Inhibition of Monkeypox virus replication by RNA interference

The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV), Vaccinia (VACV), monkeypox (MPV) and Variola (VARV) viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi) as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA). Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R) or an important gene in viral entry (E8L), inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening

Extracellular norepinephrine, norepinephrine receptor and transporter protein and mRNA levels are differentially altered in the developing rat brain due to dietary iron deficiency and manganese exposure

Abstract: Manganese (Mn) is an essential trace element, but overexposure is characterized by Parkinson's like symptoms in extreme cases. Previous studies have shown that Mn accumulation is exacerbated by dietary iron deficiency (ID) and disturbances in norepinephrine (NE) have been reported. Because behaviors associated with Mn neurotoxicity are complex, the goal of this study was to examine the effects of Mn exposure and ID-associated Mn accumulation on NE uptake in synaptosomes, extracellular NE concentrations, and expression of NE transport and receptor proteins. Sprague-Dawley rats were assigned to four dietary groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn exposure (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn (IDMn) . 3 H-NE uptake decreased significantly (R = -0.753, p=0.001) with increased Mn concentration in the locus coeruleus, while decreased Fe was associated with decreased uptake of 3 H-NE in the caudate putamen (R = 0.436, p = 0.033) and locus coeruleus (R = 0.86; p < 0.001). Extracellular concentrations of NE in the caudate putamen were significantly decreased in response to Mn exposure and ID (p < 0.001). A diverse response of Mn exposure and ID was observed on mRNA and protein expression of NE transporter (NET) and α 2 adrenergic receptor. For example, elevated brain Mn and decreased Fe caused an approximate 50% decrease in NET and α 2 adrenergic receptor protein expression in several brain regions, with reductions in mRNA expression also observed. These data suggest that Mn exposure results in a decrease in NE uptake and extracellular NE concentrations via altered expression of transport and receptor proteins

Manganese exposure inhibits the clearance of extracellular GABA and influenced taurine homeostasis in the striatum of developing rats.

