BCL11B is a general transcriptional repressor of the HIV-1 long terminal repeat in T lymphocytes through recruitment of the NuRD complex

AbstractIn this study we provide evidence that the transcription factor BCL11B represses expression from the HIV-1 long terminal repeat (LTR) in T lymphocytes through direct association with the HIV-1 LTR. We also demonstrate that the NuRD corepressor complex mediates BCL11B transcriptional repression of the HIV-1 LTR. In addition, BCL11B and the NuRD complex repressed TAT-mediated transactivation of the HIV-1 LTR in T lymphocytes, pointing to a potential role in initiation of silencing. In support of all the above results, we demonstrate that BCL11B affects HIV-1 replication and virus production, most likely by blocking LTR transcriptional activity. BCL11B showed specific repression for the HIV-1 LTR sequences isolated from seven different HIV-1 subtypes, demonstrating that it is a general transcriptional repressor for all LTRs

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

BACKGROUND: The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. RESULTS: S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. CONCLUSION: This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle

Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background: We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (Kd) and gp120-to-CD4 inhibition constants (Ki). The PA analogs were selected to satisfy Lipinski’s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity. Principal Findings: PA analog 2-bromopalmitate (2-BP) was most efficacious with Kd,74 nM and Ki,122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher Kd,140 nM and Ki,354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with Kd,364 nM and inhibiting gp120-to-CD4 binding with Ki,1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry. Significance: Considering observed differences between K i and K d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry

Studying Early Lethality of 45,XO (Turner's Syndrome) Embryos Using Human Embryonic Stem Cells

Turner's syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos

HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study

<p>Abstract</p> <p>Background</p> <p>How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE).</p> <p>Methods</p> <p>A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-κB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.</p> <p>Results</p> <p>Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-κB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-κB were abrogated by inhibitor of ERK1/2 and NF-κB.</p> <p>Conclusion</p> <p>HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-κB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.</p

GUIDELINES FOR GENETIC COUNSELLING AND TESTING FOR HEREDITARY BREAST AND OVARIAN CANCER

Tijekom posljednjih desetljeća svjedoci smo velikog napretka u izvedivosti i kliničkoj iskoristivosti genetičkog testiranja kod nasljednih karcinoma. Nasljedni karcinomi dojke i jajnika najčešće su posljedica mutacija u genima BRCA1 i BRCA2. Ovim smjernicama obuhvatili smo kriterije za upućivanje pacijenata na genetičko savjetovanje i testiranje; kriterije za upućivanje zdravih pojedinaca na prediktivno testiranje ako nije moguće testiranje oboljelog člana obitelji; postupak genetičkog savjetovanja prije i nakon testiranja; nalaz testiranja, kategorije nalaza i razine rizika; preporuke za daljnje praćenje osoba s povišenim rizikom; kemoprevenciju i profilaktičku kirurgiju kod nositelja/-ica patogenih mutacija gena BRCA 1 i BRCA 2; očuvanje reproduktivne funkcije u žena oboljelih od raka dojke i nositeljica mutacija BRCA i pristanak informiranog bolesnika na genetičko testiranje. Smjernice su namijenjene svim specijalistima koji su na bilo koji način uključeni u zbrinjavanje oboljelih od nasljednih karcinoma dojke i jajnika, a sastavila ih je radna skupina prema podacima iz relevantne medicinske literature te kliničkim iskustvima članova radne skupine.The last few decades have witnessed a great progress in feasibility and clinical utilization of genetic testing for hereditary cancers. Hereditary breast and ovarian cancers are most often the result of BRCA1 and BRCA2 gene mutations. In these guidelines we have covered: the criteria for referral of patients to genetic counselling and testing; the criteria for referral of healthy family members to predictive testing in the event when there is no possibility of testing the patient; the process of genetic counselling before and after testing; test results, their categories and risk levels; recommendations for monitoring of individuals with an increased risk; chemoprevention and prophylactic surgery for carriers of BRCA1 and BRCA2 gene mutations; preservation of reproductive function in women with breast cancer and in carriers of BRCA mutations; and informed consent for genetic testing. The guidelines are intended for all specialists who are in any way involved in the care of patients with hereditary breast and ovarian cancer, and are compiled by the working group according to the data from the relevant medical literature and from clinical experience of the members of the working group

Smjernice za genetičko savjetovanje i testiranje na nasljedni rak dojke i jajnika [Guidelines for genetic counselling and testing for hereditary breast and ovarian cancer]

The last few decades have witnessed a great progress in feasibility and clinical utilization of genetic testing for hereditary cancers. Hereditary breast and ovarian cancers are most often the result of BRCA1 and BRCA2 gene mutations. In these guidelines we have covered: the criteria for referral of patients to genetic counselling and testing; the criteria for referral of healthy family members to predictive testing in the event when there is no possibility of testing the patient; the process of genetic counselling before and after testing; test results, their categories and risk levels; recommendations for monitoring of individuals with an increased risk; chemoprevention and prophylactic surgery for carriers of BRCA1 and BRCA2 gene mutations; preservation of reproductive function in women with breast cancer and in carriers of BRCA mutations; and informed consent for genetic testing. The guidelines are intended for all specialists who are in any way involved in the care of patients with hereditary breast and ovarian cancer, and are compiled by the working group according to the data from the relevant medical literature and from clinical experience of the members of the working group

GUIDELINES FOR GENETIC COUNSELLING AND TESTING FOR HEREDITARY BREAST AND OVARIAN CANCER

Tijekom posljednjih desetljeća svjedoci smo velikog napretka u izvedivosti i kliničkoj iskoristivosti genetičkog testiranja kod nasljednih karcinoma. Nasljedni karcinomi dojke i jajnika najčešće su posljedica mutacija u genima BRCA1 i BRCA2. Ovim smjernicama obuhvatili smo kriterije za upućivanje pacijenata na genetičko savjetovanje i testiranje; kriterije za upućivanje zdravih pojedinaca na prediktivno testiranje ako nije moguće testiranje oboljelog člana obitelji; postupak genetičkog savjetovanja prije i nakon testiranja; nalaz testiranja, kategorije nalaza i razine rizika; preporuke za daljnje praćenje osoba s povišenim rizikom; kemoprevenciju i profilaktičku kirurgiju kod nositelja/-ica patogenih mutacija gena BRCA 1 i BRCA 2; očuvanje reproduktivne funkcije u žena oboljelih od raka dojke i nositeljica mutacija BRCA i pristanak informiranog bolesnika na genetičko testiranje. Smjernice su namijenjene svim specijalistima koji su na bilo koji način uključeni u zbrinjavanje oboljelih od nasljednih karcinoma dojke i jajnika, a sastavila ih je radna skupina prema podacima iz relevantne medicinske literature te kliničkim iskustvima članova radne skupine.The last few decades have witnessed a great progress in feasibility and clinical utilization of genetic testing for hereditary cancers. Hereditary breast and ovarian cancers are most often the result of BRCA1 and BRCA2 gene mutations. In these guidelines we have covered: the criteria for referral of patients to genetic counselling and testing; the criteria for referral of healthy family members to predictive testing in the event when there is no possibility of testing the patient; the process of genetic counselling before and after testing; test results, their categories and risk levels; recommendations for monitoring of individuals with an increased risk; chemoprevention and prophylactic surgery for carriers of BRCA1 and BRCA2 gene mutations; preservation of reproductive function in women with breast cancer and in carriers of BRCA mutations; and informed consent for genetic testing. The guidelines are intended for all specialists who are in any way involved in the care of patients with hereditary breast and ovarian cancer, and are compiled by the working group according to the data from the relevant medical literature and from clinical experience of the members of the working group

Characterization of lamin Mutation Phenotypes in Drosophila and Comparison to Human Laminopathies

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution