Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

Phase III clinical trial comparing the efficacy and safety of adamgammadex with sugammadex for reversal of rocuronium-induced neuromuscular block.

BACKGROUND: Preliminary clinical trials of adamgammadex, a new cyclodextrin-based selective reversal agent, have demonstrated its efficacy in reversing neuromuscular block by rocuronium. METHODS: This multicentre, randomised, double-blind, positive-controlled, non-inferiority phase III clinical trial compared the efficacy and safety of adamgammadex and sugammadex. We randomised 310 subjects to receive adamgammadex (4 mg kg-1) or sugammadex (2 mg kg-1) at reappearance of the second twitch of the train-of-four (TOF), and standard safety data were collected. RESULTS: For the primary outcome, the proportion of patients with TOF ratio ≥0.9 within 5 min was 98.7% in the adamgammadex group vs 100% in the sugammadex group, with a point estimate and 95% confidence interval (CI) of 1.3% (-4.6%, +1.3%); the lower limit was greater than the non-inferiority margin of -10%. For the key secondary outcome, the median (inter quartile range) time from the start of administration of adamgammadex or sugammadex to recovery of TOF ratio to 0.9 was 2.25 (1.75, 2.75) min and 1.75 (1.50, 2.00) min, respectively. The difference was 0.50 (95% CI: 0.25, 0.50); the upper limit was lower than the non-inferiority margin of 5 min. In addition, there were no inferior results observed in secondary outcomes. Adamgammadex had a lower incidence of adverse drug reactions compared with sugammadex (anaphylactic reaction, recurarisation, decreased heart rate, and laryngospasm; P=0.047). CONCLUSIONS: Adamgammadex was non-inferior to sugammadex with a possible lower incidence of adverse drug reactions compared with sugammadex. Adamgammadex may have a potential advantage in terms of its overall risk-benefit profile. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000039525. Registered October 30, 2020. https://www.chictr.org.cn/showproj.html?proj=56825.

Noninvasive prediction of lymph node metastasis in pancreatic cancer using an ultrasound-based clinicoradiomics machine learning model.

OBJECTIVES: This study was designed to explore and validate the value of different machine learning models based on ultrasound image-omics features in the preoperative diagnosis of lymph node metastasis in pancreatic cancer (PC). METHODS: This research involved 189 individuals diagnosed with PC confirmed by surgical pathology (training cohort: n = 151; test cohort: n = 38), including 50 cases of lymph node metastasis. Image-omics features were extracted from ultrasound images. After dimensionality reduction and screening, eight machine learning algorithms, including logistic regression (LR), support vector machine (SVM), K-nearest neighbors (KNN), random forest (RF), extra trees (ET), extreme gradient boosting (XGBoost), light gradient boosting machine (LightGBM), and multilayer perceptron (MLP), were used to establish image-omics models to predict lymph node metastasis in PC. The best omics prediction model was selected through ROC curve analysis. Machine learning models were used to analyze clinical features and determine variables to establish a clinical model. A combined model was constructed by combining ultrasound image-omics and clinical features. Decision curve analysis (DCA) and a nomogram were used to evaluate the clinical application value of the model. RESULTS: A total of 1561 image-omics features were extracted from ultrasound images. 15 valuable image-omics features were determined by regularization, dimension reduction, and algorithm selection. In the image-omics model, the LR model showed higher prediction efficiency and robustness, with an area under the ROC curve (AUC) of 0.773 in the training set and an AUC of 0.850 in the test set. The clinical model constructed by the boundary of lesions in ultrasound images and the clinical feature CA199 (AUC = 0.875). The combined model had the best prediction performance, with an AUC of 0.872 in the training set and 0.918 in the test set. The combined model showed better clinical benefit according to DCA, and the nomogram score provided clinical prediction solutions. CONCLUSION: The combined model established with clinical features has good diagnostic ability and can be used to predict lymph node metastasis in patients with PC. It is expected to provide an effective noninvasive method for clinical decision-making, thereby improving the diagnosis and treatment of PC.

TET2 Is Downregulated in Early Esophageal Squamous Cell Carcinoma and Promotes Esophageal Squamous Cell Malignant Behaviors.

