Lytic switch protein (ORF50) response element in the Kaposi’s sarcoma-associated herpesvirus K8 promoter is located within but does not require a palindromic structure

Kaposi's sarcoma-associated virus (KSHV) ORF50 protein induces lytic replication and activates the K8 promoter. We show that ORF50-induced and tetradecanoyl phorbol acetate (TPA) induced K8 transcripts initiated from the same start site. A newly identified palindrome (PAL2), containing a 12-bp response region required for ORF50-induced activation in lymphoid cells, was identified in the K8 promoter. Specific DNA binding of bacterially expressed ORF50 was not seen with the K8 promoter despite specific binding to the PAN promoter. The new palindrome shared homology with a previously described ORF50 response element (50RE(K8) and 50RE(57)). We demonstrate that the new 50RE(K8) (50RE(K8-PAL2)) is not the palindrome per se. Instead, the response element is buried within the right arm of the palindrome. We propose that the complexity of the K8 response elements reflects the complexity of mechanisms used by ORF50 during viral reactivation

DNA methylation determination by liquid chromatography–tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards

Methylation of the promoter CpG regions regulates gene transcription by inhibiting transcription factor binding. Deoxycytidine methylation may regulate cell differentiation, while aberrations in the process may be involved in cancer etiology and the development of birth defects (e.g. neural tube defects). Similarly, nutritional deficiency and certain nutragenomic interactions are associated with DNA hypomethylation. While LC-MS has been used previously to measure percentage genomic deoxycytidine methylation, a lack of a secure source of internal standards and the need for laborious and time-consuming DNA digestion protocols constitute distinct limitations. Here we report a simple and inexpensive protocol for the biosynthesis of internal standards from readily available precursors. Using these biosynthetic stable-isotopic [U-15N]-labeled internal standards, coupled with an improved DNA digestion protocol developed in our lab, we have developed a low-cost, high-throughput (>500 samples in 4 days) assay for measuring deoxycytidine methylation in genomic DNA. Inter- and intraassay variation for the assay (%RSD, n = 6) was <2.5%

High acceptance rate of anal pap screening despite limited knowledge about anal dysplasia among HIV+ MSM

Anal cancer in the general population is more prevalent in women, but in most HIV populations, MSM have the highest risk. Data suggest that screening can prevent invasive carcinoma. Use of routine cervical pap smears resulted in an 80% reduction in cervical cancer rates. The current study examines the effectiveness of a clinical intervention designed to increase anal dysplasia education, screening, and treatment for HIV+ MSM

Gene Expression from the ORF50/K8 Region of Kaposi's Sarcoma-Associated Herpesvirus

The ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 8 (KSHV), activates viral replication and is weakly homologous to the herpesvirus family of R transactivators; therefore, the transcription and translation events from this region of KSHV are key events in viral reactivation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of polypeptides: the major ones as 119 and 101 kDa, respectively. Using 3' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening, we demonstrate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcript was resistant to cyclohexamide, whereas the K8 transcript was only partially resistant to cyclohexamide at early timepoints. Both transcripts showed partial resistance after 12 h of phorbol ester induction. Using a GAL4-ORF50 fusion protein expression vector, we demonstrate that the transactivation domain of ORF50 resides within a 160-amino-acid region of the carboxyl portion of the ORF. Upstream regions of both ORF50 and K8 have basal promoter activity in KSHV-infected cells. K8, which had sequence homology to Bzip proteins, did not activate either promoter. However, both promoters were activated after cotransfection of ORF50 in BCBL-1 cells

Rhabdomyolysis: a genetic perspective

Rhabdomyolysis (RM) is a clinical emergency characterized by fulminant skeletal muscle damage and release of intracellular muscle components into the blood stream leading to myoglobinuria and, in severe cases, acute renal failure. Apart from trauma, a wide range of causes have been reported including drug abuse and infections. Underlying genetic disorders are also a cause of RM and can often pose a diagnostic challenge, considering their marked heterogeneity and comparative rarity.In this paper we review the range of rare genetic defects known to be associated with RM. Each gene has been reviewed for the following: clinical phenotype, typical triggers for RM and recommended diagnostic approach. The purpose of this review is to highlight the most important features associated with specific genetic defects in order to aid the diagnosis of patients presenting with hereditary causes of recurrent RM

Barriers to HIV Care for Women of Color Living in the Southeastern US Are Associated with Physical Symptoms, Social Environment, and Self-Determination