Abstract: Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mninduced increase in extracellular GABA (GABA EC ) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA EC of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased 3 H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA EC by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain. Article: INTRODUCTION An essential trace element and a cofactor for several enzymes (Hurley and Keen, 1987), manganese (Mn) is involved in immune function, regulation of metabolism, reproduction, digestion, bone growth, and blood clotting (see review by With the intriguing findings that striatal extracellular GABA (GABA EC ) concentrations are higher due to Mn-exposure , and uptake of 3 H-GABA is attenuated by Mn-exposure in striatal synaptosomes Taurine is an abundant non-essential amino acid in the brain formed from cysteine. Traditionally, brain taurine is thought to function as an osmoregulator in cells (cell volume regulation), but has also been implicated in neuromodulation, possibly functioning as a neurotransmitter. Data exist suggesting that taurine functions as an anxiolytic agent We chose to look at the taurine/GABA relationship in the striatum because it is a known region for Mn accumulation In addition to GABA and taurine, we felt it was prudent to examine the effect of Mn on another amino acid neurotransmitter, glycine. Glycine is an abundant inhibitor neurotransmitter, similar to GABA, and it is known that taurine is a glycine receptor agonist Within the brain, astrocytes are the primary cells that maintain the composition of the extracellular fluid MATERIALS AND METHODS Animals Male weanling (post-natal day 21) Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) (n = 8 for microdialysis study; n = 6 for PCR gene expression and metal analysis studies) were randomly divided into two dietary treatment groups used in previous studies Cell cultures Rat primary cortical astrocyte cultures were purchased from Invitrogen (Carlsbad, CA) and certified for purity with >95% staining positive for the astrocytic marker glial fibrillary acidic protein (GFAP). Cells were grown in Dulbecco's Modified Eagle Media (D-MEM) with 15% fetal bovine serum (FBS), and maintained in a humidified atmosphere of 95% air/5% CO 2 at 37 °C. Manganese treatments were delivered using 0, 100, or 300 μM Mn in the form of MnCl 2 . These dose concentrations are based on previous studies in non-human primates reporting clinical symptoms of Mn neurotoxicity at brain concentrations of 300 μM, while 100 μM concentrations appeared to be asymptomatic H-Taurine Uptake of tritiated taurine ( 3 H-Taurine) was measured as described by Stereotaxic surgery After 5 weeks of dietary treatment and 1 week prior to microdialysis experiments, rats were anesthetized with ketamine-HCl (80 mg/kg) and xylazine (12 mg/kg) and maintained on a heating pad at 37 °C. The heads of the rats were shaved and wiped with a 5% povidone-iodine solution to reduce risk of infection. Sterile instruments and gloves were used throughout the surgical procedure. The rats were secured in the stereotaxic frame and an incision was made perpendicular to the bregma. A guide cannula (CMA/12, CMA Microdialysis, Acton, MA) was implanted into the striatum using the following coordinates: 2.4 mm lateral to the midline, 7.5 mm anterior to the lambda. The cannula was lowered to a depth of 2.5 mm, positioning it in the medial area of the striatum Microdialysis During week six of the dietary protocol, a microdialysis probe (CMA/12 Elite, CMA Microdialysis, Acton, MA) was inserted into the guide cannula and the rat was perfused with artificial cerebral spinal fluid (aCSF) (155 mM Na + , 0.83 mM Mg 2+ , 2.9 mM K + , 132.76 mM Cl − , 1.1 mM Ca + , pH 7.4) for 1 h at a flow rate of 1 μL/min. After perfusion, the flow rate was adjusted to 0.5 μL/min and 30 min fractions were collected in microtubes for a total of four and a half hours (9 samples per rat) in a refrigerated fraction collector (CMA Microdialysis, Acton, MA). This protocol has been used successfully in previous studies with stable neurotransmitter recovery in the dialysate CE-LIF analysis A protocol by RNA isolation and cDNA synthesis Total RNA was isolated from astrocyte monolayers and the striatum of control and Mn-exposed rats for quantitative PCR analysis. Tissue samples were stored in 1 mL of RNAlater ® solution (Ambion Inc., Austin, TX) and kept at −80 °C until analysis. Astrocytes were cultured in 6-well plates, then treated for 24 h with media containing 0, 100, or 300 μM Mn. Astrocytes were harvested in 500 μL denaturation solution (Ambion Inc., Austin, TX). Tissue and cell culture RNA isolation were performed using the ToTALLY RNA™ system (Ambion Inc., Austin, TX), following manufacturer's instructions. RNA concentration and purity were determined by spectrophotometric analysis before carrying out cDNA synthesis. Synthesis of cDNA was performed using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA), following manufacturer's instructions. Quantitative PCR Quantitative real-time PCR analysis was utilized to determine differential mRNA expression between control and Mn-treated tissue or cell samples of the solute carrier family taurine transporter Slc6a6 (Applied Biosystems, Foster City, CA; Rn00567962_m1, Chr. 4 -125875817-125945795). Triplicate aliquots of cDNA were analyzed on 96-well plates using TaqMan ® Gene Expression assays (Applied Biosystems, Foster City, CA). Values of cDNA expression were normalized relative to the expression of β-actin (Rn00667869_m1, Chr. 12 -12047070-12050040) analyzed from the same sample on the same plate and reported as percent of control. Metal analyses Mn, Fe, and copper (Cu) concentrations were measured with graphite furnace atomic absorption spectrometry (Varian AA240, Varian, Inc., USA). Brain tissue from the striatum was digested in ultra-pure nitric acid (1:10, w/v dilution) for 48-72 h in a sand bath (60 °C). A 50 μL aliquot of digested tissue was brought to 1 mL total volume with 2% nitric acid for analysis. The extracellular striatal samples obtained via microdialysis were not diluted due to the small volume (20 μL) and the likelihood that this biological compartment has a low concentration of metals. Bovine liver (NBS Standard Reference Material, USDC, Washington, DC) (10 μg Mn/g; 184 μg Fe/g; 80 μg Cu/g) was digested in ultra-pure nitric acid and used as an internal standard for analysis (final concentration 5 μg Mn/L; 92 μg Fe/L; 10 μg Cu/L). Statistical analysis Data were analyzed using SPSS v14 for Windows (Microsoft, Redmond, WA). Metal, baseline microdialysis, and 3 H-Taurine uptake data were analyzed using paired-samples t-tests to examine the difference between Mn-treated samples and controls. Independent sample t-tests were used to examine time-point percent change differences in the microdialysis data, time-point 3 H-Taurine uptake changes, and significance between Mn-exposed versus control mRNA expression of Scl6a6. A p-value of <0.05 was considered significant. RESULTS Manganese and iron concentrations Mn-exposure resulted in significant alterations in compartmental metal concentrations. As expected, tissue Mn levels were significantly higher in Mn-exposed rats versus control (p = 0.001) ( Extracellular concentrations of taurine, GABA, and glycine Extracellular amino acid concentrations are differentially altered by Mn-exposure. Baseline levels of taurine and glycine were more abundant than GABA in the extracellular space, though Mn does not have a statistically significant effect on their levels compared to control No significant difference in baseline taurine levels was found between control and Mn-exposed animals Gly EC levels were similar in control and Mn-exposed groups, and no significant percent changes were observed between time-points within either control or Mn groups Limits of detection of the CE-LIF method employed for each neurotransmitter were found by serial dilution of derivatized standards until no discernable analyte peak could be obtained. Accordingly, limits of detection for GABA, glycine, and taurine were 6.9 ± 1.7 nM, 24 ± 5 nM, and 42 ± 21 nM, respectively, with linear dynamic ranges of 3.6 decades, 3.1 decades, and 3.3 decades, respectively. H-Taurine uptake Mn-exposure results in increased 3 H-Taurine uptake in astrocytes. After observing the unique effects of Mn-exposure on Tau EC in the striatum of rats in vivo, we decided to examine the effect of Mn-exposure on 3 H-Taurine uptake in primary rat astrocytes in vitro. Primary astrocytes exposed to Mn revealed a slight (30%) decrease in taurine uptake after 1 min, followed by a significant (219%) increase after 3 min (p = 0.034) Fig. 2: 3 H-Taurine uptake in primary astrocytes. Primary astrocytes, seeded 2 × 10 −5 in 6-well plates (n = 6) then grown to confluence, were cultured with either Mn-treated (300 μM MnCl 2 ) or control media. After 24 h cultures were exposed to 3 H-Taurine for 1, 3, or 6 min and analyzed for 3 H-Taurine retention. The inset represents percent change in uptake due to Mn-exposure expressed as percent control ± SEM. A significant (p = 0.034) increase in 3 HTaurine uptake was observed after 3 min of exposure in the Mn-treated astrocytes versus control. *p < 0.05 via independent samples t-test between Mn and control treatment groups at each time-point. Gene expression of taurine transporter Mn-exposure increased taurine transporter gene expression in the rat brain, but not cultured astrocytes. Quantitative RT-PCR analysis was conducted on primary astrocytes and striatal brain tissue to determine whether or not taurine transporter (Slc6a6) gene expression reflected the observed Mn-induced alterations in Tau EC and 3 H-uptake. Chronic Mn-exposure caused a significant (p = 0.045) increase in striatal Slc6a6 mRNA levels compared to control DISCUSSION The purpose of this study was to examine the effect of Mn on GAT-mediated GABA uptake. Knowing that glycine and taurine are important amino acid neurotransmitters that are known to modulate GABA neurochemistry Fig. 4: Working model for Mn-induced GABA and taurine alterations. The dynamic shifts in neurotransmitter concentrations observed in response to nipecotic acid (NA) (panels A, B, and C) are mitigated by Mn (panels D, E, and F). We hypothesize this lack of response in Mn-exposed rats is driven by decreased GABA transporter (GAT-1) function. (A) The control panel displays GABA EC and Tau EC under normal conditions, representing baseline microdialysis measurements. All percent change (% change) comparisons in subsequent panels are based on the % change from baseline levels, represented in the control panel. Under normal conditions GABA EC binds to GABA A receptors (GABA A -R) allowing chloride ion (Cl 2− ) movement for inhibitory hyperpolarization of post-synaptic neurons, while pre-synaptic binding to GABA B receptors (GABA B -R) regulates GABA releas

