Determining Physical Constraints in Transcriptional Initiation Complexes Using DNA Sequence Analysis

Eukaryotic gene expression is often under the control of cooperatively acting transcription factors whose binding is limited by structural constraints. By determining these structural constraints, we can understand the “rules” that define functional cooperativity. Conversely, by understanding the rules of binding, we can infer structural characteristics. We have developed an information theory based method for approximating the physical limitations of cooperative interactions by comparing sequence analysis to microarray expression data. When applied to the coordinated binding of the sulfur amino acid regulatory protein Met4 by Cbf1 and Met31, we were able to create a combinatorial model that can correctly identify Met4 regulated genes. Interestingly, we found that the major determinant of Met4 regulation was the sum of the strength of the Cbf1 and Met31 binding sites and that the energetic costs associated with spacing appeared to be minimal

A novel cortical biomarker signature for predicting pain sensitivity : protocol for the PREDICT longitudinal analytical validation study

Introduction: Temporomandibular disorder is a common musculoskeletal pain condition with development of chronic symptoms in 49% of patients. Although a number of biological factors have shown an association with chronic temporomandibular disorder in cross-sectional and case control studies, there are currently no biomarkers that can predict the development of chronic symptoms. The PREDICT study aims to undertake analytical validation of a novel peak alpha frequency (PAF) and corticomotor excitability (CME) biomarker signature using a human model of the transition to sustained myofascial temporomandibular pain (masseter intramuscular injection of nerve growth factor [NGF]). This article describes, a priori, the methods and analysis plan. Methods: This study uses a multisite longitudinal, experimental study to follow individuals for a period of 30 days as they progressively develop and experience complete resolution of NGF-induced muscle pain. One hundred fifty healthy participants will be recruited. Participants will complete twice daily electronic pain diaries from day 0 to day 30 and undergo assessment of pressure pain thresholds, and recording of PAF and CME on days 0, 2, and 5. Intramuscular injection of NGF will be given into the right masseter muscle on days 0 and 2. The primary outcome is pain sensitivity. Perspective: PREDICT is the first study to undertake analytical validation of a PAF and CME biomarker signature. The study will determine the sensitivity, specificity, and accuracy of the biomarker signature to predict an individual's sensitivity to pain

Can non‐invasive brain stimulation modulate peak alpha frequency in the human brain? A systematic review and meta‐analysis

Peak alpha frequency (PAF), the dominant oscillatory frequency within the alpha range (8–12 Hz), is associated with cognitive function and several neurological conditions, including chronic pain. Manipulating PAF could offer valuable insight into the relationship between PAF and various functions and conditions, potentially providing new treatment avenues. This systematic review aimed to comprehensively synthesise effects of non‐invasive brain stimulation (NIBS) on PAF speed. Relevant studies assessing PAF pre‐ and post‐NIBS in healthy adults were identified through systematic searches of electronic databases (Embase, PubMed, PsychINFO, Scopus, The Cochrane Library) and trial registers. The Cochrane risk‐of‐bias tool was employed for assessing study quality. Quantitative analysis was conducted through pairwise meta‐analysis when possible; otherwise, qualitative synthesis was performed. The review protocol was registered with PROSPERO (CRD42020190512) and the Open Science Framework (https://osf.io/2yaxz/). Eleven NIBS studies were included, all with a low risk‐of‐bias, comprising seven transcranial alternating current stimulation (tACS), three repetitive transcranial magnetic stimulation (rTMS), and one transcranial direct current stimulation (tDCS) study. Meta‐analysis of active tACS conditions (eight conditions from five studies) revealed no significant effects on PAF (mean difference [MD] = −0.12, 95% CI = −0.32 to 0.08, p = 0.24). Qualitative synthesis provided no evidence that tDCS altered PAF and moderate evidence for transient increases in PAF with 10 Hz rTMS. However, it is crucial to note that small sample sizes were used, there was substantial variation in stimulation protocols, and most studies did not specifically target PAF alteration. Further studies are needed to determine NIBS's potential for modulating PAF

Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection

With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents

UV Circular Polarisation in Star Formation Regions : The Origin of Homochirality?

Ultraviolet circularly polarised light has been suggested as the initial cause of the homochirality of organic molecules in terrestrial organisms, via enantiomeric selection of prebiotic molecules by asymmetric photolysis. We present a theoretical investigation of mechanisms by which ultraviolet circular polarisation may be produced in star formation regions. In the scenarios considered here, light scattering produces only a small percentage of net circular polarisation at any point in space, due to the forward throwing nature of the phase function in the ultraviolet. By contrast, dichroic extinction can produce a fairly high percentage of net circular polarisation (∼10%) and may therefore play a key role in producing an enantiomeric excessPeer reviewe

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8+ T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance

Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules

Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle

The wall shear stress (WSS) that a moving fluid exerts on a surface affects many processes including those relating to vascular function. WSS plays an important role in normal physiology (e.g. angiogenesis) and affects the microvasculature's primary function of molecular transport. Points of fluctuating WSS show abnormalities in a number of diseases; however, there is no established technique for measuring WSS directly in physiological systems. All current methods rely on estimates obtained from measured velocity gradients in bulk flow data. In this work, we report a nanosensor that can directly measure WSS in microfluidic chambers with sub-micron spatial resolution by using a specific type of virus, the bacteriophage M13, which has been fluorescently labeled and anchored to a surface. It is demonstrated that the nanosensor can be calibrated and adapted for biological tissue, revealing WSS in micro-domains of cells that cannot be calculated accurately from bulk flow measurements. This method lends itself to a platform applicable to many applications in biology and microfluidics

Lessons learned from additional research analyses of unsolved clinical exome cases

BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery. METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols. RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3). CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts

Tyrosine Kinase ETK/BMX Is Up-Regulated in Bladder Cancer and Predicts Poor Prognosis in Patients with Cystectomy

Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity