Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Rigorous benchmarking of T-cell receptor repertoire profiling methods for cancer RNA sequencing.

The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq.

The 'spiked-helmet' sign in patients with myocardial injury.

The spiked-helmet sign is a marker for high mortality in critical patients. It is characterised as a dome-shaped ST-segment elevation accompanied by an upward shift of the baseline before the onset of the QRS complex. We present two patients with the spiked-helmet sign on electrocardiogram. Patient A showed a potential relationship between the spiked-helmet sign and hyper-osmolar hyper-glycaemic state, whereas patient B had clinically suspected viral myocarditis.

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

Effects of aloe vera on burn injuries: A systematic review and meta-analysis of randomized controlled trials.

Burn injuries cause severe pain, infection risks, psychological distress, financial burdens, and mortality, necessitating effective care. Aloe vera, a traditional burn remedy, shows wound healing potential, but its analgesic effects and efficacy with varying burn severity are uncertain. This study aims to investigate aloe vera's impact on wound healing, pain management, and infection prevention in burn patients. A systematic search on PubMed, Embase, and CENTRAL was performed on 9th October 2023 for randomized controlled trials (RCTs). The risk of bias was examined using the Cochrane risk-of-bias tool (version 2), and the meta-analysis was carried out using a random-effects model. The primary outcome was wound healing time, with secondary outcomes examining pain severity and wound infection. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to assess the quality of evidence for each outcome. Nine RCTs were included in the current study, of which six provided data on the primary outcome. Aloe vera significantly reduced mean wound healing time compared to other topicals [mean difference (MD) -3.76 days; 95% confidence interval (CI) -5.69 to -1.84]. Additionally, the meta-analysis of the secondary outcomes found no significant differences in pain reduction (MD -0.76 points; 95% CI -1.53 to 0.01) and wound infection risk (risk ratio 1.10; 95% CI 0.34 to 3.59) between aloe vera and control groups. In conclusion, aloe vera expedites wound healing in second-degree burn patients without increased infection risk compared to other antimicrobial agents. The analgesic effects on burn injuries remain uncertain.

Identification of an 85-kb Heterozygous 4p Microdeletion With Full Genome Analysis in Autosomal Dominant Charcot-Marie-Tooth Disease.

Background and Objectives: Charcot-Marie-Tooth disease (CMT) is a syndrome of a hereditary neurodegenerative condition affecting the peripheral nervous system and is a single gene disorder. Deep phenotyping coupled with advanced genetic techniques is critical in discovering new genetic defects of rare genetic disorders such as CMT. Methods: We applied multidisciplinary investigations to examine the neurophysiology and nerve pathology in a family that fulfilled the diagnosis of CMT2. When phenotype-guided first-tier genetic tests and whole-exome sequencing did not yield a molecular diagnosis, we conducted full genome analysis by examining phased whole-genome sequencing and whole-genome optical mapping data to search for the causal variation. We then performed a systematic review to compare the reported patients with interstitial microdeletion in the short arm of chromosome 4. Results: In this family with CMT2, we reported the discovery of a heterozygous 85-kb microdeletion in the short arm of chromosome 4 (4p16.3)[NC_000004.12:g.1733926_1819031del] spanning 3 genes [TACC3 (intron 6-exon 16), FGFR3 (total deletion), and LETM1 (intron 10-exon14)] that cosegregated with disease phenotypes in family members. The clinical features of peripheral nerve degeneration in our family are distinct from the well-known 4p microdeletion syndrome of Wolf-Hirschhorn syndrome, in which brain involvement is the major phenotype. Discussion: In summary, we used the full genome analysis approach to discover a new microdeletion in a family with CMT2. The deleted segment contains 3 genes (TACC3, FGFR3, and LETM1) that likely play a role in the pathogenesis of nerve degeneration.

Retrospective analyzing the effects of nerve block on postoperative pain management after total knee arthroplasty.

