A new species of Macrobrachium Bate, 1868 (Decapoda, Palaemonidae) from the dolphin trenches of Kulsi River, N. India, possibly under threat

The Kulsi River, a major tributary of Brahmaputra River, N. India is reported to have resident population of Ganges river dolphin, Platanista gangetica (Roxburgh), which feeds on fishes and prawns. While surveying for the dolphins of the river, a number of fishes and prawns were collected. On identification, one of the prawns was found to be undescribed, and hence is described herein. The ecology of the river consisted of: temperature fluctuating widely from 15 to 28 °C, depth from 0.8 to 10 m, turbidity of 11-19 cm, sand mining @ 12,500 MT annually, and fish catch of 300-800 kg (from 1.5 km area). All these factors pose a great threat to the fish and prawn wealth of the river.Macrobrachium kulsiense sp. nov. is a very small sized prawn (maximum size - 34.5 mm in total length), exhibiting species-specific characters, such as highly elevated and moderately long rostrum with 9-12 dorsal teeth, a single ventral tooth, and percentage ratios of ischium, merus, carpus, palm, dactylus of first and second chelipeds (19.05:28.57:33.33:09.52:09.52 and 21.43:25.00:21.43:14.28:17.86, respectively). The species shows close similarity with Macrobrachium mirabile (Kemp). The females are larger than males, eggs are large in size (1.2×0.9 mm) and fecundity is low (15-20).     

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia ^1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens ⁴), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs ^{4, 5}. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions³. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers ^{6, 7}, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane ^{8, 9} and mask RBC surface antigens^{10, 11}.     

Time-resolved Analysis of the Matrix Metalloproteinase 10 Substrate Degradome

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.Historically regarded as a mechanism for unspecific degradation of proteins, proteolysis is now recognized as a specific irreversible post-translational modification that affects major intra- and intercellular signaling processes (1, 2). Proteases specifically process bioactive proteins, their receptors, and associated proteins in an interconnected interaction network termed the protease web (3). Dysregulation of the protease web might cause or result from pathologies, such as impaired tissue repair, cancer and neurodegenerative diseases. Therefore, a better understanding of the functions of individual proteases and their interconnections within proteolytic networks is a prerequisite for exploiting proteases as targets for therapeutic intervention (4).To address this issue, several powerful technologies have been developed for the system-wide discovery of protease substrates, i.e. substrate degradomes, in complex and active proteomes (5, 6). A common principle of these mass spectrometry-based methods is the enrichment and monitoring of N-terminal peptides (protein neo-N termini) that are newly generated by a test protease (7). Protein N termini are enriched from complex proteomes either by chemical tagging and affinity resins (positive selection) or by depletion of internal peptides (negative selection) (7). Both principles have been successfully applied in various studies to characterize N-terminomes and to identify protease substrates using in vitro or cell-based systems and more recently also in vivo (8, 9). Negative enrichment approaches were further extended to the analysis of protein C termini (10, 11) and have the general advantage of recording data on naturally blocked (e.g. acetylated) N termini and internal peptides in the same experiment (8).Even if successful in identifying novel proteolytic cleavage events, which could also be validated by orthogonal methods, high-throughput substrate discovery approaches potentially suffer from high numbers of false positive identifications, particularly when employing in vitro systems (12). These have been reduced by monitoring abundances of N-terminal peptides at multiple time points after incubation of a proteome with a test protease (12). In this SILAC-based approach the authors efficiently distinguished critical from bystander cleavages, but it was limited to three time points. Therefore, it did not allow recording kinetic profiles of the relative abundance of N-terminal peptides that are required for determination of apparent kinetic parameters for processing events. Agard et al. elegantly overcame this limitation by use of selected reaction monitoring (SRM)¹ in combination with a positive N-terminal enrichment platform and determined apparent catalytic efficiencies for hundreds of caspase cleavage events in parallel (13). In a similar approach the same group characterized cellular responses to pro-apoptotic cancer drugs by recording time-courses for caspase-generated neo-N termini (14). Although very powerful and highly accurate in quantification, this method strongly exploited the canonical cleavage specificity of caspases after aspartate residues and required a two-stage process involving two types of mass spectrometers. Hence, it would be desirable to monitor the time-resolved generation of neo-N termini in complex proteomes in a single experiment by a simple and robust workflow in an unbiased manner.The development of such an analysis platform would require a reliable method for the system-wide characterization of protein N termini that is easy to perform, fast and highly multiplexible. All these criteria are met by iTRAQ-terminal amine isotopic labeling of substrates (TAILS), a multiplex N-terminome analysis technique that has been applied in 2plex and 4plex experiments to map the matrix metalloproteinase (MMP) 2 and MMP9 substrate degradomes in vitro (15) and most recently to quantitatively analyze the proteome and N-terminome of inflamed mouse skin in the presence or absence of the immune-modulatory protease MMP2 in vivo (8).Here, we exploited the multiplex capabilities of iTRAQ-TAILS by use of 8plex-iTRAQ reagents to monitor the generation of neo-N-terminal peptides by a test protease in complex samples over time. First, using GluC as a test protease with canonical cleavage specificity, we established a workflow for time-resolved substrate degradomics. Recording kinetic profiles significantly increased the confidence in identified cleavage events compared with binary systems and categorized primary cleavage specificities as well as secondary structure elements based on clusters of processing events with different efficiencies. By including data from before N-terminal enrichment, we extended our analysis to neo-C-terminal peptides and concomitantly monitored the generation of neo-N termini and neo-C termini as well as the decrease in abundance of the tryptic peptides spanning the cleavage sites in the same experiment. Next, we applied this approach to the time-resolved analysis of the hardly elucidated substrate degradome of matrix metalloproteinase 10 (MMP10). This important wound- and tumor-related protease is secreted by proliferating and migrating keratinocytes at the wound edge in close proximity to dermal fibroblasts and is also highly expressed in aggressive tumor cells (16–18). Our analysis revealed MMP10-dependent shedding of the platelet-derived growth factor receptor alpha (PDGFRα), processing of ADAMTS-like protein 1 (ADAMTSL1) and multiple cleavages of type I collagen, which could be validated and classified by time-resolved abundance profiles of their corresponding neo-N termini.     

