HCV infective virions can be carried by human platelets

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.     

人为活动对舟山群岛大中型兽的影响：大中型兽受威胁状态分析

本文根据历史记载对比和皮张收购资料分析研究了舟山群岛大中型兽的受威胁状态。结果显示,舟山群岛曾有大中型兽16种,5种已绝灭,3种处于濒危状态,3种分布区缩小（一种种群数量正在下降）,3种种群保持稳定,2种种群动态不明;食肉动物的绝灭率大于食草动物;大型动物绝灭率较高;岛屿物种绝灭率高于邻近大陆。文章还比较了确定种群趋势的几种方法。     

Intraspecific genotypic characterization of Lactobacillus rhamnosus strains intended for probiotic use and isolates of human origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

Can resource use be the link between productivity and species richness in mammals?

Species richness has been shown to be affected by primary productivity at various spatial scales. However, the mechanisms behind this effect are still poorly understood. Under the assumption that primary productivity is directly related to energy availability for an animal assemblage, the hypotheses are that (i) primary productivity will affect the number of ways different food resources can be combined into species specific niches and (ii) it will also determine the number of specialist species that can be supported. These hypotheses were tested on the herbivorous mammalian fauna of Australia. Productivity only had a weak effect on the number of food resource combinations in this case. Specialization was most common at low and high productivity. In addition there were significant differences in where different types of specialization was the most common. This study seeks an energetic mechanism to explain an energetic relationship and though no clear effect was found for resource use it identified a possible link between productivity and species richness.     

Protein adsorbents intended for use in aqueous two-phase systems

Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry to carry proteins away from the insoluble cell fragments generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume.     

Porcine endothelial cells,unlike human endothelial cells,can be killed by human CTL via Fas ligand and cannot be protected by Bcl-2

In clinical transplantation host CTL are major effectors of acute rejection, and graft endothelial cells (EC) are major targets of the CTL response. It is unclear what roles CTL will play in pig-into-human xenotransplantation. We compared the mechanisms of killing used by human CTL (huCTL) vs allogeneic and pig xenogeneic EC targets. Both responses show MHC class I restriction of target cell recognition. A granzyme B inhibitor peptide completely blocks anti-human and partially blocks anti-pig responses, while inhibitory Fas ligand Ab only blocks killing of porcine cells despite similar levels of Fas expression in both target cell types. Transduction of Bcl-2 completely protects human EC from huCTL, but has no effect on huCTL-mediated killing of porcine EC despite its efficacy vs drug-induced apoptosis. Bcl-2 effectively protects human EC rendered sensitive to Fas ligand by overexpressing Fas from huCTL, yet fails to protect porcine aortic endothelial cells from huCTL in the presence of anti-Fas ligand Ab. These data reveal differences in the susceptibility of human and porcine targets to huCTL.     

Functional human insulin-degrading enzyme can be expressed in bacteria

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.     

Ribavirin can be mutagenic for arenaviruses

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.     

Physiological conditions can be reflected in human urine proteome and metabolome

Biomarkers are the measurable changes associated with physiological or pathophysiological processes. Urine, unlike blood, lacks mechanisms for maintaining homeostasis: it is therefore an ideal source of biomarkers that can reflect systemic changes. Urinary proteome and metabolome have been studied for their diagnostic capabilities, ability to monitor disease and prognostic utility. In this review, the effects of common physiological conditions such as gender, age, diet, daily rhythms, exercise, hormone status, lifestyle and extreme environments on human urine are discussed. These effects should be considered when biomarker studies of diseases are conducted. More importantly, if physiological changes can be reflected in urine, we have reason to expect that urine will become widely used to detect small and early changes in pathological and/or pharmacological conditions.     

Aging by telomere loss can be reversed

Recently in Nature, Jaskelioff et al. (2010) demonstrated that multiple aging phenotypes in a mouse model of accelerated telomere loss can be reversed within 4 weeks of reactivating telomerase. This raises the major question of whether physiological aging, likely caused by a combination of molecular defects, may also be reversible.     

Reflex muscle contractions can be elicited by valgus positional perturbations of the human knee

Experimental evidence on the reflex responses of thigh muscles to valgus mechanical perturbations at the human knee are presented. Random step positional deflections, ranging from 5 degrees to 12 degrees at 60 degrees /s, were applied to the fully extended knees of seven healthy subjects. Subjects were instructed to maintain a constant background co-activation ( approximately 2-11% MVC) of the quadriceps and hamstring muscles prior to and during the mechanical stimulus. We found that the reflex response to sustained valgus joint deflection in the vasti muscles had longer onset latencies (range: 83-92ms) than did the stretch reflex in the same muscles (latencies: 29-31ms). This reflex EMG response consisted typically of a peak followed by sustained muscle activity throughout the step perturbation. The sustained EMG activity was dependent on the amplitude of the perturbing stimulus, but in a nonlinear manner. The long latency of the valgus response suggests that the reflex originates in nonmuscular sensory pathways, potentially from mechanoreceptors lying in periarticular tissues such as joint ligaments and capsule. Analysis of the spatial distribution of reflex responses showed an asymmetrical pattern with preferential activation of medial vs. lateral muscles of the knee. We assess whether these asymmetric reflex contractions could promote joint stability, either by inducing generalized joint stiffening, or by preferential activation of those muscles that are best suited to resist induced ligament strain.     

Boundaries between soil compartments formed by microporous hydrophobic membranes (GORE-TEXR) can be crossed by vesicular-arbuscular mycorrizal fungi but not by ions in the soil solution

Various container systems have been described in which soil regions available to hyphae only are separated from the mycorrhizal root region by 30–60 m mesh screens to study nutrient exchange between plants and fungi in the mycorrhizal symbiosis. The screens designed up to now prevent penetration by roots but allow easy passage of fungal hyphae as well as diffusion or mass flow of water and nutrient solutions. We tested hydrophobic microporous polytetrafluorethylene (PTFE) membranes (GORE-TEX^R) with 5 to 15 m diameter pores in an attempt to obtain a better seal between compartments and to prevent uncontrolled nutrient transport by diffusion or mass flow. We found that these membranes completely prevented diffusion or mass flow of ions between two soil compartments but could be penetrated easily by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae, as demonstrated by the rapid colonization of soybean roots (Glycine max L.) from an inoculum across the membranes.     

Haloperidol reduction can be assayed in human red blood cells

One metabolite of haloperidol present in plasma is "reduced haloperidol." This study demonstrates that human red blood cells are capable of converting haloperidol to reduced haloperidol in vitro. The reductase involved requires NADPH, as does haloperidol (ketone) reductase in human liver cytosol.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double‐strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell‐specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20–80‐fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat‐induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non‐homologous end‐joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT‐rich PAM broadens the range of ipGT drastically, particularly when targeting in CG‐deserts like promoters and introns.     

Domestic dogs and puppies can use human voice direction referentially

Domestic dogs are particularly skilled at using human visual signals to locate hidden food. This is, to our knowledge, the first series of studies that investigates the ability of dogs to use only auditory communicative acts to locate hidden food. In a first study, from behind a barrier, a human expressed excitement towards a baited box on either the right or left side, while sitting closer to the unbaited box. Dogs were successful in following the human''s voice direction and locating the food. In the two following control studies, we excluded the possibility that dogs could locate the box containing food just by relying on smell, and we showed that they would interpret a human''s voice direction in a referential manner only when they could locate a possible referent (i.e. one of the boxes) in the environment. Finally, in a fourth study, we tested 8–14-week-old puppies in the main experimental test and found that those with a reasonable amount of human experience performed overall even better than the adult dogs. These results suggest that domestic dogs’ skills in comprehending human communication are not based on visual cues alone, but are instead multi-modal and highly flexible. Moreover, the similarity between young and adult dogs’ performances has important implications for the domestication hypothesis.     

Metabolic properties of human acetylcholine receptors can be characterized on cultured human muscle

Experiments examining acetylcholine receptor (AChR) metabolism in tissue culture have hitherto been limited to animal systems. For many studies, the human AChR on human skeletal muscle provides a more physiologic target. However, previous studies suggested that the levels of AChR produced on cultured human muscle were inadequate for metabolic studies. We demonstrate here that the metabolism of human acetylcholine receptors can be analysed on pure human muscle fibers that develop in tissue culture. Degradation of AChR follows first-order kinetics and is inhibited 85% by leupeptin, demonstrating that proteolysis of human AChR occurs in the lysosome. New AChR continue to appear on the cell surface for 3 h in the presence of cycloheximide, indicating the existence of a pool of intracellular AChR destined for the cell membrane. This pool is equivalent to approximately one-third of the AChR present on the surface of the cell. At any given time, the rate of AChR accumulation on the cell surface can be quantitatively accounted for by the rates of synthesis and degradation. Our results demonstrate that studies on the effects of hormones, neurotoxins or antibodies from patients with autoimmune neuromuscular diseases are now possible with human AChR which develop on intact human muscle myotubes formed in tissue culture.     

Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.     

AA-amyloidosis can be transferred by peripheral blood monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils ('amyloid enhancing factor') combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis.