Influenza epidemics occur worldwide annually. The incidence of influenza shows a seasonal pattern in temperate areas, but little is known about influenza seasonality in tropical regions. The ...objective of this study was to determine the prevalence and the seasonal pattern of influenza infections in children living in the city of Fortaleza in northeastern Brazil. An indirect immunofluorescence assay was performed on nasopharyngeal aspirates collected from children attending in ambulatories, emergency rooms, and wards of the Hospital Infantil Albert Sabin with suspicion of acute respiratory infection during 7 consecutive years (2001-2007). Influenza viruses were detected in 6.3% (234/3,708) of specimens. Laboratory-based surveillance data showed a clear annual epidemic cycle of influenza, with a peak usually occurring in the rainy periods. In Fortaleza, flu infections occurred at a low level throughout the year but exhibit a marked seasonal increase during the rainy season.
Somatic cells can be reprogrammed into an embryonic stem cell-like pluripotent state by Oct-3/4, Sox2, c-Myc, and Klf4. Sox2 as an essential reprogramming factor also contributes to the development ...of the eye and the retina. This study was conducted to determine whether induced pluripotent stem (iPS) cells express retinal progenitor cell (RPC)-related genes and whether iPS cells can directly differentiate into retinal ganglion cells (RGCs).
Mouse iPS cells were induced by the ectopically expressed four factors in tail-tip fibroblasts (TTFs). The expression of RPC-related genes in iPS cells was analyzed by RT-PCR and immunofluorescence. iPS cells were induced to differentiate into RGCs by the addition of Dkk1 + Noggin (DN) + DAPT and overexpression of Math5. iPS-derived retinal ganglion (RG)-like cells were injected into the retina, and the eyes were analyzed by immunohistochemistry.
iPS cells inherently express RPC-related genes such as Pax6, Rx, Otx2, Lhx2, and Nestin. Overexpression of Math5 and addition of DN can directly differentiate iPS into retinal ganglion-like cells. These iPS-derived RG-like cells display long synapses and gene expression patterns, including Math5, Brn3b, Islet-1, and Thy1.2. Furthermore, inhibiting Hes1 by DAPT increases the expression of RGC marker genes. In addition, iPS-derived RG-like cells were able to survive but were unable to be integrated into the normal retina after transplantation.
The four factor iPS cell inherently expressed RPC-related genes, and the iPS cell could be further turned into RG-like cells by the regulation of transcription factor expression. These findings demonstrate that iPS cells are valuable for regeneration research into retinal degeneration diseases.
In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally ...triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.
Background: Lipid droplets have been implicated in HCV virion assembly.
Results: Inhibitors of the synthesis of triacylglycerol and cholesterol ester, the main components of lipid droplets, impair virion assembly.
Conclusion: Production of infectious HCV is linked integrally with the biosynthesis of the major components of lipid droplets.
Significance: Inhibitors of enzymes that generate acylglycerols and cholesterol esters have antiviral activity.
The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have ...neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58, CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.
Adipose-derived stem cells are capable of differentiating into multiple cell types and thus considered useful for regenerative medicine. However, this differentiation feature seems to be associated ...with tumor initiation and metastasis raising safety concerns, which requires further investigation. In this study, we isolated adipose-derived stem cells from subcutaneous as well as from visceral adipose tissues of the same donor and systematically compared their features. Although being characteristic of mesenchymal stem cells, subcutaneous adipose-derived stem cells tend to be spindle form-like and are more able to home to cancer cells, whereas visceral adipose-derived stem cells incline to be "epithelial"-like and more competent to differentiate. Moreover, compared to subcutaneous adipose-derived stem cells, visceral adipose-derived stem cells are more capable of promoting proliferation, inducing the epithelial-to-mesenchymal transition, enhancing migration and invasion of breast cancer cells by cell-cell contact and by secreting interleukins such as IL-6 and IL-8. Importantly, ASCs affect the low malignant breast cancer cells MCF-7 more than the highly metastatic MDA-MB-231 cells. Induction of the epithelial-to-mesenchymal transition is mediated by the activation of multiple pathways especially the PI3K/AKT signaling in breast cancer cells. BCL6, an important player in B-cell lymphoma and breast cancer progression, is crucial for this transition. Finally, this transition fuels malignant properties of breast cancer cells and render them resistant to ATP competitive Polo-like kinase 1 inhibitors BI 2535 and BI 6727.
Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown antifibrotic effects on several diseases. The aims of the present in vitro study were to investigate the antifibrotic effects of bromfenac (a ...kind of NSAID) on primary human pterygium fibroblasts (HPFs) and primary human conjunctival fibroblasts (HConFs), as well as to explore the possible mechanisms of these effects.
The cells used in this study were primary HPFs and HConFs, and profibrotic activation was induced by transforming growth factor-beta1 (TGF-β1). Western blot, quantitative real-time PCR, and immunofluorescence (IF) assays were used to detect the effects of TGF-β1 and bromfenac on the synthesis of fibronectin (FN), type III collagen (COL3), and alpha-smooth muscle actin (α-SMA) in HPFs and HConFs; the changes of signaling pathways were detected by Western blot; cell migration ability was detected by wound healing assay; cell proliferation ability was detected by CCK-8 assay; and pharmaceutical inhibitions of the downstream signaling pathways of TGF-β1 were used to assess their possible associations with the effects of bromfenac.
Bromfenac suppressed the TGF-β1-induced protein expression of FN (0.59 ± 0.07 folds, P = 0.008), COL3 (0.48 ± 0.08 folds, P = 0.001), and α-SMA (0.61 ± 0.03 folds, P = 0.008) in HPFs. Bromfenac also attenuated TGF-β1-induced cell migration (0.30 ± 0.07 folds, P < 0.001), cell proliferation (0.64 ± 0.03 folds, P = 0.002) and the expression levels of p-AKT (0.66 ± 0.08 folds, P = 0.032), p-ERK1/2 (0.69 ± 0.11 folds, P = 0.003), and p-GSK-3β-S9 (0.65 ± 0.10 folds, P = 0.002) in HPFs. PI3K/AKT inhibitor (wortmannin) and MEK/ERK inhibitor (U0126) reduced the TGF-β1-induced synthesis of FN, COL3, and α-SMA in HPFs. All the results were similar in HConFs.
Bromfenac protects against TGF-β1-induced synthesis of FN, α-SMA, and COL3 in HPFs and HConFs at least in part by inactivating the AKT and ERK pathways.
In the diagnostic work up of autoimmune gastritis several immunological methods are available for the detection of antibodies against Intrinsic Factor (IF) and Parietal Cells (PC). However, there are ...no recent reports directly comparing all the available assays and methods. The objective of this study was to compare the performance of several commercially available anti-IF and anti-PC antibody assays from different manufacturers in a multi-center multi-cohort setting.
Sera were used from 5 different cohorts consisting of samples from 25 healthy elderly, 20 HCV or HIV positive patients and 150 patients positive for anti-IF or anti-PC antibodies or in whom these antibodies were requested. These cohorts were tested for anti-IF antibodies with 6 different assays (IIF, ELISA, DIA and EliA) and for anti-PC antibodies with 7 different assays (IIF, ELISA, DIA and EliA). Performance was evaluated by calculating the concordance and relative sensitivity and specificity.
Good concordance was found between the assays for both antibody specificities, ranging from 81 to 100% and 91–100% for anti-IF and anti-PC antibodies, respectively. Highest relative sensitivity was found with the (automated) ELISA based methods. However, all assays had a relative sensitivity between 85 and 100% for anti-IF antibodies and between 95 and 100% for anti-PC antibodies. The relative specificity ranged between 76 and 100% for anti-IF antibodies and between 96 and 100% for anti-PC antibodies.
We conclude that most assays perform well and are concordant to each other, despite the methodological differences and the different sources of antigen used. However, the method used affects the sensitivity and specificity. The (automated) ELISA based assays have the highest relative sensitivity and relative specificity. Care should be taken in the interpretation of positive results by IIF and negative results by the Blue Diver when testing for anti-IF antibodies.
Adenosine 5'-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been ...examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.