Effects of Moderate Caloric Restriction on Cortical Microvascular Density and Local Cerebral Blood Flow in Aged Rats

The present study was designed to assess the impact of moderate caloric restriction (60% of ad libitum fed animals) on cerebral vascular density and local cerebral blood flow. Vascular density was assessed in male Brown–Norway rats from 7–35 months of age using a cranial window technique. Arteriolar density, arteriole-arteriole anastomoses, and venular density decreased with age and these effects were attenuated by moderate caloric restriction. Analysis of local cerebral blood using [14C] iodoantipyrine indicated that basal blood flow decreased with age in CA1, CA3 and dentate gyrus of hippocampus; similar trends were evident in cingulate, retrosplenal, and motor cortex. Basal blood flow was increased in all brain regions of moderate caloric restricted old animals (compared to old ad libitum fed animals) and no differences were observed between ad libitum fed young and caloric restricted older animals. In response to a CO2 challenge to maximally dilate vessels, blood flow increased in young and old ad libitum fed animals, but a similar increase was not observed in caloric restricted old animals. We conclude that a decrease in cerebral vasculature is an important contributing factor in the reduction in blood flow with age. Nevertheless, vessels from young and old animals have the capacity to dilate in response to a CO2 challenge and, after CO2, no differences are observed between the two age-groups. These results are consistent with the hypothesis that aged animals fail to adequately regulate local cerebral blood flow in response to physiological stimuli. Moderate caloric restriction increases microvascular density and cerebral blood flow in aged animals but tissues exhibit little or no increase in blood flow in response to CO2 challenge. The cause of this deficient response may indicate that vessels are maximally dilated in aged calorically restricted animals or that they fail to exhibit normal regulatory control

