The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes

AbstractThe phenotypic changes that are induced by immune activation in CD4+ T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4+ T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4+ T lymphocytes

Micro-Vesicles of Moringa oleifera Seeds in Heterozygous Rats for DAT Gene: Effects of Oral Intake on Behavioral Profile and Hematological Parameters

Previous studies have shown multiple biological properties of Moringa oleifera, a plant native to Africa and Asia. In the present study, potential physiological properties of microvesicles extracted from Moringa oleifera seeds were assessed. For this purpose, we investigated behavioral profile and hematological parameters in a recent rat model characterized by dysregulation in dopamine transporter, a key regulator of dopaminergic system. Experimental design consisted of male Wistar-DAT rats aged between two and four months: wild-type (WT) (n = 5) and heterozygous (DATHET) (n = 4) control groups, which drank tap water; WT (n = 5) and DATHET (n = 6) groups which drank a solution of Moringa microvesicles and water (2: 68 mL per day), which was orally administered for two months. Rats were monitored for spontaneous locomotor activity on a 24/7 basis. In the early lit hours, treated DATHET subjects showed higher locomotor activity, proposing a sleep-delay effect of Moringa. In forced swimming test, WT subjects who took Moringa exhibited more depressive behavior. In DATHET rats, Moringa seemed to potentiate the struggle to find a way out, counteracting an initial panic. Hemoglobin and hematocrit underwent opposite changes in either genotype, supporting the opposite effects on behavioral phenotype observed. Future work is clearly needed to further explore these preliminary profiles

Plant microRNAs from Moringa oleifera Regulate Immune Response and HIV Infection

Traditional medicine is often chosen due to its affordability, its familiarity with patient's cultural practices, and its wider access to the local community. Plants play an important role in providing indispensable nutrients, while specific small RNAs can regulate human gene expression in a cross-kingdom manner. The aim of the study was to evaluate the effects of plant-enriched purified extract microRNAs from Moringa oleifera seeds (MO) on the immune response and on HIV infection. Bioinformatic analysis shows that plant microRNAs (p-miRs) from MO belonging to 18 conserved families, including p-miR160h, p-miR166, p-miR482b, p-miR159c, p-miR395d, p-miR2118a, p-miR393a, p-miR167f-3p, and p-miR858b are predicted to target with high affinity BCL2, IL2RA, TNF, and VAV1, all these being involved in the cell cycle, apoptosis, immune response and also in the regulation of HIV pathogenesis. The effects of MO p-miRs transfected into HIV+ PBMCs were analyzed and revealed a decrease in viability associated with an increase of apoptosis; an increase of T helper cells expressing Fas and a decrease of intracellular Bcl2 protein expression. Meanwhile no effects were detected in PBMCs from healthy donors. In CD4(+) T cells, transfection significantly reduced cell activation and modified the T cell differentiation, thereby decreasing both central and effector memory cells while increasing terminal effector memory cells. Interestingly, the p-miRs transfection induces a reduction of intracellular HIV p24 protein and a reduction of viral DNA integration. Finally, we evaluated the effect of synthetic (mimic) p-miR858b whose sequence is present in the MO p-miR pool and predicted to target VAV1, a protein involved in HIV-Nef binding. This protein plays a pivotal role in T cell antigen receptor (TCR) signaling, so triggering the activation of various pathways. The transfection of HIV+ PBMCs with the synthetic p-miR858b showed a reduced expression of VAV1 and HIV p24 proteins. Overall, our evidence defines putative mechanisms underlying a supplementary benefit of traditional medicine, alongside current antiretroviral therapy, in managing HIV infection in resource-limited settings where MO remains widely available

CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression

The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1

Micro-Vesicles of Moringa oleifera Seeds in Heterozygous Rats for DAT Gene: Effects of Oral Intake on Behavioral Profile and Hematological Parameters

Previous studies have shown multiple biological properties of Moringa oleifera, a plant native to Africa and Asia. In the present study, potential physiological properties of microvesicles extracted from Moringa oleifera seeds were assessed. For this purpose, we investigated behavioral profile and hematological parameters in a recent rat model characterized by dysregulation in dopamine transporter, a key regulator of dopaminergic system. Experimental design consisted of male Wistar-DAT rats aged between two and four months: wild-type (WT) (n = 5) and heterozygous (DATHET) (n = 4) control groups, which drank tap water; WT (n = 5) and DATHET (n = 6) groups which drank a solution of Moringa microvesicles and water (2: 68 mL per day), which was orally administered for two months. Rats were monitored for spontaneous locomotor activity on a 24/7 basis. In the early lit hours, treated DATHET subjects showed higher locomotor activity, proposing a sleep-delay effect of Moringa. In forced swimming test, WT subjects who took Moringa exhibited more depressive behavior. In DATHET rats, Moringa seemed to potentiate the struggle to find a way out, counteracting an initial panic. Hemoglobin and hematocrit underwent opposite changes in either genotype, supporting the opposite effects on behavioral phenotype observed. Future work is clearly needed to further explore these preliminary profiles

