New conformationally restricted analog of the immunosuppressory mini-domain of HLA-DQ and its biological properties

Our previous studies revealed that the nonapeptide fragment of HLA-DQ located in the beta 164-172 loop of the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr sequence suppresses the immune humoral and cellular responses [30]. Based on the crystal structure of HLA-class II molecules we designed and synthesized a cyclic analog with restricted conformation, cyclo(Suc-Thr-Pro-Gln-Arg-Gly-Asp-Val-Lys)-Thr-OH (Suc = succinyl) by reacting a Lys side chain with a succinylated N-terminus. The cyclization product more potently suppresses the cellular immune response than its linear counterparts and is efficiently cleaved by trypsin. The results indicate that the beta 164-172 loop may serve as a functional epitope on the HLA class II surface for intermolecular binding.     

Cholesterol sulphate sulphohydrolase from human placenta microsomes--purification and properties of the dephosphorylated form of enzyme

The procedure for purification of cholesterol sulphate sulphohydrolase (ChS-ase) from human placenta microsomes was elaborated. The highy purified enzyme preparation (specific activity 2000 nmol×min⁻¹×mg protein⁻¹) exhibited optimal activity at pH 9.0. The K_m value was established to be 1.5±0.85×10⁻⁵ M. The high molecular weight form (200 kDa) and the low molecular weight form (20 kDa) of the enzyme were separated. The interconversion of the high molecular weight variant into the low one occurs under the influence of dephosphorylation. Both forms exhibited typical Michaelis–Menten saturation kinetics. The effect of different compounds on the enzyme activity was tested.     

8-N(3)-3'-biotinyl-ATP, a novel monofunctional reagent: differences in the F(1)- and V(1)-ATPases by means of the ATP analogue

A novel photoaffinity label, 8-N(3)-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N(3)-3'-biotinyl-ATP.     

A Standardized Reference Data Set for Vertebrate Taxon Name Resolution

Taxonomic names associated with digitized biocollections labels have flooded into repositories such as GBIF, iDigBio and VertNet. The names on these labels are often misspelled, out of date, or present other problems, as they were often captured only once during accessioning of specimens, or have a history of label changes without clear provenance. Before records are reliably usable in research, it is critical that these issues be addressed. However, still missing is an assessment of the scope of the problem, the effort needed to solve it, and a way to improve effectiveness of tools developed to aid the process. We present a carefully human-vetted analysis of 1000 verbatim scientific names taken at random from those published via the data aggregator VertNet, providing the first rigorously reviewed, reference validation data set. In addition to characterizing formatting problems, human vetting focused on detecting misspelling, synonymy, and the incorrect use of Darwin Core. Our results reveal a sobering view of the challenge ahead, as less than 47% of name strings were found to be currently valid. More optimistically, nearly 97% of name combinations could be resolved to a currently valid name, suggesting that computer-aided approaches may provide feasible means to improve digitized content. Finally, we associated names back to biocollections records and fit logistic models to test potential drivers of issues. A set of candidate variables (geographic region, year collected, higher-level clade, and the institutional digitally accessible data volume) and their 2-way interactions all predict the probability of records having taxon name issues, based on model selection approaches. We strongly encourage further experiments to use this reference data set as a means to compare automated or computer-aided taxon name tools for their ability to resolve and improve the existing wealth of legacy data.     

Habitat displacement effect between two competing owl species in fragmented forests

Many owl species use the same nesting and food resources, which causes strong interspecific competition and spatio-temporal niche separation. We made use of a recent colonisation of Ural Owls (Strix uralensis) in southern Poland to compare habitat preferences of Tawny Owls (Strix aluco) allopatry and sympatry with Ural Owls. We investigated spatial niche segregation of Ural Owl and the Tawny Owl in sympatry and compared habitat preferences of Tawny Owls breeding in allopatry and sympatry. Tawny Owls breeding in sympatry with Ural Owls occupied forests with higher canopy compactness, sites located closer to forest border and to built-up areas, as well as stands with a higher share of fir and spruce and a lower share of beech as compared to sites occupied by Ural Owls. Allopatric Tawny Owls occupied sites with lower canopy compactness and bred at sites located further from forest borders and in stands with lower share of fir and spruce and a higher share of deciduous as compared to sympatric Tawny Owls. As Ural owls are dominant in relation to Tawny Owls, this indicates that the presence of Ural Owls prevents Tawny Owls from occupying deciduous-dominated and old stands located in forest interior areas, far from buildings and forest edges. The results support habitat displacement between the two species when breeding in sympatry. We also show that protection of large forest patches is crucial for the Ural Owl, a species still rare in central Europe, while small patches are occupied by the abundant Tawny Owl.     

The GBIF Integrated Publishing Toolkit: Facilitating the Efficient Publishing of Biodiversity Data on the Internet

The planet is experiencing an ongoing global biodiversity crisis. Measuring the magnitude and rate of change more effectively requires access to organized, easily discoverable, and digitally-formatted biodiversity data, both legacy and new, from across the globe. Assembling this coherent digital representation of biodiversity requires the integration of data that have historically been analog, dispersed, and heterogeneous. The Integrated Publishing Toolkit (IPT) is a software package developed to support biodiversity dataset publication in a common format. The IPT’s two primary functions are to 1) encode existing species occurrence datasets and checklists, such as records from natural history collections or observations, in the Darwin Core standard to enhance interoperability of data, and 2) publish and archive data and metadata for broad use in a Darwin Core Archive, a set of files following a standard format. Here we discuss the key need for the IPT, how it has developed in response to community input, and how it continues to evolve to streamline and enhance the interoperability, discoverability, and mobilization of new data types beyond basic Darwin Core records. We close with a discussion how IPT has impacted the biodiversity research community, how it enhances data publishing in more traditional journal venues, along with new features implemented in the latest version of the IPT, and future plans for more enhancements.     

Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions

Mammalian oocytes are long-lived cells in the human body. They initiate meiosis already in the embryonic ovary, arrest meiotically for long periods in dictyate stage, and resume meiosis only after extensive growth and a surge of luteinizing hormone which mediates signaling events that overcome meiotic arrest. Few mitochondria are initially present in the primordial germ cells while there are mitogenesis and structural and functional differentiation and stage-specific formation of functionally diverse domains of mitochondria during oogenesis. Mitochondria are most prominent cell organelles in oocytes and their activities appear essential for normal spindle formation and chromosome segregation, and they are one of the most important maternal contributions to early embryogenesis. Dysfunctional mitochondria are discussed as major factor in predisposition to chromosomal nondisjunction during first and second meiotic division and mitotic errors in embryos, and in reduced quality and developmental potential of aged oocytes and embryos. Several lines of evidence suggest that damage by oxidative stress/reactive oxygen species in dependence of age, altered antioxidative defence and/or altered environment and bi-directional signaling between oocyte and the somatic cells in the follicle contribute to reduced quality of oocytes and blocked or aberrant development of embryos after fertilization. The review provides an overview of mitogenesis during oogenesis and some recent data on oxidative defence systems in mammalian oocytes, and on age-related changes as well as novel approaches to study redox regulation in mitochondria and ooplasm. The latter may provide new insights into age-, environment- and cryopreservation-induced stress and mitochondrial dysfunction in oocytes and embryos.     

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca²⁺ sensitivity and cardiomyocyte contractility without changes in intracellular Ca²⁺ transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.     

Detection of HIV-1 Neutralizing Antibodies in a Human CD4+/CXCR4+/CCR5+ T-Lymphoblastoid Cell Assay System

Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α₄β₇+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens.     

Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.