: 0.80-0.99) and SPEC = 1.00(95%CI: 0.60-1.00). Subgroup analysis showed that NIR imaging, imaging performed 20 minutes after intraoperative injection, preoperative injection (especially for FI imaging), stained with immunohistochemistry (IHC) (+hematoxylin-eosin [HE]), cT1 stage, submucosa injection (especially for cT1), mean number of SLN ≥ 5, study size > 26 were associated with higher SLN sensitivity. In terms of ICG concentration, diluted ICG concentration that 0.5 mg/mL (compared with 5 mg/mL) in NIR imaging and 0.05 mg/mL (compared with 0.5 mg/mL) in FI system showed higher sensitivities. However, the differences in tumor diameter (≤30 mm vs >30 mm), gastrectomy methods (opening vs laparoscopy), lymphadenectomy methods (LBD vs pick-up), and publication year (≥2010 vs <2010) did not achieve statistical significance. CONCLUSION: ICG combined with NIR or FI guided SLN mapping is technically feasible for GC. Based on the small sample size evidence, the IREE and IRLS devices may have higher sensitivity than FI in current clinical studies; and there may be an excessive ICG concentration used for current SLN mapping in GC. However, well-designed further studies with large sample size are needed to confirm the best procedure and suitable criteria. MINI-ABSTRACT: This meta-analysis was registered at PROSPERO. Clinical studies on this topic were retrieved from the electronic database Pubmed, Embase, Medline, Web of Science, and the Cochrane Library. The NIR or FI involved IREE, IRLS, and FI techniques. A total of 13 clinical studies (971 patients) were included. Based on the small sample size evidence, NIR or FI ICG guided SLN mapping in GC is technically feasible.

Cross-reactive saliva IgA antibodies to oxidized LDL and periodontal pathogens in humans.

AIM: Oxidized low-density lipoproteins (oxLDL) are formed as a result of lipid peroxidation and are highly immunogenic and proatherogenic. In this study, saliva antibodies binding to oxLDL, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were characterized and their cross-reactivity was evaluated. MATERIALS AND METHODS: Resting and stimulated saliva samples were collected from 36 healthy adults (mean age 26 years). Saliva IgA, IgG and IgM autoantibody levels to copper oxidized LDL (CuOx-LDL) and malondialdehyde acetaldehyde-modified LDL (MAA-LDL) were determined with chemiluminescence immunoassay. RESULTS: Saliva IgA and IgG antibodies binding to MAA-LDL and CuOx-LDL were detected in all samples and they were associated with the saliva levels of IgA and IgG to P. gingivalis and A. actinomycetemcomitans. Competitive immunoassay showed that saliva antibodies to MAA-LDL cross-reacted specifically with P. gingivalis. The autoantibody levels to oxLDL in saliva were not associated with the autoantibody levels to oxLDL in plasma or with saliva apolipoprotein B 100 levels. CONCLUSIONS: Saliva contains IgA and IgG binding to oxLDL, which showed cross-reactive properties with the periodontal pathogens Porphyromonas gingivalis (P.g). The data suggest that secretory IgA to P.g may participate in immune reactions involved in LDL oxidation through molecular mimicry.

Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human α9α10 Nicotinic Acetylcholine Receptor.

Conotoxins are a pool of disulfide-rich peptide neurotoxins produced by cone snails for predation and defense. They are a rich reservoir of novel ligands for ion channels, neurotransmitter receptors and transporters in the nervous system. In this study, we identified a novel conotoxin component, O-conotoxin GeXXVIIA, from the venom of Conus generalis. The native form of this component is a disulfide-linked homodimer of a 5-Cys-containing peptide. Surprisingly, our electrophysiological studies showed that, in comparison to the folded monomers, the linear peptide of this toxin had the highest inhibitory activity at the human α9α10 nicotinic acetylcholine receptor (nAChR), with an IC50 of 16.2 ± 1.4 nM. The activities of the N-terminal and C-terminal halves of the linear toxin are markedly reduced compared with the full-length toxin, suggesting that the intact sequence is required to potently inhibit the hα9α10 nAChR. α9α10 nAChRs are expressed not only in the nervous system, but also in a variety of non-neuronal cells, such as cochlear hair cells, keratinocytes, epithelial and immune cells. A potent inhibitor of human α9α10 nAChRs, such as GeXXVIIA, would facilitate unraveling the functions of this nAChR subtype. Furthermore, this unusual nAChR inhibitor may lead to the development of novel α9α10 nAChR-targeting drugs.

A Novel Lid-Covering Peptide Inhibitor of Nicotinic Acetylcholine Receptors Derived from αD-Conotoxin GeXXA.

