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SRX246, an orally available CNS penetrant vasopressin (VP) V1a receptor antagonist, was studied in Huntington's disease (HD) patients with irritability and aggressive behavior in the exploratory phase 2 trial, Safety, Tolerability, and Activity of SRX246 in Irritable HD patients (STAIR). This was a dose-escalation study; subjects received final doses of 120 mg BID, 160 mg BID, or placebo. The compound was safe and well tolerated. In this paper, we summarize the results of exploratory analyses of measures of problematic behaviors, including the Cohen–Mansfield Agitation Inventory (CMAI), Aberrant Behavior Checklist (ABC), Problem Behaviors Assessment-short form (PBA-s), Irritability Scale (IS), Clinical Global Impression (CGI), HD Quality of Life (QoL), and Caregiver Burden questionnaires. In addition to these, we asked subjects and caregivers to record answers to short questions about mood, irritability, and aggressive conduct in an eDiary. STAIR was the first rigorously designed study of behavioral endpoints like these in HD. The exploratory analyses showed that SRX246 reduced aggressive acts. Readily observed behaviors should be used as trial endpoints. [ABSTRACT FROM AUTHOR]

Rationale: Arginine vasopressin (AVP) is a neuropeptide that modulates both physiological and emotional responses to threat. Until recently, drugs that target vasopressin receptors (V1a) in the human central nervous system were unavailable. The development of a novel V1a receptor antagonist, SRX246, permits the experimental validation of vasopressin's role in the regulation of anxiety and fear in humans. Objectives: Here, we examined the effects of SRX246 in a proof-of-concept translational paradigm of fear (phasic response to imminent threat) and anxiety (prolonged response to potential threat). Methods: Healthy volunteers received both SRX246 and placebo in a randomized, double-blind, counter-balanced order separated by a 5–7-day wash-out period. Threat consisted of unpleasant electric shocks. The "NPU" threat test probed startle reactivity during predictable threat (i.e., fear-potentiated startle) and unpredictable threat (i.e., anxiety-potentiated startle). Results: As predicted, SRX246 decreased anxiety-potentiated startle independent of fear-potentiated startle. Conclusions: As anxiety-potentiated startle is elevated in anxiety and trauma-associated disorders and decreased by traditional anxiolytics such as SSRIs and benzodiazepines, the V1a receptor is a promising novel treatment target. [ABSTRACT FROM AUTHOR]

For nearly two centuries, developmental biologists have known that body organs are derived from distinct germlayers. They have argued that adult stemcells formed in one of these, mesoderm for example, cannot give rise to cells that originate in another. We disagree. An exception to this rule has been described in crayfish recently. In this species, hemocytes appear to replenish neurogenic cells. This may happen in humans as well. In women who were given male bone marrow-derived cells, Y chromosome positive cheek cells and brain neurons were detected.While repopulation of these tissues by bone marrow derived cells may not occu rnormally, and while it does not appear to be terribly efficient, the phenomenon should be studied in more detail. Perhaps cells in the marrow could be used to regenerate tissues elsewhere. [ABSTRACT FROM AUTHOR]

SRX246 is a vasopressin (AVP) 1a receptor antagonist that crosses the blood-brain barrier. It reduced impulsive aggression, fear, depression and anxiety in animal models, blocked the actions of intranasal AVP on aggression/fear circuits in an experimental medicine fMRI study and demonstrated excellent safety in Phase 1 multiple-ascending dose clinical trials. The present study was a 3-arm, multicenter, randomized, placebo-controlled, double-blind, 12-week, dose escalation study of SRX246 in early symptomatic Huntington's disease (HD) patients with irritability. Our goal was to determine whether SRX246 was safe and well tolerated in these HD patients given its potential use for the treatment of problematic neuropsychiatric symptoms. Participants were randomized to receive placebo or to escalate to 120 mg twice daily or 160 mg twice daily doses of SRX246. Assessments included standard safety tests, the Unified Huntington's Disease Rating Scale (UHDRS), and exploratory measures of problem behaviors. The groups had comparable demographics, features of HD and baseline irritability. Eighty-two out of 106 subjects randomized completed the trial on their assigned dose of drug. One-sided exact-method confidence interval tests were used to reject the null hypothesis of inferior tolerability or safety for each dose group vs. placebo. Apathy and suicidality were not affected by SRX246. Most adverse events in the active arms were considered unlikely to be related to SRX246. The compound was safe and well tolerated in HD patients and can be moved forward as a candidate to treat irritability and aggression. [ABSTRACT FROM AUTHOR]

