Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Long-Term Performance of Modern Coronary Sinus Leads in Cardiac Resynchronization Therapy

Background

Cardiac resynchronization therapy (CRT) has become an important pillar of contemporary heart failure therapy. The efficacy of CRT, however, critically relies on proper LV lead placement and performance, which is why data regarding the long-term performance of CS leads are of considerable interest. Available studies are limited by a restricted variety of lead vendors, earlier lead models and / or very short follow-up periods. In the current study, we therefore investigated the long-term performance of modern LV leads in a large "real world" cohort of patients undergoing CRT implantation.

Methods and Results

All 193 patients who had successfullyundergone CRT implantation at the University Hospital Zurich between September 2003 and January 2010 were included in the study. An overall stable course of stimulation energy was observed over time; neither ischemic etiology, lead configuration, or severely reduced EF had an influence on the evolution of energy thresholds over time. Interestingly, patients with a high energy threshold at baseline experienced a significant reduction during follow-up. In contrast, a significant drop in impedance was seen following implantation, followed by a steady course for the rest of the observation period. Only 15 patients (9.7%) showed an impedance > 1000 Ohm at any time during their follow-up. Seven lead dislocations were observed during follow up.

Conclusion

The current comprehensive analysis of long-term performance of modern coronary sinus leads demonstrates excellent stability, performance and safety. These data may have important implications for physicians involved in biventricular pacemaker implantations and in the follow-up care of these patients.     

Stimulation of Symbiotic N2 Fixation in Trifolium repens L. under Elevated Atmospheric pCO2 in a Grassland Ecosystem

Symbiotic N2 fixation is one of the main processes that introduces N into terrestrial ecosystems. As such, it may be crucial for the sequestration of the extra C available in a world of continuously increasing atmospheric CO2 partial pressure (pCO2). The effect of elevated pCO2 (60 Pa) on symbiotic N2 fixation (15N-isotope dilution method) was investigated using Free-Air-CO2-Enrichment technology over a period of 3 years. Trifolium repens was cultivated either alone or together with Lolium perenne (a nonfixing reference crop) in mixed swards. Two different N fertilization levels and defoliation frequencies were applied. The total N yield increased consistently and the percentage of plant N derived from symbiotic N2 fixation increased significantly in T. repens under elevated pCO2. All additionally assimilated N was derived from symbiotic N2 fixation, not from the soil. In the mixtures exposed to elevated pCO2, an increased amount of symbiotically fixed N (+7.8, 8.2, and 6.2 g m-2 a-1 in 1993, 1994, and 1995, respectively) was introduced into the system. Increased N2 fixation is a competitive advantage for T. repens in mixed swards with pasture grasses and may be a crucial factor in maintaining the C:N ratio in the ecosystem as a whole.     

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10⁸ 50% tissue culture infective doses (TCID₅₀) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 10⁵ copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 10⁴ copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.To date, the most promising approach to inducing sterilizing immunity in the macaque model has been through the use of live attenuated virus (LAV) vaccines based on simian immunodeficiency virus (SIV). A major advantage of an attenuated virus strategy for the development of a human immunodeficiency virus (HIV) vaccine is the ability of attenuated viruses to induce broad and persistent immunity (29, 51). In particular, SIV strains engineered with deletions of nef (SIVΔnef) have afforded the most significant protection upon challenge with pathogenic SIV (13, 14, 29, 60, 65, 72). Numerous SIV-derived live attenuated vaccine models have been developed, many of which employ deletions in the viral accessory genes (3, 12, 14, 15, 25, 29, 30, 53, 64, 72). In many cases, vaccinations have been shown to substantially decrease viral burden during the acute phase of infection, maintain low to undetectable levels of virus during the chronic phase of infection, and limit the progression to AIDS. Although promising, a major caveat to the live attenuated virus vaccine approach is the potential for compensatory reversion and the observations that incompletely attenuated viruses may harbor residual pathogenicity (5, 10, 14). Even SIV constructs containing multiple deletions in nef, vpr, and the negative regulatory element (NRE) can cause AIDS-like disease in adult macaques and particularly in neonates (4, 5, 27, 53). This may be analogous to some human long-term nonprogressors infected by nef-deleted HIV variants in whom a slowly increasing viral burden has been accompanied by disease progression (22, 34, 37). Additional mutations can be engineered into vaccine vectors to generate highly attenuated viruses, but this often comes at the expense of their protective efficacy (8, 23, 30).We previously made two series of novel live attenuated SIV vaccine models (25) in which the simplified SIV constructs retain all the structural viral proteins but have inactivating mutations for all viral accessory genes. These constructs retain significant antigenicity, without the pathogenic effects associated with accessory viral factors, thus limiting or eliminating the potential for reversion (25).Whether administered parenterally or mucosally, conventional challenge trials in macaques have often utilized artificially high single-dose inocula in an effort to ensure that most, if not all, of the naive or placebo-immunized animal subjects become infected following a single exposure. The rationale for using a single massive challenge has been reconsidered in light of the possibility that vaccines with protective efficacy under physiologic challenge conditions may not identified. This practice is now being replaced by an approach designed to better approximate the relatively low in vivo acquisition rates following a single sexual exposure to HIV (21, 45, 69) and should provide a more realistic assessment of vaccine efficacy in “real-world” situations. Importantly, recent studies using this approach have demonstrated viremia of magnitude and kinetics comparable to that seen following single high-dose mucosal inocula (47), and this approach has been used successfully in more recent challenge trials (31, 70). Here we are assessing the safety, immunogenicity, and protective efficacy of two hyperattenuated SIV vaccine candidates following a multi-low-dose intrarectal challenge with highly pathogenic SIVmac239 in the cynomolgus macaque model.SIV-specific humoral immune responses were assessed at various time points postvaccination and postchallenge by Western blotting. Cellular immunogenicity was monitored by evaluation of peripheral T-cell responses (via gamma interferon [IFN-γ] enzyme-linked immunospot [ELISPOT] assay) following stimulation with peptide pools spanning the entire SIVmac239 proteome. The protective efficacy of the different vaccine candidates was assessed by classical endpoints, such as quantitative analysis of plasma viral load, quantitative immunophenotyping of lymphocytes, and clinical markers of disease progression. Even using extremely attenuated SIV constructs with only minimal evidence of replication, a modest immune response that can impact long-term disease progression is generated.     