OBJECTIVE: To explore the expression of the ten eleven translocation (TET) 2 protein in early esophageal squamous cell carcinoma (EESCC), precancerous lesions, and cell lines and to evaluate the effect of TET2 on the functional behavior of EC109 esophageal cancer cells. METHODS: Thirty-one samples of EESCC and precancerous lesions collected via endoscopic submucosal dissection at Taihe Hospital, Shiyan, from February 1, 2017, to February 1, 2019, were analyzed. The study involved evaluating TET2 expression levels in lesion tissue and adjacent normal epithelium, correlating these with clinical pathological features. Techniques including 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, cell scratch assays, flow cytometry for propidium iodide (PI) staining, Hoechst 333258/PI double staining, and nude mouse tumorigenesis experiments were employed to assess the effect of TET2 on the proliferation, migration, cell cycle, apoptosis, and tumorigenic ability of esophageal cancer cells. RESULTS: TET2 expression was notably reduced in early esophageal cancer tissue and correlated with tumor invasion depth (P < 0.05). Overexpression of TET2 enhanced the proliferation and migration of esophageal cancer cells, increased the cell population in the G0 phase, decreased it in the S phase, and intensified cell necrosis (P < 0.05). There was a partial increase in tumorigenic ability (P = 0.087). CONCLUSION: TET2 downregulation in ESCC potentially influences the necrosis, cell cycle, and tumorigenic ability of esophageal cancer cells, suggesting a role in the onset and progression of esophageal cancer.

Impact of delirium on acute stroke outcomes: A systematic review and meta-analysis.

Delirium is a common complication in acute stroke patients. A 2011 meta-analysis showed an increased risk of in-hospital mortality and mortality within 12 months post-stroke, longer hospitalization durations, and increased likelihood of being discharged to a nursing home for patients experiencing post-stroke delirium. There is a need for an updated meta-analysis with several new studies having been since published. The PubMed and Scopus databases were screened for relevant studies. Inclusion criteria were as follows: retrospective or prospective studies reporting on the effects of delirium accompanying acute stroke on mortality, functional outcomes, length of hospital stay and need for re-admission. Strength of association was presented as pooled adjusted relative risk (RR) for categorical outcomes and weighted mean difference (WMD) for continuous outcomes. Statistical analysis was done using STATA version 16.0. The meta-analysis included 22 eligible articles. Eighteen of the 22 studies were prospective follow ups. Included studies were of good quality. Post-stroke delirium was associated with increased risk of in-hospital mortality, as well as mortality within 12 months post-stroke. Patients with delirium experienced increased hospital stay durations, were at greater risk for hospital readmission, and showed elevated risk for poor functional outcome. Compared to those who did not have delirium, stroke patients with delirium were 42% less likely to be discharged to home. Acute stroke patients with delirium are at an increased risk for poor short- and long-term outcomes. More research is needed to identify the best set of interventions to manage such patients and improve outcomes.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Elucidation of the Anti-Lung Cancer Mechanism of Xiao'ai Jiedu Prescription Based on Network Pharmacology and Molecular Docking.

Objective: Network pharmacology is an emerging discipline that applies computational methods to understand drug actions and interactions with multiple molecular targets. Xiao'ai Jiedu is a valued traditional Chinese medicine preparation for which the mechanism of action is not yet established. This study aims to explore the mechanism of Xiao'ai Jiedu in treating lung cancer through network pharmacology. Methods: First, the Traditional Chinese Medicine Systems Pharmacology (TCMSP) data platform was used to analyze the target treatment results of different medicinal materials in Mr. Zhou's cancer prescriptions. Then, functional enrichment analysis was performed to conduct a secondary analysis of the dissemination of cancer biological and pharmacological information in the human body. The Cancer Genome Atlas (TCGA) was used to obtain several cancer-aggressive target groups, and their transcription RNA was extracted for collection. The CIBERSORT evaluation method was used to conduct a Spearman correlation analysis on the data processing results. Then the matching degree between the experimental cells and the principle of drug treatment was analyzed to improve the statistical analysis. Results: Pharmacology research results showed that the network can accurately eliminate cancer detoxification targeted target correlation set, and through the data interpretation found that four different gene transcription have significant influence on lung cancer. The findings also confirmed that the degree of immune cell infiltration has a key role in lung cancer The study summarizes the active ingredients and their targets and mechanisms of action of the elimination of Xiao'ai Jiedu formula for the treatment of lung cancer. Conclusion: Network pharmacology can carry on the processing of the data, find the key to conform to the goal of research data, and the corresponding results are obtained, and the development of network pharmacology is not limited to, the study of lung cancer.