Nicotinic acetylcholine receptors (nAChRs) play a fundamental role in nervous signal transmission, therefore various antagonists and agonists are highly desired to explore the structure and function of nAChRs. Recently, a novel dimeric αD-conotoxin GeXXA was identified to inhibit nAChRs by binding at the top surface of the receptors, and the monomeric C-terminal domain (CTD) of αD-GeXXA retains some inhibitory activity. In this study, the internal dimeric N-terminal domain (NTD) of this conopeptide was further investigated. We first developed a regio-selective protection strategy to chemically prepare the anti-parallel dimeric NTD, and found that the isolated NTD part of GeXXA possesses the nAChR-inhibitory activity, the subtype-dependence of which implies a preferred binding of NTD to the ß subunits of nAChR. Deletion of the NTD N-terminal residues did not affect the activity of NTD, indicating that the N-terminus is not involved in the interaction with nAChRs. By optimizing the sequence of NTD, we obtained a fully active single-chain cyclic NTD, based on which 4 Arg residues were found to interact with nAChRs. These results demonstrate that the NTD part of αD-GeXXA is a "lid-covering" nAChR inhibitor, displaying a novel inhibitory mechanism distinct from other allosteric ligands of nAChRs.

Immune status of children with obstructive sleep apnea/hypopnea syndrome.

OBJECTIVE: We aimed to evaluate the immune status of children with obstructive sleep apnea/hypopnea syndrome (OSAHS). METHODS: Fifty children with OSAHS having the symptoms of "snoring, mouth breathing and suffocating during sleep", who were admitted in our hospital from May 2014 to May 2016, were randomly selected. Another 52 healthy, age- and gender-matched children were enrolled as control subjects after taking informed consent. After admission, the peripheral venous blood was collected. T cell subsets and cytokines were analyzed by flow cytometry. Immunoglobulin and complement levels were detected by immunoassay analyzer. RESULTS: The percentage of CD8+ T lymphocytes in children with OSAHS was (26.47 ± 1.52)% which was significantly higher than that of control group ((21.94 ± 1.92)%) (P<0.05). OSAHS group had a significantly lower CD4+/CD8+ ratio (1.24 ± 0.12) than that of control group (1.45 ± 0.11) (P<0.05). The two groups had similar percentages of CD3+ and CD4+ T lymphocytes (P>0.05). OSAHS group had significantly higher serum levels of IL-4, IL-6, IL-10 and IFN-γ than those of control group (P<0.05), but their IL-2 and TNF-α levels were similar (P>0.05). The serum IgA and C3 levels of OSAHS group significantly exceeded those of control group (P<0.05), but their IgG, IgM and C4 levels were similar (P>0.05). CONCLUSION: Children with OSAHS had increased percentage of CD8+ T lymphocytes and decreased CD4+/CD8+ ratio, suggesting this group had poor immune function. Increase in humoral immune-related indices IL-4, IL-6, IL-10 and IFN-γ indicated the occurrence of oxidative stress and systemic inflammatory status.

New Insights into the Coupling between Microtubule Depolymerization and ATP Hydrolysis by Kinesin-13 Protein Kif2C.

Kinesin-13 proteins depolymerize microtubules in an ATP hydrolysis-dependent manner. The coupling between these two activities remains unclear. Here, we first studied the role of the kinesin-13 subfamily-specific loop 2 and of the KVD motif at the tip of this loop. Shortening the loop, the lysine/glutamate interchange and the additional Val to Ser substitution all led to Kif2C mutants with decreased microtubule-stimulated ATPase and impaired depolymerization capability. We rationalized these results based on a structural model of the Kif2C-ATP-tubulin complex derived from the recently determined structures of kinesin-1 bound to tubulin. In this model, upon microtubule binding Kif2C undergoes a conformational change governed in part by the interaction of the KVD motif with the tubulin interdimer interface. Second, we mutated to an alanine the conserved glutamate residue of the switch 2 nucleotide binding motif. This mutation blocks motile kinesins in a post-conformational change state and inhibits ATP hydrolysis. This Kif2C mutant still depolymerized microtubules and yielded complexes of one Kif2C with two tubulin heterodimers. These results demonstrate that the structural change of Kif2C-ATP upon binding to microtubule ends is sufficient for tubulin release, whereas ATP hydrolysis is not required. Overall, our data suggest that the conformation reached by kinesin-13s upon tubulin binding is similar to that of tubulin-bound, ATP-bound, motile kinesins but that this conformation is adapted to microtubule depolymerization.

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.

BACKGROUND: Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations. RESULTS: Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active. CONCLUSIONS: Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.