Xenon and sevoflurane provide analgesia during labor and fetal brain protection in a perinatal rat model of hypoxia-ischemia

It is not possible to identify all pregnancies at risk of neonatal hypoxic-ischemic encephalopathy (HIE). Many women use some form of analgesia during childbirth and some anesthetic agents have been shown to be neuroprotective when used as analgesics at subanesthetic concentrations. In this study we sought to understand the effects of two anesthetic agents with presumptive analgesic activity and known preconditioning-neuroprotective properties (sevoflurane or xenon), in reducing hypoxia-induced brain damage in a model of intrauterine perinatal asphyxia. The analgesic and neuroprotective effects at subanesthetic levels of sevoflurane (0.35%) or xenon (35%) were tested in a rat model of intrauterine perinatal asphyxia. Analgesic effects were measured by assessing maternal behavior and spinal cord dorsal horn neuronal activation using c-Fos. In separate experiments, intrauterine fetal asphyxia was induced four hours after gas exposure; on post-insult day 3 apoptotic cell death was measured by caspase-3 immunostaining in hippocampal neurons and correlated with the number of viable neurons on postnatal day (PND) 7. A separate cohort of pups was nurtured by a surrogate mother for 50 days when cognitive testing with Morris water maze was performed. Both anesthetic agents provided analgesia as reflected by a reduction in the number of stretching movements and decreased c-Fos expression in the dorsal horn of the spinal cord. Both agents also reduced the number of caspase-3 positive (apoptotic) neurons and increased cell viability in the hippocampus at PND7. These acute histological changes were mirrored by improved cognitive function measured remotely after birth on PND 50 compared to control group. Subanesthetic doses of sevoflurane or xenon provided both analgesia and neuroprotection in this model of intrauterine perinatal asphyxia. These data suggest that anesthetic agents with neuroprotective properties may be effective in preventing HIE and should be tested in clinical trials in the future.     

Case studies on the use of biotechnologies and on biosafety provisions in four African countries

This review is based on a study commissioned by the European Commission on the evaluation of scientific, technical and institutional challenges, priorities and bottlenecks for biotechnologies and regional harmonisation of biosafety in Africa. Biotechnology was considered within four domains: agricultural biotechnologies (‘Green’), industrial biotechnologies and biotechnologies for environmental remediation (‘White’), biotechnologies in aquaculture (‘Blue’) and biotechnologies for healthcare (‘Red’). An important consideration was the decline in partnerships between the EU and developing countries because of the original public antipathy to some green biotechnologies, particularly genetically modified organisms (GMOs) and food from GM crops in Europe. The study focus reported here was West Africa (Ghana, Senegal, Mali and Burkina Faso).The overall conclusion was that whereas high-quality research was proceeding in the countries visited, funding is not sustained and there is little evidence of practical application of biotechnology and benefit to farmers and the wider community. Research and development that was being carried out on genetically modified crop varieties was concentrating on improving food security and therefore unlikely to have significant impact on EU markets and consumers. However, there is much non-controversial green biotechnology such as molecular diagnostics for plant and animal disease and marker-assisted selection for breeding that has great potential application. Regarding white biotechnology, it is currently occupying only a very small industrial niche in West Africa, basically in the sole sector of the production of liquid biofuels (i.e., bio-ethanol) from indigenous and locally planted biomass (very often non-food crops). The presence of diffused small-scale fish production is the basis to develop and apply new (Blue) aquaculture technologies and, where the research conditions and the production sector can permit, to increase this type of production and the economy of this depressed areas. However, the problems bound to environmental protection must not be forgotten; priority should be given to monitor the risks of introduction of foreign species. Red biotechnologies potentially bring a vast domain of powerful tools and processes to achieve better human health, most notably improved diagnostics by molecular techniques, better targeting of pathogens and a better knowledge of their sensitivities to drugs to permit better treatment.Biosafety regulatory frameworks had been initiated in several countries, starting with primary biosafety law. However, disparate attitudes to the purpose of biosafety regulation (e.g., fostering informed decision-making versus ‘giving the green-light for a flood of GMOs’) currently prevent a needed consensus for sub-regional harmonisation. To date, most R&D funding has come from North America with some commercial interests from Asia, but African biotechnology workers expressed strong desire for (re-)engagement with interested parties from the European Union. Although in some of the visited countries there are very well qualified personnel in molecular biology and biosafety/regulation, the main message received is that human resources and capacity building in-house are still needed. This could be achieved through home-based courses and capacity-building including funds for post-degree research to motivate and retain trained staff.     

Dysfunction of Inflammation-Resolving Pathways Is Associated with Exaggerated Postoperative Cognitive Decline in a Rat Model of the Metabolic Syndrome

The cholinergic antiinflammatory pathway (CAP), which terminates in the spleen, attenuates postoperative cognitive decline (PCD) in rodents. Surgical patients with metabolic syndrome exhibit exaggerated and persistent PCD that is reproduced in postoperative rats selectively bred for easy fatigability and that contain all features of metabolic syndrome (low-capacity runners [LCRs]). We compared the CAP and lipoxin A₄ (LXA₄), another inflammation-resolving pathway in LCR, with its counterpart high-capacity runner (HCR) rats. Isoflurane-anesthetized LCR and HCR rats either underwent aseptic trauma involving tibial fracture (surgery) or not (sham). At postoperative d 3 (POD3), compared with HCR, LCR rats exhibited significantly exaggerated PCD (trace fear conditioning freezing time 43% versus 57%). Separate cohorts were killed at POD3 to collect plasma for LXA4 and to isolate splenic mononuclear cells (MNCs) to analyze CAP signaling, regulatory T cells (Tregs) and M2 macrophages (M2 Mφ). Under lipopolysaccharide (LPS) stimulation, tumor necrosis factor (TNF)-α produced by splenic MNCs was 117% higher in LCR sham and 52% higher in LCR surgery compared with HCR sham and surgery rats; LPS-stimulated TNF-α production could not be inhibited by an α7 nicotinic acetylcholine receptor agonist, whereas inhibition by the β₂ adrenergic agonist, salmeterol, was significantly less (−35%) than that obtained in HCR rats. Compared to HCR, sham and surgery LCR rats had reduced β₂ adrenergic receptor–expressing T lymphocytes (59%, 44%), Tregs (47%, 54%) and M2 Mφ (45%, 39%); surgical LCR rats’ hippocampal M2 Mφ was 66% reduced, and plasma LXA4 was decreased by 120%. Rats with the metabolic syndrome have ineffective inflammation-resolving mechanisms that represent plausible reasons for the exaggerated and persistent PCD.     

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.     

Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.     

Musculotendon variability influences tissue strains experienced by the biceps femoris long head muscle during high-speed running

The hamstring muscles frequently suffer injury during high-speed running, though the factors that make an individual more susceptible to injury remain poorly understood. The goals of this study were to measure the musculotendon dimensions of the biceps femoris long head (BFlh) muscle, the hamstring muscle injured most often, and to use computational models to assess the influence of variability in the BFlh’s dimensions on internal tissue strains during high-speed running. High-resolution magnetic resonance (MR) images were acquired over the thigh in 12 collegiate athletes, and musculotendon dimensions were measured in the proximal free tendon/aponeurosis, muscle and distal free tendon/aponeurosis. Finite element meshes were generated based on the average, standard deviation and range of BFlh dimensions. Simulation boundary conditions were defined to match muscle activation and musculotendon length change in the BFlh during high-speed running. Muscle and connective tissue dimensions were found to vary between subjects, with a coefficient of variation (CV) of 17±6% across all dimensions. For all simulations peak local strain was highest along the proximal myotendinous junction, which is where injury typically occurs. Model variations showed that peak local tissue strain increased as the proximal aponeurosis width narrowed and the muscle width widened. The aponeurosis width and muscle width variation models showed that the relative dimensions of these structures influence internal muscle tissue strains. The results of this study indicate that a musculotendon unit’s architecture influences its strain injury susceptibility during high-speed running.     

Absorbent Markov chains as a model for the study of the evolution of proteins

The formalism of absorbent Markov chains, previously developed by Kemeny & Snell (1960) is used as a model for the study of the evolution of proteins. Within the limits of statistical analysis used, the amino acid substitution frequencies of McLachlan (1972) are explained by the numerical values derived from the model used. In addition, the amino acid composition of proteins is partially explained and the relative mutability of amino acids receives a new interpretation in the light of the above mentioned stochastic model. The results show that some basic aspect of protein evolution can be predicted by a stochastic model and therefore a significant component of protein evolution is driven by a random element.     

Activation and aponeurosis morphology affect in vivo muscle tissue strains near the myotendinous junction

Hamstring strain injury is one of the most common injuries in athletes, particularly for sports that involve high speed running. The aims of this study were to determine whether muscle activation and internal morphology influence in vivo muscle behavior and strain injury susceptibility. We measured tissue displacement and strains in the hamstring muscle injured most often, the biceps femoris long head muscle (BFLH), using cine DENSE dynamic magnetic resonance imaging. Strain measurements were used to test whether strain magnitudes are (i) larger during active lengthening than during passive lengthening and (ii) larger for subjects with a relatively narrow proximal aponeurosis than a wide proximal aponeurosis. Displacement color maps showed higher tissue displacement with increasing lateral distance from the proximal aponeurosis for both active lengthening and passive lengthening, and higher tissue displacement for active lengthening than passive lengthening. First principal strain magnitudes were averaged in a 1cm region near the myotendinous junction, where injury is most frequently observed. It was found that strains are significantly larger during active lengthening (0.19 SD 0.09) than passive lengthening (0.13 SD 0.06) (p<0.05), which suggests that elevated localized strains may be a mechanism for increased injury risk during active as opposed to passive lengthening. First principal strains were higher for subjects with a relatively narrow aponeurosis width (0.26 SD 0.15) than wide (0.14 SD 0.04) (p<0.05). This result suggests that athletes who have BFLH muscles with narrow proximal aponeuroses may have an increased risk for BFLH strain injuries.