Alterations in Insulin-like Growth Factor-1 Gene and Protein Expression and Type 1 Insulin-like Growth Factor Receptors in the Brains of Ageing Rats

Ageing in mammals is characterized by a decline in plasma levels of insulin-like growth factor-1 that appears to contribute to both structural and functional changes in a number of tissues. Although insulin-like growth factor-1 has been shown to provide trophic support for neurons and administration of insulin-like growth factor-1 to ageing animals reverses some aspects of brain ageing, age-related changes in insulin-like growth factor-1 or type 1 insulin-like growth factor receptors in brain have not been well documented. In this series of studies, insulin-like growth factor-1 messenger RNA and protein concentrations, and type 1 insulin-like growth factor receptor levels were analysed in young (three to four- and 10–12-month-old), middle-aged (19–20-month-old) and old (29–32-month-old) Fisher 344× Brown Norway rats. Localization of insulin-like growth factor-1 messenger RNA throughout the lifespan revealed that expression was greatest in arteries, arterioles, and arteriolar anastomoses with greater than 80% of these vessels producing insulin-like growth factor-1 messenger RNA. High levels of expression were also noted in the meninges. No age-related changes were detected by either in situ hybridization or quantitative dot blot analysis of cortical tissue. However, analysis of insulin-like growth factor-1 protein levels in cortex analysed after saline perfusion indicated a 36.5% decrease between 11 and 32 months-of-age (P\u3c0.05). Similarly, analysis of type 1 insulin-like growth factor receptor messenger RNA revealed no changes with age but levels of type 1 insulin-like growth factor receptors indicated a substantial decrease with age (31% in hippocampus and 20.8 and 27.3% in cortical layers II/III and V/VI, respectively). Our results indicate that (i) vasculature and meninges are an important source of insulin-like growth factor-1 for the brain and that expression continues throughout life, (ii) there are no changes in insulin-like growth factor-1 gene expression with age but insulin-like growth factor-1 protein levels decrease suggesting that translational deficiencies or deficits in the transport of insulin-like growth factor-1 through the blood–brain barrier contribute to the decline in brain insulin-like growth factor-1 with age, and (iii) type 1 insulin-like growth factor receptor messenger RNA is unchanged with age but type 1 insulin-like growth factor receptors decrease in several brain regions. We conclude that significant perturbations occur in the insulin-like growth factor-1 axis with age. Since other studies suggest that i.c.v. administration of insulin-like growth factor-1 reverses functional and cognitive deficiencies with age, alterations within the insulin-like growth factor-1 axis may be an important contributing factor in brain ageing

Acute Exposure to High-Fat Diets Increases Hepatic Expression of Genes Related to Cell Repair and Remodeling in Female Rats

High-fat diets (HFD) promote the development of both obesity and fatty liver disease through the up-regulation of hepatic lipogenesis. Insulin resistance, a hallmark of both conditions, causes dysfunctional fuel partitioning and increases in lipogenesis. Recent work has demonstrated that systemic insulin resistance occurs in as little as the first 72 hours of an HFD, suggesting the potential for hepatic disruption with HFD at this time point. The current study sought to determine differences in expression of lipogenic genes between sexes in 3-month-old male and female Long-Evans rats after 72 hours of a 40% HFD or a 17% fat (chow) diet. Owing to the response of estrogen on hepatic signaling, we hypothesized that a sexual dimorphic response would occur in the expression of lipogenic enzymes, inflammatory cytokines, apoptotic, and cell repair and remodeling genes. Both sexes consumed more energy when fed an HFD compared with their low fat-fed controls. However, only the males fed the HFD had a significant increase in body fat. Regardless of sex, HFD caused down-regulation of lipogenic and inflammatory genes. Interestingly, females fed an HFD had up-regulated expression of apoptotic and cell repair-related genes compared with the males. This may suggest that females are more responsive to the acute hepatic injury effects caused by HFDs. In summary, neither male nor female rats displayed disrupted hepatic metabolic pathways after 72 hours of the HFD treatment. In addition, female rats appear to have protection from increases in fat deposition, possibly due to increased caloric expenditure; male rats fed an HFD were less active, as demonstrated by distance traveled in their home cage