Genetic polymorphisms and resistance mutations of HIV type 2 in antiretroviral-naive patients in Burkina Faso

Natural polymorphisms in the pol gene of HIV-2 may influence the susceptibility to antiretroviral drugs and the choice of treatment. We collected samples in centers for anonymous HIV testing in Ouagadougou, Burkina Faso, in patients supposedly naive for any antiretroviral treatment. Eighty-four samples were first tested as HIV-2 positive in Burkina Faso and then shipped to Brussels, Belgium, for confirmation of the serological status and plasma viral load. Fifty-two samples were confirmed as HIV-2 positive in Belgium. Twelve others were HIV-1 positive and 20 were dually reactive. Twenty-one of HIV-2 confirmed samples had an HIV-2 plasma viral load higher than 1000 copies/ml. These viruses were sequenced in the protease and reverse trancriptase genes and 17 sequences of the pol gene were obtained. Highly polymorphic positions were identified in protease and RT genes. Two samples harbored known resistance mutations: M184V RT mutation in one and Q151M with M184V in the other. Phylogenetic analysis showed that viruses in Burkina Faso did not cluster separately from published sequences from neighboring countries. The two resistant strains were unrelated. Our findings imply either that resistant viruses are circulating in Burkina Faso or that some individuals take unsupervised treatment. Both hypotheses present problems.     

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.Eukaryotic DNA replication initiates from multiple replication origins within each chromosome to duplicate the large genome efficiently. To ensure DNA synthesis occurs once and only once across the genome, cells adopt a two-step process to activate replication origins during two separate stages of the cell-division cycle. The first step is licensing of replication origins, which occurs only when cyclin-dependent kinase (CDK) activity is low. In Saccharomyces cerevisiae, origins of DNA replication are licensed in G1 by the formation of a prereplicative complex (pre-RC). The process begins with the origin recognition complex binding to replication origins and recruiting the licensing factor Cdc6, which facilitates loading of the Cdt1-bound minichromosome maintenance (MCM) complex composed of Mcm2–Mcm7 (Mcm2–7). The hexameric Mcm2–7 is the core of the replicative helicase that unwinds DNA during replication. Within the pre-RC Mcm2–7 is loaded as an inactive double hexamer. The next step, activation of licensed origins (origin firing), occurs throughout the S phase and requires the continuous presence of two kinases, the S phase CDKs and the Dbf4-dependent Cdc7 kinase (DDK). CDK phosphorylates Sld2 and Sld3 to allow their binding to Dpb11 (1, 2), facilitating recruitment of Cdc45 and GINS (composed of protein subunits Sld5, Psf1, Psf2 and Psf3; Go, Ichi, Nii, and San stand for five, one, two, and three in Japanese, respectively) to Mcm2–7 to create an active helicase. DDK phosphorylates Mcm2–7 and blocks an intrinsic initiation inhibitory activity residing in the N terminus of the Mcm4 subunit (3). The concerted action of these S-phase kinases transforms the inactive Mcm2–7 double hexamer into the active helicase complex composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (4–6). Upon initiation, DNA polymerases and other components of the replication machinery are recruited to form replisomes and establish replication forks, where DNA synthesis ensues.Kinase-signaling pathways target various components of the replication machinery. Both CDK and DDK target replication proteins in addition to their essential targets described above. Furthermore, Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related (ATM/ATR) signaling targets components of the CMG helicase complex under replication stress (7–10). In the yeast S. cerevisiae, DNA damage activates the checkpoint kinase Rad53, which phosphorylates both Sld3 and Dbf4 to inhibit late origin firing (11, 12). The yeast ATM/ATR homolog Mec1 also targets Mcm4 (13). The stress-activated protein kinase Hog1 targets an auxiliary replisome component Mrc1 to regulate both origin firing and fork progression (14). Although we now have a better understanding of the essential functions of protein kinases in controlling the initiation of replication, we do not completely understand how the separate kinase signaling pathways are coordinated to regulate both initiation and replication fork progression.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 is a target of multiple kinases, including DDK, CDK, and Mec1 (3, 13, 15, 16). Within this region we have identified two functionally distinct domains that exert different functions and are regulated by different kinase systems even though they overlap extensively in primary amino acid sequences. The segment of the Mcm4 NSD proximal to the DDK-docking domain (DDD) (15), and hence termed “proximal NSD,” blocks initiation until it is phosphorylated by DDK. In contrast, the distal segment of the NSD at the N terminus, away from the DDD, is targeted by additional kinases and contributes positively to promote S-phase progression. In this study we present a comprehensive analysis of the pattern of origin activation, replication fork progression, and the checkpoint response in cells under replication stress caused by the inhibition of ribonucleotide reductase (RNR). We show that the distal and proximal NSD segments contribute differently to origin activation and DNA replication fork progression. Furthermore, they exert opposing effects on checkpoint signaling under replication stress. All these effects are regulated by phosphorylation. We suggest that the Mcm4 NSD, a regulatory domain intrinsic to the replicative helicase, mediates the control of multiple aspects of DNA replication. Our data reveal a sophisticated mechanism to fine-tune S-phase progression in response to changing environments.     

