Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

Effectiveness of structured teaching programme on transmission and prevention of human immunodeficiency virus/acquired immune deficiency syndrome among adolescent girls in Lowry Memorial High School, Bengaluru.

BACKGROUND: Adolescence is a critical stage in human development. Most young people become sexually active during adolescence and are more likely to have multipartner and unprotected sex with high-risk behavior that predisposes them to sexually transmitted infections such as human immunodeficiency virus (HIV). OBJECTIVES: The objective of the study was to evaluate the effectiveness of a structured teaching programme on transmission and prevention of HIV/acquired immune deficiency syndrome (HIV/AIDS) among adolescent girls. METHODS: An evaluative research approach was adopted, in which a preexperimental, one group pre- and post-test research design was used to evaluate the effectiveness of the structured teaching programme on transmission and prevention of HIV/AIDS among adolescent girls studying at Lowry Memorial High School, Bengaluru. A self-administered structured questionnaire was used for data collection. Data were presented in frequency tables and statistical graphs (bar charts) and analyzed using descriptive statistics (mean, standard deviation) and inferential statistical methods (Chi-square and paired "t"-tests) using SPSS version 21. RESULTS: The findings of the study revealed that the mean percentage difference in the pre- and post-test knowledge scores was statistically significant at 5% level (P < 0.05). The overall mean post-test knowledge score of adolescent girls on transmission and prevention of HIV/AIDS was 88.83%. It is apparently higher compared to the pretest knowledge score, which was 67.67% with enhancement of 21.16%. This implies that the structured teaching programme was effective in gaining knowledge of adolescent girls regarding transmission and prevention of HIV/AIDS. CONCLUSION: Our study suggests that structured teaching programme enhanced the knowledge of the adolescent girls on transmission and prevention of HIV/AIDS. We, therefore, recommend that structured teaching programmes on transmission and prevention of HIV/AIDS should be encouraged among adolescents and youths to reduce the spread of HIV infection.

The Perception of People Living with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome toward Human Immunodeficiency Virus Infection: A Single-Center Experience.

BACKGROUND: The prevention and control of human immunodeficiency virus (HIV) infection depend on the prevention of new infections as well as treating currently infected individuals. Adequate knowledge of HIV infection among person living with HIV/acquired immunodeficiency syndrome (AIDS) (PLWHA) may be an important tool in reducing spread of the virus. OBJECTIVE: The objective of the study was to evaluate knowledge and attitude of PLWHA on HIV infection. METHODOLOGY: This was a cross-sectional study conducted at the Chukwuemeka Odumegwu Ojukwu Teaching Hospital. Knowledge of infection, spread, control, and effect was sought from HIV-positive respondents using a structured questionnaire. Information about their attitude and beliefs was also obtained. Collected data were analyzed using the Statistical Package for Social Sciences for Windows, Version 21.0. RESULTS: A total of 70 HIV-positive patients, including 23 (32.9%) males and 47 (67.1%) females with a mean age of 37.7 years were participated. The overall knowledge on HIV transmission, clinical effects, complications, and controls was good in 15.7%, average in 72.9%, and poor in 11.4%. Knowledge of means of transmission was appropriate in majority of them. Majority of 66 (94.3%) patients showed a positive attitude to life. CONCLUSION: Most of the HIV-positive patients had average knowledge on HIV, and majority had a positive attitude to life.

Intervención educativa sobre virus de inmunodeficiencia humana en la comunidad habitante de calle que asiste a un centro de acogida en la ciudad de Bogotá D.C. - Colombia / Educational intervention on Human Immunodeficiency Virus in the homeless community that attends a reception center in the city of Bogotá D.C. - Colombia

Resumen Objetivo. Evaluar el impacto de capacitaciones impartidas por estudiantes de la Universidad Colegio Mayor de Cundinamarca (UCMC) referentes al VIH en población habitante de calle asistente a un centro de acogida en la ciudad de Bogotá D.C. Materiales y método. Se realizaron encuestas en las que se definieron los factores socio-demográficos, los hábitos y los conocimientos sobre el VIH de la población. Se examinaron 47 individuos de los cuales el 92% eran hombres y el 12,78% de toda la población fue seropositiva para VIH, así mismo, de ésta población el 33,33% de los individuos no recordaban el número de parejas sexuales que habían tenido en el último año. Respecto a las charlas sobre VIH por parte de los estudiantes de la UCMC el 61,40% las calificaron como buenas. Resultados. Con base en el estudio se pudo evidenciar que en la población habitante de calle es necesaria una intervención educativa, dado que esto reduce el riesgo de infección por VIH y el desarrollo de la enfermedad, a la vez que se puede promover la reintegración social de esta comunidad.

Abstract HIV is a world problem, according to the World Health Organization 36.7 million people infected by this virus, of which the greatest risk occurs in people who are in a state of neglect, by example the inhabitant street. It is estimated in Bogotá D.C., the inhabitant street population near 9,614 characters. The objective was to evaluate the Impact of training given by students of the Universidad Colegio Mayor de Cundinamarca (UCMC) concerning HIV Population Inhabitants street attending the reception Centre in the City of Bogotá D.C. To collect information conducted surveys that defined the socio-demographic factors, Habits, and Knowledge About HIV population. 47 individuals of which 92% were men and 12.78% of the entire population was seropositive HIV, also, of this population they were examined 33.33% of individuals did not remember the number of sexual partners in the last year. Regarding the talks on HIV by students UCMC the 61.40% rated this as the good. Base with the study itself was evident that's inhabitant street population is required educational intervention, given a this Risk of HIV infection and disease development reduce the time you can promote reintegration this type of social community.