Expression of growth hormone gene in the baboon eye Pérez-Ibave, Diana Cristina; Rodríguez-Sánchez, Irám Pablo; Garza-Rodríguez, María Lourdes ...
Experimental eye research,
April 2018, 2018-04-00, 20180401, Letnik:
169
Journal Article
Recenzirano
The human growth hormone (GH) locus is comprised by two GH (GH1 and GH2) genes and three chorionic somatomammotropin (CSH1, CSH2 and CSH-L) genes. While GH1 is expressed in the pituitary gland, the ...rest are expressed in the placenta. However, GH1 is also expressed in several extrapituitary tissues, including the eye. So to understand the role of this hormone in the eye we used the baboon (Papio hamadryas), that like humans has a multigenic GH locus; we set up to investigate the expression and regulation of GH locus in adult and fetal baboon ocular tissues.
We searched in baboon ocular tissues the expression of GH1, GH2, CSH1/2, Pit1 (pituitary transcription factor 1), GHR (growth hormone receptor), GHRH (growth hormone releasing hormone), GHRHR (growth hormone releasing hormone receptor), SST (somatostatin), SSTR1 (somatostatin receptor 1), SSTR2 (somatostatin receptor 2), SSTR3 (somatostatin receptor 3), SSTR4 (somatostatin receptor 4), and SSTR5 (somatostatin receptor 5) mRNA transcripts and derived proteins, by qPCR and immunofluorescence assays, respectively. The transcripts found were characterized by cDNA cloning and sequencing, having found only the one belonging to GH1 gene, mainly in the retina/choroid tissues. Through immunofluorescence assays the presence of GH1 and GHR proteins was confirmed in several retinal cell layers. Among the possible neuroendocrine regulators that may control local GH1 expression are GHRH and SST, since their mRNAs and proteins were found mainly in the retina/choroid tissues, as well as their corresponding receptors (GHRH and SSTR1-SSTR5). None of the ocular tissues express Pit1, so gene expression of GH1 in baboon eye could be independent of Pit1. We conclude that to understand the regulation of GH in the human eye, the baboon offers a very good experimental model.
•This is the first report of the presence of GH in the baboon eye.•The GH transcript isolated from the baboon eye confirms a new expression site for this hormone.•GH and GHR proteins were present not only in retinal ganglion cells (RGC) but also in the entire retina.•GH and its GHR were both seen localized in the cytoplasm as well as in the nucleus by immunohistochemistry.•GH may trigger both autocrine and paracrine specific action in different retinal cell lines in the baboon.
virus (MCV) is an infection caused by the
. Antiviral medications used to treat MCV infections have several problems, including drug-resistant and toxicity. As a result, improving safe, innovative, ...and effective antiviral drugs is critical. Therefore the current study aimed to investigate ZnO-NPs effects on
infection and
replication, among the main exciting viruses that menace human health. The antiviral activity of zinc oxide nanoparticles (ZnO-NPs) against MCV infection was investigated in this work. FESEM and TEM electron microscopy were used to examine the nanoparticles. The cytotoxicity of the nanoparticles was assessed using the MTT assay, and anti-influenza effects were detected using RT-PCR and TCID50. An indirect immunofluorescence experiment was used to investigate the inhibitory effect of nanoparticles on viral antigen expression. In all tests, acyclovir was employed as a control. Compared to virus control, post-exposure of MCV with ZnO nanoparticles at the highest dose but is not toxic (100 g/mL) resulted in 0.2, 0.9, 1.9, and 2.8 log10 TCID50 reductions in infectious diseases virus titer (P=0.0001). This ZnO-nanoparticles level was accompanied by an inhibition percentage (17.8%, 27.3%, 53.3%, 62.5 %, and 75.9%), respectively, measured based on viral load compared with the virus control. Compared to the positive control, fluorescence emission intensity in virally infected cells that administrated ZnO nanoparticles was statically decreased. Our findings demonstrated that ZnO-NPs have antiviral effects against the MCV. This property indicates that ZnO-NP has a high potential for usage in topical formulations to treat facial and labial lesions.
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