Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules

Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water–acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.     

Pretreatment CD4 Cell Slope and Progression to AIDS or Death in HIV-Infected Patients Initiating Antiretroviral Therapy—The CASCADE Collaboration: A Collaboration of 23 Cohort Studies

Background

CD4 cell count is a strong predictor of the subsequent risk of AIDS or death in HIV-infected patients initiating combination antiretroviral therapy (cART). It is not known whether the rate of CD4 cell decline prior to therapy is related to prognosis and should, therefore, influence the decision on when to initiate cART.

Methods and Findings

We carried out survival analyses of patients from the 23 cohorts of the CASCADE (Concerted Action on SeroConversion to AIDS and Death in Europe) collaboration with a known date of HIV seroconversion and with at least two CD4 measurements prior to initiating cART. For each patient, a pre-cART CD4 slope was estimated using a linear mixed effects model. Our primary outcome was time from initiating cART to a first new AIDS event or death. We included 2,820 treatment-naïve patients initiating cART with a median (interquartile range) pre-cART CD4 cell decline of 61 (46–81) cells/µl per year; 255 patients subsequently experienced a new AIDS event or death and 125 patients died. In an analysis adjusted for established risk factors, the hazard ratio for AIDS or death was 1.01 (95% confidence interval 0.97–1.04) for each 10 cells/µl per year reduction in pre-cART CD4 cell decline. There was also no association between pre-cART CD4 cell slope and survival. Alternative estimates of CD4 cell slope gave similar results. In 1,731 AIDS-free patients with >350 CD4 cells/µl from the pre-cART era, the rate of CD4 cell decline was also not significantly associated with progression to AIDS or death (hazard ratio 0.99, 95% confidence interval 0.94–1.03, for each 10 cells/µl per year reduction in CD4 cell decline).

Conclusions

The CD4 cell slope does not improve the prediction of clinical outcome in patients with a CD4 cell count above 350 cells/µl. Knowledge of the current CD4 cell count is sufficient when deciding whether to initiate cART in asymptomatic patients. Please see later in the article for the Editors'' Summary     

Comparison of joint kinetics during free weight and flywheel resistance exercise

The most common modality for resistance exercise is free weight resistance. Alternative methods of providing external resistance have been investigated, in particular for use in microgravity environments such as space flight. One alternative modality is flywheel inertial resistance, which generates resistance as a function of the mass, distribution of mass, and angular acceleration of the flywheel. The purpose of this investigation was to characterize net joint kinetics of multijoint exercises performed with a flywheel inertial resistance device in comparison to free weights. Eleven trained men and women performed the front squat, lunge, and push press on separate days with free weight or flywheel resistance, while instrumented for biomechanical analysis. Front squats performed with flywheel resistance required greater contribution of the hip and ankle, and less contribution of the knee, compared to free weight. Push presses performed with flywheel resistance had similar impulse requirements at the knee compared to free weight, but greater impulse requirement at the hip and ankle. As used in this investigation, flywheel inertial resistance increases the demand on the hip extensors and ankle plantarflexors and decreases the mechanical demand on the knee extensors for lower extremity exercises such as the front squat and lunge. Exercises involving dynamic lower and upper extremity actions, such as the push press, may benefit from flywheel inertial resistance, due to the increased mechanical demand on the knee extensors.     

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.     

Drug-metabolizing enzymes and praziquantel bioavailability in mice harboring Schistosoma mansoni isolates of different drug susceptibilities

The level of drug-metabolizing enzymes (cytochrome P450 [CYP450] and cytochrome b5 [cyt b5]) and the bioavailability of praziquantel (PZQ) were investigated in batches of mice infected with Schistosoma mansoni displaying either a decreased susceptibility to PZQ ("EE2" and "BANL"-isolates), or a normal susceptibility to the drug ("CD" isolate). Each batch was divided into 2 groups. The first group was further subdivided into 5 subgroups. Subgroups 1 to 4 were treated 7 wk postinfection (PI) with oral PZQ at 25, 50, 100, and 200 mg/kg for 5 consecutive days, whereas the fifth subgroup was administered the vehicle only as control. Animals were perfused 9 wk PI, and worms were counted to estimate PZQ ED50. CYP450 and cyt b5 were examined in hepatic microsomes of infected untreated mice and of infected mice treated with 25 and 200 mg/ kg PZQ. The second group was given PZQ 7 wk PI and was further subdivided into 11 subgroups, killed at 2, 5, 15, 30, 60, 90, 120, 150, 180, 240, and 360 min postdosing to study pharmacokinetic parameters of PZQ. Mice harboring S. mansoni isolates having higher PZQ ED50 (170.3 mg/kg for EE2 and 249.9 mg/kg for BANL vs. 82.96 mg/kg for CD) had higher levels of CYP450 and cyt b5, a PZQ Cmax decreased by 19-30% and area under the serum concentration-time curve0-6 hr decreased by 57-74%. Data suggest that S. mansoni isolates that are less sensitive to PZQ induce a lower inhibition of hepatic drug-metabolizing enzymes, with a consequently higher metabolic transformation of PZQ.     

Comparison of barley stripe mosaic virus strains

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.     

Thyroid-stimulating hormone increases active transport of perchlorate into thyroid cells

Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct measurement techniques. To demonstrate intracellular perchlorate accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1-10 microM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 microM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 microM) did not inhibit cell proliferation after 72 h of incubation. In conclusion, perchlorate is actively transported into thyroid cells and does not inhibit cell proliferation.     

Influence of age on pain sensitivity in response to paw pressure and formalin injection in rats: a role of nitric oxide

The effect of age on pain response to paw pressure and intraplantar formalin injection in rats is elucidated. Pain responses evoked by mechanical pressure on hind paw and intraplantar injection of formaldehyde (5%) into the hind paw were evaluated in groups of adult, young and aged male Sprague Dawley rats, after intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) injection of L-arginine or NG-nitro-L-arginine methyl ester (L-NAME). Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining was done in the two groups. The results show that pain response was reduced in the aged rats and enhanced pain response to paw pressure in aged rats only. L-arginine (i.c.v.) had no effect on pain response to paw pressure in the two groups but enhanced biphasic pain response to formalin. L-NAME (i.p. and i.c.v.) suppressed pain response to paw pressure in the two groups. L-NAME (i.c.v.) suppressed pain response to formalin during the acute phase and enhanced it during the late phase. NADPH-diaphorase activity was significantly greater in young rats. In conclusion, pain response is blunted in the aged rats. NO might be involved in mechanical nociception in aged rats and in formalin-induced nociception in both groups. NO blockade has an antinociceptive effect on pain response. Central NO has dual role in pain response evoked by formalin.     

Oxidative Stress in a Model of Toxic Demyelination in Rat Brain: The Effect of Piracetam and Vinpocetine

We studied the role of oxidative stress and the effect of vinpocetine (1.5, 3 or 6 mg/kg) and piracetam (150 or 300 mg/kg) in acute demyelination of the rat brain following intracerebral injection of ethidium bromide (10 μl of 0.1%). Results: ethidium bromide caused (1) increased malondialdehyde (MDA) in cortex, hippocampus and striatum; (2) decreased total antioxidant capacity (TAC) in cortex, hippocampus and striatum; (3) decreased reduced glutathione (GSH) in cortex and hippocampus (4); increased serum nitric oxide and (5) increased striatal (but not cortical or hippocampal) acetylcholinesterase (AChE) activity. MDA decreased in striatum and cortex by the lower doses of vinpocetine or piracetam but increased in cortex and hippocampus and in cortex, hypothalamus and striatum by the higher dose of vinpocetine or piracetam, respectively along with decreased TAC. GSH increased by the higher dose of piracetam and by vinpocetine which also decreased serum nitric oxide. Vinpocetine and piracetam displayed variable effects on regional AChE activity.