Miracle-dz/UniFAR-FTUs huggingface dataset:
Standardization procedure for plasma biomarker analysis in rat models of epileptogenesis: Focus on circulating microRNAs

The World Health Organization estimates that globally 2.4 million people are diagnosed with epilepsy each year. In nearly 30% of these cases, epilepsy cannot be properly controlled by antiepileptic drugs. More importantly, treatments to prevent or modify epileptogenesis do not exist. Therefore, novel therapies are urgently needed. In this respect, it is important to identify which patients will develop epilepsy and which individually tailored treatment is needed. However, currently, we have no tools to identify the patients at risk, and diagnosis of epileptogenesis remains as a major unmet medical need, which relates to lack of diagnostic biomarkers for epileptogenesis. As the epileptogenic process in humans is typically slow, the use of animal models is justified to speed up biomarker discovery. We aim to summarize recommendations for molecular biomarker research and propose a standardized procedure for biomarker discovery in rat models of epileptogenesis. The potential of many phylogenetically conserved circulating noncoding small RNAs, including microRNAs (miRNAs), as biomarkers has been explored in various brain diseases, including epilepsy. Recent studies show the feasibility of detecting miRNAs in blood in both experimental models and human epilepsy. However, the analysis of circulating miRNAs in rodent models is challenging, which relates both to the lack of standardized sampling protocols and to analysis of miRNAs. We will discuss the issues critical for preclinical plasma biomarker discovery, such as documentation, blood and brain tissue sampling and collection, plasma separation and storage, RNA extraction, quality control, and RNA detection. We propose a protocol for standardization of procedures for discovery of circulating miRNA biomarkers in rat models of epileptogenesis. Ultimately, we hope that the preclinical standardization will facilitate clinical biomarker discovery for epileptogenesis in man.
New approaches for physical mapping of small genomes

Although linear chromosomal DNAs of fungi and protozoa (up to about 10 megabases [Mb] in size) (10, 23, 35) are fractionated by pulsed-field gel electrophoresis (PFG; 28), circular bacterial chromosomal DNAs (1 to 20 Mb) are not. Thus, methods were developed that use agarose as a protective matrix (32) to purify intact bacterial chromosomes that were enzymatically convertible to linear molecules. The first application was the construction of a complete low-resolution NotI restriction map of the Escherichia coli chromo-
The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.
Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.
Cortico‐Subcortical Interactions in Behavioral Sensitization: Differential Effects of Daily Nicotine and Morphine

Acute systemic injections of morphine and nicotine elicit increased locomotion in rats and repeating these injections produces an enhanced or sensitized effect.1J Sensitization of locomotion has also been reported for arnphetarnir~e~. ~ and cocaine.5 Different lines of evidence suggest that mesolimbic dopaminergic (DA) activation underlies the acute locomotor effects of these drugs and that enhanced activity in this system may be responsible for sensitization to their locomotor effects.4,bs Further evidence implicating DA system activation in locomotor sensitization also comes from experiments in which morphine or a p-opioid receptor agonist was injected directly into the ventral tegmental area (VTA). 9-11 Indeed, this mesencephalic structure contains a high density of DA perikarya12 innervating cortical and subcortical fbrebrain areas such as the prefiontal cortex and the nucleus accumbens, one of the main targets of the mesolimbic DA system.13 Supporting the view that sensitization of the locomotor hyperactivity induced by repeated enkephalin injections into the VTA is associated with an activation of the mesolimbic DA pathway, an enhanced or sensitized metabolism of DA was fbund in the nucleus accumbens in response to the drug. " However, when morphine or a p-opioid receptor agonist was repeatedly injected into the nucleus accumbens, no sensitization occurred.15 Qualitatively similar findings were obtained with amphetamine: repeatedly injecting amphetamine into the VTA produces sensitized locomotor responses to peripheral administrations of either amphetamine, cocaine or morphine, whereas amphetamine repeatedly injected into the nucleus accumbens elicits locomotor hyperactivity but no sensitization.16J7 It has been proposed therefore, that sensitization involves changes in a local neuronal loop which mul t in altered modulation of DA neurons in the VTA, rendering them more easily excitable by subsequent pharmacological challenge. A diminished somatodendritic release of DA fiom mesolimbic DA cell bodies, shown to occur in sensitized animals, could account fbr the enhanced release of DA in the nucleus accumbens.15J6J8 Alternatively, but not exdusively, it
Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.

BACKGROUND & AIMS Intestinal-type gastric cancer is often preceded by intestinal metaplasia in humans. The genetic events responsible for the transdifferentiation that occurs in intestinal metaplasia are not well understood. Cdx2, a transcription factor whose expression is normally limited to the intestine, has been detected in gastric intestinal metaplasia. Cdx2 induces differentiation of intestinal epithelial cells in vitro; therefore, we sought to establish whether a causal relationship exists between Cdx2 activation and intestinal metaplasia. METHODS Cdx2 expression was directed to the gastric mucosa in transgenic mice using cis-regulatory elements of Foxa3 (Hnf3gamma). Transgenic mice were analyzed for histologic and gene expression changes. RESULTS Histologic examination of the gastric mucosa of the Foxa3/Cdx2 mice revealed the presence of alcian blue-positive intestinal-type goblet cells, a hallmark of intestinal metaplasia. In addition, Cdx2 induced the expression of intestine-specific genes. CONCLUSIONS Gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice. These mice represent a powerful tool to investigate the molecular mechanisms that promote intestinal metaplasia. Moreover, as gastric cancer in humans is often preceded by intestinal metaplasia, the phenotype described here strongly suggests involvement of Cdx2 in the initiation of the process leading to intestinal neoplasia of the gastric mucosa.
Bacterial protease inhibitors

Serine‐, cysteine‐, and metalloproteases are widely spread in many pathogenic bacteria, where they play critical functions related to colonization and evasion of host immune defenses, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. Since all the antibiotics used clinically at the moment share a common mechanism of action, acting as inhibitors of the bacterial cell wall biosynthesis or affecting protein synthesis on ribosomes, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using new generation of antibiotics, possessing a different mechanism of action. Bacterial protease inhibitors constitute an interesting such possibility, due to the fact that many specific as well as ubiquitous proteases have recently been characterized in some detail in both gram‐positive as well as gram‐negative pathogens. Few potent, specific inhibitors for such bacterial proteases have been reported at this moment except for some signal peptidase, clostripain, Clostridium histolyticum collagenase, botulinum neurotoxin, and tetanus neurotoxin inhibitors. No inhibitors of the critically important and ubiquitous AAA proteases, degP or sortase have been reported, although such compounds would presumably constitute a new class of highly effective antibiotics. This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 4, 329–372, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10007
Mexiletine block of wild‐type and inactivation‐deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium‐aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na+ channels when cells are stimulated infrequently and produces additional use‐dependent block during repetitive pulses. We examined the state‐dependent block by mexiletine in human skeletal muscle hNav1.4 wild‐type and inactivation‐deficient mutant Na+ channels (hNav1.4‐L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC50) for the inactivated‐state block and the resting‐state block of wild‐type Na+ channels by mexiletine were measured as 67.8 ± 7.0 μm and 431.2 ± 9.4 μm, respectively (n= 5). In contrast, the IC50 for the block of open inactivation‐deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μm (n= 5), which was within the therapeutic plasma concentration range (2.8–11 μm). Estimated on‐ and off‐rates for the open‐state block by mexiletine at +30 mV were 10.4 μm−1 s−1 and 54.4 s−1, respectively. Use‐dependent block by mexiletine was greater in inactivation‐deficient mutant channels than in wild‐type channels during repetitive pulses. Furthermore, the IC50 values for the block of persistent late hNav1.4 currents in chloramine‐T‐pretreated cells by mexiletine was 7.5 ± 0.8 μm (n= 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open‐channel block of persistent late Na+ currents, which may arise during various pathological conditions.
Characterization of the Body-Area Propagation Channel for Monitoring a Subject Sleeping