The Beagle Dog as an Animal Model for a Bioavailability Study of Controlled-Release Theophylline Under the Influence of Food

Beagle dogs were evaluated as an animal model to study the effect of food on the bioavailability of two commercially available oral controlled-release theophylline products. The products were administered with and without food in single doses, and the bioavailability parameters were compared with those following an i.v. aminophylline dose. The total plasma theophylline clearance in dogs following an i.v. dose was 0.128 liter/hr/kg and the volume of distribution was 0.8 liter/kg using a one-compartment model. The absolute bioavailabilities of these two products under fasting conditions were 31 and 48%, respectively. The food increased the bioavailability of one product and decreased the bioavailability of the other. The overall trends in relative bioavailability of these two products with and without food appeared to be similar to those reported in humans.     

Multicenter Study of 19 Aortopulmonary Window Parathyroid Tumors: The Challenge of Embryologic Origin

Background  

Ectopic abnormal parathyroid glands are relatively common in the superior mediastinum but are rarely situated in the aortopulmonary window (APW). The embryological origin of these abnormal parathyroid glands is controversial. The purpose of this investigation was to investigate the embryological origin and the surgical management of abnormal parathyroid glands situated in the APW.     

Relation between mean arterial pressure and renal function in the early phase of shock: a prospective, explorative cohort study

Introduction

Because of disturbed renal autoregulation, patients experiencing hypotension-induced renal insult might need higher levels of mean arterial pressure (MAP) than the 65 mmHg recommended level in order to avoid the progression of acute kidney insufficiency (AKI).

Methods

In 217 patients with sustained hypotension, enrolled and followed prospectively, we compared the evolution of the mean arterial pressure (MAP) during the first 24 hours between patients who will show AKI 72 hours after inclusion (AKI_h72) and patients who will not. AKI_h72 was defined as the need of renal replacement therapy or "Injury" or "Failure" classes of the 5-stage RIFLE classification (Risk, Injury, Failure, Loss of kidney function, End-stage renal disease) for acute kidney insufficiency using the creatinine and urine output criteria. This comparison was performed in four different subgroups of patients according to the presence or not of AKI at the sixth hour after inclusion (AKI_h6 as defined as a serum creatinine level above 1.5 times baseline value within the first six hours) and the presence or not of septic shock at inclusion.The ability of MAP averaged over H6 to H24 to predict AKI_h72 was assessed by the area under the receiver operating characteristic curve (AUC) and compared between groups.

