Mature Region Research Articles

Insertions and Deletions Play an Important Role in the Diversity of Conotoxins.

Previous studies have indicated that each conotoxin precursor has a hyperconserved signal region, a rather conserved pro region and a hypervariable mature region, and nucleotide mutations are the main driven factor. However, in this study, we made an in-depth analysis on the M-superfamily conotoxin precursors and found that the diversity of the signal, pro and mature regions are more complicated than previous findings. Different conotoxin precursors can have same signal, pro and/or mature regions, especially different conotoxin precursors with same mature region but different signal and pro regions. In addition, insertions and deletions (indels) were detected in conotoxin precursors. Indels are infrequent in the signal region but frequent in the pro and mature regions. In contrast to deletions that dominate in the pro region, insertions dominate in the mature region. The number of amino acids is crucial for the physiological functions of mature conotoxins, therefore indels, especially insertions in the mature region, play an important role in the sequence and function diversity of conotoxins.

Increased Expression of Growth Hormone–Releasing Hormone in Fibrinous Inflammation of Proliferative Diabetic Retinopathy

To investigate the involvement of growth hormone-releasing hormone (GHRH) - growth hormone (GH) signaling in pathogenesis of proliferative diabetic retinopathy (PDR). Experimental laboratory study. Vitreous humor, aqueous humor, and serum were obtained from 36 eyes of 36 patients with or without type 2 diabetes from 2017 to 2019. For histologic examination, 6 fibrovascular membranes were excised from eyes with active PDR. Three fibrovascular membranes were excised from nondiabetic patients with proliferative vitreoretinopathy (PVR) as controls. In PDR, the fibrovascular tissues consisted of a mature region containing fibrocytes, and an immature region populated by abundant polymorphonuclear leukocytes in a fibrinogen meshwork. Clusters of leukocytes were found adhering to the vascular walls. In PVR, no fibrinogen and polymorphonuclear leukocyte was observed in the fibrovascular membranes. The levels of GHRH and GH in PDR were significantly increased (P < .001), with 1.8-fold and 72.8-fold in vitreous humor, and 2-fold and 4.9-fold in aqueous humor, respectively, when compared with corresponding levels in controls. No significant difference was detected for insulin-like growth factor-1. Immunohistochemistry showed intense expression of GHRH and its receptor GHRH-R in polymorphonuclear leukocytes, vascular endothelial cells, and fibrocytes in fibrovascular membranes of PDR. GHRH staining was not detectable in infiltrating cells within the fibrovascular membrane of PVR. These findings reveal a possible involvement of GHRH/GHRH-R in fibrinous inflammation that might contribute to the formation of fibrovascular membrane in PDR through mediating activities of leukocytes, vascular endothelial cells, and fibrocytes. Targeting GHRH/GHRH-R may be considered as a potential therapeutic approach for the treatment of PDR.

An integrative analysis of genome-wide association study and regulatory SNP annotation datasets identified candidate genes for bipolar disorder

BackgroundBipolar disorder (BD) is a complex mood disorder. The genetic mechanism of BD remains largely unknown.MethodsWe conducted an integrative analysis of genome-wide association study (GWAS) and regulatory SNP (rSNP) annotation datasets, including transcription factor binding regions (TFBRs), chromatin interactive regions (CIRs), mature microRNA regions (miRNAs), long non-coding RNA regions (lncRNAs), topologically associated domains (TADs) and circular RNAs (circRNAs). Firstly, GWAS dataset 1 of BD (including 20,352 cases and 31,358 controls) and GWAS dataset 2 of BD (including 7481 BD patients and 9250 controls) were integrated with rSNP annotation database to obtain BD associated SNP regulatory elements and SNP regulatory element-target gene (E–G) pairs, respectively. Secondly, a comparative analysis of the two datasets results was conducted to identify the common rSNPs and also their target genes. Then, gene sets enrichment analysis (FUMA GWAS) and HumanNet-XC analysis were conducted to explore the functional relevance of identified target genes with BD.ResultsAfter the integrative analysis, we identified 52 TFBRs target genes, 44 TADs target genes, 55 CIRs target genes and 21 lncRNAs target genes for BD, such as ITIH4 (Pdataset1 = 6.68 × 10−8, Pdataset2 = 6.64 × 10−7), ITIH3 (Pdataset1 = 1.09 × 10−8, Pdataset2 = 2.00 × 10−7), SYNE1 (Pdataset1 = 1.80 × 10−6, Pdataset2 = 4.33 × 10−9) and OPRM1 (Pdataset1 = 1.80 × 10−6, Pdataset2 = 4.33 × 10−9).ConclusionWe conducted a large-scale integrative analysis of GWAS and 6 common rSNP information datasets to explore the potential roles of rSNPs in the genetic mechanism of BD. We identified multiple candidate genes for BD, supporting the importance of rSNP in the development of BD.

