UNC-98 and UNC-96 interact with paramyosin to promote its incorporation into thick filaments of Caenorhabditis elegans

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.     

Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats

To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > or = 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.     

The Implication of Early Chromatin Changes in X Chromosome Inactivation

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Quality of life of HIV-infected persons in Croatia

The aim of this study was to examine health-related quality of life among Croatian HIV infected individuals, and to assess the impact of socio-demographic and disease-related variables on health-related quality of life. This was a crosssectional study of 111 HIV-infected adults who received care at the University Hospital for Infectious Diseases in Zagreb, Croatia. The World Health Organization Quality of Life Questionnaire for HIV brief version (WHOQOL-HIV BREF) was used to assess each patient's quality of life. Ratings of quality of life differed across age, marital status, level of education, health status and "currently ill" status. Subjects who perceived themselves as not ill and their health status as better reported better quality of life for all quality of life domains (p < 0.01). Subjects who were in relationships gave higher ratings for the social relationships domain (p < 0.01). Subjects with higher level of education gave significantly higher ratings within the independence (p < 0.05) and environment (p < 0.01) domains. Younger subjects perceived their psychological state as better (p < 0.05). The findings demonstrate a moderate degree of overall quality of life (X = 112.2; SD = 18.92) with main concerns related to social relationships. These results highlight the need for better access to psychosocial support and medical and legal services for people living with HIV/AIDS in Croatia.     

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Resistance outside the substrate envelope: hepatitis C NS3/4A protease inhibitors

Direct acting antivirals have dramatically increased the efficacy and tolerability of hepatitis C treatment, but drug resistance has emerged with some of these inhibitors, including nonstructural protein 3/4?A protease inhibitors (PIs). Although many co-crystal structures of PIs with the NS3/4A protease have been reported, a systematic review of these crystal structures in the context of the rapidly emerging drug resistance especially for early PIs has not been performed. To provide a framework for designing better inhibitors with higher barriers to resistance, we performed a quantitative structural analysis using co-crystal structures and models of HCV NS3/4A protease in complex with natural substrates and inhibitors. By comparing substrate structural motifs and active site interactions with inhibitor recognition, we observed that the selection of drug resistance mutations correlates with how inhibitors deviate from viral substrates in molecular recognition. Based on this observation, we conclude that guiding the design process with native substrate recognition features is likely to lead to more robust small molecule inhibitors with decreased susceptibility to resistance.     

Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.     

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.     

Interferon-γ receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus

Summary Interferon-γ can facilitate the spinal nociceptive flexor reflex and elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-γ receptor (IFN-γR) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-γR in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-γR was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-γR with markers for astrocytes and microglial cells. Ultrastructurally, the IFN-γR was found predominantly in axon terminals in the lateral spinal nucleus but also at postsynaptic sites in dendrites in laminae I and II. The IFN-γR expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-γR in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not effect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-γR in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-γR to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-γ can modulate nociception at the spinal cord level.     

Consequences of Cathodal Stimulation for Behavior: When Does It Help and When Does It Hurt Performance?

Cathodal Transcranial Direct Current Stimulation (C-tDCS) has been reported, across different studies, to facilitate or hinder performance, or simply to have no tangible effect on behavior. This discrepancy is most prominent when C-tDCS is used to alter a cognitive function, questioning the assumption that cathodal stimulation always compromises performance. In this study, we aimed to study the effect of two variables on performance in a simple cognitive task (letter Flanker), when C-tDCS was applied to the left prefrontal cortex (PFC): (1) the time of testing relative to stimulation (during or after), and (2) the nature of the cognitive activity during stimulation in case of post-tDCS testing. In three experiments, we had participants either perform the Flanker task during C-tDCS (Experiment 1), or after C-tDCS. When the Flanker task was administered after C-tDCS, we varied whether during stimulation subjects were engaged in activities that posed low (Experiment 2) or high (Experiment 3) demands on the PFC. Our findings show that the nature of the task during C-tDCS has a systematic influence on the outcome, while timing per se does not.