OBJECTIVE: Total knee arthroplasty (TKA) is usually associated with moderate-to-severe postoperative pain. Our study investigated the possible benefits of the use of nerve blocks (NBs), including pain score reduction, the rescuing dosage of morphine, the timing of ambulation, and the length of stay (LOS) in the hospital. MATERIALS AND METHODS: We included patients who underwent unilateral primary TKA due to primary knee osteoarthritis under general anesthesia with laryngeal mask airway. The control group only received oral pain medication with rescuing morphine injections, whereas the NB group received oral pain medication with an NB and rescuing morphine injections. We collected data on the patients' basic characteristics, postoperative visual analog scale (VAS), the dosage of rescuing morphine over 3 days, time to ambulation, and LOS in the hospital. RESULTS: The NB group received significantly fewer morphine dose compared with the control group during postoperative days 1 to 3. There were no statistically significant differences between the NB and control groups on days 1 and 2 in the VAS score, and the VAS score was significantly lower in the NB group on postoperative day 3. The NB group had a significantly shorter time to ambulation compared with the control group. LOS did not differ significantly between the NB and control groups. CONCLUSION: Patients, who underwent TKA under general anesthesia with laryngeal mask airway (LMAGA) receiving NB for postoperative pain, needed less dosage of morphine and had the trend of having lower VAS. There was no association with LOS between two groups, but time to ambulation might be decreased with NB group. Some limitations might need to be further investigated in future study, such as NB regimens, knee function after TKA, muscle power, information after discharge, and NB-related complications.

Development of Whole Slide Imaging on Smartphones and Evaluation With ThinPrep Cytology Test Samples: Follow-Up Study.

BACKGROUND: The smartphone-based whole slide imaging (WSI) system represents a low-cost and effective alternative to automatic scanners for telepathology. In a previous study, the development of one such solution, named scalable whole slide imaging (sWSI), was presented and analyzed. A clinical evaluation of its iOS version with 100 frozen section samples verified the diagnosis-readiness of the produced virtual slides. OBJECTIVE: The first aim of this study was to delve into the quantifying issues encountered in the development of an Android version. It should also provide insights into future high-resolution real-time feedback medical imaging apps on Android and invoke the awareness of smartphone manufacturers for collaboration. The second aim of this study was to further verify the clinical value of sWSI with cytology samples. This type is different from the frozen section samples in that they require finer detail on the cellular level. METHODS: During sWSI development on Android, it was discovered that many models do not support uncompressed camera pixel data with sufficient resolution and full field of view. The proportion of models supporting the optimal format was estimated in a test on 200 mainstream Android models. Other factors, including slower processing speed and camera preview freezing, also led to inferior performance of sWSI on Android compared with the iOS version. The processing speed was mostly determined by the central processing unit frequency in theory, and the relationship was investigated in the 200-model simulation experiment with physical devices. The camera preview freezing was caused by the lag between triggering photo capture and resuming preview. In the clinical evaluation, 100 ThinPrep cytology test samples covering 6 diseases were scanned with sWSI and compared against the ground truth of optical microscopy. RESULTS: Among the tested Android models, only 3.0% (6/200) provided an optimal data format, meeting all criteria of quality and efficiency. The image-processing speed demonstrated a positive relationship with the central processing unit frequency but to a smaller degree than expected and was highly model-dependent. The virtual slides produced by sWSI on Android and iOS of ThinPrep cytology test samples achieved similar high quality. Using optical microscopy as the ground truth, pathologists made a correct diagnosis on 87.5% (175/200) of the cases with sWSI virtual slides. Depending on the sWSI version and the pathologist in charge, the kappa value varied between .70 and .82. All participating pathologists considered the quality of the sWSI virtual slides in the experiment to be adequate for routine usage. CONCLUSIONS: Limited by hardware and operating system support, the performance of sWSI on mainstream Android smartphones did not fully match the iOS version. However, in practice, this difference was not significant, and both were adequate for digitizing most of the sample types for telepathology consultation.

Clinical significance of TM4SF1 as a tumor suppressor gene in gastric cancer.