Intra-Molecular Chaperone Mediated Folding of a Peptide Hormone in Molecular Evolution

Uroguanylin, an endogenous ligand of guanylyl cyclase C, consists of 16 amino acid residues and contains two intra-molecular disulfide bonds. The folding of uroguanylin is regulated by the assistance of the pro-peptide region that functions as an intra-molecular chaperone. Our previous studies suggested that a mis-bridged disulfide isomer of prouroguanylin is formed at an early stage of the refolding reaction and the molecule is then converted to the native conformation. Importantly, this folding mechanism involves a change in the secondary structure from an α-helix to a β-sheet structure at the processing site and at the mature region, respectively. However, eel-prouroguanylin possesses three Pro residues at the processing site, which inhibits α-helix formation during folding. To investigate the folding mechanism and quality control during molecular evolution, a cassette mutation was incorporated into eel and human prouroguanylin and its folding examined.The cassette mutants were expressed in E. coli cells using the T7-expression system, and produced as inclusion bodies. Therefore, the proteins were solubilized in 8 M urea containing dithiothreitol and purified by a reversed-phase high performance liquid chromatography as the reduced forms. The oxidative refolding reactions of the cassette mutants were then examined under the conditions of kinetic or thermodynamic control. The folding mechanism of the mutants and wild type prouroguanylins were compared among several species of prourogaunylin and the findings were evaluated from the standpoint of molecular evolution. The results are discussed in this paper.

Enzyme Activation Mechanism of Cocoonase

Cocoonase, a protein produced by the silkworm (Bombyx mori), consists of a pro-peptide and a mature region with three disulfide bonds. Cocoonase is known to specifically digest the sericin protein which is a coating protein of the fibroin of silkworm cocoons. However, the specificity of the protease activity and the mechanism responsible for its activation related to folding have not yet been determined in detail although its primary structure shows a high homology to that of trypsin. Therefore, to further study the activation mechanism and biological activity of cocoonase, we prepared recombinant cocoonase using an E. coli expression system, and examined its folding and enzymatic activity.For this purpose, a recombinant pro-cocoonase (pro-CCN), cocoonase (CCN), and its mutant proteins were expressed using the T7 expression system in E. coli cells. The recombinant proteins were expressed in the forms of inclusion bodies. Therefore, the refolding reaction was carried out in the presence of 2 mM GSH/1 mM GSSG under several different conditions after purification. In general, pro-CCN was treated with trypsin to eliminate the pro-peptide region for its enzyme activation. To avoid contamination by the trypsin activity in the final product, the refolded pro-CCN was further processed to the mature form, CCN, by the autolysis of the pro-peptide region in the absence of trypsin at several temperatures. The enzyme activity of the recombinant CCN derived from pro-CCN was then estimated, either by a casein assay or the hydrolysis of Bz-Arg-OEt as a substrate. In addition, to eliminate the non-specific degradation of CCN during the refolding reaction, a protease-resistant mutant was also prepared and its folding examined.

The Cenozoic magmatism of East Africa: Part III – Rifting of the craton

Magmatic activity in the Tanzania Craton-influenced regions of the East African Rift System (EARS) exhibits clear evidence of derivation from a variety of metasomatic sources. These metasomatic sources, which may span a range of ages, have likely been preserved within the lithospheric mantle. The influence of these metasomes on the composition of the rift magmas represents the single greatest heterogeneity in the composition of erupted lavas within the EARS. The earliest widespread magmatic event in the craton-influenced regions of the EARS south of Lake Turkana is the Samburu Phase (ca. 20–17 Ma), which manifests as alkaline volcanics derived from the lithospheric mantle. This phase represents the initial destabilization of the continental lithosphere as a precursor of rift development. The Flood Phonolite Phase (ca. 16–12 Ma) was most pronounced in regions south of those impacted by the prior Samburu Phase, and likely represent the continued southward expansion of lithospheric destabilization. The Mid-Miocene Resurgence Phase (ca. 12–9 Ma) is poorly represented in the Southern Kenya Rift, though magmatic activity is recorded in the Western Branch of the EARS at Kivu during this time. In contrast, the Early Rift Development Phase (ca. 9–4 Ma) is particularly well-represented in the Kenya Rift. Large-scale magmatic events include: commencement of activity in the Northern Tanzania Divergence (NTD) and Mt. Kenya, Rungwe Volcanic Province, and the Southern Kenya Rift. The Stratoid Phase (ca. 4 Ma – 0.5 Ma) commences with significant magmatic events at ca. 3 to 2.5 Ma within the Southern Kenya Rift and NTD. This phase manifests as basalts and trachytes that flood the newly formed graben in the Southern Kenya Rift. This phase also correlates with the main phase of magmatism in the NTD, the commencement of volcanism at Virunga, and the intermediate shield phase in the Rungwe Volcanic Province. The Axial Phase (0.5 Ma to present) is defined by narrow axial grabens in the Kenya Rift with central volcanoes and interposing basaltic cinder cones. In the less mature regions of the EARS, alkaline volcanoes dominate. Three distinct pulses of magmatism and extension initiated in Turkana commencing at ca. 23 Ma, 12 Ma, and 4 Ma; the consistent temporal lag in magmatic activity in the Southern EARS following each pulse reveals the southward expansion of these extensional events. Lavas at any given location within the rift exhibit a consistent temporal sequence characterized by a shift from Type II lavas (lithospheric mantle derived) to Type IV lavas (hybrid lithospheric mantle – sub-lithosphere derived) to Type III lavas (sub-lithosphere derived), requiring a progressive change in the reservoirs contributing to rift magmatism from lithospheric to sub-lithospheric. When examined in aggregate, these data paint a picture of progressive rift development and attendant lithospheric thinning, mediated by pulses of extension originating in Turkana.

Basin structure and prospectivity of the NE Atlantic volcanic rifted margin: cross-border examples from the Faroe–Shetland, Møre and Southern Vøring basins

Abstract The NE Atlantic volcanic rifted margins include vast underexplored basin areas neighbouring mature petroleum-producing regions. We appraise the cross-border prospectivity of the outer and central Faroe–Shetland, Møre and southern Vøring basins and present insights from extensive new 3D seismic surveys. Regional seismic surfaces are used to compile a cross-border seismic profile highlighting key discoveries from the UK Rosebank field in the SW to the Norwegian Ormen Lange field in the NE. Cretaceous to Paleocene reservoirs remain the main exploration focus seaward of the platform areas, and the presence of several large untested structures presents important exploration targets in the mid-Norway region. Improved imaging of the areas affected by Paleogene igneous rocks reveals major untested sub-basalt structures including some regions on the marginal highs where the basalt cover has been entirely removed by erosion, revealing sub-basalt stratigraphy and structures with pre-Cretaceous potential prospectivity. The influence of igneous rocks on both discovered and prospective hydrocarbon systems is discussed. Neogene sand injectite fields and Quaternary glacial sand bodies are extremely well imaged in the Møre Basin, documenting shallow prospectivity supported by the presence of successful regional analogue plays. New 3D seismic data are revealing previously unseen prospectivity in frontier and underexplored regions.

Entrepreneurship in Emerging Economies

Research Summary: The economic center of gravity is shifting from mature markets to emerging regions. This shift provides a good opportunity to broaden and deepen our theoretical base of concepts and frameworks because emerging and mature regions differ significantly in their institutional regimes. Hence entrepreneurial resource mobilization in emerging regions could differ significantly because of theoretical differences in actors’ action logics and resource governance. The eight papers in this special issue provide new empirical evidence on antecedents and consequences of entrepreneurial resource mobilization efforts in emerging regions. Here, we briefly summarize the state of the field, introduce the articles by situating them in a novel theoretical framework on entrepreneurial resource mobilization, and finally using our framework, we suggest opportunities for future research on entrepreneurship in emerging regions. Managerial Summary: Entrepreneurship research has advanced mainly using empirical data from the developed economies of North America and Western Europe. Because emerging economies differ markedly in their institutional development from developed economies, this prior research is less likely to be useful to understand entrepreneurship in emerging regions - which are increasingly crucial components of the global economy. This special issue contains eight articles addressing different aspects of the entrepreneurial resource mobilization process using diverse research methods on empirical data drawn from a broad range of emerging economies. This introduction describes the state of the field prior to the special issue, introduces the special issue articles and identifies topics that still need further investigation. We distill the current state of knowledge and offer a roadmap for future scholarship.

Assessing the Local Developmental Impact of Hydrocarbon Exploitation in a Mature Region: A Random Forest Approach

The impact of natural resource exploitation has been a controversial topic, subject to intense debate. The literature has traditionally focused on its consequences on national socioeconomic development. More recently, scholars concentrated on local effects following greater availability of data at the subnational and project level. We add to the literature by concentrating on Romanian oil and gas operations, a mature region with a long history of hydrocarbon activities. Such regions have seldom been studied and we argue that in light of the ongoing energy transition these should garner greater interest, particularly those located within the European Union where environmental pressure is significant. Our methodology consists of testing the ability of the random forest classification algorithm to distinguish between local communities with oil and gas operations present and those without on a number of indicators which could be broadly considered developmental. The algorithm fails to accurately classify hydrocarbon-intensive communities, indicating that there are no significant differences between these and the rest. We argue that this is likely due to the limited tax collection powers of local governments, with royalties going directly to the central government with no specific distribution provision at the local level. Another potential explanation may be the diversification of local economies and existing related manufacturing and services activities.

Is The Oil Price a Determinant of Employment in Oil Intensive Romanian Communities?

Purpose: The purpose of this study is to determine the resilience of oil intensive Romanian communities to oil price fluctuations. Design/methodology/approach: The methodology employed is based on identifying the communities with the most extensive oil extraction activities using GIS (Geographic Information System) software. We then apply a random effects panel regression model to check the significance of the oil price as a predictor of employment. Finding: Results indicate that the effect of oil price fluctuations is limited, with employment in the identified communities following the national trend. Research limitations/implications: Unfortunately, our study is hindered by data availability issues and a short time series. Nevertheless, conclusions are backed by the country’s characteristics - a diversified economy, integration with refining operations, and the presence of related manufacturing and services activities. Originality/value: The study adds to the existing literature by focusing on a mature region with a long history of hydrocarbon extraction activities. We argue that the study of local communities in mature regions is of great importance in the context of the ongoing energy transition, particularly of those located within the European Union.

Analytical Study Examines Production of Multistage Horizontal Wells in the Bakken

This article, written by JPT Technology Editor Chris Carpenter, contains highlights of paper SPE 191455-18IHFT-MS, “Twelve Years and 12,000 Multistage Horizontal Wells in the Bakken: How Is Industry Continuing To Increase the Cumulative Production per Well?” by C. Mark Pearson, SPE, Larry G. Griffin, SPE, Stacy L. Strickland, SPE, and Paul M. Weddle, SPE, Liberty Resources, prepared for the 2018 SPE International Hydraulic Fracturing Technology Conference and Exhibition, Muscat, Oman, 16–18•October. The paper has not been peer reviewed. Multistage horizontal well designs were first implemented in the Bakken in 2007. Since then, more than 12,000 wells have been completed in either the Middle Bakken or Three Forks zones. Early-time production rates, as measured by 180-day state-reported cumulative production, have increased fourfold during this period, during which industry has pursued a program of innovation and continuous improvement in completions technology, with production per well increasing in 10 of 12 years. Through a big data analytical study comparing geological data, completions parameters, and state-reported production results, the authors have evaluated the fundamental changes that have guided industry to these production rates during the period discussed. Geology The Bakken Formation is Mississippian and Devonian in age and consists of Upper, Middle, and Lower members. The focus of this paper is to review work in both the Middle Bakken member and the lower Three Forks intervals contained within the Bakken Petroleum System. The Upper and Lower Bakken shales are the prolific source rocks for the petroleum system. The Middle member consists of distinct lithofacies that range from silty sandstone on the east flank of the basin to silty dolomite on the west flank. Porosities in the Middle member range from 4–10% and permeabilities are generally less than 0.1 md. Fig. 1 shows the basin depth structure map with a color fill of the thermal maturity of the Upper Bakken shale. The onset of oil generation occurred at a pyrolysis temperature (Tmax) of greater than 430°F as indicated in the figure by shades of green and warmer colors. Oil generation in this thermally mature window created overpressure and resulted in oil mobilization into less- mature and immature regions toward the extremities of the basin. The Bakken is a basin-centered system and thus the increased pore pressure that occurred with the process of kerogen conversion and the generation of oil has resulted in pore pressure gradients as high as 0.9•psi/ft in the deepest part of the basin. Unsurprisingly, in the deepest part of the basin, water cut is the lowest, with increasing water cut with distance from the areas of greatest thermal•maturity.• The complete paper provides a detailed description of the Bakken development history, which the authors divide into seven periods beginning with first production in 1953 to the present. Production Bakken production has grown historically, but as recently as early 2004 it yielded only approximately 25,000 BOPD. The increase in drilling activity then drove production growth, with an initial peak in December 2014 of 1.22 million BOPD. The rapid decline in industry activity then resulted in a significant production drop. However, the decline reversed, and new production records of 1.23 and 1.24 million BOPD were set in June and July of 2018. The return to peak production levels has been driven by companies using more-effective•completions.

Association between MicroRNA-4669 Polymorphism and Ischemic Stroke in a Korean Population.

Recent studies have explored the association between single-nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) and ischemic stroke (IS). In particular, the associations of rs2910164 (miRNA-146A), rs11614913 (miRNA-196A2), and rs3746444 (miRNA-499A) were intensively studied in IS. In this study, we investigated the associations between SNPs in miRNAs and IS including rs2910164, rs11614913, and rs3746444 in a Korean population. For a pilot study, we selected 19 SNPs in pre-miRNA region (including mature miRNA region) and genotyped in 140 IS patients and 240 control subjects using the Fluidigm Dynamic Array. Our pilot study showed a weak association of rs79402775 in miRNA-933 (p = 0.044) and a relatively strong association of rs35196866 in miRNA-4669 (p = 0.016) with IS. From the pilot study, we selected rs79402775, rs35196866, and rs7202008 (miRNA-2117; p = 0.055) as candidate miRNA SNPs on IS and further genotyped these SNPs in 264 IS patients and 455 control subjects using direct sequencing. In addition, we further analyzed the associations of rs2910164, rs11614913, and rs3746444 that have been intensively studied in previous studies. In the further analysis, we found the significant association between rs35196866 and IS (p = 0.0014 in additive model and p = 0.00015 in dominant model; p = 0.00037 in allele frequency analysis). However, the association between rs2910164, rs11614913, rs3746444, rs79402775, and rs7202008 and IS was not shown. These results suggest that miRNA-4669 may be involved in the susceptibility of IS.

Conotoxin Diversity in the Venom Gland Transcriptome of the Magician's Cone, Pionoconus magus.

The transcriptomes of the venom glands of two individuals of the magician’s cone, Pionoconus magus, from Okinawa (Japan) were sequenced, assembled, and annotated. In addition, RNA-seq raw reads available at the SRA database from one additional specimen of P. magus from the Philippines were also assembled and annotated. The total numbers of identified conotoxin precursors and hormones per specimen were 118, 112, and 93. The three individuals shared only five identical sequences whereas the two specimens from Okinawa had 30 sequences in common. The total number of distinct conotoxin precursors and hormones for P. magus was 275, and were assigned to 53 conotoxin precursor and hormone superfamilies, two of which were new based on their divergent signal region. The superfamilies that had the highest number of precursors were M (42), O1 (34), T (27), A (18), O2 (17), and F (13), accounting for 55% of the total diversity. The D superfamily, previously thought to be exclusive of vermivorous cones was found in P. magus and contained a highly divergent mature region. Similarly, the A superfamily alpha 4/3 was found in P. magus despite the fact that it was previously postulated to be almost exclusive of the genus Rhombiconus. Differential expression analyses of P. magus compared to Chelyconus ermineus, the only fish-hunting cone from the Atlantic Ocean revealed that M and A2 superfamilies appeared to be more expressed in the former whereas the O2 superfamily was more expressed in the latter.

Transcriptomic Analysis of Marine Gastropod Hemifusus tuba Provides Novel Insights into Conotoxin Genes.

The marine gastropod Hemifusus tuba is served as a luxury food in Asian countries and used in traditional Chinese medicine to treat lumbago and deafness. The lack of genomic data on H. tuba is a barrier to aquaculture development and functional characteristics of potential bioactive molecules are poorly understood. In the present study, we used high-throughput sequencing technologies to generate the first transcriptomic database of H. tuba. A total of 41 unique conopeptides were retrieved from 44 unigenes, containing 6-cysteine frameworks belonging to four superfamilies. Duplication of mature regions and alternative splicing were also found in some of the conopeptides, and the de novo assembly identified a total of 76,306 transcripts with an average length of 824.6 nt, of which including 75,620 (99.1%) were annotated. In addition, simple sequence repeats (SSRs) detection identified 14,000 unigenes containing 20,735 SSRs, among which, 23 polymorphic SSRs were screened. Thirteen of these markers could be amplified in Hemifusus ternatanus and seven in Rapana venosa. This study provides reports of conopeptide genes in Buccinidae for the first time as well as genomic resources for further drug development, gene discovery and population resource studies of this species.

Cortex cell hydraulic conductivity, endodermal apoplastic barriers and root hydraulics change in barley (Hordeum vulgare L.) in response to a low supply of N and P.

Mineral nutrient limitation affects the water flow through plants. We wanted to test on barley whether any change in root-to-shoot ratio in response to low supply of nitrogen and phosphate is accompanied by changes in root and cell hydraulic properties and involves changes in aquaporin (AQP) gene expression and root apoplastic barriers (suberin lamellae, Casparian bands). Plants were grown hydroponically on complete nutrient solution or on solution containing only 3.3 % or 2.5 % of the control level of nutrient. Plants were analysed when they were 14-18 d old. Nutrient-limited plants adjusted water flow to an increased root-to-shoot surface area ratio through a reduction in root hydraulic conductivity (Lp) as determined through exudation analyses. Cortex cell Lp (cell pressure probe analyses) decreased in the immature but not the mature region of the main axis of seminal roots and in primary lateral roots. The aquaporin inhibitor HgCl2 reduced root Lp most in nutrient-sufficient control plants. Exchange of low-nutrient for control media caused a rapid (20-80 min) and partial recovery in Lp, though cortex cell Lp did not increase in any of the root regions analysed. The gene expression level (qPCR analyses) of five plasma membrane-localized AQP isoforms did not change in bulk root extracts, while the formation of apoplastic barriers increased considerably along the main axis of root and lateral roots in low-nutrient treatments. Decrease in root and cortex cell Lp enables the adjustment of root water uptake to increased root-to-shoot area ratio in nutrient-limited plants. Aquaporins are the prime candidate to play a key role in this response. Modelling of water flow suggests that some of the reduction in root Lp is due to increased formation of apoplastic barriers.

Management of mature pineal region teratomas in pediatric age group.

Mature pineal region teratomas differ from other pineal tumors in terms of their characteristic radiological appearance and their clinical outcome after gross total excision. Our aim is to share our clinical experience and treatment outcomes in pediatric patients with mature pineal region teratoma. In this retrospective study, we reviewed clinical, radiological, and surgical data of ten patients who had radiologically predicted diagnosis of pineal region teratoma and pathologically confirmed diagnosis of mature pineal region teratoma between years 2004 and 2017 in our clinic. Statistical analysis was performed with SPSS 20. All patients were male. Ages of patients ranged between 5 and 17 (median age was 9.5). All of them presented with headache. Magnetic resonance imaging showed characteristic appearance of teratomas with variable degree of hydrocephalus. All patients had negative results for AFP and b-HCG levels. All patients had gross total resection of pineal tumor through occipital transtentorial approach with no permanent neurological deficit. Pathological results of all tumor samples were consistent with mature teratoma. None of them had adjuvant chemotherapy or radiotherapy. Follow-up periods ranged between 3 and 170months (median follow-up period was 60.5months). All patients are alive with no tumor recurrence. Pediatric mature pineal region teratomas are benign tumors with characteristic MRI appearance and negative tumor markers. Their definitive treatment is gross total surgical excision. Occipital transtentorial approach is a safe procedure for treatment of pediatric mature pineal teratomas.

High-Throughput Identification and Analysis of Novel Conotoxins from Three Vermivorous Cone Snails by Transcriptome Sequencing.

The venom of each Conus species consists of a diverse array of neurophysiologically active peptides, which are mostly unique to the examined species. In this study, we performed high-throughput transcriptome sequencing to extract and analyze putative conotoxin transcripts from the venom ducts of 3 vermivorous cone snails (C. caracteristicus, C. generalis, and C. quercinus), which are resident in offshore waters of the South China Sea. In total, 118, 61, and 48 putative conotoxins (across 22 superfamilies) were identified from the 3 Conus species, respectively; most of them are novel, and some possess new cysteine patterns. Interestingly, a series of 45 unassigned conotoxins presented with a new framework of C-C-C-C-C-C, and their mature regions were sufficiently distinct from any other known conotoxins, most likely representing a new superfamily. O- and M-superfamily conotoxins were the most abundant in transcript number and transcription level, suggesting their critical roles in the venom functions of these vermivorous cone snails. In addition, we identified numerous functional proteins with potential involvement in the biosynthesis, modification, and delivery process of conotoxins, which may shed light on the fundamental mechanisms for the generation of these important conotoxins within the venom duct of cone snails.

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BackgroundTolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3–5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen.Methods/Principle findingsGenome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis.Conclusion/SignificanceThis study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.

Determination of Evolution Stages of Landform: A Comparative Study of Mountain-Plain (Kosi, Bihar) and Plateau-Plain (Kangsabati, WB) Regions of Tropical India

The major aim of quantitative geomorphology is to understand the geomorphological stages of evolution of any area. The quantitative revolution and the development of remote sensing and GIS techniques have brought greater attention to this field of analysis. We have used a morphometric analysis, which is an important indicator to understand the geomorphic stages of evolution of any drainage basin, to compare the drainage basin characteristics and related stages of evolution of a mountain-plain and plateau-plain drainage basin in tropical India. The Kosi basin has been selected for the mountain-plain area and Kangsabati basin is the chosen region for the plateau-plain area. Different drainage morphometric parameters and measurements related to linear, areal, relief characteristics have been determined through the use of SRTM (Shuttle Radar Topographic Mission) GeoDEM and ARC GIS 10.1. Area-altitude relationship and hypsometric characteristics have also been accessed to identify the stages of geomorphic evolution. All the relief characteristics indicate Kosi in a young or rejuvenated stage when compared to the mature plateau region of the river of Kangsabati. Morphometric characteristics also indicate that there are high geologic and geomorphological controls on river basin characteristics.

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.