Irritability, a common neuropsychiatric symptom in Huntington's disease (HD), lacks a standardized measurement. The Irritability Scale (IS), tailored for HD, has patient and informant versions, but variable interrater agreement has been reported frequently in previous studies. To enhance the clinical utility of the IS, this study aimed to identify the most reliable components estimating the underlying construct and develop a shortened version for time-limited contexts. Participant and informant/observer concordance and the relationship of individual items to the complete IS scale were assessed. The short-form (SF) items were selected based on interrater agreement, exploratory factor analysis (EFA), and Item Response Theory (IRT) analysis results. Pair-wise correlation and covariance models were used to examine how SF predicted total IS score in 106 participants from the STAIR (Safety, Tolerability, and Activity of SRX246 in Irritable Subjects with Huntington's Disease) trial. Item Response Theory (IRT) analysis was used to evaluate the range and function of the selected items. IS interrater agreement was statistically significant (r = 0.33, p =.001). In combination with EFA factors and IRT analyses, five items were identified that showed good reliability and performance in differentiating levels of irritability. The proposed 5-item SF IS provided a reliable measure of the full scale and may be less burdensome for use in a clinical setting. • Irritability is a key feature of Huntington's disease (HD). • Informant reporting may enhance assessment of irritability in HD. • A short-form of the Irritability Scale (IS) may have utility in HD. [ABSTRACT FROM AUTHOR]

The author reflects on two papers describing the molecular anatomy and formation of one member of the inflammasome family, the NAIP2/NLRC4 inflammasome, produced in response to intracellular PrgJ protein. He references the articles "Structural and biochemical basis for induced self-propagation of NLRC4" by Z. Hu and colleagues and "Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization" by L. Zhang and colleagues, which both appeared in "Science" in 2015.

Abstract: The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC50 45nM at the human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing hormone receptor. Structure–activity relationships for the series were explored to optimize receptor affinity and pharmacokinetic properties, resulting in compounds with K i values <1nM and brain levels after oral dosing ∼100-fold higher than receptor affinities. Display Omitted [Copyright &y& Elsevier]

• Chronic rhinosinusitis can be very difficult to treat. • Many patients have recurrent infections for years. • Antibiotics and surgery sometimes are not enough to cure the patient. • Phage therapy is a promising approach for difficult-to-treat infections. We present a case of refractory methicillin-resistant Staphylococcus aureus that was successfully treated with a combination of antibiotics, systemic phage and intranasal phage therapy. [ABSTRACT FROM AUTHOR]

Treatment options for Achromobacter xylosoxidans are limited. Eight cystic fibrosis patients with A. xylosoxidans were treated with 12 cefiderocol courses. Pretreatment in vitro resistance was seen in 3 of 8 cases. Clinical response occurred after 11 of 12 treatment courses. However, microbiologic relapse was observed after 11 of 12 treatment courses, notably without emergence of resistance. [ABSTRACT FROM AUTHOR]

Background Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. Methods The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient's isolate and determined by safranin staining. Results Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P  = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections. [ABSTRACT FROM AUTHOR]

• A single, mild head impact causes a rise in vasogenic edema 2−6 hrs post impact. • Areas most affected, like the cerebellum, are remote from the site of impact. • The edema peaks within 6 h of head impact but resolves within 24 h. • A vasopressin V1a receptor antagonist blocks much of the early vasogenic edema. Does minor head impact without signs of structural brain damage cause short-term changes in vasogenic edema as measured by an increase apparent diffusion coefficient (ADC) using diffusion weighted imaging? If so, could the increase in vasogenic edema be treated with a vasopressin V1a receptor antagonist? We hypothesized that SRX251, a highly selective V1a antagonist, would reduce vasogenic edema in response to a single minor head impact. Lightly anesthetized male rats were subjected to a sham procedure or a single hit to the forehead using a closed skull, momentum exchange model. Animals recovered in five min and were injected with saline vehicle (n = 8) or SRX251 (n = 8) at 15 min post head impact and again 7−8 hrs later. At 2 h, 6 h, and 24 h post injury, rats were anesthetized and scanned for increases in ADC, a neurological measure of vasogenic edema. Sham rats (n = 6) were exposed to anesthesia and scanned at all time points but were not hit or treated. Images were registered to and analyzed using a 3D MRI rat atlas providing site-specific data on 150 different brain areas. These brain areas were parsed into 11 major brain regions. Untreated rats with brain injury showed a significant increase in global brain vasogenic edema as compared to sham and SRX251 treated rats. Edema peaked at 6 h in injured, untreated rats in three brain regions where changes in ADC were observed, but returned to sham levels by 24 h. There were regional variations in the time course of vasogenic edema and drug efficacy. Edema was significantly reduced in cerebellum and thalamus with SRX251 treatment while the basal ganglia did not show a response to treatment. A single minor impact to the forehead causes regional increases in vasogenic edema that peak at 6 h but return to baseline within a day in a subset of brain regions. Treatment with a selective V1a receptor antagonist can reduce much of the edema. [ABSTRACT FROM AUTHOR]

A patient with a trauma-related left tibial infection associated with extensively drug-resistant Acinetobacter baumannii and multidrug-resistant Klebsiella pneumoniae was treated with bacteriophages and antibiotics. There was rapid tissue healing and positive culture eradication. As a result, the patient's leg did not have to be amputated and he is undergoing rehabilitation. [ABSTRACT FROM AUTHOR]

Renal ischemia-reperfusion injury (IRI) is a severe complication of major surgery and a risk factor for increased morbidity and mortality. Here, we investigated mechanisms that might contribute to IRIinduced progression to chronic kidney disease (CKD). Acute kidney injury (AKI) was induced by unilateral IRI for 35 min in CD1 and C57BL/6 (B6) mice. Unilateral IRI was used to overcome early mortality. Renal morphology, NGAL upregulation, and neutrophil infiltration as well as peritubular capillary density were studied by immunohistochemistry. The composition of leukocyte infiltrates in the kidney after IRI was investigated by flow cytometry. Systemic blood pressure was measured with a tail cuff, and renal perfusion was quantified by functional magnetic resonance imaging (fMRI). Mesangial matrix expansion was assessed by silver staining. Following IRI, CD1 and B6 mice developed similar morphological signs of AKI and increases in NGAL expression, but neutrophil infiltration was greater in CD1 than B6 mice. IRI induced an increase in systemic blood pressure of 20 mmHg in CD1, but not in B6 mice; and CD1 mice also had a greater loss of renal perfusion and kidney volume than B6 mice (P 0.05). CD1 mice developed substantial interstitial fibrosis and decreased peritubular capillary (PTC) density by day 14 while B6 mice showed only mild renal scarring and almost normal PTC. Our results show that after IRI, CD1 mice develop more inflammation, hypertension, and later mesangial matrix expansion than B6 mice do. Subsequently, CD1 animals suffer from CKD due to impaired renal perfusion and pronounced permanent loss of peritubular capillaries. [ABSTRACT FROM AUTHOR]