Breath ethane in dialysis patients and control subjects

Oxidant stress may play a role in the accelerated pathology of patients on dialysis, especially in the development of cardiovascular disease, which is a frequent condition in end-stage renal disease (ESRD) patients. Measurement of hydrocarbons can be employed to assess oxidant stress since breath hydrocarbons have been directly traced to in vivo breakdown of lipid hydroperoxides. We undertook to measure ethane, a major breath hydrocarbon, in 15 control subjects, 13 patients on peritoneal dialysis (PD), and 35 patients on hemodialysis (HD). Within the HD group, we separately examined 12 diabetic and 23 nondiabetic patients. Breath samples were collected after patients had breathed purified air for 4 min, and ethane content was measured by GC and expressed as pmoles/kg-body weight-minute (pmol/kg-min). As the data for the hemodialysis patients appeared skewed, nonparametric statistical techniques were employed to analyze these data, which are reported as median and interquartile range (IQR). Ethane levels were similar in 15 control subjects (median, 2.50 pmol [1.38-3.30]/kg-min] and 13 PD patients (median, 2.51 pmol [1.57-3.17]/kg-min). Breath ethane was significantly elevated in a portion (18 of 35 patients, 52%) of the HD patients (median, 6.16 pmol [4.46-8.88]/kg-min) (p <.001 vs. control, Mann-Whitney U test). Two of the diabetic HD patients showed extremely high values of breath ethane. Breath ethane was not altered by a single hemodialysis session, suggesting that long-term metabolic processes contribute to its elevation. Measurement of breath ethane may provide insight into severity of oxidant stress and metabolic disturbances, and provide guidance for optimal therapy and prevention of pathology in patients on long-term hemodialysis.     

Effect of high-pressure-induced ice I-to-ice III phase transitions on inactivation of Listeria innocua in frozen suspension

The inactivation of Listeria innocua BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices. The state of aggregation of water was taken into account. The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect. The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition. This effect was independent of the applied pressure holding time. Flow cytometric analyses supported the assumption of different mechanisms of inactivation of L. innocua in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture). Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Sequence and peptide-binding motif for a variant of HLA-A*0214 (A*02142) in an HIV-1-resistant individual from the Nairobi Sex Worker cohort

As part of the ongoing study of natural HIV-1 resistance in the women of the Nairobi Sex Workers' study, we have examined a resistance-associated HLA class I allele at the molecular level. Typing by polymerase chain reaction using sequence-specific primers determined that this molecule is closely related to HLA-A*0214, one of a family of HLA-A2 supertype alleles which correlate with HIV-1 resistance in this population. Direct nucleotide sequencing shows that this molecule differs from A*0214, having a silent nucleotide substitution. We therefore propose to designate it HLA-A*02142. We have determined the peptide-binding motif of HLA-A*0214/02142 by peptide elution and bulk Edman degradative sequencing. The resulting motif, X-[Q,V]-X-X-X-K-X-X-[V,L], includes lysine as an anchor at position 6. The data complement available information on the peptide-binding characteristics of this molecule, and will be of use in identifying antigenic peptides from HIV-1 and other pathogens.