HIV-infected women of color (WOC) face particular barriers to accessing HIV medical care. To understand the impact of physical symptoms, social support, and self-determination on barriers to care, we interviewed HIV-infected women of color. HIV-infected WOC (N=141), attending an academic infectious disease clinic for HIV care in North Carolina, completed the Barriers to Care scale and were categorized as reporting a history of low (less than four of eleven barriers) or high (five or more) barriers to care. Binomial regression was used to estimate prevalence ratios and risk differences of reported barriers to care and its correlates such as depression, anxiety, illness-severity, psychological abuse, social support, treatment-specific social support, and self-determination (autonomy, relatedness, competency). A lower risk of reporting five or more barriers to care was associated with higher levels of autonomy (PR=0.93, 95% CI: 0.89, 0.96), relatedness (PR=0.92, 95% CI: 0.89, 0.94), competency (PR=0.93, 95% CI: 0.87, 0.98), and social support (PR=0.24, 95% CI: 0.81, 0.81). Depression, illness severity, and psychological abuse were associated with a greater risk of having five or more barriers to care. There are multiple social and psychological factors that contribute to perceived barriers to HIV care among WOC in the southeastern USA. Interventions that promote social support and increase individual self-determination have the potential to improve access to HIV care for WOC

Subclinical VZV reactivation in immunocompetent children hospitalized in the ICU associated with prolonged fever duration*

AbstractA prospective observational study was conducted to examine whether asymptomatic VZV reactivation occurs in immunocompetent children hospitalized in an ICU and its impact on clinical outcome. A secondary aim was to test the hypothesis that vaccinated children have a lower risk of reactivation than naturally infected children. Forty immunocompetent paediatric ICU patients and healthy controls were enrolled. Patients were prospectively followed for 28 days. Clinical data were collected and varicella exposure was recorded. Admission serum levels of TNF-a, cortisol and VZV-IgG were measured. Blood and saliva samples were collected for VZV-DNA detection via real-time PCR. As a comparison, the detection of HSV-DNA was also examined. Healthy children matched for age and varicella exposure type (infection or vaccination) were also included. VZV reactivation was observed in 17% (7/39) of children. Children with VZV reactivation had extended duration of fever (OR = 1.17; 95% CI, 1.02–1.34). None of the varicella-vaccinated children or healthy controls had detectable VZV-DNA in any blood or saliva samples examined. HSV-DNA was detected in saliva from 33% of ICU children and 2.6% of healthy controls. Among children with viral reactivation, typing revealed wild-type VZV and HSV-1. In conclusion, VZV reactivation occurs in immunocompetent children under severe stress and is associated with prolonged duration of fever

Substance Abuse, Violence, and HIV/AIDS (SAVA) Syndemic Effects on Viral Suppression Among HIV Positive Women of Color

The combined epidemics of substance abuse, violence, and HIV/AIDS, known as the SAVA syndemic, contribute to the disproportionate burden of disease among people of color in the US. To examine the association between HIV viral load suppression and SAVA syndemic variables, we used baseline data from 563 HIV+ women of color treated at nine HIV medical and ancillary care sites participating in HRSA's Special Project of National Significance Women of Color (WOC) Initiative. Just under half the women (n=260) were virally suppressed. Five psychosocial factors contributing to the SAVA syndemic were examined in this study: substance abuse, binge drinking, intimate partner violence, poor mental health, and sexual risk taking. Associations among the psychosocial factors were assessed and clustering confirmed. A SAVA score was created by summing the dichotomous (present/absent) psychosocial measures. Using generalized estimating equation (GEE) models to account for site-level clustering and individual-covariates, a higher SAVA score (0 to 5) was associated with reduced viral suppression; OR (adjusted)=0.81, 95% CI: 0.66, 0.99. The syndemic approach represents a viable framework for understanding viral suppression among HIV positive WOC, and suggests the need for comprehensive interventions that address the social/environmental contexts of patients' lives

Multisystem mitochondrial disease caused by a rare m.10038G>A mitochondrial tRNAGly (MT-TG) variant

Most pathogenic mitochondrial DNA (mtDNA) variants occur in the 22 mtDNA-encoded tRNA (mt-tRNA) genes. However, despite more than 270 reported mt-tRNA gene mutations, only 5 reside within mt-tRNAGly (MT-TG). We report a rare MT-TG variant and evaluate this, in addition to all previously reported MT-TG variants, against the published criteria used to help determine the pathogenicity of the mt-tRNA variants