A dynamic characterization of the wireless body-area communication channel for monitoring a sleeping person is presented. The characterization uses measurements near the 2.4 GHz ISM band with measurements of eight adult subjects each over a period of at least 2 hours. Numerous transmit-receive pair (Tx-Rx) locations on and off the body for a typical body-area-network (BAN) are used. Three issues are addressed: 1) modeling of channel gain, 2) outage probability, and 3) outage duration. It is shown that over very large durations (far in excess of a delay requirement of 125 ms that is typical for many IEEE 802.15.6 medical BAN applications) there is not a reliable communications channel for star-topology BAN. The best case outage probability, with 0 dBm Tx power and - 100 dBm Rx sensitivity, is in excess of a packet-error-rate of 10%. Following from these issues the feasibility of using alternate on-body or off-body links as relays is demonstrated.
arly ( 2008 – 2010 ) hospital outbreak of Klebsiella pneumoniae roducing OXA-48 carbapenemase in the UK laire

OXA-48 -lactamase is one of the several emerging carbapenemases. Pre-2007 reports were almost exclusively from Turkey, but subsequently its distribution has expanded. We report an early and prolonged outbreak in the UK of Klebsiella pneumoniae producing OXA-48 carbapenemase affecting a predominantly renal cohort in a West London hospital. Carbapenemase production was detected by the modified Hodge test, with confirmation by PCR for blaOXA-48. Isolates were typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Risk factors for acquisition were determined. Between January 2008 and April 2010, 20 K. pneumoniae isolates with reduced susceptibility to carbapenems were identified from 13 patients, comprising 12 renal cases and 1 oncology patient; 8 were outpatients and 5 were inpatients; 7 were deemed to be colonised and 6 infected, including 2 with bacteraemia, 1 of whom died. Hodge tests were positive for all isolates and all had blaOXA-48. PFGE showed strain similarity in isolates from nine patients, whereas four patients’ isolates were distinct, representing three further PFGE profiles and suggesting horizontal spread of blaOXA-48. Most patients had received antibiotics in the preceding 3 months and all had healthcare contact, but none had recent travel to areas with endemic OXA-48 Enterobacteriaceae. The renal cohort was screened and a prevalence rate of 0.17% was found. Interventions that collectively brought the outbreak under control included strict infection control precautions, screening, improved laboratory detection protocols and antibiotic stewardship rounds. © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
GABAergic Interneuron Differentiation in the Basal Forebrain Is Mediated through Direct Regulation of Glutamic Acid Decarboxylase Isoforms by Dlx Homeobox Transcription Factors

GABA is the key inhibitory neurotransmitter in the cortex but regulation of its synthesis during forebrain development is poorly understood. In the telencephalon, members of the distal-less (Dlx) homeobox gene family are expressed in, and regulate the development of, the basal ganglia primodia from which many GABAergic neurons originate and migrate to other forebrain regions. The Dlx1/Dlx2 double knock-out mice die at birth with abnormal cortical development, including loss of tangential migration of GABAergic inhibitory interneurons to the neocortex (Anderson et al., 1997a). We have discovered that specific promoter regulatory elements of glutamic acid decarboxylase isoforms (Gad1 and Gad2), which regulate GABA synthesis from the excitatory neurotransmitter glutamate, are direct transcriptional targets of both DLX1 and DLX2 homeoproteins in vivo. Further gain- and loss-of-function studies in vitro and in vivo demonstrated that both DLX1 and DLX2 are necessary and sufficient for Gad gene expression. DLX1 and/or DLX2 activated the transcription of both Gad genes, and defects in Dlx function disrupted the differentiation of GABAergic interneurons with global reduction in GABA levels in the forebrains of the Dlx1/Dlx2 double knock-out mouse in vivo. Identification of Gad genes as direct Dlx transcriptional targets is significant; it extends our understanding of Dlx gene function in the developing forebrain beyond the regulation of tangential interneuron migration to the differentiation of GABAergic interneurons arising from the basal telencephalon, and may help to unravel the pathogenesis of several developmental brain disorders. SIGNIFICANCE STATEMENT GABA is the major inhibitory neurotransmitter in the brain. We show that Dlx1/Dlx2 homeobox genes regulate GABA synthesis during forebrain development through direct activation of glutamic acid decarboxylase enzyme isoforms that convert glutamate to GABA. This discovery helps explain how Dlx mutations result in abnormal forebrain development, due to defective differentiation, in addition to the loss of tangential migration of GABAergic inhibitory interneurons to the neocortex. Reduced numbers or function of cortical GABAergic neurons may lead to hyperactivity states such as seizures (Cobos et al., 2005) or contribute to the pathogenesis of some autism spectrum disorders. GABAergic dysfunction in the basal ganglia could disrupt the learning and development of complex motor and cognitive behaviors (Rubenstein and Merzenich, 2003).
SCROTAL ULTRASONOGRAPHY IN THE ASSESSMENT OF SUBFERTILE MALES

Subfertility is defined as a failure to conceive after one year of unprotected regular sexual intercourse. A prospective study was conducted to identity the role of ultrasonography in assessing the cause of male subfertility in a Sri Lankan population. The objective of the study was to identify the role of ultrasonography in assessing the cause of male subfertility in a Sri Lankan population. The sample consists of 86 patients referred for scrotal ultrasonography for the assessment of male subfertility. Ultrasound scan of the scrotum was done for the assessment of the testicular volume, testicular pathologies and extra testicular pathologies. The patients who were azoospermic or had seminal fluid volume less than 2.5ml were subjected to Trans Rectal Ultrasound Scan (TRUS) to exclude any obstruction to the seminal fluid pathway. As the results mean duration of subfertility was 4.47 (SD=2.63). 91.9% of the study population had primary subfertility while 8.1% had secondary subfertility. Testicular volume was calculated using three measurements and the formula of an ellipsoid. The mean ultrasonic testicular volume of the right testis was 7.66 cm (SD=4.24), and that of the left was 7.43 cm (SD=4.50). 44.2% of patients had some abnormality detected by ultrasound scan while 55.8% had normal ultrasonography. 26.8 % (n = 23) of the sample had varicocele and of which 73.91 % (n = 17) was on left side only, 17.39 % (n = 4) had only right sided varicocele and 8.69 % (n = 2) had bilateral disease. 4.7% of patients had epididymal cysts and 4.7% had testicular microlithiasis. When considering the echo pattern of the testis 77.9 % (n = 67) had normal echo pattern but 16.3% (n = 14) had heterogeneous echo pattern. As a conclusion the commonest abnormality detected in sub fertile men was a left sided varicocele. However there was a significant difference in the mean testicular size of Sri Lankan sub fertile men.
Perceptions of clinical utility of an Augmented Reality musical software among health care professionals

Abstract Purpose: Augmented Reality musical software (GenVirtual) is a technology, which primarily allows users to develop music activities for rehabilitation. This study aimed to analyse the perceptions of health care professionals regarding the clinical utility of GenVirtual. A second objective was to identify improvements to GenVirtual software and similar technologies. Method: Music therapists, occupational therapists, physiotherapists and speech and language therapist who assist people with physical and cognitive disabilities were enrolled in three focus groups. The quantitative and qualitative data were collected through inductive thematic analysis. Results: Three main themes were identified: the use of GenVirtual in health care areas; opportunities for realistic application of GenVirtual; and limitations in the use of GenVirtual. The registration units identified were: motor stimulation, cognitive stimulation, verbal learning, recreation activity, musicality, accessibility, motivation, sonic accuracy, interference of lighting, poor sound, children and adults. Conclusion: This research suggested that the GenVirtual is a complementary tool to conventional clinical practice and has great potential to motor and cognitive rehabilitation of children and adults. Implications for Rehabilitation Gaining health professional’ perceptions of the Augmented Reality musical game (GenVirtual) give valuable information as to the clinical utility of the software. GenVirtual was perceived as a tool that could be used as enhancing the motor and cognitive rehabilitation process. GenVirtual was viewed as a tool that could enhance clinical practice and communication among various agencies, but it was suggested that it should be used with caution to avoid confusion and replacement of important services.