Transmembrane-4-L-six-family member-1 (TM4SF1), a tumor-associated antigen, is overexpressed in most epithelial cell carcinomas and a potential target for antibody-mediated therapy. However, the role of TM4SF1 in gastric cancer has not been elucidated. The aim of this study was to investigate the clinical significance of TM4SF1 expression in gastric carcinoma (GC) tissues using 152 GC tissue samples and matched adjacent nontumor tissue samples analyzed by immunohistochemistry, and 13 fresh GC tissue samples analyzed by Western blotting. The results showed that TM4SF1 was heterogeneously expressed in normal gastric mucosa, with a high expression rate in fundus mucosa. Higher levels and strong expression rate of TM4SF1 were associated with GC tissues of higher-grade differentiation. TM4SF1 levels were lower in gastric cancer tissues than gastric noncancerous tissues. Expression of TM4SF1 was not correlated with USP10 (P = 0.157), S100A12 (P = 0.479), p53 (P = 0.249), or Ki67 (P = 0.166) in GC. The expression of TM4SF1 was significantly and negatively correlated with depth of invasion (P = 0.031), nodal metastasis (P = 0.042), TNM stage (P = 0.030), and Lauren classification (P = 0.026). There was no significant correlation between TM4SF1 expression and age, gender, tumor size, or distant metastasis (P > 0.05). The expression of TM4SF1 was associated with well overall survival (P = 0.0164). The 5-year survival rate for patients with GC showing TM4SF1 positive was 58.82% (10/17), and the median survival time was 78 months, higher than that (12.90%, 12/93) of patients who were TM4SF1 negative, whose median survival time was 62 months. These data suggested that low expression of TM4SF1 is associated with carcinogenesis and development, tumor progression and invasion of gastric cancer, and poor overall survival of patients with GC. TM4SF1 is a tumor suppressor for GC and a novel prognostic marker for patients with GC.

The NF-κB p65/miR-23a-27a-24 cluster is a target for leukemia treatment.

p65 is a transcription factor that is involved in many physiological and pathologic processes. Here we report that p65 strongly binds to the miR-23a-27a-24 cluster promoter to up-regulate its expression. As bone marrow-derived cells differentiate into red blood cells in vitro, p65/miR-23a-27a-24 cluster expression increases sharply and then declines before the appearance of red blood cells, suggesting that this cluster is negatively related to erythroid terminal differentiation. Bioinformatic and molecular biology experiments confirmed that the miR-23a-27a-24 cluster inhibited the expression of the erythroid proteome and contributed to erythroleukemia progression. In addition, high level of the p65/miR-23a-27a-24 cluster was found in APL and AML cell lines and in nucleated peripheral blood cells from leukemia patients. Furthermore, anti-leukemia drugs significantly inhibited the expression of the p65/miR-23a-27a-24 cluster in leukemia cells. Administration of the p65 inhibitor parthenolide significantly improved hematology and myelogram indices while prolonging the life span of erythroleukemia mice. Meanwhile, stable overexpression of these three miRNAs in mouse erythroleukemia cells enhanced cell malignancy. Our findings thus connect a novel regulation pathway of the p65/miR-23a-27a-24 cluster with the erythroid proteome and provide an applicable approach for treating leukemia.

Reynolds stress model involving the mean spin tensor.

In this work, we develop a Reynolds stress model along the line of the approach presented by Commun. Nonlinear Sci. Numer. Simul. 9, 543 (2004)]], aiming to assess the role and contribution of the mean spin tensor in turbulence modeling. Here, the constitutive functional for the Reynolds stress depends on the mean spin tensor as well as the mean stretching tensor and its Jaumann derivative, the turbulent kinetic energy K , and the turbulent dissipation rate epsilon , which is at the complexity level of p=1,m=1 , and n=0 of a rate-type constitutive equation for the Reynolds stress proposed in the aforementioned paper. The explicit form for the Reynolds stress is obtained with recourse to the representation theorem and the theory of invariants developed in modern rational continuum mechanics, and, as an approximation, a nonlinear cubic K-epsilon model is worked out in which the model coefficients are analytically identified based on the experimental results of Tavoularis and Corrsin [J. Fluid Mech. 104, 311 (1981)]]. In addition, numerical results based on this model, in the forms of employing the Jaumann derivative and the Oldroyd derivative, respectively, for homogeneous turbulent shear flow and fully developed turbulent flow over a backward-facing step, are presented in comparison with those obtained based on a few previously proposed linear and nonlinear K-epsilon models, showing reasonably good agreement with the experimental results and the DNS data concerned and a better performance than the previously developed quadratic models.

Crystallization and preliminary X-ray diffraction analysis of a novel ß-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum.

The ß-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the ß-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3x21, with unit-cell parameters a = b = 75.9, c = 254.0Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78Å resolution. A BLASTP search (http://blast.ncbi.nlm.nih.gov/) of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress.