A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus

RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15-bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P = 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV.     

Mechanisms of Prostate Permeability Triggered by Microbubble-Mediated Acoustic Cavitation

The objective of this research was to study the mechanisms of opening of the blood?Cprostate barrier and increased permeability of prostate tissue induced by microbubble cavitation. Thirty-five rabbits were randomly divided into four study groups: (1) control group and groups exposed to (2) microbubble alone, (3) ultrasound alone, or (4) combined intervention (ultrasound?+?microbubble group). Evans blue (EB) tracer was used to gauge the changes of permeability of prostate tissue. Furthermore, light and electron microscopy analyses were conducted, as well as the western blot analysis of expression of gap junction (Cx43) protein. We observed that EB concentration in prostate tissue was significantly greater in the ultrasound?+?microbubble group compared with either intervention alone (p?<?0.05, both comparisons). Furthermore, light microscopy of tissue samples from animals exposed to ultrasound?+?microbubble showed epithelial cell disarrangement, loss of interstitial structure, and thickness of fibrous stroma. In line with these findings, electron microscopy analysis demonstrated widening of cell gaps and broken cell connections, as well as more dense lysosomes and secretary granules, and mitochondrial swelling. These changes were absent in the animals exposed to microbubble or ultrasound alone. Finally, only combined treatment with microbubble or ultrasound significantly elevated expression of Cx43 (p?<?0.05 vs. control group). In conclusion, increases of permeability of prostate tissue by acoustic cavitation appear to involve opening of tight junctions, widening of intracellular spaces, changes in the structure of acinar cell membrane, enhancement of vesicular transport, and loosening of fibrous stroma. Increased expression of cell gap junction protein will help to restore normal connections between cells and the blood?Cprostate barrier after the treatment.     

Usefulness of Speckle Tracking Imaging to Assess Myocardial Contractility in Intra-Abdominal Hypertension: Study in a Mini-Pig Model

This study evaluated the usefulness of speckle tracking imaging (STI) in assessment of myocardial contractility in intra-abdominal hypertension experimentally induced in mini-pigs. To this effect, 12 mini-pigs were anesthetized with intravenous injection of 3?% sodium pentobarbital, hemorrhaged to reach the shock status, and resuscitated with excessive volume of lactated Ringer??s solution. The animals were either sham-operated (study group 1) or underwent treatment with intra-abdominal volume increment (study group 2). Observations were made prior to induction of shock, 1?h after shock, 2?h after induction of intra-abdominal hypertension, and 8 and 12?h after treatment. The heart rate and mean artery pressure were conventionally measured. STI was used to assess radial and circumferential strains of segmental ventricular wall. The results obtained demonstrated that myocardial contractility, as manifested by radial and circumferential strains of different ventricular wall segments, was decreased after induction of intra-abdominal hypertension. Treatment with intra-abdominal volume increment was able to decrease heart rate and intra-bladder pressure (indicator of effectiveness of treatment) and significantly improved myocardial contractility of involved ventricular wall segments. In conclusion, STI is a useful method to assess myocardial regional functions.     

Migratory Responses of Benthic Diatoms to Light and Temperature Monitored by Chlorophyll Fluorescence

As an important adaptation for survival in the sediments of intertidal flats, benthic diatoms move up and down in response to a wide range of environmental stimuli. We investigated the vertical migration of two diatoms—Cylindrotheca closterium (Agradh) Kützing (B-25) and Nitzschia sp. (B-3)—under different combinations of light intensity and temperature conditions. An imaging pulse amplitude modulated (PAM) fluorometer was used to measure the minimum fluorescence (F ₀) in order to monitor variations in diatom biomass in surface sediments. Rapid light curves (RLCs) were applied to assess their photosynthetic activities. Both species had increased motility under higher temperatures, with the longer valved C. closterium being twice as fast as the shorter valved Nitzschia sp. The former was also influenced by exposure to light intensities of 100 or 250 μmol m⁻² s⁻¹, whereas the latter was not. Consequently, no light/temperature interaction effect was associated with the vertical migration of Nitzschia sp., perhaps because of its lower photosynthetic capacity and smaller cell size. Therefore, we conclude that motile benthic diatoms exhibit species-specific responses to light and temperature due to differences in their photosynthetic capability and morphological characteristics.     

The Lipid-Bound Apolipoprotein A-I Cysteine Mutant (N74C) Inhibits the Activation of NF-κB,JNK and p38 in Endotoxemic Mice and RAW264.7 Cells

Our previous studies showed that recombinant high-density lipoprotein (rHDL) rHDL74 exhibited higher anti-inflammatory capabilities compared to wild-type rHDL (rHDLwt), while rHDL228 showed hyper-proinflammation. In this paper, we further investigated the potential mechanisms involved in their different inflammatory functions using two models: endotoxemic mice and the RAW264.7 inflammation model. Our results showed that 24 h after the injection of lipopolysaccharide (LPS), mice treated with rHDL74 had a significant decrease in plasma CRP (P<0.01 vs. rHDLwt; P<0.01 vs. LPS), MCP-1 (P<0.05 vs. rHDLwt; P<0.01 vs. LPS) and CD14 (P<0.01 vs. LPS) compared with the mice treated with rHDLwt or the controls that received LPS only. Similar to our previous study, rHDL228 increased the plasma level of CRP (P<0.05 vs. LPS) and MCP-1 (P<0.01 vs. LPS). Our immunohistochemistry and western blot analysis showed that rHDL74 inhibited the activation of NF-κB in endotoxemic mice and JNK and p38 in the RAW264.7 inflammation model, while rHDL228 exacerbated the activation of NF-κB and ERK. In summary, our data suggest that rHDL74 exhibits higher anti-inflammatory activity by decreasing inflammatory factors and inhibiting the activation of NF-κB, JNK and p38, while rHDL228 appears to be hyper-proinflammation by increasing these inflammatory factors and aggravating the activation of NF-κB and ERK.     

Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens

It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity.     

Upregulation of Protein Phosphatase 2A and NR3A-Pleiotropic Effect of Simvastatin on Ischemic Stroke Rats

Ca²⁺ influxes are regulated by the functional state of N-methyl-D-aspartate receptors (NMDARs). Dephosphorylation of NMDARs subunits decreases Ca²⁺ influxes. NR3, a novel subunit of NMDARs, also decreases Ca²⁺ influxes by forming new NMDARs with NR1 and NR2. It is meaningful to uncover whether protein phosphatase 2A (PP2A) and NR3A play a role in the protective effect of Simvastatin on ischemic stroke. In the present study, the Sprague-Dawley rats were pretreated with Simvastatin for 7 days before middle cerebral artery occlusion was performed to mimic ischemic stroke. The results showed that Simvastatin decreased brain ischemic infarct area significantly while increasing the expression levels of PP2A and NR3A, thus dephosphorylating the serine sites of NR1 (ser896 and ser897) along with increased enzymatic activities of PP2A. The protein levels of NR3A decreased as the enzymatic activities of PP2A were inhibited by okadaic acid. The results indicated that Simvastatin could protect the cerebrum from ischemic injury through a signaling mechanism involving elevated levels of PP2A and NR3A, and that PP2A might involve in the regulatory mechanism of NR3A expression.     

Transforming Growth Factor-β, Macrophage Colony-Stimulating Factor and C-Reactive Protein Levels Correlate with CD14highCD16+ Monocyte Induction and Activation in Trauma Patients

Severe injury remains a leading cause of death and morbidity in patients under 40, with the number of annual trauma-related deaths approaching 160,000 in the United States. Patients who survive the initial trauma and post-traumatic resuscitation are at risk for immune dysregulation, which contributes to late mortality and accounts for approximately 20% of deaths after traumatic injury. This post-traumatic immunosuppressed state has been attributed to over-expression of anti-inflammatory mediators in an effort to restore host homeostasis. We measured a panel of monocyte markers and cytokines in 50 severely injured trauma patients for 3 days following admission. We made the novel observation that the subpopulation of monocytes expressing high levels of CD14 and CD16 was expanded in the majority of patients. These cells also expressed CD163 consistent with differentiation into alternatively activated macrophages with potential regulatory or wound-healing activity. We examined factors in trauma plasma that may contribute to the generation and activation of these cells. The percentage of CD14^highCD16⁺ monocytes after trauma correlated strongly with plasma C-reactive protein (CRP) transforming growth factor-β (TGF-β), and macrophage colony-stimulating factor (M-CSF) levels. We demonstrate a role for TGF-β and M-CSF, but not CRP in generating these cells using monocytes from healthy volunteers incubated with plasma from trauma patients. CD16 is a receptor for CRP and IgG, and we showed that monocytes differentiated to the CD14^highCD16⁺ phenotype produced anti-inflammatory cytokines in response to acute phase concentrations of CRP. The role of these cells in immunosuppression following trauma is an area of ongoing investigation.