An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis

AbstractPrevious studies by Papermaster and coworkers introduced the use of rhodopsin–green fluorescent protein (rho–GFP) fusion proteins in the construction of transgenic Xenopus laevis with retinal rod photoreceptor cell-specific transgene expression [Moritz et al., J. Biol. Chem. 276 (2001) 28242–28251]. These pioneering studies have helped to develop the Xenopus system not only for use in the investigation of rhodopsin biosynthesis and targeting, but for studies of the phototransduction cascade as well. However, the rho–GFP fusion protein used in the earlier work had only 50% of the specific activity of wild-type rhodopsin for activation of transducin and only 10% of the activity of wild-type in rhodopsin kinase assays. While not a problem for the biosynthesis studies, this does present a problem for investigation of the phototransduction cascade. We report here an improved rhodopsin/EGFP fusion protein in which placement of the EGFP domain at the C-terminus of rhodopsin results in wild-type activity for activation of transducin, wild-type ability to serve as a substrate for rhodopsin kinase, and wild-type localization of the protein to the rod photoreceptor cell outer segment in transgenic X. laevis

Impact of nitric oxide synthase 2 gene variant on risk of anti-tuberculosis drug- induced liver injury in the Malaysian population

Liver injury is a great threat associated with anti-tuberculosis (anti-TB) medication. Genetic variations in genes encoding drug-metabolising enzymes further enhance this threat. We aimed to explore genetic contributions by evaluating the impact of single nucleotide polymorphisms (SNPs) within the anti-tuberculosis (AT) metabolism pathway genes and within their respective chromosomes on anti-tuberculosis drug- induced liver injury (AT-DILI). Patients (n= 90) were recruited and 170 SNPs were genotyped using Illumina array and validated using Sanger Sequencing. The well-studied N-acetyltransferase 2 (NAT2*6) rs1799930 and cytochrome P450 2E1 (CYP2E1) C1/C1 were not significantly associated with AT-DILI in our cohort but nitric oxide synthase (NOS2A) rs11080344-C was found to be significantly higher in the cases than the controls (OR 2.73, 95% CI 1.12-6.64, P= 0.027). Association studies on all other SNPs within the anti-tuberculosis metabolism pathway genes and within their respective chromosomes also found no significant report. Our study suggests that genetic variation in NOS2A could influence the occurrence of AT-DILI

Interleukin-35 Dampens CD8+ T Cells Activity in Patients With Non-viral Hepatitis-Related Hepatocellular Carcinoma

Interleukin (IL)-35 is a newly identified IL-12 cytokine family member, which has been demonstrated to induce immunotolerance by suppression of CD8+ T cells function in chronic viral hepatitis. However, the role of IL-35 in modulating CD8+ T cells activity in non-viral hepatitis-related hepatocellular carcinoma (HCC) was not fully elucidated. Forty-four patients with non-viral hepatitis-related HCC and 20 healthy individuals were enrolled. Serum IL-35 concentration was measured by ELISA. CD8+ T cells were purified from peripheral bloods and liver tissues. mRNA expression of cytotoxic/inhibitory molecules in CD8+ T cells with IL-35 stimulation was semi-quantified by real-time PCR. Direct and indirect contact co-culture systems of CD8+ T cells and HCC cell lines were set up. The modulatory function of IL-35 on peripheral and liver-resident CD8+ T cells was assessed by measurement of lactate dehydrogenase release and cytokine production in the co-culture supernatants. Serum IL-35 was notably elevated in HCC patients, while effective anti-tumor therapies down-regulated IL-35 concentration. Recombinant IL-35 stimulation suppressed cytotoxicity and proinflammatory cytokine secretion of peripheral and liver-resident CD8+ T cells in direct and indirect contact co-culture systems. This process was accompanied by reduction of perforin expression and interferon-γ production, as well as programmed death-1 and cytotoxic T-lymphocyte-associated protein 4 elevation in CD8+ T cells. The current data suggested that IL-35 inhibited both cytolytic and non-cytolytic function of CD8+ T cells to non-viral hepatitis-related HCC probably via repression of perforin expression. IL-35 might be considered to be one of the therapeutic targets for patients with HCC

Clinical-radiomic analysis for non-invasive prediction of liver steatosis on non-contrast CT: A pilot study

Purpose: Our aim is to build and validate a clinical-radiomic model for non-invasive liver steatosis prediction based on non-contrast computed tomography (CT).Methods: We retrospectively reviewed 342 patients with suspected NAFLD diagnoses between January 2019 and July 2020 who underwent non-contrast CT and liver biopsy. Radiomics features from hepatic and splenic regions-of-interests (ROIs) were extracted based on abdominal non-contrast CT imaging. The radiomics signature was constructed based on reproducible features by adopting the least absolute shrinkage and selection operator (LASSO) regression. Then, multivariate logistic regression analysis was applied to develop a combined clinical-radiomic nomogram integrating radiomics signature with several independent clinical predictors in a training cohort of 124 patients between January 2019 and December 2019. The performance of models was determined by the area under the receiver operating characteristic curves and calibration curves. We conducted an internal validation during 103 consecutive patients between January 2020 and July 2020.Results: The radiomics signature was composed of four steatosis-related features and positively correlated with pathologic liver steatosis grade (p < 0.01). In both subgroups (Group One, none vs. steatosis; Group Two, none/mild vs. moderate/severe steatosis), the clinical-radiomic model performed best within the validation cohort with an AUC of 0.734 and 0.930, respectively. The calibration curve confirmed the concordance of excellent models.Conclusion: We developed a robust clinical-radiomic model for accurate liver steatosis stage prediction in a non-invasive way, which may improve the clinical decision-making ability

