Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Chemodiversity and molecular plasticity: recognition processes as explored by property spaces.

In the last few years, a need to account for molecular flexibility in drug-design methodologies has emerged, even if the dynamic behavior of molecular properties is seldom made explicit. For a flexible molecule, it is indeed possible to compute different values for a given conformation-dependent property and the ensemble of such values defines a property space that can be used to describe its molecular variability; a most representative case is the lipophilicity space. In this review, a number of applications of lipophilicity space and other property spaces are presented, showing that this concept can be fruitfully exploited: to investigate the constraints exerted by media of different levels of structural organization, to examine processes of molecular recognition and binding at an atomic level, to derive informative descriptors to be included in quantitative structure--activity relationships and to analyze protein simulations extracting the relevant information. Much molecular information is neglected in the descriptors used by medicinal chemists, while the concept of property space can fill this gap by accounting for the often-disregarded dynamic behavior of both small ligands and biomacromolecules. Property space also introduces some innovative concepts such as molecular sensitivity and plasticity, which appear best suited to explore the ability of a molecule to adapt itself to the environment variously modulating its property and conformational profiles. Globally, such concepts can enhance our understanding of biological phenomena providing fruitful descriptors in drug-design and pharmaceutical sciences.

Biological materials in colorectal surgery: current applications and potential for the future.

Biological materials are increasingly used in abdominal surgery for ventral, pelvic and perineal reconstructions, especially in contaminated fields. Future applications are multi-fold and include prevention and one-step closure of infected areas. This includes prevention of abdominal, parastomal and pelvic hernia, but could also include prevention of separation of multiple anastomoses, suture- or staple-lines. Further indications could be a containment of infected and/or inflammatory areas and protection of vital implants such as vascular grafts. Reinforcement patches of high-risk anastomoses or unresectable perforation sites are possibilities at least. Current applications are based mostly on case series and better data is urgently needed. Clinical benefits need to be assessed in prospective studies to provide reliable proof of efficacy with a sufficient follow-up. Only superior results compared with standard treatment will justify the higher costs of these materials. To date, the use of biological materials is not standard and applications should be limited to case-by-case decision.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Rapid evolution of cancer/testis genes on the X chromosome.

BACKGROUND: Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole. RESULTS: To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes. CONCLUSION: Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and biological activity analysis of specific antibodies

Background: Plasmodium falciparum(P. falciparum) merozoite surfaceprotein 2 (MSP-2) is one of bloodstage proteins that are associated withprotection from malaria. MSP-2 consistsof a highly polymorphic centralrepeat region flanked by a dimorphicregion that defines the two allelicfamilies, 3D7 and FC27; N- and Cterminalregions are conserved domains.Long synthetic peptides (LSP)representing the two allelic familiesof MSP-2 and constant regions arerecognized by sera from donors livingin endemic areas; and specific antibodies(Abs) are associated with protectionand active in antibody dependentcellular inhibition (ADCI) in vitro.However, the fine specificity ofAb response to the two allelic familiesof MSP-2 is unknown. Methods: Peptidesrepresenting dimorphic regionof 3D7 and FC27 families and theirC-terminal (common fragment to thetwo families) termed 3D7-D (88 aa),FC27-D (48 aa) and C (40 aa) respectivelywere synthesized. Overlapping20 mer peptides covering dimorphicand constant regions of two familieswere also synthesized for epitopemapping. Human sera were obtainedfrom donors living in malaria endemicareas. SpecificDand CregionsAbs were purified from single or poolhuman sera. Sera from mice were obtainedafter immunization with thetwo families LSP mixture in three differentadjuvants: alhydrogel (Alum),Glucopyranosyl Lipid Adjuvant-Stableoil-in-water Emulsion (GLA-SE)and Virosome. For ADCI, P. falciparum(strain 3D7) parasite wasmaintained in culture at 0.5% parasitemiaand 4% hematocrit in air tightbox at love oxygen (2%) and 37 ºC.Results: We identified several epitopesfrom the dimorphic and constantregions of both families of MSP-2, inmice and humans (adults and children).In human, most recognizedepitopes were the same in differentendemic regions for each domain ofthe two families of MSP-2. In mice,the differential recognition of epitopewas depending on the strain of mouseand interestingly on the adjuvantused. GLA-SE and alum as adjuvantswere more often associated with therecognition of multiple epitopes thanvirosomes. Epitope-specific Abs recognizednative merozoites of P.falciparum and were active in ADCIto block development of parasite.Conclusion: The delineation of a limitednumber of epitopes could be exploitedto develop MSP-2 vaccinesactive on both allelic families ofMSP-2.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models.