Proteomics profiling of nontumor liver tissues identifies prognostic biomarkers in hepatitis B-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often occurs following chronic hepatitis B virus (HBV) infection, leading to high recurrence and a low 5-year survival rate. We developed an overall survival (OS) prediction model based on protein expression profiles in HBV-infected nontumor liver tissues. We aimed to demonstrate the feasibility of using protein expression profiles in nontumor liver tissues for survival prediction. A univariate Cox and differential expression analysis were performed to identify candidate prognostic factors. A multivariate Cox analysis was performed to develop the liver gene prognostic index (LGPI). The survival differences between the different risk groups in the training and validation cohorts were also estimated. A total of 363 patients, 159 in the training cohort, and 204 in the validation cohort were included. Of the 6478 proteins extracted from nontumor liver tissues, we identified 1275 proteins altered between HCC and nontumor liver tissues. A total of 1090 out of 6478 proteins were significantly related to OS. The prognostic values of the proteins in nontumor tissues were mostly positively related to those in the tumor tissues. Protective proteins were mainly enriched in the metabolism-related pathways. From the differentially expressed proteins, the top 10 most significant prognosis-related proteins were submitted for LGPI construction. In the training and validation cohorts, this LGPI showed a great ability for distinguishing patients' OS risk stratifications. After adjusting for clinicopathological features, the LGPI was an independent prognostic factor in the training and validation cohorts. We demonstrated the prognostic value of protein expression profiling in nontumor liver tissues. The proposed LGPI was a promising predictive model for estimating OS in HBV-related HCC.

IgG memory B cells expressing IL4R and FCER2 are associated with atopic diseases.

BACKGROUND: Atopic diseases are characterized by IgE antibody responses that are dependent on cognate CD4 T cell help and T cell-produced IL-4 and IL-13. Current models of IgE cell differentiation point to the role of IgG memory B cells as precursors of pathogenic IgE plasma cells. The goal of this work was to identify intrinsic features of memory B cells that are associated with IgE production in atopic diseases. METHODS: Peripheral blood B lymphocytes were collected from individuals with physician diagnosed asthma or atopic dermatitis (AD) and from non-atopic individuals. These samples were analyzed by spectral flow cytometry, single cell RNA sequencing (scRNAseq), and in vitro activation assays. RESULTS: We identified a novel population of IgG memory B cells characterized by the expression of IL-4/IL-13 regulated genes FCER2/CD23, IL4R, IL13RA1, and IGHE, denoting a history of differentiation during type 2 immune responses. CD23+ IL4R+ IgG+ memory B cells had increased occurrence in individuals with atopic disease. Importantly, the frequency of CD23+ IL4R+ IgG+ memory B cells correlated with levels of circulating IgE. Consistently, in vitro stimulated B cells from atopic individuals generated more IgE+ cells than B cells from non-atopic subjects. CONCLUSIONS: These findings suggest that CD23+ IL4R+ IgG+ memory B cells transcribing IGHE are potential precursors of IgE plasma cells and are linked to pathogenic IgE production.

Curative effect of medicine cake sticking ultrasound drug penetration combined with body training on hemiplegia after stroke: An in vitro ultrasound targeted drug controlled release technology.

To explore the efficacy of ultrasound drugs in the treatment of hemiplegia after stroke. The evaluation included clinical symptoms and signs, the Stroke Scale, activities of daily living, sensory disorder Fugl-Meyer and Lindmark, electromyography sensory nerve amplitude, and conduction velocity indexes in both groups. There was no significant difference in the improved Fugl-Meyer and Lindmark score between treatment (26.97 ± 2.78) and the control group (27.45 ± 3.1) (t = 14.528, P = 0.593). After treatment, the observation group (37.10 ± 4.2) was significantly different from the control group (34.76 ± 4.36) (t = 11.259, P = 0.005) and (t = 10.15 ± 1.69), (40.87 ± 6.58) (t = 7.943,9.538, P = 0.564,0.826). After treatment, the observation group the Stroke Scale (4.27 ± 0.57), activities of daily living score (76.15 ± 12.38) and the control group (5.36 ± 0.89), (58.41 ± 9.69) (t = 16.274,5.379, P = 0.035,0.000) after treatment and F wave and M wave. The cure rate of the observation group was 77.50% (31/40), which was significantly better than that of the control group, 47.50% (19/40), with a significant difference (χ2 = 11.724,P = 0.000). After comparison, the total response rate of the observed group reached 92.500% (37 / 40), which was significantly higher than the 80.00% (32 / 40) of the control group. This difference was statistically significant (χ 2 = 9.458, P = 0.015). This therapy closely links the theoretical knowledge of modern medicine with the theoretical knowledge of traditional Chinese medicine, and uses the meridian theory to give full play to the unique advantages of traditional Chinese medicine.