A Survey on Service Route and Time Prediction in Instant Delivery: Taxonomy, Progress, and Prospects

Instant delivery services, such as food delivery and package delivery, have achieved explosive growth in recent years by providing customers with daily-life convenience. An emerging research area within these services is service Route\&Time Prediction (RTP), which aims to estimate the future service route as well as the arrival time of a given worker. As one of the most crucial tasks in those service platforms, RTP stands central to enhancing user satisfaction and trimming operational expenditures on these platforms. Despite a plethora of algorithms developed to date, there is no systematic, comprehensive survey to guide researchers in this domain. To fill this gap, our work presents the first comprehensive survey that methodically categorizes recent advances in service route and time prediction. We start by defining the RTP challenge and then delve into the metrics that are often employed. Following that, we scrutinize the existing RTP methodologies, presenting a novel taxonomy of them. We categorize these methods based on three criteria: (i) type of task, subdivided into only-route prediction, only-time prediction, and joint route\&time prediction; (ii) model architecture, which encompasses sequence-based and graph-based models; and (iii) learning paradigm, including Supervised Learning (SL) and Deep Reinforcement Learning (DRL). Conclusively, we highlight the limitations of current research and suggest prospective avenues. We believe that the taxonomy, progress, and prospects introduced in this paper can significantly promote the development of this field

Placenta-Derived Fetal Specific mRNA Is More Readily Detectable in Maternal Plasma than in Whole Blood

BACKGROUND:Placental mRNA was detected in maternal whole blood, raising the possibility of using maternal blood for noninvasive prenatal diagnosis. We investigated fetal mRNA detection in maternal whole blood and determined if it offered advantages over maternal plasma analysis. METHODOLOGY:The concentrations of placental expressed genes, CSH1, KISS1, PLAC4 and PLAC1 in plasma and whole blood from healthy pregnant and non-pregnant individuals were compared by real-time quantitative reverse-transcriptase polymerase chain reaction analysis. Their fetal specificity was investigated by comparing the transcript concentrations in pre- and post-delivery samples and through SNP genotyping by matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry. The gene expression profiles of pregnant and non-pregnant whole blood were investigated by microarray analysis. Upregulated genes in pregnant whole blood were selected for further quantitative analysis. PRINCIPAL FINDINGS:The concentrations of the four transcripts were significantly higher in third trimester maternal whole blood than corresponding plasma without significant correlations. KISS1, PLAC4 and PLAC1 were detected in non-pregnant whole blood but not plasma. The transcripts remained detectable in some postpartum whole blood samples. The PLAC4 mRNA in maternal plasma showed fetal genotype while that in corresponding whole blood indicated both fetal and maternal contributions. Microarray analysis revealed upregulation of genes involved in neutrophil functions in pregnant whole blood including DEFA4, CEACAM8, OLFM4, ORM1, MMP8 and MPO. Though possibly pregnancy-related, they were not pregnancy-specific as suggested by the lack of post-delivery reduction in concentrations. CONCLUSIONS:Maternal plasma is preferred over maternal whole blood for placenta-derived fetal RNA detection. Most studied 'placental' mRNA molecules in maternal whole blood were of maternal origin and might be derived from processes such as 'illegitimate transcription'

New therapeutic directions in type II diabetes and its complications: mitochondrial dynamics

As important organelles of energetic and metabolism, changes in the dynamic state of mitochondria affect the homeostasis of cellular metabolism. Mitochondrial dynamics include mitochondrial fusion and mitochondrial fission. The former is coordinated by mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy 1 (Opa1), and the latter is mediated by dynamin related protein 1 (Drp1), mitochondrial fission 1 (Fis1) and mitochondrial fission factor (MFF). Mitochondrial fusion and fission are generally in dynamic balance and this balance is important to preserve the proper mitochondrial morphology, function and distribution. Diabetic conditions lead to disturbances in mitochondrial dynamics, which in return causes a series of abnormalities in metabolism, including decreased bioenergy production, excessive production of reactive oxygen species (ROS), defective mitophagy and apoptosis, which are ultimately closely linked to multiple chronic complications of diabetes. Multiple researches have shown that the incidence of diabetic complications is connected with increased mitochondrial fission, for example, there is an excessive mitochondrial fission and impaired mitochondrial fusion in diabetic cardiomyocytes, and that the development of cardiac dysfunction induced by diabetes can be attenuated by inhibiting mitochondrial fission. Therefore, targeting the restoration of mitochondrial dynamics would be a promising therapeutic target within type II diabetes (T2D) and its complications. The molecular approaches to mitochondrial dynamics, their impairment in the context of T2D and its complications, and pharmacological approaches targeting mitochondrial dynamics are discussed in this review and promise benefits for the therapy of T2D and its comorbidities