Comparison of the sensitivities of WaterLOGSY and saturation transfer difference NMR experiments

The WaterLOGSY (WL) and saturation transfer difference (STD) NMR experiments have proven to be extremely useful techniques to characterize interactions between small molecules and large biomolecules. In this work we compare the relative sensitivities of WL and STD NMR using 3 experimental systems: ketoprofen (KET)–bovine serum albumin (BSA), tert-butyl hydroquinone (TBHQ)–hemagglutinin (HA), and chloramphenicol (CAM)–ribosome (70S). In all cases we find that WL is more sensitive than STD for a given experimental time with the ratios ranging from 3.2 for KET–BSA to 16 for TBHQ–HA and CAM–70S. We attribute the increased sensitivity of WL to be due to simultaneous saturation of multiple sources of cross correlation, including direct NOEs of ¹H of water and exchangeable groups and indirect NOEs of ¹H–C groups. We suggest that the outstanding sensitivity of WL make it ideally suited for drug screening efforts targeting very large biomolecules at relatively low concentrations.     

Environmental evaluation and comparison of selected industrial scale biomethane production facilities across Europe

Purpose

The two main reasons for producing biomethane as renewable fuel are reduction of climate impacts and depletion of fossil resources. Biomethane is expected to be sustainable, but how sustainable is it actually? This article contributes to the clarification. Therefore, the environmental impacts of several biomethane facilities all over Europe were assessed. A special focus is put on the differences between the facilities as they follow different production routes.

Methods

The method used for evaluation is life cycle assessment (LCA) applied in a well-to-wheel approach. This enables to show the overall performance in terms of global warming potential (GWP), acidification potential (AP), eutrophication potential (EP), photochemical ozone creation potential (POCP) and PE fossil. The system boundary includes the entire chain from biogas production to upgrading, distribution and use. For evaluating the different production routes several years of measuring data, calculating and improving the LCA models in close cooperation with the plant operators were carried out.

Results and discussion

The evaluation of the production routes shows a high reduction potential compared to fossil fuels. Regarding the depletion of fossil resources, the amounts vary between the sites, but the reduction is at least 50 % and reaches almost 100 % reductions at some sites. The reduction of GWP is at least 65 %, because waste flows free of environmental burdens are used almost exclusively as substrate. Other dominant factors are power and heat demand, methane losses to the environment and the use of by-products, e.g. fertilizer.

Conclusions

Despite this caveat, the evaluated systems demonstrate the possible positive results of renewable fuel production if done properly.     

Epigenetic regulation of glycosylation is the quantum mechanics of biology

Background

Most proteins are glycosylated, with glycans being integral structural and functional components of a glycoprotein. In contrast to polypeptides, which are fully encoded by the corresponding gene, glycans result from a dynamic interaction between the environment and a network of hundreds of genes.

Scope of review

Recent developments in glycomics, genomics and epigenomics are discussed in the context of an evolutionary advantage for higher eukaryotes over microorganisms, conferred by the complexity and adaptability which glycosylation adds to their proteome.

Major conclusions

Inter-individual variation of glycome composition in human population is large; glycome composition is affected by both genes and environment; epigenetic regulation of “glyco-genes” has been demonstrated; and several mechanisms for transgenerational inheritance of epigenetic marks have been documented.

General significance

Epigenetic recording of acquired characteristics and their transgenerational inheritance could be important mechanisms used by higher organisms to compete or collaborate with microorganisms.     

MCM9 Mutations Are Associated with Ovarian Failure,Short Stature,and Chromosomal Instability

Premature ovarian failure (POF) is genetically heterogeneous and manifests as hypergonadotropic hypogonadism either as part of a syndrome or in isolation. We studied two unrelated consanguineous families with daughters exhibiting primary amenorrhea, short stature, and a 46,XX karyotype. A combination of SNP arrays, comparative genomic hybridization arrays, and whole-exome sequencing analyses identified homozygous pathogenic variants in MCM9, a gene implicated in homologous recombination and repair of double-stranded DNA breaks. In one family, the MCM9 c.1732+2T>C variant alters a splice donor site, resulting in abnormal alternative splicing and truncated forms of MCM9 that are unable to be recruited to sites of DNA damage. In the second family, MCM9 c.394C>T (p.Arg132^∗) results in a predicted loss of functional MCM9. Repair of chromosome breaks was impaired in lymphocytes from affected, but not unaffected, females in both families, consistent with MCM9 function in homologous recombination. Autosomal-recessive variants in MCM9 cause a genomic-instability syndrome associated with hypergonadotropic hypogonadism and short stature. Preferential sensitivity of germline meiosis to MCM9 functional deficiency and compromised DNA repair in the somatic component most likely account for the ovarian failure and short stature.     

Transgenic Expression of the 3D Polymerase Inhibits Theiler's Virus Infection and Demyelination

The RNA-dependent RNA polymerase 3D^pol is required for the elongation of positive- and negative-stranded picornavirus RNA. During the course of investigating the effect of the transgenic expression of viral genes on the host immune response, we evaluated the viral load present in the host after infection. To our surprise, we found that 3D transgenic expression in genetically susceptible FVB mice led to substantially lower viral loads after infection with Theiler''s murine encephalomyelitis virus (TMEV). As a result, spinal cord damage caused by chronic viral infection in the central nervous system was reduced in FVB mice that expressed 3D. This led to the preservation of large-diameter axons and motor function in these mice. The 3D transgene also lowered early viral loads when expressed in FVB-D^b mice resistant to persistent TMEV infection. The protective effect of 3D transgenic expression was not altered in FVB-Rag^−/−.3D mice that are deficient in T and B cells, thus ruling out a mechanism by which the overexpression of 3D enhanced the adaptive immune clearance of the virus. Understanding how endogenously overexpressed 3D polymerase inhibits viral replication may lead to new strategies for targeting therapies to all picornaviruses.Picornavirus infection is a major contributor to worldwide disease. Diseases such as poliomyelitis and hand-foot-and-mouth disease can be fatal. Other picornaviruses, such as rhinovirus, are partly responsible for upper respiratory tract infections. There are no drugs to treat picornavirus illness. However, some therapies show promise in vitro and in animal models. Winthrop compounds, which bind to hydrophobic sites on the surface of the virion that are important in viral attachment to the host and uncoating, decreased the number of upper respiratory symptoms following challenge with coxsackie virus 21 (45). A similar pocket binding drug, pleconaril (VP63843), showed 95% inhibition against 215 non-polio enteroviruses (39). A phase II trial of this drug against enteroviral meningitis decreased disease duration compared to the placebo (1). However, these drugs have side effects and were less effective in larger studies. Another limitation is that mutant viruses arose that sterically inhibited binding by substituting a bulky amino acid in the binding pocket (21). The administration of small interfering RNA (siRNA) has shown some promise in controlling picornavirus infections; however, the development of a delivery system is a major hurdle (8-10, 44).The picornavirus Theiler''s murine encephalomyelitis virus (TMEV) is a member of the Cardiovirus genus. TMEV is divided into two groups based on disease in mice after intracerebral injection (12, 15). The highly virulent GDVII subgroup causes fatal encephalitis, and the lowly virulent Theiler''s original (TO) subgroup, which includes BeAn and DA strains, causes a persistent infection in the white matter of the central nervous system (CNS), leading to chronic inflammation and demyelination in genetically susceptible mice. Chronic inflammation and demyelination leads to secondary axonal dysfunction and paralysis. Therefore, the BeAn and DA strains of TMEV infection in mice are used as animal models of demyelinating diseases such as multiple sclerosis (4, 11, 13).Inbred strains of mice differ in their susceptibility to TMEV (14, 18). Resistance to persistent infection depends on the haplotype of the major histocompatibility complex (H-2). Mice of the H-2^b,d,k haplotype are resistant to persistent infection, whereas mice of the H-2^f,p,q,r,s,v haplotype are susceptible to persistent infection (32). Resistance to persistent infection has been further defined to the D locus of H-2 (33, 35). The mice used in this paper all are on an FVB/NJ background. Due to the prominent pronuclei in their fertilized eggs and large litter size, FVB/NJ mice commonly are used for transgenic injection (43). These mice are of the H-2^q haplotype and are susceptible to persistent TMEV infection. Persistent infection leads to chronic spinal cord inflammation and demyelination in all inbred mice that are genetically susceptible to TMEV. FVB-D^b mice contain the H-2D^b transgene, which confers resistance to persistent TMEV infection (2). FVB mice and FVB-D^b mice have similar early acute disease in the brain at 7 days postinfection (dpi); however, unlike FVB mice, FVB-D^b mice control the virus in the brain and spinal cord by day 45 and do not develop demyelination.Picornaviruses perform multiple tasks inside host cells for successful viral replication, with very few gene products being responsible for these tasks. The single-stranded RNA picornavirus genome has, on average, 7,500 nucleotides and produces a single polyprotein that is cleaved by its own virus-encoded proteases. One of these proteins, the RNA-dependent RNA-polymerase 3D^pol, is required for the elongation of positive- and negative-stranded viral RNA. 3D^pol oligomerizes, which favors elongation and binding to RNA (16). 3D^pol forms a membranous replication complex with VPg and precursor proteins 3AB and 3CD to initiate VPg uridylylation, which serves as a primer for positive- and negative-strand RNA replication by 3D^pol (25, 40, 42). The stimulatory effect of 3AB on RNA replication by 3D^pol is inhibited by increasing concentrations of 3D (26, 31).During the course of investigating the effect of the transgenic expression of viral genes on the host immune response to TMEV, we evaluated the viral load present in the host after infection. Mice expressing the 3D transgene were used as control mice, since previous studies have shown that the 3D protein was ignored by T and B cells in the immune system (3, 22, 29). To our surprise, we found that the 3D transgenic mice substantially reduced TMEV in vivo. Therefore, we set out to study the effect of 3D overexpression in a transgenic mouse model of TMEV infection.     

M-LDH physically associated with sarcolemmal KATP channels mediates cytoprotection in heart embryonic H9C2 cells

Muscle form of lactate dehydrogenase (M-LDH) physically associate with K_ATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K⁺ (K_ATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the K_ATP channel protein complex. The K⁺ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes K_ATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal K_ATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.     

Distinct Regions of Human eIF3 Are Sufficient for Binding to the HCV IRES and the 40S Ribosomal Subunit

Translation of the hepatitis C virus (HCV) genomic RNA initiates from an internal ribosome entry site (IRES) in its 5′ untranslated region and requires a minimal subset of translation initiation factors to occur, namely eukaryotic initiation factor (eIF) 2 and eIF3. Low-resolution structural information has revealed how the HCV IRES RNA binds human eIF3 and the 40S ribosomal subunit and positions the start codon for initiation. However, the exact nature of the interactions between the HCV IRES RNA and the translational machinery remains unknown. Using limited proteolysis and mass spectrometry, we show that distinct regions of human eIF3 are sufficient for binding to the HCV IRES RNA and the 40S subunit. Notably, the eIF3 subunit eIF3b is protected by HCV IRES RNA binding, yet is exposed in the complex when compared to subunits eIF3e, eIF3f, eIF3h, and eIF3l. Limited proteolysis reveals that eIF3 binding to the 40S ribosomal subunit occurs through many redundant interactions that can compensate for each other. These data suggest how the HCV IRES binds to specific regions of eIF3 to target the translational machinery to the viral genomic RNA and provide a framework for modeling the architecture of intact human eIF3.     

The NIH Roadmap Epigenomics Mapping Consortium

The NIH Roadmap Epigenomics Mapping Consortium aims to produce a public resource of epigenomic maps for stem cells and primary ex vivo tissues selected to represent the normal counterparts of tissues and organ systems frequently involved in human disease.     

Influence of instability and muscular weakness in ethiopathogenesis of hip fractures

The aim of our study was to, in accordance with the presented theoretical presumptions, analyze the possible reasons for hip fractures of the older population in the north-eastern part of Croatia. A group of 2,696 persons (1,936 women and 760 men) with hip fractures has been analyzed during a 12 year period (from 1993. until 2005. year) in the Clinical Hospital Osijek. The date of admittance, age, gender and fracture location were recorded. In men, the incidence of total hip fracture number on the left side was greater 23.5% (p<0.01) compared to the right side, while in women this difference does not exist. Men have a greater incidence of trochanteric fractures than fractures of femoral neck on both sides, while in women this difference could be shown on the right side only. In women, a 30.5% (p<0.001) higher fracture incidence occurred in the winter compared to the summer. It has been concluded that in men the impaired neuromuscular function on the left body side caused the greater incidence of falls on this side. The more frequent multifragmental fractures of the trochanteric massive in men indicate the possible role of preserved pelvitrochanteric muscle tension in fracture characterization. Increased incidence of falls and fractures in the older female population can be interpreted with a more pronounced weakness of pelvitrochanteric muscles and consequent walking instability. Furthermore, a smaller incidence of hip fractures was noticed in the summer compared with winter. This is explained by a reduced exhaustion of pelvic muscles in summer (primarily pelvitrochanteric) and decrease in fall frequency.