Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection

Objective

To evaluate minority variant drug resistance mutations detected by the oligonucleotide ligation assay (OLA) but not consensus sequencing among subjects with primary HIV-1 infection.

Design/Methods

Observational, longitudinal cohort study. Consensus sequencing and OLA were performed on the first available specimens from 99 subjects enrolled after 1996. Survival analyses, adjusted for HIV-1 RNA levels at the start of antiretroviral (ARV) therapy, evaluated the time to virologic suppression (HIV-1 RNA<50 copies/mL) among subjects with minority variants conferring intermediate or high-level resistance.

Results

Consensus sequencing and OLA detected resistance mutations in 5% and 27% of subjects, respectively, in specimens obtained a median of 30 days after infection. Median time to virologic suppression was 110 (IQR 62–147) days for 63 treated subjects without detectable mutations, 84 (IQR 56–109) days for ten subjects with minority variant mutations treated with ≥3 active ARVs, and 104 (IQR 60–162) days for nine subjects with minority variant mutations treated with <3 active ARVs (p = .9). Compared to subjects without mutations, time to virologic suppression was similar for subjects with minority variant mutations treated with ≥3 active ARVs (aHR 1.2, 95% CI 0.6–2.4, p = .6) and subjects with minority variant mutations treated with <3 active ARVs (aHR 1.0, 95% CI 0.4–2.4, p = .9). Two subjects with drug resistance and two subjects without detectable resistance experienced virologic failure.

Conclusions

Consensus sequencing significantly underestimated the prevalence of drug resistance mutations in ARV-naïve subjects with primary HIV-1 infection. Minority variants were not associated with impaired ARV response, possibly due to the small sample size. It is also possible that, with highly-potent ARVs, minority variant mutations may be relevant only at certain critical codons.     

Environmental influences on the spatial ecology of juvenile smalltooth sawfish (Pristis pectinata): results from acoustic monitoring

To aid recovery efforts of smalltooth sawfish (Pristis pectinata) populations in U.S. waters a research project was developed to assess how changes in environmental conditions within estuarine areas affected the presence, movements, and activity space of this endangered species. Forty juvenile P. pectinata were fitted with acoustic tags and monitored within the lower 27 km of the Caloosahatchee River estuary, Florida, between 2005 and 2007. Sawfish were monitored within the study site from 1 to 473 days, and the number of consecutive days present ranged from 1 to 125. Residency index values for individuals varied considerably, with annual means highest in 2005 (0.95) and lowest in 2007 (0.73) when several P. pectinata moved upriver beyond detection range during drier conditions. Mean daily activity space was 1.42 km of river distance. The distance between 30-minute centers of activity was typically <0.1 km, suggesting limited movement over short time scales. Salinity electivity analysis demonstrated an affinity for salinities between 18 and at least 24 psu, suggesting movements are likely made in part, to remain within this range. Thus, freshwater flow from Lake Okeechobee (and its effect on salinity) affects the location of individuals within the estuary, although it remains unclear whether or not these movements are threatening recovery.     

Stabilization of the human beta2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure

G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu122^3.41 in the well-studied β₂-adrenergic receptor (β₂AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu122^3.41 with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded β₂AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the β₂AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 °C. The β₁AR was also stabilized by the introduction of tryptophan at Glu147^3.41, and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro211^5.50 and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val206^5.45. Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4-TM3-TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.     

Phylogenetic and expression analysis of the basic helix-loop-helix transcription factor gene family: genomic approach to cellular differentiation

A phylogenetic analysis of seven different species (human, mouse, rat, worm, fly, yeast, and plant) utilizing all (541) basic helix-loop-helix (bHLH) genes identified, including expressed sequence tags (EST), was performed. A super-tree involving six clades and a structural categorization involving the entire coding sequence was established. A nomenclature was developed based on clade distribution to discuss the functional and ancestral relationships of all the genes. The position/location of specific genes on the phylogenetic tree in relation to known bHLH factors allows for predictions of the potential functions of uncharacterized bHLH factors, including EST's. A genomic analysis using microarrays for four different mouse cell types (i.e. Sertoli, Schwann, thymic, and muscle) was performed and considered all known bHLH family members on the microarray for comparison. Cell-specific groups of bHLH genes helped clarify those bHLH genes potentially involved in cell specific differentiation. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique aspects of the evolution and functional relationships of the different genes in the bHLH gene family.     

Expression of adiponectin receptor 1 in olfactory mucosa of mice

AdipoR1 and AdipoR2 are receptors for the adipocyte-derived hormone adiponectin, which is an important regulator of glucose and lipid metabolism, and which has also been implicated in the control of food intake and energy homeostasis. In the present study, we have demonstrated that AdipoR1 is expressed in mature sensory neurons of the olfactory mucosa of mice, in a pattern reminiscent of the olfactory marker protein. AdipoR1 expression levels in the olfactory mucosa have been observed to increase gradually during late embryogenesis until adulthood. No local expression of adiponectin has been detected in nasal tissues, indicating that serum adiponectin is the ligand for AdipoR1 in olfactory sensory neurons. As the serum adiponectin concentration is regulated depending on adipose tissue mass, with a reduction of adiponectin levels being seen in obesity, AdipoR1 function in the olfactory epithelium seems to be directly linked to the nutritional status of the body, suggesting a potential modulatory role for AdipoR1 in the adjustment of the olfactory system to energy balance requirements. This work was supported by the Forschungsfonds ZEM Tübingen/Hohenheim. Nicole Hass is recipient of a Peter und Traudl Engelhorn Stiftung scholarship.     

Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.     

Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.     

Effectiveness of sucrose analgesia in newborns undergoing painful medical procedures

Background

Sucrose is widely used to manage procedural pain in term newborns despite a lack of evidence of its effectiveness for different procedures and infant populations. Our objectives were to evaluate the effectiveness and safety of sucrose in newborns undergoing various medical procedures within 2 days of birth.

Methods

We performed a double-blind, randomized controlled trial. We included newborns (≥ 36 weeks gestation) of diabetic mothers and nondiabetic mothers. Each newborn received 2 mL of a 24%-sucrose or placebo solution before all procedures. We used the Premature Infant Pain Profile to assess pain during intramuscular injection of vitamin K, venipuncture for the newborn screening test and the first 3 heel lances for glucose monitoring (newborns of diabetic mothers only). Scores ranged from from 0 (no pain) to 18 (maximum pain).

Results

We included 240 newborns (120 from diabetic mothers, 120 from nondiabetic mothers). The overall mean pain score was lower among newborns who received sucrose than among those who received a placebo (mean difference –1.3, 95% confidence interval [CI] –2.0 to –0.6). We found that pain scores during intramuscular injection did not differ significantly between the sucrose and placebo groups for newborns of diabetic or nondiabetic mothers (newborns of nondiabetic mothers: mean difference –1.1, 95% CI –2.4 to 0.2; newborns of diabetic mothers: mean difference –1.0, 95% CI –2.4 to 0.4). During venipuncture, newborns who received sucrose had lower pain scores compared with those who received a placebo (newborns of nondiabetic mothers: mean difference –3.2, 95% CI –4.6 to –1.8; newborns of diabetic mothers: mean difference –2.4, 95% CI –3.8 to –1.0). Among newborns of diabetic mothers, there was no difference in pain during the first 3 heel lances or mean glucose levels between the sucrose and placebo groups (p = 0.94 and p = 0.29 respectively).

Interpretation

We found a modest reduction of pain in newborns of both diabetic and nondiabetic mothers when sucrose was used for all medical procedures performed in the first 2 days after birth. However, when each procedure was analyzed separately, we found that the effectiveness of sucrose was limited to venipuncture for the newborn screening test. (http://Clinicaltrials.gov trial register no. {"type":"clinical-trial","attrs":{"text":"NCT00213213","term_id":"NCT00213213"}}NCT00213213.)All newborns experience pain from medical procedures in the first days after birth. At the very least, newborns receive an intramuscular injection of vitamin K and venipuncture or heel-lance for newborn screening tests. Newborns of diabetic mothers, about 4% of newborns, undergo additional heel-lances for monitoring glucose levels.^1,2 Untreated pain from these procedures can have long-lasting effects.^3,4 We previously reported that newborns of diabetic mothers who received an average of 10 heel-lances within 2 days of birth exhibited a heightened pain response during subsequent venipunctures compared with newborns from nondiabetic mothers.²Studies aimed at reducing pain and its consequences in newborns are clinically important.^5–7 Sucrose (table sugar) has great potential for routine use.⁶ Sucrose is a naturally occurring sweetener with analgesic effects in newborns. Although not fully understood, the mechanism of action is thought to involve activation of the endogenous opioid system through taste.⁸ This is supported by the presence of opioid receptors on the tongue and animal studies showing that analgesia can be reversed by opioid antagonists during noxious stimulation.⁹ Moreover, chronic consumption of sweeteners has been shown to negate the analgesic effects of opioids.¹⁰In systematic reviews, single doses of sucrose have been reported to reduce pain in newborns undergoing commonly performed medical procedures, including heel-lancing and venipuncture.^11,12 Based on these data, national and international pain management guidelines promote the widespread use of sucrose.^13–16 However, sucrose has not been evaluated for all commonly performed procedures in healthy term newborns, and there are concerns that sucrose may raise blood glucose levels in newborns of diabetic mothers. It is necessary to demonstrate that sucrose is effective and safe for varied procedures, repeated use and diverse infant populations.We sought to evaluate the effectiveness and safety of sucrose in newborns of diabetic mothers and nondiabetic mothers undergoing routine painful medical procedures during the first 2 days after birth. Our hypothesis was that sucrose reduces pain during intramuscular injection of vitamin K, venipuncture for the newborn screening test and heel lance for blood glucose monitoring without serious adverse effects.