Hereditary sensory and autonomic neuropathy type III, or familial dysautonomia [FD; Online Mendelian Inheritance in Man (OMIM) 223900], affects the development and long-term viability of neurons in the peripheral nervous system (PNS) and retina. FD is caused by a point mutation in the gene IKBKAP/ELP1 that results in a tissue-specific reduction of the IKAP/ELP1 protein, a subunit of the Elongator complex. Hallmarks of the disease include vasomotor and cardiovascular instability and diminished pain and temperature sensation caused by reductions in sensory and autonomic neurons. It has been suggested but not demonstrated that mitochondrial function may be abnormal in FD. We previously generated an Ikbkap/Elp1 conditional-knockout mouse model that recapitulates the selective death of sensory (dorsal root ganglia) and autonomic neurons observed in FD. We now show that in these mice neuronal mitochondria have abnormal membrane potentials, produce elevated levels of reactive oxygen species, are fragmented, and do not aggregate normally at axonal branch points. The small hydroxylamine compound BGP-15 improved mitochondrial function, protecting neurons from dying in vitro and in vivo, and promoted cardiac innervation in vivo. Given that impairment of mitochondrial function is a common pathological component of neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer’s, Parkinson’s, and Huntington’s diseases, our findings identify a therapeutic approach that may have efficacy in multiple degenerative conditions. [ABSTRACT FROM AUTHOR]

Dementia with Lewy bodies (DLB) patients frequently experience well formed recurrent complex visual hallucinations (RCVH). This is associated with reduced blood flow or hypometabolism on imaging of the primary visual cortex. To understand these associations in DLB we used pathological and biochemical analysis of the primary visual cortex to identify changes that could underpin RCVH. Alpha-synuclein or neurofibrillary tangle pathology in primary visual cortex was essentially absent. Neurone density or volume within the primary visual cortex in DLB was also unchanged using unbiased stereology. Microarray analysis, however, demonstrated changes in neuropeptide gene expression and other markers, indicating altered GABAergic neuronal function. Calcium binding protein and GAD65/67 immunohistochemistry showed preserved interneurone populations indicating possible interneurone dysfunction. This was demonstrated by loss of post synaptic GABA receptor markers including gephyrin, GABARAP, and Kif5A, indicating reduced GABAergic synaptic activity. Glutamatergic neuronal signalling was also altered with vesicular glutamate transporter protein and PSD-95 expression being reduced. Changes to the primary visual cortex in DLB indicate that reduced GABAergic transmission may contribute to RCVH in DLB and treatment using targeted GABAergic modulation or similar approaches using glutamatergic modification may be beneficial. [ABSTRACT FROM AUTHOR]

SRX246 is a potent, highly selective, orally bioavailable vasopressin 1a receptor antagonist that represents a novel mechanism of action for the treatment of mood disorders. The compound previously showed efficacy in animal models of mood disorders and excellent safety and tolerability in healthy volunteers in phase I clinical trials. In this study, SRX246 was further characterized in rats and dogs. In vitro determinations of permeability, protein binding, hepatocyte metabolism, and cytochrome P450 enzyme inhibition and in vivo assessments of pharmacokinetics were conducted. In parallel artificial membrane permeability assay (PAMPA) and PAMPA-blood-brain barrier models, SRX246 was comparable to highly permeable, orally active pharmaceuticals. SRX246 hydrochloride salt was 95.5 ± 1.7%, 95.9 ± 1.3%, and 98.6 ± 0.4% bound to rat, dog, and human serum proteins, respectively, and was stable in serum after a 4 h incubation at 37°C. P450 enzyme inhibition results showed a very low potential for drug-drug interactions. Metabolism in primary hepatocytes demonstrated that SRX246 was stable in humans and moderately metabolized in dogs and rats. Plasma pharmacokinetics findings showed a half-life ( T½) of 2 and 6 h in rat and dog, respectively. Rat brain levels following a single oral dose were approximately 20% of plasma values with a T½ of 6 h. The observed profile for SRX246 supports further development. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2033-2043, 2013 [ABSTRACT FROM AUTHOR]

Abstract: SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood–brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed. [Copyright &y& Elsevier]

