DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.     

The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Health Service Use and Quality of Life Recovery 12 Months Following Major Osteoporotic Fracture: Latent Class Analyses of the International Costs and Utilities Related to Osteoporotic Fractures Study (ICUROS)

Major osteoporotic fractures (MOFs) are associated with a rapid decline in health-related quality of life (HRQoL); however, there is limited knowledge about which healthcare services positively affect HRQoL postfracture. This study aimed to identify specific combinations of health service use associated with recovery of HRQoL 12 months post-MOF. The analyses included 4126 adults aged ≥50 years with an MOF (1657 hip, 1354 distal forearm, 681 vertebrae, 434 humerus) participating in the International Costs and Utilities Related to Osteoporotic fractures Study (ICUROS), a multinational observational study (Australia, Austria, Estonia, France, Italy, Lithuania, Mexico, Russia, Spain, United Kingdom, and United States). HRQoL at prefracture and 12 months postfracture was measured using the EuroQoL questionnaire (EQ-5D-3L). Health service use data were collected via participant interviews and medical record reviews including in-hospital care; outpatient care; community services; and medication use. Data analyses involved two stages: (i) latent class analyses to identify different combinations of health service use (“classes”); and (ii) logistic regression to assess effects of classes on HRQoL recovery. Analyses were repeated excluding hip fractures (non-hip MOFs). Overall, 2057 MOF participants (49.9%) recovered to their prefracture HRQoL at 12-month follow-up; this proportion was higher for non-hip MOFs (n = 1439; 58.3%). Several distinct classes were identified across countries (range, 2–5 classes). Classes that were associated with increased odds of HRQoL recovery were characterized by a combination of hospital presentations without admission; outpatient department visits; allied health visits; vitamin D/calcium supplementation; and/or non-opioid analgesic use. Similar classes were observed for non-hip MOFs. Understanding country-specific healthcare service pathways that influence greater recovery of HRQoL, particularly services that are uncommon in some countries and routine in others, could improve postfracture care on a global scale. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Two screening instruments for collecting alcohol-related information from expectant mothers and fathers: Testing the reliability of the Parent Alcohol Screening Questionnaire and the Social Support for an Alcohol-Free Pregnancy Questionnaire

The aim is to test the reliability of two alcohol screening instruments: (1) The Parent Alcohol Screening Questionnaire (PASQ5), and (2) the Social Support for an Alcohol-free Pregnancy (SSAFP) questionnaire. This is a cohort study from the south of Sweden using repeated surveys during pregnancy. To examine if responses differed according to different data collection methods, two cohorts consisting of 289 expectant mothers and 141 fathers completed the PASQ5 both verbally (weeks 6–7) and in writing (week 12) within regular antenatal visits. One of the cohorts (n = 137/64) also completed the SSAFP in week 12 and later in week 33. The third cohort, consisting of 179 and 133 expectant mothers and fathers, respectively, completed the PASQ5 and the SSAFP twice in late pregnancy (week 31 + 33). Eight of 10 items in the PASQ5 were stable for both expectant mothers and expectant fathers when comparing verbal versus written-delivered formats. Eight of 10 questions in the PASQ5 were stable when assessing the items in a test–retest analysis in late pregnancy for expectant mothers and nine of 10 questions were stable for fathers. The SSAFP items showed high internal consistency (0.86) for expectant mothers and excellent internal consistency (0.94) for expectant fathers. Most SSAFP items (17 of 21 for expectant mothers and 18 of 22 for expectant fathers) were also stable in a test–retest scenario in late pregnancy. Both the PASQ5 and SSAFP are reliable tools and may be helpful for clinicians who aim to have a deeper dialogue about alcohol consumption during pregnancy. These tools may also be helpful for researchers aiming to better understand a person's changes in alcohol intake and/or their social support network.     

Nerve-induced release of nitric oxide in the rabbit gastrointestinal tract as measured by in vivo microdialysis

1. Nitric oxide (NO) has been suggested as a gastrointestinal neurotransmitter, mediating the gastric receptive relaxation and the relaxation in the peristaltic reflex. The aim of the present study was to measure nerve-induced NO formation in vivo in the gastrointestinal tract.
2. Formation of the nitric oxide oxidation products nitrite and nitrate during vagal nerve stimulation were measured in the anaesthetized rabbit. Microdialysis probes were inserted into the wall of the stomach and proximal colon, and nitrite and nitrate in dialysate measured by capillary electrophoresis.
3. During bilateral vagal nerve stimulation there was an increase in nitrite and nitrate formation at the level of the stomach and in nitrite formation at the level of the colon. This increase was inhibited by intravenous administration of the NO synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME 30 mg kg⁻¹). Furthermore, L-NAME significantly increased nerve-induced gastric and colonic contractions, as well as spontaneous colonic contractions.
4. In summary, we present a new methodological procedure for quantification of small changes in nitric oxide formation in vivo. This study provides evidence that nitric oxide is released in the stomach and colonic wall during vagal nerve activity, at concentrations able to cause inhibition of smooth muscle contractions in vivo.