Exposure to Moringa oleifera microRNAs induces proteomic changes linked to tumorigenesis and epithelial-mesenchymal transition in HeLa cells

Cervical cancer (CC) is one of the most frequent cancers in women worldwide. The epithelial-mesenchymal transition (EMT) and the extracellular release of TGF-β are phenomena typically associated with different tumorigenic processes, including tumour cell proliferation and metastatization. Specific human microRNAs (miRNAs; miRs) involved in these tumorigenic processes have been identified, becoming important diagnostic and prognostic markers, and even potential therapeutic targets. In parallel, different studies have also shown that plant miRNAs can mediate a cross-kingdom regulation (CKR) of mammalian genes and modulate host's gene expression under pathological conditions, restoring the regulatory activity of endogenous miRNAs lost in cancer. In our previous studies, the miRNome from Moringa oleifera Lam. (henceforth moringa or mol) has been sequenced, showing the presence of several conserved miRNAs in the plant kingdom, whose ability to differentially regulate proliferation and apoptosis in healthy and cancer cells has been demonstrated. Furthermore, the effects of mol-miR treatment on tumorigenesis and EMT have been proved in liver tumour cells. According to these premises, we here investigated the proteomic profile of CC-derived HeLa cells exposed to a mol-miRNA pool, demonstrating the down-representation of specific factors involved in tumorigenesis. The treatment with plant miRs was able to modulate proteins involved in several biological processes linked to EMT. Furthermore, it reduced the expression of TGF-β and significantly inhibited cell motility, as observed following Scratch test and cell viability measurements, with a significant increase of apoptotic events. In conclusion, our results suggest and pave the way for the development of new potential therapeutic approaches based on CKR mediated by plant miRNAs for contrasting human cervical cancer, even in the form of adjuvants to classic treatments for limiting their side effects

Nutraceutical plant derived microrna elements for treatment of leukemia.

The present invention relates to the fields of molecular biology and medicine. The invention is based on the innovative concept of "cross-kingdom functional sequence homology" between plant and mammalian microRNAs (miR- NAs), where plant miRNAs explicit a regulation mechanism into the host mammalian cell, comparable to the endogenous one. More specifically, the invention relates to identification of specific plant miRNA from Olea europea, Moringa oleifera and other Mediterranean and medicinal plants which inhibits in vitro proliferation of human acute leukemia cells, and is fit for the nutraceutical compositions comprising the miRN

Exogenous miRNAs from Moringa oleifera Lam. recover a dysregulated lipid metabolism

A balanced diet is critical for human health, and edible plants play an important role in providing essential micronutrients as well as specific microRNAs (miRNAs) that can regulate human gene expression. Here we present the effects of Moringa oleifera (MO) miRNAs (mol-miRs) on lipid metabolism. Through in silico studies we identified the potential genes involved in lipid metabolism targeted by mol-miRs. To this end, we tested the efficacy of an aqueous extract of MO seeds (MOES), as suggested in traditional African ethnomedicine, or its purified miRNAs. The biological properties of MO preparations were investigated using a human derived hepatoma cell line (HepG2) as a model. MOES treatment decreased intracellular lipid accumulation and induced apoptosis in HepG2. In the same cell line, transfection with mol-miRs showed similar effects to MOES. Moreover, the effect of the mol-miR pool was investigated in a pre-obese mouse model, in which treatment with mol-miRs was able to prevent dysregulation of lipid metabolism.1

Modern flow visualization techniques

The subject of this bachelor thesis is an overview of the flow visualization methods applied in the wind tunnels and fly tests. An overview of flow visualization methods is presented in the first part of the work. The particular methods are presented and evaluated with respect of its complexity, requirements on the instrumentation and expanses. The pros and cons are mentioned for each method finally. The second part of the work deals with selection of the flow visualization method suitable for application in the wind tunnel of the Aeronautical department of the VUT Brno

Most highly conserved miRNAs among selected plants.

<p>Most highly conserved miRNAs among selected plants.</p