Use of inert gas jets to measure the forces required for mechanical gene transfection

BACKGROUND: Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. METHODS: We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. RESULTS: The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. CONCLUSIONS: The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods

Prospects for terahertz imaging the human skin cancer with the help of gold-nanoparticles-based terahertz-to-infrared converter

The design is suggested, and possible operation parameters are discussed, of an instrument to inspect a skin cancer tumour in the terahertz (THz) range, transferring the image into the infrared (IR) and making it visible with the help of standard IR camera. The central element of the device is the THz-to-IR converter, a Teflon or silicon film matrix with embedded 8.5 nm diameter gold nanoparticles. The use of external THz source for irradiating the biological tissue sample is presumed. The converter's temporal characteristics enable its performance in a real-time scale. The details of design suited for the operation in transmission mode (in vitro) or on the human skin in reflection mode {in vivo) are specified.Comment: To be published in the proceedings of the FANEM2018 workshop - Minsk, 3-5 June 201

Torsion of the Gallbladder

A 77-year-old woman was seen with progressive abdominal pain. A CT scan was made and showed a large gallbladder extending into the right lower abdomen. Ultrasound was performed but demonstrated no gallstones. Laparoscopy showed a tordated, necrotic gallbladder that was attached to the liver only by the cystic artery and cystic duct. Cholecystectomy was performed. Torsion of the gallbladder is a rare but clinically important condition in which the diagnosis seldom is made preoperatively. In radiological and clinical signs of cholecystitis without gallstones, this condition should be considered

Arsenic Metabolism by Human Gut Microbiota upon in Vitro Digestion of Contaminated Soils

BACKGROUND: Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. However, it is unclear whether human microbiota display similar As metabolism, especially when present in a contaminated matrix. OBJECTIVES: We evaluated the metabolic potency of in vitro cultured human colon microbiota toward inorganic As (iAs) and As-contaminated soils. METHODS: A colon microbial community was cultured in a dynamic model of the human gut. These colon microbiota were incubated with iAs and with As-contaminated urban soils. We determined As speciation analysis using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. RESULTS: We found a high degree of methylation for colon digests both of iAs (10 mu g methylarsenical/g biomass/hr) and of As-contaminated soils (up to 28 mu g/g biomass/hr). Besides the formation of monomethylarsonic acid (MMA(V)), we detected the highly toxic monomethylarsonous acid (MMA(III)). Moreover, this is the first description of microbial thiolation leading to monomethylmonothioarsonic acid (MMMTA(V)). MMMTA(V), the toxicokinetic properties of which are not well known, was in many cases a major metabolite. CONCLUSIONS: Presystemic As metabolism is a significant process in the human body. Toxicokinetic studies aiming to completely elucidate the As metabolic pathway would therefore benefit from incorporating the metabolic potency of human gut microbiota. This will result in more accurate risk characterization associated with As exposures

Homozygous microdeletion of exon 5 in ZNF277 in a girl with specific language impairment

Peer reviewedPublisher PD

Evaluation of miCRovascular rarefaction in vascUlar Cognitive Impairment and heArt faiLure (CRUCIAL): Study protocol for an observational study

INTRODUCTION: Microvascular rarefaction, the functional reduction in perfused microvessels and structural reduction of microvascular density, seems to be an important mechanism in the pathophysiology of small blood vessel related disorders including vascular cognitive impairment (VCI) due to cerebral small vessel disease and heart failure with preserved ejection fraction (HFpEF). Both diseases share common risk factors including hypertension, diabetes mellitus, obesity, and ageing; in turn, these co-morbidities are associated with microvascular rarefaction. Our consortium aims to investigate novel non-invasive tools to quantify microvascular health and rarefaction in both organs, as well as surrogate biomarkers for cerebral and/or cardiac rarefaction (via sublingual capillary health, vascular density of the retina, and RNA content of circulating extracellular vesicles), and to determine whether microvascular density relates to disease severity. METHODS/DESIGN: The clinical research program of CRUCIAL consists of four observational cohort studies. We aim to recruit 75 VCI patients, 60 HFpEF patients, 60 patients with severe aortic stenosis (AS) undergoing surgical aortic valve replacement as a pressure overload HFpEF model, and 200 elderly participants with mixed comorbidities to serve as controls. Data collected will include medical history, physical examination, cognitive testing, advanced brain and cardiac MRI, ECG, echocardiography, sublingual capillary health, optical coherence tomography angiography (OCTa), extracellular vesicles RNA analysis and myocardial remodelling-related serum biomarkers. The AS cohort undergoing surgery will also have myocardial biopsy for histological microvascular assessment. DISCUSSION: CRUCIAL will examine the pathophysiological role of microvascular rarefaction in VCI and HFpEF using advanced brain and cardiac MRI techniques. Furthermore, we will investigate surrogate biomarkers for non-invasive, faster, easier, and cheaper assessment of microvascular density since these are more likely to be disseminated into widespread clinical practice. If microvascular rarefaction is an early marker of developing small vessel diseases, then measuring rarefaction may allow pre-clinical diagnosis, with implications for screening, risk stratification, and prevention. Further knowledge of the relevance of microvascular rarefaction and its underlying mechanisms may provide new avenues for research and therapeutic targets

Head Position in Stroke Trial (HeadPoST)- sitting-up vs lying-flat positioning of patients with acute stroke: study protocol for a cluster randomised controlled trial

Background Positioning a patient lying-flat in the acute phase of ischaemic stroke may improve recovery and reduce disability, but such a possibility has not been formally tested in a randomised trial. We therefore initiated the Head Position in Stroke Trial (HeadPoST) to determine the effects of lying-flat (0°) compared with sitting-up (≥30°) head positioning in the first 24 hours of hospital admission for patients with acute stroke. Methods/Design We plan to conduct an international, cluster randomised, crossover, open, blinded outcome-assessed clinical trial involving 140 study hospitals (clusters) with established acute stroke care programs. Each hospital will be randomly assigned to sequential policies of lying-flat (0°) or sitting-up (≥30°) head position as a ‘business as usual’ stroke care policy during the first 24 hours of admittance. Each hospital is required to recruit 60 consecutive patients with acute ischaemic stroke (AIS), and all patients with acute intracerebral haemorrhage (ICH) (an estimated average of 10), in the first randomised head position policy before crossing over to the second head position policy with a similar recruitment target. After collection of in-hospital clinical and management data and 7-day outcomes, central trained blinded assessors will conduct a telephone disability assessment with the modified Rankin Scale at 90 days. The primary outcome for analysis is a shift (defined as improvement) in death or disability on this scale. For a cluster size of 60 patients with AIS per intervention and with various assumptions including an intracluster correlation coefficient of 0.03, a sample size of 16,800 patients at 140 centres will provide 90 % power (α 0.05) to detect at least a 16 % relative improvement (shift) in an ordinal logistic regression analysis of the primary outcome. The treatment effect will also be assessed in all patients with ICH who are recruited during each treatment study period. Discussion HeadPoST is a large international clinical trial in which we will rigorously evaluate the effects of different head positioning in patients with acute stroke. Trial registration ClinicalTrials.gov identifier: NCT02162017 (date of registration: 27 April 2014); ANZCTR identifier: ACTRN12614000483651 (date of registration: 9 May 2014). Protocol version and date: version 2.2, 19 June 2014

Pro-apoptotic and antiproliferative activity of human KCNRG, a putative tumor suppressor in 13q14 region

Deletion of 13q14.3 and a candidate gene KCNRG (potassium channel regulating gene) is the most frequent chromosomal abnormality in B-cell chronic lymphocytic leukemia and is a common finding in multiple myeloma (MM). KCNRG protein may interfere with the normal assembly of the K+ channel proteins causing the suppression of Kv currents. We aimed to examine possible role of KCNRG haploinsufficiency in chronic lymphocytic leukemia (CLL) and MM cells. We performed detailed genomic analysis of the KCNRG locus; studied effects of the stable overexpression of KCNRG isoforms in RPMI-8226, HL-60, and LnCaP cells; and evaluated relative expression of its transcripts in various human lymphomas. Three MM cell lines and 35 CLL PBL samples were screened for KCNRG mutations. KCNRG exerts growth suppressive and pro-apoptotic effects in HL-60, LnCaP, and RPMI-8226 cells. Direct sequencing of KCNRG exons revealed point mutation delT in RPMI-8226 cell line. Levels of major isoform of KCNRG mRNA are lower in DLBL lymphomas compared to normal PBL samples, while levels of its minor mRNA are decreased across the broad range of the lymphoma types. The haploinsufficiency of KCNRG might be relevant to the progression of CLL and MM at least in a subset of patients