     

Cerebral uptake and metabolism of (11C) Vinpocetine in monkeys: PET studies]

Vinpocetine, a vinca alkaloid, is a therapeutic agent widely used in the treatment of acute and chronic stroke patients. To explore the uptake and distribution of vinpocetine in the primate brain, vinpocetine was labelled with 11C and positron emission tomography (PET) was used to measure the uptake and distribution of 11C-vinpocetine in the brain and the trunk of a cynomolgous monkey. HPLC was used to determine the concentration of vinpocetine and its labelled metabolites in blood and plasma. Following the radioligand's intravenous administration, after an initial peak, the total concentration of radioactivity in blood was relatively stable with time. The uptake of 11C-vinpocetine into the brain was rapid and about 5% of the total injected radioactivity was present in the brain two minutes after drug administration. These facts indicate that the compound passes the blood-brain barrier readily and enters the brain. The radioactivity uptake was heterogeneously distributed among brain regions. The highest concentrations were found in the thalamus, the basal ganglia and certain neocortical regions. In an earlier PET investigation on chronic stroke patients the highest increases in cerebral blood flow and glucose metabolism after intravenous vinpocetine treatment occurred in these anatomical structures. The heterogenous regional distribution of vinpocetine and the observation that the highest uptake values in brain structures go parallel with the greatest regional blood flow and glucose metabolic rate increases indicate that direct CNS effects of vinpocetine should be considered as an explanation for the therapeutic effects. The confirmation of this suggestion requires further investigations.     

Release of intracellular enzymes from cutaneous cells after non-necrotizing damage by ultraviolet light

Summary Experimental blisters were produced with suction on normal human skin and simultaneously on skin inflamed after exposure to middle wave ultraviolet light. Total proteins and marker enzymes for the plasma membrane, cytosol, lysosomes, peroxisomes, mitochondria, and microsomes were assayed in the blister fluid. In blisters on erythematous skin, a large increase of lactate dehydrogenase from cytosol was noted. A small increase of the plasma membrane marker phosphodiesterase I and some increase of -mannosidase from lysosomes was also found. No significant increase in total proteins or in the markers for peroxisomes was observed, whereas mitochondrial and microsomal marker enzymes were not detectable.It is concluded that cutaneous cells to some extent may lose intracellular enzymes without visible signs of irreversible damage (necrosis), but that an UVB-induced injury/regeneration cycle probably explains the enzyme release.     

Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer.

PURPOSE: The RNASEL gene has been proposed as a candidate gene for the HPC1 locus through a positional cloning and candidate gene approach. Cosegregation between the truncating mutation E265X and disease in a hereditary prostate cancer (HPC) family and association between prostate cancer risk and the common missense variant R462Q has been reported. To additionally evaluate the possible role of RNASEL in susceptibility to prostate cancer risk, we performed a comprehensive genetic analysis of sequence variants in RNASEL in the Swedish population. EXPERIMENTAL DESIGN: Using 1624 prostate cancer cases and 801 unaffected controls, the truncating mutation E265X and five common sequence variants, including the two missense mutations R462Q and D541E, were evaluated for association between genotypes/haplotypes and prostate cancer risk. RESULTS: The prevalence of E265X carriers among unaffected controls and prostate cancer patients was almost identical (1.9 and 1.8% in controls and cases, respectively), and evidence for segregation of E265X with disease was not observed within any HPC family. Overall, the analyses of common sequence variants provided limited evidence for association with prostate cancer risk. We found a marginally significant inverse association between the missense mutation D541E and sporadic prostate cancer risk (odds ratio, 0.77; 95% confidence interval, 0.59-1.00) and reduced risk of prostate cancer in carriers of two different haplotypes being completely discordant. CONCLUSIONS: Considering the high quality in genotyping and the size of this study, these results provide solid evidence against a major role of RNASEL in prostate cancer etiology in Sweden.