BACKGROUND: Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS: We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS: We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.

Oxidative potential for fine/ultrafine particles in occupational situations

Introduction : The redox properties of fine/ultrafine particles as well as nanoparticles (NP) are suggested to be important to explain their biological activity and could constitute a novel and promising metric for hazard evaluation. The acellular in vitro dithiothreitol (DTT) assay allows measuring this property. Objectives : (1) to evaluate sampling requirements for fine/ultrafine particle allowing measurement of their oxidative potential (2) to apply the methodology to occupational situations where particle from combustion sources are generated. Material and method : Sampling parameters (type of filters and loaded amount) and storage duration affecting the DTT measurements were evaluated. Based on these results, a methodological approach was defined and applied in two occupational situations where diesel and other combustion particles are present (toll station in a tunnel and mechanical yard for bus reparation). Results : Teflon filters loaded with diesel particles were found more suitable for the DTT assay, due to their better chemical inertness compared to quartz filters: after storage durations larger than 150 hours, an increased reactivity toward DTT was observed only with quartz filters. Reactivity was linearly correlated to the loaded mass until about 1000 μg/filter. Different redox reactivities were determined in both working places, with the mechanical yard presenting a higher DTT consumption rate. Discussion and conclusions : These results demonstrate the feasibility of this method to determine the oxidative potential of fine/ultrafine particles in occupational situations. We propose to include this approach for hazard assessment of work places with exposure to manufactured and other NP.

Roles of aldehyde dehydrogenase (ALDH) isoforms and retinoic acids in the growth and differentiation potential of human adult cardiac progenitor cells

Aim: We have studied human adult cardiac progenitor cells (CPCs) based on high aldehyde dehydrogenase activity (ALDH-hi), a property shared by many stem cells across tissues and organs. However, the role of ALDH in stem cell function is poorly known. In humans, there are 19 ALDH isoforms with different biological activities. The isoforms responsible for the ALDH-hi phenotype of stem cells are not well known but they may include ALDH1A1 and ALDH1A3 isoforms, which function in all-trans retinoic acid (RA) cell signaling. ALDH activity has been shown to regulate hematopoietic stem cell function via RA. We aimed to analyze ALDH isoform expression and the role of RA in human CPC function. Methods: Human adult CPCs were isolated from atrial appendage samples from patients who underwent heart surgery for coronary artery or valve disease. Atrial samples were either cultured as primary explants or enzymatically digested and sorted for ALDH activity by FACS. ALDH isoforms were determined by qRT-PCR. Cells were cultured in the presence or absence of the specific ALDH inhibitor DEAB, with or without RA. Induction of cardiac-specific genes in cells cultured in differentiation medium was measured by qRT-PCR. Results: While ALDH-hi CPCs grew in culture and could be expanded, ALDH-low cells grew poorly. CPC isolated as primary explant outgrowths expressed high levels of ALDH1A3 but not of other isoforms. CPCs isolated from cardiospheres expressed relatively high levels of all the 11 isoforms tested. In contrast, expanded CPCs and cardiosphere-derived cells expressed low levels of all ALDH isoforms. DEAB inhibited CPC growth in a dose-dependent manner, whereas RA rescued CPC growth in the presence of DEAB. In differentiation medium, ALDH-hi CPCs expressed approximately 300-fold higher levels of cardiac troponin T compared with their ALDH-low counterparts. Conclusions: High ALDH activity identifies human adult cardiac cells with high growth and cardiomyogenic potential. ALDH1A3 and, possibly, ALDH1A1 isoforms account for high ALDH activity and RA-mediated regulation of CPC growth.

The clinical and biological impact of new pathogen inactivation technologies on platelet concentrates.

