Short Communication: Analysis of Minor Populations of Human Immunodeficiency Virus by Primer Identification and Insertion-Deletion and Carry Forward Correction Pipelines

Accurate analysis of minor populations of drug-resistant HIV requires analysis of a sufficient number of viral templates. We assessed the effect of experimental conditions on the analysis of HIV pol 454 pyrosequences generated from plasma using (1) the ‘‘Insertion-deletion (indel) and Carry Forward Correction’’ (ICC) pipeline, which clusters sequence reads using a nonsubstitution approach and can correct for indels and carry forward errors, and (2) the ‘‘Primer Identification (ID)’’ method, which facilitates construction of a consensus sequence to correct for sequencing errors and allelic skewing. The Primer ID and ICC methods produced similar estimates of viral diversity, but differed in the number of sequence variants generated. Sequence preparation for ICC was comparably simple, but was limited by an inability to assess the number of templates analyzed and allelic skewing. The more costly Primer ID method corrected for allelic skewing and provided the number of viral templates analyzed, which revealed that amplifiable HIV templates varied across specimens and did not correlate with clinical viral load. This latter observation highlights the value of the Primer ID method, which by determining the number of templates amplified, enables more accurate assessment of minority species in the virus population, which may be relevant to prescribing effective antiretroviral therapy

Resistance Mutations to Zidovudine and Saquinavir in Patients Receiving Zidovudine plus Saquinavir or Zidovudine and Zalcitabine plus Saquinavir in AIDS Clinical Trials Group 229

The relationships among treatment regimens, plasma human immunodeficiency virus (HIV) RNA levels, and resistance mutations to saquinavir (codons 48 and 90) and zidovudine (codon 215) were examined in a cohort of 144 patients from the AIDS Clinical Trials Group 229 study. After 24-40 weeks of therapy, no patients who had received the two-drug combination (zidovudine plus saquinavir) had only codon 48 mutations, 45.8% had only codon 90 mutations, and 8.3% had both codon 48 and 90 mutations. Mutations developed by patients who had received the three-drug combination (zidovudine and zalcitabine plus saquinavir) were codon 48 alone in 1.4%, codon 90 alone in 33.3%, and both codons 48 and 90 in 4.2%. The difference between the groups showed a trend toward reduced mutations with three versus two drugs but did not reach significance (p = .11, two-sided χ2). Higher baseline HIV RNA levels correlated with the development of protease mutations. Mutations at codon 215 were present in 82% of all patients at baseline and in 87% after therap

Effects of Early Postnatal Exposure to Ethanol on Retinal Ganglion Cell Morphology and Numbers of Neurons in the Dorsolateral Geniculate in Mice

Background: The adverse effects of fetal and early postnatal ethanol intoxication on peripheral organs and the central nervous system are well documented. Ocular defects have also been reported in about 90% of children with fetal alcohol syndrome, including microphthalmia, loss of neurons in the retinal ganglion cell (RGC) layer, optic nerve hypoplasia, and dysmyelination. However, little is known about perinatal ethanol effects on retinal cell morphology. Examination of the potential toxic effects of alcohol on the neuron architecture is important because the changes in dendritic geometry and synapse distribution directly affect the organization and functions of neural circuits. Thus, in the present study, estimations of the numbers of neurons in the ganglion cell layer and dorsolateral geniculate nucleus (dLGN), and a detailed analysis of RGC morphology were carried out in transgenic mice exposed to ethanol during the early postnatal period

Cervical HIV-1 RNA shedding after cryotherapy among HIV-positive women with cervical intraepithelial neoplasia stage 2 or 3