Effect of downregulation of FBI-1 on proliferation of human breast carcinoma cell line and its mechanism / 中国癌症杂志

Background and purpose: Factor that binds to the inducer of short transcripts of human immuno-deficiency virus-1 (FBI-1) in a variety of malignant tumors showed high expression levels, which may be closely related to tumor proliferation and differentiation, angiogenesis, metastasis, but its relationship with breast cancer has not been fully elucidated. The purpose of this study was to investigate the expression of FBI-1 in breast cancer cells, and to study the effect of FBI-1 gene expression on the proliferation of breast cancer cells and its possible mechanism. Methods:Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot analysis were applied to detect FBI-1 expression in normal human mammary epithelial cell line MCF-10A and breast cancer cell MCF-7. RNA interference method was used to down-regulate FBI-1 expression in MCF-7 cells. The cell proliferation was measured by CCK-8 kit and colony formation assay. RTFQ-PCR and Western blot were used to detect the expression of FBI-1 and NF-κBp65 in MCF-7 cells before and after the interference of FBI-1 expression. Results: The expression of FBI-1 was higher in breast cancer cells than that in normal human mammary epithelial cells (P<0.05). The effects of FBI-1 down-regulation inhibited proliferation in MCF-7 cells (P<0.05). At the same time, after inhibition of FBI-1, the NF-κBp65 mRNA and protein expression levels were significantly decreased (P<0.05). Conclusion: FBI-1 is highly expressed in breast cancer cells. Down-regulated FBI-1 expression can inhibit the proliferation of breast cancer cells,and its mechanism may be related to the inhibition of NF-κB signaling pathway.

Lung function abnormalities in HIV-infected adults and children.

Despite the advent of antiretroviral therapy (ART), the human immunodeficiency virus (HIV) epidemic remains a global health crisis with a high burden of respiratory disease among infected persons. While the early complications of the epidemic were dominated by opportunistic infections, improved survival has led to the emergence of non-infectious conditions that are associated with chronic respiratory symptoms and pulmonary disability. Obstructive ventilatory defects and reduced diffusing capacity are common findings in adults, and the association between HIV and chronic obstructive pulmonary disease is increasingly recognized. There is synergism between viral factors, opportunistic infections, conventional influences like tobacco smoke and biomass fuel exposure, and potentially, the immunological effects of ART on the development of HIV-associated chronic obstructive lung disease. Pulmonary function data for HIV-infected infants and children are scarce, but shows that bronchiectasis and obliterative bronchiolitis with severe airflow limitation are major problems, particularly in the developing world. However, studies from these regions are sorely lacking. There is thus a major unmet need to understand the influences of chronic HIV infection on the lung in both adults and children, and to devise strategies to manage and prevent these diseases in HIV-infected individuals. It is important for clinicians working with HIV-infected individuals to have an appreciation of their effects on measurements of lung function. This review therefore summarizes the lung function abnormalities described in HIV-positive adults and children, with an emphasis on obstructive lung disease, and examines potential pathogenic links between HIV and the development of chronic pulmonary disability.

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide.

BACKGROUND: Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process. METHODS: In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles. RESULTS: The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence. CONCLUSIONS: These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently. GENERAL SIGNIFICANCE: Understanding the mechanism behind Fl-CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.

Different Effects of Regulatory Genes (tat, nef) of Human Immunodeficiency Virus Type 1 (HIV-1) on the Proliferation and Differentiation of Mouse Embryonic Stem Cells in vitro.

To examine the effects of the tat and nef regulatory genes of human immunodeficiency virus (HIV-1) on cell differentiation we used the mouse embryonic stem cells (ESC) as a model. Proliferation, embryoid bodies (EB) formation and subsequent differentiation into cardiomyocytes, glial and neuronal cells were investigated in ESC lines transfected with these genes. It has been shown that the transfection of ESC by the tat gene increased their proliferating activity, whereas the nef gene transfected ESC showed its decrease. The number of embryoid bodies formed was higher in the cultures of ESC transfected by the nef and lower in the cells transfected by the tat in comparison with controls. The percentage of embryoid bodies with contracting cardiomyocytes was higher against control in the nef transfected cells and lower in ESC transfected with the tat. There were no reliable differences in the appearance of glial cells between control and the nef and tat transfected cell lines. Spontaneous differentiation of ESC into neuronal cells was almost not observed in the nef transfected cells, in contrast to control and the tat transfected cells. However, addition of retinoic acid (RA) to the nef transfected cells caused even a slight increase in neuron formation as compared to control ESC treated with RA. Thus, for the first time we have shown that the tat and nef regulatory genes of HIV-1 had a visible effect on proliferation of ESC and some first steps of their differentiation. In general, the reverse correlation between the effects of these two viral genes on ESC proliferation and differentiation were observed.