Since 1990, several techniques have been developed to photochemically inactivate pathogens in platelet concentrates, potentially leading to safer transfusion therapy. The three most common methods are amotosalen/UVA (INTERCEPT Blood System), riboflavin/UVA-UVB (MIRASOL PRT), and UVC (Theraflex-UV). We review the biology of pathogen inactivation methods, present their efficacy in reducing pathogens, discuss their impact on the functional aspects of treated platelets, and review clinical studies showing the clinical efficiency of the pathogen inactivation methods and their possible toxicity.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil.

BACKGROUND: The FIFA has implemented an important antidoping programme for the 2014 FIFA World Cup. AIM: To perform the analyses before and during the World Cup with biological monitoring of blood and urine samples. METHODS: All qualified players from the 32 teams participating in the World Cup were tested out-of-competition. During the World Cup, 2-8 players per match were tested. Over 1000 samples were collected in total and analysed in the WADA accredited Laboratory of Lausanne. RESULTS: The quality of the analyses was at the required level as described in the WADA technical documents. The urinary steroid profiles of the players were stable and consistent with previously published papers on football players. During the competition, amphetamine was detected in a sample collected on a player who had a therapeutic use exemption for attention deficit hyperactivity disorder. The blood passport data showed no significant difference in haemoglobin values between out-of-competition and postmatch samples. CONCLUSIONS: Logistical issues linked to biological samples collection, and the overseas shipment during the World Cup did not impair the quality of the analyses, especially when used as the biological passport of football players.

Clinical and biological determinants of kidney outcomes in a population-based cohort study

BACKGROUND/AIMS: Prospective studies on factors associated with adverse kidney outcomes in European general populations are scant. Also, few studies consider the potential confounding effect of baseline kidney function. METHODS: We used baseline (2003-2006) and 5-year follow-up data of adults from the general population to evaluate the effect of baseline kidney function and proteinuria on the association of clinical, biological (e.g. uric acid, homocysteine, cytokines), and socioeconomic factors with change in kidney function, rapid decline in kidney function, and incidence of chronic kidney disease (CKD). Estimated glomerular filtration rate (eGFR) and urinary albuminuria-to-creatinine ratio (UACR) were collected. Kidney outcomes were modeled using multivariable regressions. RESULTS: A total of 4,441 subjects were included in the analysis. Among participants without CKD at baseline, 11.4% presented rapid decline in eGFR and/or incident CKD. After adjustment for baseline eGFR and log UACR, only age (Odds Ratio; 1.25 [95%CI 1.18-1.33]), diabetes (OR 1.48 [1.03-2.13]), education (OR middle vs. high 1.51 [1.08-2.11]) and log ultrasensitive CRP (OR 1.16 [1.05-1.22]) were associated with rapid decline in eGFR or incident CKD. Baseline log UACR (OR 1.18 [1.06-1.32]) but not eGFR was associated with rapid decline in eGFR and/or incident CKD. CONCLUSION: In addition to age and diabetes, education and CRP levels are associated with adverse kidney outcomes independently of baseline kidney function.

Endogenous steroid profiling in the athlete biological passport.

The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.

The Crocidura fuliginosa species complex (Mammalia, Insectivora) in Peninsular Malaya: biological, karyological and genetical evidence

Four West Malaysian shrew populations of the genus Crocidura were investigated through their karyotype and allozyme variations, and, in part, by interfertility experiments. Two different karyotypes characterize these shrews. The first, restricted to the Cameron Highlands (Peninsular Malaysia), invariably has 2n = 40 chromosomes but a varying fundamental number (FN = 54-58). The second karyotype shows a fundamental number of 62-68 and a polymorphic chromosomal number of 2n = 38, 39 or 40, a rare event in the genus Crocidura. Thus both can be distinguished by either a low or a higher number of meta- and submetacentric elements. In heterospecific breeding experiments, mutual avoidance was observed suggesting prezygotic barriers, whereas intraspecific pairs produced 13 liters (mean 2.1 young). Furthermore, our biochemical results indicate that both karyotypes correspond to a relatively ancient separation (Nei's D = 0.354), an amount of genetic differentiation comparable to the distance separating them from the West Palearctic C. russula (D = 0.429-0.583). In contrast, conspecific island and mainland Malaysian shrews possessing the second karyotype had only one fixed allelic difference over the 35 loci surveyed. The problem of naming the two biological species remains unsolved and requires further comparative investigations.