The H3 loop of antibodies shows unique structural characteristics

The H3 loop in the Complementarity Determining Region of antibodies plays a key role in their ability to bind the diverse space of potential antigens. It is also exceptionally difficult to model computationally causing a significant hurdle for in silico development of antibody biotherapeutics. In this article, we show that most H3s have unique structural characteristics which may explain why they are so challenging to model. We found that over 75% of H3 loops do not have a sub‐Angstrom structural neighbor in the non‐antibody world. Also, in a comparison with a nonredundant set of all protein fragments over 30% of H3 loops have a unique structure, with the average for all of other loops being less than 3%. We further observed that this structural difference can be seen at the level of four residue fragments where H3 loops present numerous novel conformations, and also at the level of individual residues with Tyrosine and Glycine often found in energetically unfavorable conformations. Proteins 2017; 85:1311–1318. © 2017 Wiley Periodicals, Inc.     

Gelatinous invertebrate zooplankton of the South Adriatic: species composition and vertical distribution

The species composition and vertical distribution of gelatinousinvertebrate zooplankton were investigated from April 1993 toJune 1994 in the 0–1000 m water column at a deep-sea stationin the northern part of the South Adriatic Pit. Fifty-sevenspecies were identified: 11 hydromedusae, 13 calycophores, 3ctenophores, 3 heteropods, 10 pteropods, 8 polychaetes and 9chaetognaths. The pteropod Desmopterus papilio and the heteropodProtatlanta mediterranea were recorded for the first time inthe Adriatic Sea. Data from this study differed from those ofprevious investigations in the South Adriatic as regards thenumerically dominant polychaete and pteropod species. All investigatedgroups generally were more abundant in the upper 100 m and decreasedwith depth. Different vertical distributions of life stageswere observed for those species that occupy a wide depth range:Persa incolorata, Solmissus albescens, Limacina inflata, Cymbuliaperoni, Pelagobia longicirrata, Sagitta lyra and Sagitta decipiens.     

The epidemiological investigation of Trichinella infection in brown rats (Rattus norvegicus) and domestic pigs in Croatia suggests that rats are not a reservoir at the farm level

Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering.     

Secular change in body height and cephalic index of Croatian medical students (University of Rijeka)

An investigation of body height and cephalic measurements was performed among five groups of first-year medical students of the University of Rijeka School of Medicine (Rijeka, Croatia). Body height and different cephalic measurements showed normal distribution, both in male and female students. Differences between measured variables were statistically analyzed by ANOVA. No significant difference with regard to year of birth was found in either males or females. The cephalic index showed no statistically significant difference between sexes or with regard to body height, while head breadth and length correlated significantly with birth year and body height, both in males and females. Head breadth decreased within the study period, while head length increased. Results were compared with those of similar studies from the mid-20th century. Student's t-test showed a significant change of cephalic indices and other head measurements, but not of body height, in males. The frequency difference between various head shapes was tested using the chi-square test. A significant increase of dolichocephalic and mesocephalic and a significant decrease of brachycephalic head shape were found in both sexes. These results suggest a continuity of the debrachycephalization process observed in our population at the past midcentury.     

In mammalian skeletal muscle,phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2β-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.     

Structure and characterization of a high affinity C5a monoclonal antibody that blocks binding to C5aR1 and C5aR2 receptors

C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a–C5aR1 receptor are well defined, whereas C5a–C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement–mediated bacterial cell killing. Unlike other anti–C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a–C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia–reperfusion injury.     

ECM Cross-Linking Regulates Invadopodia Dynamics

Invadopodia are membrane protrusions dynamically assembled by invasive cancer cells in contact with the extracellular matrix (ECM). Invadopodia are enriched by the structural proteins actin and cortactin as well as metalloproteases such as MT1-MMP, whose function is to degrade the surrounding ECM. During metastasis, invadopodia are necessary for cancer cell intravasation and extravasation. Although signaling pathways involved in the assembly and function of invadopodia are well studied, few studies address invadopodia dynamics and how the cell-ECM interactions contribute to cell invasion. Using iterative analysis based on time-lapse microscopy and mathematical modeling of invasive cancer cells, we found that cells oscillate between invadopodia presence and cell stasis—termed the “invadopodia state”—and invadopodia absence during cell translocation—termed the “migration state.” Our data suggest that β1-integrin-ECM binding and ECM cross-linking control the duration of each of the two states. By changing the concentration of cross-linkers in two-dimensional and three-dimensional cultures, we generate an ECM in which 0–0.92 of total lysine residues are cross-linked. Using an ECM with a range of cross-linking degrees, we demonstrate that the dynamics of invadopodia-related functions have a biphasic relationship to ECM cross-linking. At intermediate levels of ECM cross-linking (0.39), cells exhibit rapid invadopodia protrusion-retraction cycles and rapid calcium spikes, which lead to more frequent MT1-MMP delivery, causing maximal invadopodia-mediated ECM degradation. In contrast, both extremely high or low levels of cross-linking lead to slower invadopodia-related dynamics and lower ECM degradation. Additionally, β1-integrin inhibition modifies the dynamics of invadopodia-related functions as well as the length of time cells spend in either of the states. Collectively, these data suggest that β1-integrin-ECM binding nonlinearly translates small physical differences in the extracellular environment to differences in the dynamics of cancer cell behaviors. Understanding the conditions under which invadopodia can be reduced by subtle environment-targeting treatments may lead to combination therapies for preventing metastatic spread.     

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation.     

Come to the dark side! The role of functional traits in shaping dark diversity patterns of south-eastern European hoverflies

1. Dark diversity represents the set of species that can potentially inhabit a given area under particular ecological conditions, but are currently ‘missing’ from a site. This concept allows characterisation of the mechanisms determining why species are sometimes absent from an area that seems ecologically suitable for them. 2. The aim of this study was to determine the dark diversity of hoverflies in south-eastern Europe and to discuss the role of different functional traits that might increase the likelihood of species contributing to dark diversity. Based on expert opinion, the Syrph the Net database and known occurrences of species, the study estimated species pools, and observed and dark diversities within each of 11 defined vegetation types for 564 hoverfly species registered in south-eastern Europe. To detect the most important functional traits contributing to species being in dark diversity across different vegetation types, a random forest algorithm and respective statistics for variable importance were used. 3. The highest dark diversity was found for southwest Balkan sub-Mediterranean mixed oak forest type, whereas the lowest was in Mediterranean mixed forest type. Three larval feeding modes (saproxylic, and phytophagous on bulbs or roots) were found to be most important for determining the probability of a species contributing to hoverfly dark diversity, based on univariate correlations and random forest analysis. 4. This study shows that studying dark diversity might provide important insights into what drives community assembly in south-eastern European hoverflies, especially its missing components, and contributes to more precise conservation prioritisation of both hoverfly species and their habitats.