Abstract: Dehydroepiandrosterone (DHEA) is the most abundant steroid in humans and a multi-functional neuroactive steroid that has been implicated in a variety of biological effects in both the periphery and central nervous system. Mechanistic studies of DHEA in the periphery have emphasized its role as a prohormone and those in the brain have focused on effects exerted at cell surface receptors. Recent results demonstrated that DHEA is intrinsically androgenic. It competes with DHT for binding to androgen receptor (AR), induces AR-regulated reporter gene expression in vitro, and exogenous DHEA administration regulates gene expression in peripheral androgen-dependent tissues and LnCAP prostate cancer cells, indicating genomic effects and adding a level of complexity to functional models. The absence of information about the effect of DHEA on gene expression in the CNS is a significant gap in light of continuing clinical interest in the compound as a hormone replacement therapy in older individuals, patients with adrenal insufficiency, and as a treatment that improves sense of well-being, increases libido, relieves depressive symptoms, and serves as a neuroprotective agent. In the present study, ovariectomized CF-1 female mice, an established model for assessing CNS effects of androgens, were treated with DHEA (1mg/day), dihydrotestosterone (DHT, a potent androgen used as a positive control; 0.1mg/day) or vehicle (negative control) for 7 days. The effects of DHEA on gene expression were assessed in two regions of the CNS that are enriched in AR, hypothalamus and hippocampus, using DNA microarray, real-time RT-PCR, and immunohistochemistry. RIA of serum samples assessed treatment effects on circulating levels of major steroids. In hypothalamus, DHEA and DHT significantly up-regulated the gene expression of hypocretin (Hcrt; also called orexin), pro-melanin-concentrating hormone (Pmch), and protein kinase C delta (Prkcd), and down-regulated the expression of deleted in bladder cancer chromosome region candidate 1 (Dbccr1) and chitinase 3-like 3 (Chi3l3). Two-step real-time RT-PCR confirmed changes in the expression of three genes (Pmch, Hcrt and Prkcd) using the same RNA sample employed in the microarray experiment. Immunohistochemistry showed augmentation of prepro-hypocretin (pHcrt) neuropeptide protein expression by DHEA and DHT in hypothalamus, consistent with the localization of orexin neurons. In hippocampus, DHT down-regulated the expression of Prkcd, while DHEA did not have significant effects. RIA results supported the view that DHEA-induced effects were mediated through AR. The current study identified neurogenomic effects of DHEA treatment on a subset of genes directly implicated in the regulation of appetite, energy utilization, alertness, apoptosis, and cell survival. These changes in gene expression in the CNS represent a constellation of effects that may help explain the diverse benefits attributed to replacement therapy with DHEA. The data also provide a new level of detail regarding the genomic mechanism of action of DHEA in the CNS and strongly support a central role for the androgen receptor in the production of these effects. More broadly, the results may be clinically significant because they provide new insights into processes that appear to mediate the diverse CNS effects attributed to DHEA. [Copyright &y& Elsevier]

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2′-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP‐38 administered i.v. or i.c.v. 1 h after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 h later in wild‐type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP‐deficient than in wild‐type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP‐deficient mice to the same level of improvement as in wild‐type mice. A 35,546‐clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild‐type and PACAP‐deficient mice, and with PACAP treatment after MCAO in wild‐type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild‐type mice. 49 transcripts were significantly up‐regulated only at 1 h post‐MCAO (acute response transcripts), 142 were up‐regulated only at 24 h post‐MCAO (delayed response transcripts) and 37 transcripts were up‐regulated at both times (sustained response transcripts). More than half of these are transcripts not previously reported to be altered in ischemia. A larger percentage of genes up‐regulated at 24 hr than at 1 hr required endogenous PACAP, suggesting a more prominent role for PACAP in later response to injury than in the initial response. This is consistent with a neuroprotective role for PACAP in late response to injury, i.e., even when administered 1 hr or more after MCAO. Putative injury effector transcripts regulated by PACAP include β‐actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP‐regulated in other contexts. Prominent among these were transcripts encoding the PACAP‐regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin. [Copyright &y& Elsevier]

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins—known as the ‘Piwi family’—is required for germ- and stem-cell development in invertebrates, and two Piwi members—MILI and MIWI—are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5′ uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26–31 nucleotides (nt) in length—clearly distinct from the 21–23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)—and we refer to them as ‘Piwi-interacting RNAs’ or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis. [ABSTRACT FROM AUTHOR]

Oxytocin- and vasopressin-producing magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system are the only neuronal phenotypes present in the rat supraoptic nucleus (SON). Laser microdissection of the SON, extraction and T7-based amplification of its RNAs, and analysis of the resulting cDNAs by hybridization on a 35, 319 element DNA microarray have provided a detailed composite view of the gene expression profile of the MCNs. The genes expressed in the SON were compared with those expressed in a reference tissue consisting of total hypothalamus, and this "expression ratio" indicated which genes were preferentially expressed in the SON. Of the 26,000 unique genes on the array, 1385 were found to be expressed in the SON at levels more than two times greater than in the hypothalamus as a whole. Of these, 123 were expressed ≥3.4-fold higher in the SON versus hypothalamus. Most of these preferentially expressed genes were not previously known to be expressed in the MCNs. Quantitative and double-label in situ hybridization histochemistry was used selectively to confirm a number of these microarray observations and to evaluate the osmotic regulation and cell-specific expression of these genes, respectively. [ABSTRACT FROM AUTHOR]

Neurogenesis is one of the most complex events in embryonic development. However, little information is available regarding the molecular events that occur during neurogenesis. To identify regulatory genes and underlying mechanisms involved in the differentiation of embryonic stem (ES) cells to neurons, gene expression profiling was performed using cDNA microarrays. In mouse ES cells, we compared the gene expression of each differentiated cell stage using a five-stage lineage selection method. Of 10,368 genes, 1633 (16%) known regulatory genes were differentially expressed at least 2-fold or greater at one or more stages. At stage 3, during which ES cells differentiate into neural stem cells, modulation of nearly 1000 genes was observed. Most of transcription factors (Otx2, Ebf-3, Ptx3, Sox4, 13, 18, engrailed, Irx2, Pax8, and Lim3), signaling molecules (Wnt, TGF, and Shh family members), and extracellular matrix/adhesion molecules (collagens, MAPs, and NCAM) were up-regulated. However, some genes which may play important roles in maintaining the pluripotency of ES cells (Kruppel-like factor 2, 4, 5, 9, myeloblast oncogene like2, ZFP 57, and Esg-1) were down-regulated. The many genes identified with this approach that are modulated during neurogenesis will facilitate studies of the mechanisms underlying ES cell differentiation, neural induction, and neurogenesis. [Copyright &y& Elsevier]

Familial dysautonomia (FD) is the best-known and most common member of a group of congenital sensory/autonomic neuropathies characterized by widespread sensory and variable autonomic dysfunction. As opposed to the sensory/motor neuropathies, little is known about the causes of neuronal dysfunction and loss in the sensory/autonomic neuropathies. FD involves progressive neuronal degeneration, has a broad impact on the operation of many of the body’s systems, and leads to a markedly reduced quality of life and premature death. In 2001, we identified two mutations in the IKBKAP gene that result in FD. IKBKAP encodes IKAP, a member of the putative human holo-Elongator complex, which may facilitate transcription by RNA polymerase II. Whether or not the Elongator plays this role is moot. The FD mutation found on >99.5% of FD chromosomes does not cause complete loss of function. Instead, it results in a tissue-specific decrease in splicing efficiency of the IKBKAP transcript; cells from patients retain some capacity to produce normal mRNA and protein. To better understand the relationship between the genotype of FD patients and their phenotype, we have used in situ hybridization histochemistry to map the IKAP mRNA in sections of whole rat embryos. The mRNA is widely distributed. Highest levels are in the nervous system, but substantial amounts are also present in peripheral organs. [Copyright &y& Elsevier]

Summary: Background: Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in-vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. Methods: To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. Findings: When examined 4-6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0.8-12.7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0.01%) and one XXY cell (0.01%), both of which could have arisen when an XY cell fused with an XX cell. Interpretation: Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine. [ABSTRACT FROM AUTHOR]

Examines the development of a highly informative mouse simple sequence length polymorphism marker set and construction of a Mouse family tree using parsimony analysis. Selection of multiplexing; Rate of the informative markers per cross; Reconstruction of phylogenetic history of the laboratory mouse.

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 µg of total RNA or 2 µg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 µg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5′ ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simpie to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers. [ABSTRACT FROM AUTHOR]

Examines the importance of acid-sensing ion channel 3 (ASIC3) in pain signaling in knockout mice. Identification of mouse genotypes; Composition of neuronal cell of dorsal root ganglia from ASIC3; Inactivation of the ASIC3 gene.

Presents information on a study which investigated the molecular basis of familial dysautonomia. Role of tissue-specific expression of a splicing mutation in the IKBKAP gene; Methodology; Results and discussion.

Successful joint replacement is a life-enhancing procedure with significant growth in the past decade. Prosthetic joint infection occurs rarely; it is a biofilm-based infection that is poorly responsive to antibiotic alone. Recent interest in bacteriophage therapy has made it possible to treat some biofilm-based infections, as well as those caused by multidrug-resistant pathogens, successfully when conventional antibiotic therapy has failed. Here, we describe the case of a 61-year-old woman who was successfully treated after a second cycle of bacteriophage therapy administered at the time of a two-stage exchange procedure for a persistent methicillin-sensitive Staphylococcus aureus (MSSA) prosthetic knee-joint infection. We highlight the safety and efficacy of both intravenous and intra-articular infusions of bacteriophage therapy, a successful outcome with a single lytic phage, and the development of serum neutralization with prolonged treatment. [ABSTRACT FROM AUTHOR]

A microprocessor-controlled system has been developed to automatically scan tissue culture dishes in order to detect and locate fluorescently labeled cell colonies for subsequent cloning. An X-Y recorder mechanism moves an 80-mm-diam dish in a raster scan through a stationary laser beam adjacent to a photomultiplier tube equipped with an emission filter. Front-end electronics processes the PMT signal to screen out small-scale fluorescent artifacts of selectable size (e.g., <0.2 mm). Appropriate signals are input to the computer which then interrupts the raster scan to perform a limited fine scan of the dish to accurately localize the fluorescent spot position. An additional artifact test using a different filter is then performed. The underside of the dish is scribed at the location of spots that pass this test. Using fluorescein as a label, fluorescence as low as 25% above intrinsic cell background fluorescence can be detected. The fluorescence signal level threshhold can be set (e.g., 70% above intrinsic cell background) to within an accuracy of approximately 15% of intrinsic cell background fluorescence, and the system will reliably find colonies with a signal exceeding that level. With the above typical values, the number of false positives is typically less than five per dish and the time to completely scan an 80-mm-diam tissue culture dish is 2–4 min. [ABSTRACT FROM AUTHOR]

G protein–coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 μg of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines. [ABSTRACT FROM AUTHOR]

Examines the localization of a gene for Duane Retraction Syndrome (DRS) to chromosome 2q31. Diseases caused by undiagnosed DRS in children; Nononuclear anomalies associated with DRS; Use of genomewide search with highly polymorphic microsatellite markers to determine genetic link of DRS.

The article presents information on hereditary nephrogenic diabetes, a rare, X-linked disorder manifested by an inability to concentrate the urine despite high plasma concentrations of arginine vasopressin. Patients with nephrogenic diabetes insipidus have a defect in the V2-receptor signaling pathway, but their V1-receptor signaling is normal. The results of a study on a family with nephrogenic diabetic patients suggest that the mutation within the vasopressin V2-receptor gene is the cause of the hereditary disease.