The effect of adjuvant transarterial chemoembolization for hepatocellular carcinoma after liver resection based on risk stratification.

BACKGROUND: There is currently no standard adjuvant treatment proven to prevent hepatocellular carcinoma (HCC) recurrence. Recent studies suggest that postoperative adjuvant transarterial chemoembolization (PA-TACE) is beneficial for patients at high risk of tumor recurrence. However, it is difficult to select the patients. The present study aimed to develop an easy-to-use score to identify these patients. METHODS: A total of 4530 patients undergoing liver resection were recruited. Independent risk factors were identified by Cox regression model in the training cohort and the Primary liver cancer big data transarterial chemoembolization (PDTE) scoring system was established. RESULTS: The scoring system was composed of ten risk factors including alpha-fetoprotein (AFP), albumin-bilirubin (ALBI) grade, operative bleeding loss, resection margin, tumor capsular, satellite nodules, tumor size and number, and microvascular and macrovascular invasion. Using 5 points as risk stratification, the patients with PA-TACE had higher recurrence-free survival (RFS) compared with non-TACE in > 5 points group (P < 0.001), whereas PA-TACE patients had lower RFS compared with non-TACE in ≤ 5 points group (P = 0.013). In the training and validation cohorts, the C-indexes of PDTE scoring system were 0.714 [standard errors (SE) = 0.010] and 0.716 (SE = 0.018), respectively. CONCLUSIONS: The model is a simple tool to identify PA-TACE for HCC patients after liver resection with a favorable performance. Patients with > 5 points may benefit from PA-TACE.

Prediction of clinical efficacy of subcutaneous immunotherapy for Artemisia sieversiana pollen allergic rhinitis by serum metabolomics.

BACKGROUND/PURPOSE: Specific immunotherapy is the only effective etiological treatment for allergic rhinitis, but subcutaneous immunotherapy has a slow onset and poor compliance. Predicting the clinical efficacy of subcutaneous immunotherapy in advance can reduce unnecessary medical costs and resource waste. This study aimed to identify metabolites that could predict the efficacy of subcutaneous immunotherapy on seasonal allergic rhinitis by serum metabolomics. METHODS: Patients (n = 43) with Artemisia sieversiana pollen allergic rhinitis were enrolled and treated with subcutaneous immunotherapy for one year. Patients were divided into the ineffective group (n = 10) and effective group (n = 33) according to the therapeutic index. Serum samples were collected before treatment. Metabolomics was determined by liquid chromatography-mass spectrometry combined with gas chromatography-mass spectrometry and analyzed differential compounds and related metabolic pathways. RESULTS: A total of 129 differential metabolites (P < 0.05) were identified and 4 metabolic pathways, namely taurine and hypotaurine metabolism, pentose and glucuronate interconversions, pentose phosphate pathway, and alanine, aspartate, and glutamate metabolism, were involved. CONCLUSION: Some metabolites, such as hypotaurine, taurine, and l-alanine, have the potential to become predictive biomarkers for effective subcutaneous immunotherapy.

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

Mg2TiO4 spinel modified by nitrogen doping as a Visible-Light-Active photocatalyst for antibacterial activity.

Nitrogen doped Mg2TiO4 spinel, i.e. Mg2TiO4-xNy, has been synthesized and investigated as a photocatalyst for antibacterial activity. Mg2TiO4-xNy demonstrates superior photocatalytic activity for E. coli disinfection under visible light illumination (λ ≥ 400 nm). Complete disinfection of E. coli at a bacterial cell density of 1.0 × 107 CFU mL-1 can be achieved within merely 60 min. Mg2TiO4-xNy is capable of generating superoxide radicals (•O2 -) under visible light illumination which are the reactive oxygen species (ROSs) for bacteria disinfection. DFT calculations have verified the importance of nitrogen dopants in improving the visible light sensitivity of Mg2TiO4-xNy. The facile synthesis, low cost, good biocompatibility and high disinfection activity of Mg2TiO4-xNy warrant promising applications in the field of water purification and antibacterial products.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.

DNMT3a in the hippocampal CA1 is crucial in the acquisition of morphine self-administration in rats.

Drug-reinforced excessive operant responding is one fundamental feature of long-lasting addiction-like behaviors and relapse in animals. However, the transcriptional regulatory mechanisms responsible for the persistent drug-specific (not natural rewards) operant behavior are not entirely clear. In this study, we demonstrate a key role for one of the de novo DNA methyltransferase, DNMT3a, in the acquisition of morphine self-administration (SA) in rats. The expression of DNMT3a in the hippocampal CA1 region but not in the nucleus accumbens shell was significantly up-regulated after 1- and 7-day morphine SA (0.3 mg/kg/infusion) but not after the yoked morphine injection. On the other hand, saccharin SA did not affect the expression of DNMT3a or DNMT3b. DNMT inhibitor 5-aza-2-deoxycytidine (5-aza) microinjected into the hippocampal CA1 significantly attenuated the acquisition of morphine SA. Knockdown of DNMT3a also impaired the ability to acquire the morphine SA. Overall, these findings suggest that DNMT3a in the hippocampus plays an important role in the acquisition of morphine SA and may be a valid target to prevent the development of morphine addiction.

Regional Citrate Anticoagulation for Continuous Renal Replacement Therapy - A Safe and Effective Low-Dose Protocol.

AIMS: Regional citrate anticoagulation (RCA) is the preferred mode of anticoagulation for continuous renal replacement therapy (CRRT). Conventional RCA-CRRT citrate dose ranges from 3 to 5 mmol/L of blood. This study explored the effectiveness of an RCA protocol with lower citrate dose and its impact on citrate-related complications. METHODS: This prospective observational study compared two RCA-CRRT protocols in the intensive care unit. RCA Protocol 1 used an initial citrate dose of 3.0 mmol/L while Protocol 2 started with 2.5 mmol/L. The citrate dose was titrated by sliding scale to target circuit-iCa 0.26-0.40 mmol/L. Calcium was re-infused post-dialyzer and titrated by protocol to target systemic-iCa 1.01-1.20 mmol/L. RESULTS: Two hundred RCA-CRRT sessions were performed (81 Protocol 1; 119 Protocol 2). The median age was 65.4 years and median APACHE-II score was 23. Citrate dose for Protocol 1 was significantly higher than Protocol 2 in the first 12 h. The circuit clotting rate was similar in both arms (Protocol 1: 9.9%; Protocol 2: 9.2%; P = 0.881). With Protocol 2, circuit-iCa levels were 2.42 times more likely to be on target (P = 0.003) while the odds of hypocalcaemia was 4.67 times higher with Protocol 1 (P < 0.001). There was a wider anion gap was noted with Protocol 1, which suggests a propensity for citrate accumulation with higher citrate exposure. CONCLUSION: The RCA protocol with a lower initial citrate dose of 2.5 mmol/L blood had less citrate-related complications with no loss of efficacy. A more precise RCA prescription at the start of treatment avoids unnecessary citrate exposure and improves safety.

DEK terminates diapause by activation of quiescent cells in the crustacean Artemia.

To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.

Identification and characterization of a symbiotic agglutination-related C-type lectin from the hydrothermal vent shrimp Rimicaris exoculata.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species of hydrothermal vent communities, which inside its gill chamber harbors ectosymbioses with taxonomic invariability while compositional flexibility. Several studies have revealed that the establishment of symbiosis can be initiated and selected by innate immunity-related pattern recognition receptors (PRRs), such as C-type lectins (CTLs). In this research, a CTL was identified in R. exoculata (termed RCTL), which showed high expression at both mRNA and protein levels in the scaphognathite, an organ where the ectosymbionts are attached outside its setae. Linear correlationships were observed between the relative quantities of two major symbionts and the expression of RCTL based on analyzing different shrimp individuals. The recombinant protein of RCTL could recognize and agglutinate the cultivable γ-proteobacterium of Escherichia coli in a Ca2+-dependent manner, obeying a dose-dependent and time-cumulative pattern. Unlike conventional crustacean CTLs, the involvement of RCTL could not affect the bacterial growth, which is a key issue for the successful establishment of symbiosis. These results implied that RCTL might play a critical role in symbiotic recognition and attachment to R. exoculata. It also provides insights to understand how R. exoculata adapted to such a chemosynthesis-based environment.