Results

The MAP averaged over H6 to H24 or over H12 to H24 was significantly lower in patients who showed AKI_h72 than in those who did not, only in septic shock patients with AKI_h6, whereas no link was found between MAP and AKI_h72 in the three others subgroups of patients. In patients with septic shock plus AKI_h6, MAP averaged over H6 to H24 or over H12 to H24 had an AUC of 0.83 (0.72 to 0.92) or 0.84 (0.72 to 0.92), respectively, to predict AKI_h72 . In these patients, the best level of MAP to prevent AKI_h72 was between 72 and 82 mmHg.

Conclusions

MAP about 72 to 82 mmHg could be necessary to avoid acute kidney insufficiency in patients with septic shock and initial renal function impairment.     

Thermosensitive and Mucoadhesive Pluronic-Hydroxypropylmethylcellulose Hydrogel Containing the Mini-CD4 M48U1 Is a Promising Efficient Barrier against HIV Diffusion through Macaque Cervicovaginal Mucus

To be efficient, vaginal microbicide hydrogels should form a barrier against viral infections and prevent virus spreading through mucus. Multiple particle tracking was used to quantify the mobility of 170-nm fluorescently labeled COOH-modified polystyrene particles (COOH-PS) into thermosensitive hydrogels composed of amphiphilic triblock copolymers with block compositions EO_n-PO_m-EO_n (where EO refers to ethylene oxide and PO to propylene oxide) containing mucoadhesive hydroxypropylmethylcellulose (HPMC). COOH-PS were used to mimic the size and the surface charge of HIV-1. Analysis of COOH-PS trajectories showed that particle mobility was decreased by Pluronic hydrogels in comparison with cynomolgus macaque cervicovaginal mucus and hydroxyethylcellulose hydrogel (HEC; 1.5% by weight [wt%]) used as negative controls. Formulation of the peptide mini-CD4 M48U1 used as an anti-HIV-1 molecule into a mixture of Pluronic F127 (20 wt%) and HPMC (1 wt%) did not affect its anti-HIV-1 activity in comparison with HEC hydrogel. The 50% inhibitory concentration (IC₅₀) was 0.53 μg/ml (0.17 μM) for M48U1-HEC and 0.58 μg/ml (0.19 μM) for M48U1-F127-HPMC. The present work suggests that hydrogels composed of F127-HPMC (20/1 wt%, respectively) can be used to create an efficient barrier against particle diffusion in comparison to conventional HEC hydrogels.     

Six-month prognosis of patients with lung cancer admitted to the intensive care unit

Background

Intensive care unit (ICU) admission of patients with lung cancer remains debated because of the poor short-term prognosis. However, ICU admission of such patients should also be assessed on the possibility to administer specific anticancer treatment and the long-term outcome thereafter.

Objectives

To identify predictive factors of hospital and 6-month mortality in critically ill lung-cancer patients.

Design and setting

Retrospective study conducted in the ICU of a university hospital.

Patients

One hundred five consecutive lung-cancer patients included between 1 January 1997 and 31 December 2006.

Interventions

None.

Results

Of the 105 patients (mean age 64.8 years), 87 (83%) had a non-small cell lung cancer (NSCLC). Extensive disease was diagnosed in 85 patients (83%) (NSCLC stages IIIB and IV or disseminated small cell lung cancer). The main reasons for ICU admission were acute respiratory failure (59%) and/or hemoptysis (45%). Forty-three patients (41%) needed mechanical ventilation (MV). The ICU, hospital and 6-month mortality rates were 43, 54 and 73%, respectively. A performance status (PS) ≥2 [odds ratio OR = 3.6 (95% confidence interval CI (1.5–8.7)] and acute respiratory failure [OR = 3.5 (95% CI (1.5–8.4)] predicted hospital mortality. In a multivariate Cox model, the cancer progression [hazard ratio HR = 6.1 (95% CI 2.2–17)] and the need for MV [HR = 3.6 (95% CI 1.35–9.4)] were independently associated with 6-month mortality. Two-thirds of the ICU survivors were able to receive anticancer treatment.

Conclusions

ICU admission should be considered in selected patients with lung cancer (PS <2, no cancer disease progression).