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.     

Analysis of the role of oligosaccharides in the apoptotic activity of glycodelin A

Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy. It is a known suppressor of T cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation (Asn(28) and Asn(63)) to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at Asn(28) appears to play a role in the extracellular secretion of the molecule, as mutation of Asn(28) resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.     

How large is an alpha-helix? Studies of the radii of gyration of helical peptides by small-angle X-ray scattering and molecular dynamics

Using synchrotron radiation and the small-angle X-ray scattering technique we have measured the radii of gyration of a series of alanine-based alpha-helix-forming peptides of the composition Ace-(AAKAA)(n)-GY-NH(2), n=2-7, in aqueous solvent at 10(+/-1) degrees C. In contrast to other techniques typically used to study alpha-helices in isolation (such as nuclear magnetic resonance and circular dichroism), small-angle X-ray scattering reports on the global structure of a molecule and, as such, provides complementary information to these other, more sequence-local measuring techniques. The radii of gyration that we measure are, except for the 12-mer, lower than the radii of gyration of ideal alpha-helices or helices with frayed ends of the equivalent sequence-length. For example, the measured radius of gyration of the 37-mer is 14.2(+/-0.6)A, which is to be compared with the radius of gyration of an ideal 37-mer alpha-helix of 17.6A. Attempts are made to analyze the origin of this discrepancy in terms of the analytical Zimm-Bragg-Nagai (ZBN) theory, as well as distributed computing explicit solvent molecular dynamics simulations using two variants of the AMBER force-field. The ZBN theory, which treats helices as cylinders connected by random walk segments, predicts markedly larger radii of gyration than those measured. This is true even when the persistence length of the random walk parts is taken to be extremely short (about one residue). Similarly, the molecular dynamics simulations, at the level of sampling available to us, give inaccurate values of the radii of gyration of the molecules (by overestimating them by around 25% for longer peptides) and/or their helical content. We conclude that even at the short sequences examined here (< or =37 amino acid residues), these alpha-helical peptides behave as fluctuating semi-broken rods rather than straight cylinders with frayed ends.     

An ultra-stable redox-controlled self-assembling polypeptide nanotube for targeted imaging and therapy in cancer

We introduce a self-assembling polypeptide-based nanotube system having the ability to specifically target cancer cells. The nanotubes target the cancer cell surface through integrin engagement with the help of multiple RGD units present along their surface. While the nanotubes are non-toxic towards cells in general, they can be loaded with suitable drugs to be released in a sustained manner in cancer cells. In addition, the nanotubes can be utilized for cellular imaging using any covalently tagged fluorescent dye. They are stable over a wide range of temperature due to intermolecular disulphide bonds formed during the self-assembly process. At the same time, presence of disulphide bonds provides a redox molecular switch for their degradation. Taken together this system provides a unique avenue for multimodal formulation in cancer therapy.

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<Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> – a potential biological control agent for the invasive ‘old world climbing fern’ (<Emphasis Type="Italic">Lygodium microphyllum</Emphasis>)

One of the greatest threats to the native ecosystems in any part of the world is the invasion and permanent colonization of ecosystems by non-native species. Florida is no exception to this biological invasion, and is currently colonized by an extensive variety of exotic plant species. Originally imported from Asia over 30 years ago, Old World Climbing Fern Lygodium microphyllum (Cavanilles) R. Brown) has become one of the most invasive and destructive weeds in southern Florida. To date different effective control measures of its growth and spread have not been successful; fire and herbicide applications that are currently employed are neither cost effective nor environmentally friendly. In light of the highly delicate ecosystem that is being affected by L. microphyllum, we tested the soil fungus Myrothecium verrucaria (Albertini and Schwein) Ditmar: Fr. for its pathogenicity on the invasive fern. In greenhouse studies the effect of two conidial concentrations of M. verrucaria on L. microphyllum was investigated. Plants were spray inoculated with M. verrucaria which resulted in successful disease development with leaf necrosis symptoms. The higher conidial concentration (1 × 10⁸ ml⁻¹) produced a disease index of approximately 3 on a scale of 0 to 4, day 24 postinitial inoculation, demonstrating the efficacy of this fungus as a severe retardant of Lygodium growth. Preliminary screening of selected native plant species for susceptibility to M. verrucaria showed low disease indices after repeated spray inoculations; the highest index attained was 0.4 by Slash pine (Pinus elliottii).