Objective: To determine the effect of cryotherapy on HIV-1 cervical shedding. Design: Prospective cohort study. Methods: Five hundred HIV-positive women enrolled at an HIV treatment clinic in Nairobi, Kenya were screened for cervical cancer. Women diagnosed with cervical intraepithelial neoplasia stage 2 or 3 (CIN 2/3) by histology were offered cryotherapy treatment. The first 50 women had cervical swabs taken at baseline and at 2 and 4 weeks following treatment. Swabs were analyzed for HIV-1 RNA and compared using General Estimating Equation (GEE) with binomial or Gaussian links. Results: Of the 50 women enrolled, 40 were receiving antiretroviral therapy (ART) and 10 were not receiving ART at the time of cryotherapy and during study follow-up. Among all women, the odds of detectable cervical HIV-1 RNA did not increase at 2 weeks [odds ratio (OR) 1.18; 95% confidence interval (CI) 0.65-2.13] or 4 weeks (OR 1.29; 95% CI 0.71-2.33) following cryotherapy. Among 10 women not receiving ART, the OR of detectable shedding at 2 weeks was higher, but not statistically significant (OR 4.02; 95% CI 0.53-30.79; P = 0.2), and at 4 weeks remained unchanged (OR 1.00; 95% CI 0.27-3.74). Conclusion: There was no increase in detectable cervical HIV-1 RNA among HIV-positive women after cryotherapy. The risk of HIV-1 transmission after cryotherapy may not be significant, particularly among women already on ART at the time of cervical treatment. However, further investigation is needed among women not receiving ART

Range expansion and the origin of USA300 north american epidemic methicillin-resistant Staphylococcus aureus

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation. IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone’s geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone’s spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone’s geographic spread

Impact of combination antiretroviral therapy on cerebrospinal fluid HIV RNA and neurocognitive performance

Determine if antiretroviral (ARV) regimens with good central nervous system (CNS) penetration control HIV in cerebrospinal fluid (CSF) and improve cognition

Observation of an Excited Bc+ State

Using pp collision data corresponding to an integrated luminosity of 8.5 fb-1 recorded by the LHCb experiment at center-of-mass energies of s=7, 8, and 13 TeV, the observation of an excited Bc+ state in the Bc+π+π- invariant-mass spectrum is reported. The observed peak has a mass of 6841.2±0.6(stat)±0.1(syst)±0.8(Bc+) MeV/c2, where the last uncertainty is due to the limited knowledge of the Bc+ mass. It is consistent with expectations of the Bc∗(2S31)+ state reconstructed without the low-energy photon from the Bc∗(1S31)+→Bc+γ decay following Bc∗(2S31)+→Bc∗(1S31)+π+π-. A second state is seen with a global (local) statistical significance of 2.2σ (3.2σ) and a mass of 6872.1±1.3(stat)±0.1(syst)±0.8(Bc+) MeV/c2, and is consistent with the Bc(2S10)+ state. These mass measurements are the most precise to date

Measurement of the inelastic pp cross-section at a centre-of-mass energy of 13TeV

The cross-section for inelastic proton-proton collisions at a centre-of-mass energy of 13TeV is measured with the LHCb detector. The fiducial cross-section for inelastic interactions producing at least one prompt long-lived charged particle with momentum p > 2 GeV/c in the pseudorapidity range 2 < η < 5 is determined to be ϭ acc = 62:2 ± 0:2 ± 2:5mb. The first uncertainty is the intrinsic systematic uncertainty of the measurement, the second is due to the uncertainty on the integrated luminosity. The statistical uncertainty is negligible. Extrapolation to full phase space yields the total inelastic proton-proton cross-section ϭ inel = 75:4 ± 3:0 ± 4:5mb, where the first uncertainty is experimental and the second due to the extrapolation. An updated value of the inelastic cross-section at a centre-of-mass energy of 7TeV is also reported

The HOPE fixation technique - a promising alternative to common prostate cancer biobanking approaches

<p>Abstract</p> <p>Background</p> <p>The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes.</p> <p>Methods</p> <p>10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. <it>ERG </it>rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, β-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies.</p> <p>Results</p> <p>Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses.</p> <p>Conclusions</p> <p>This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.</p