Open Source Tool for VH-replacement Products Discovery and Analysis

In this project an open source tool for discovery and analysis of abnormal immunoglobulin genes rearrangements was developed. The main goal is in finding so-called “footprints” - small parts of incompletely rearranged V-segments in IGH genes. Joint usage of publicly available databases, on-line markup IMGT V-Quest and clonal families analyzer Partis allowed to prepare an informative dataset, which was processed via the developed tool. A few dependencies between patient phenotype and footprints statistics were discovered

B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma

LuxO controls luxR expression in Vibrio harveyi : evidence for a common regulatory mechanism in Vibrio

Quorum-sensing control of luminescence in Vibrio harveyi , which involves an indirect autoinducer-mediated phosphorelay signal transduction system, contrasts with the prototypical quorum-sensing system of Vibrio fischeri , in which the autoinducer and the transcriptional activator LuxR directly activate lux operon expression. In V. harveyi , a regulator not homologous to V. fischeri LuxR and also designated LuxR (LuxR vh ), binds specifically to the lux operon promoter region and activates the expression of luminescence. A direct connection has not been identified previously between V. harveyi LuxR vh and the autoinducer-mediated phosphorelay system. Here, we demonstrate by mobility shift assays and measurement of luxR vh mRNA levels with luxO + and luxO – cells that the central response regulator of the V. harveyi phosphorelay system (LuxO) represses the level of LuxR vh . Expression of a luxR vh -bearing plasmid strongly stimulated luminescence of a luxO – mutant but had no effect on luminescence of wild-type luxO + cells, indicating tight regulation of luxR vh by LuxO. Furthermore, luxO null mutants of V. fischeri MJ-1 and two autoinducer mutants, MJ-211 ( luxI – ) and MJ-215 ( luxI – ainS – ), emitted more light and exhibited more elevated levels of litR , a newly identified V. harveyi luxR vh homologue, than their luxO + counterparts. These results suggest that activity of the autoinducer-mediated phosphorelay system is coupled to LuxR vh /LitR control of luminescence through LuxO in V. harveyi and V. fischeri . The presence of homologues of V. harveyi LuxR vh , LuxO and other phosphorelay system proteins in various Vibrio species and the control of LuxR vh and its homologues by LuxO identified here in V. harveyi and V. fischeri and recently in Vibrio cholerae suggest that the luxO–luxR vh couple is a central feature of this quorum-sensing system in members of the genus Vibrio .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72700/1/j.1365-2958.2003.03453.x.pd

Development of a Dusky kob scFv gene phage display library for the discovery of antibodies to Brome mosaic virus - a proxy for a novel, emerging fish pathogen

Fish farming is rapidly becoming the world's fastest growing production sector, achieving an annual growth rate of approximately 8.9% since the early 1970s. However, high stocking densities result in elevated stress levels in farmed fish, leading to increased susceptibility to infection by opportunistic pathogens and parasites. Antibody phage display is a method that allows foreign peptides or proteins to be expressed on the phage surface through translational fusion with phage coat proteins. Consequently, antibodies expressed by a diverse repertoire of genes coding for the single chain variable fragment (scFv) of immunoglobulin M can be isolated and screened for affinity to a specific infectious agent or parasite. In this study, a phage display library displaying scFvs derived from combination pairings of Dusky kob (Argyrosomus japonicas) variable heavy and light chain fragments, sourced from the splenic B cells of healthy Dusky kob, was constructed. The library was subjected to two rounds of biopanning against brome mosaic virus (BMV), a grass virus to which Dusky kob would have no prior exposure that served as a proxy for an emerging fish pathogen. Five clones were identified as having high affinity and specificity to BMV, as determined by phage enzymelinked immunosorbent assay (ELISA) and phage western blot analysis, respectively. To validate the diagnostic and therapeutic potential of antibody fragments isolated from this phage display library, the gene encoding the antibody fragment of the clone displaying the highest affinity to BMV was selected and expressed using a yeast surface display system. ELISA analysis of serum sampled from Dusky kob exposed to BMV by injection demonstrated that the yeast displayed anti-BMV antibody could successfully detect BMV in the blood serum of BMV-infected Dusky kob with similar sensitivity to a commercially available counterpart. Similarly, this study demonstrated the neutralising effect of yeast displayed anti-BMV antibodies which were found to successfully reduce BMV infection in barley. Overall, these findings demonstrate the feasibility of a Dusky kob phage display library as a source of diagnostically and therapeutically important antibodies against emerging fish pathogens or parasites that threaten the fish farming industry of South Africa

Positive Influence of a Probiotic Mixture on the Intestinal Morphology and Microbiota of Farmed Guinea Fowls (Numida meleagris)

To understand the effectiveness of a probiotic mixture on intestinal morphology, mucus layer composition, and cecal microbiota diversity, 40 10-day-old Guinea fowls (Numida meleagris) were assigned to two groups: the control group (C), receiving drinking water, and the treated group (P), receiving water plus a commercial multi-strain probiotic (Slab51®, 2 × 1011 CFU/L). Birds were slaughtered after 4 months, and the intestines were collected. Samples from the duodenum, ileum, and cecum were processed for morphological and morphometric studies, and conventional glycohistochemistry. Cecal samples were also used to assess the microbiota by 16S metataxonomic approach. Group P showed significant increase in the villus height (p < 0.001 in the duodenum and p < 0.05 in the ileum and cecum), villus width (p < 0.05 in all investigated tracts), depth of crypts (p < 0.001 in the duodenum and cecum; p < 0.05 in the ileum), and goblet cells per villus (p < 0.001 in all investigated tracts) compared with group C. Cecal microbiota of the birds varied considerably and comparing the relative abundance of the main observational taxonomic units (OTUs), a positive enrichment of several beneficial taxa, such as Oscillospira, Eubacterium, Prevotella, and members of the Ruminococcaceae, was observed. The enrichment of those taxa can improve microbiota stability and resilience facing environmental stresses, enhancing its resistance against invading pathogens. Ruminococcaceae, which represent the most important taxon in both groups, and Prevotella have a key role in the gut physiology due to the production of short-chain fatty acids (SCFAs), which are a vital energy source for enterocytes, improve glucose metabolism, and exert an overall anti-inflammatory effect. Probiotic administration enriches the presence of Coprococcus, Oscillospira, and Eubacterium taxa that produce butyrate, which exerts a beneficial effect on growth performance, structure of villi, and pathogen control and has anti-inflammatory properties too. This study indicates that Slab51® supplementation positively affects the morphology and microbiota diversity of the guinea fowl intestine

Light dark Higgs boson in minimal sub-GeV dark matter scenarios

Minimal scenarios with light (sub-GeV) dark matter whose relic density is obtained from thermal freeze-out must include new light mediators. In particular, a very well-motivated case is that of a new "dark" massive vector gauge boson mediator. The mass term for such mediator is most naturally obtained by a "dark Higgs mechanism" which leads to the presence of an often long-lived dark Higgs boson whose mass scale is the same as that of the mediator. We study the phenomenology and experimental constraints on two minimal, self-consistent dark sectors that include such a light dark Higgs boson. In one the dark matter is a pseudo-Dirac fermion, in the other a complex scalar. We find that the constraints from BBN and CMB are considerably relaxed in the framework of such minimal dark sectors. We present detection prospects for the dark Higgs boson in existing and projected proton beam-dump experiments. We show that future searches at experiments like Xenon1T or LDMX can probe all the relevant parameter space, complementing the various upcoming indirect constraints from astrophysical observations.Comment: 32 pages, 10 figures, 4 tables. An error in the estimation of the dark matter annihilation rates has been corrected and all figures as well as section 3.2 have been accordingly updated. References updated, typos corrected, matches version published in JHE

Smart Sustainable Manufacturing Systems

With the advent of disruptive digital technologies, companies are facing unprecedented challenges and opportunities. Advanced manufacturing systems are of paramount importance in making key enabling technologies and new products more competitive, affordable, and accessible, as well as for fostering their economic and social impact. The manufacturing industry also serves as an innovator for sustainability since automation coupled with advanced manufacturing technologies have helped manufacturing practices transition into the circular economy. To that end, this Special Issue of the journal Applied Sciences, devoted to the broad field of Smart Sustainable Manufacturing Systems, explores recent research into the concepts, methods, tools, and applications for smart sustainable manufacturing, in order to advance and promote the development of modern and intelligent manufacturing systems. In light of the above, this Special Issue is a collection of the latest research on relevant topics and addresses the current challenging issues associated with the introduction of smart sustainable manufacturing systems. Various topics have been addressed in this Special Issue, which focuses on the design of sustainable production systems and factories; industrial big data analytics and cyberphysical systems; intelligent maintenance approaches and technologies for increased operating life of production systems; zero-defect manufacturing strategies, tools and methods towards online production management; and connected smart factories

Sixth Åland Island Conference on von Willebrand disease

Introduction The sixth angstrom land Islands Conference on von Willebrand disease (VWD) on the angstrom land Islands, Finland, was held from 20 to 22 September 2018. Aim The meeting brought together experts in the field of VWD from around the world to share the latest advances and knowledge in VWD. Results and discussion The topics covered both clinical aspects of disease management, and biochemical and laboratory insights into the disease. The clinical topics discussed included epidemiology, diagnosis and treatment of VWD in different countries, management of children with VWD, bleeding control during surgery, specific considerations for the management of type 3 VWD and bleeding control in women with VWD. Current approaches to the management of acquired von Willebrand syndrome were also discussed. Despite significant advances in the understanding and therapeutic options for VWD, there remain many challenges to be overcome in order to optimise patient care. In comparison with haemophilia A, there are very few registries of VWD patients, which would be a valuable source of data on the condition and its management. VWD is still underdiagnosed, and many patients suffer recurrent or severe bleeding that could be prevented. Awareness of VWD among healthcare practitioners, including non-haematologists, should be improved to allow timely diagnosis and intervention. Diagnosis remains challenging, and the development of fast, simple assays may help to facilitate accurate and rapid diagnosis of VWD.Peer reviewe

Sustainability in Peripheral and Ultra-Peripheral Rural Areas through a Multi-Attribute Analysis: The Case of the Italian Insular Region

Italy has adopted the strategy of inner areas, mainly based on physical distance from public services. The strategy promotes a multi-level and multi-fund governance approach and the local partnership of mayors. Our paper focuses on rural areas, identified by the national strategy of inner areas, as peripheral and ultra-peripheral, in the Italian insular region (Sicily and Sardinia). It analyzes, at the municipality level, socio-demographic, economic, and environmental sustainability using appropriate indicators. Aiming at discovering the underlying relationship portrayed by multi-attribute data in an information system, we applied rough set theory. The inductive decision rules obtained through this data mining methodology reveal the simultaneous presence or absence of important characteristics aiming at reaching different levels of sustainability. Without the requirement of statistical assumptions regarding data distribution or structures for collecting data, such as functions or equations, this method ensures the description of patterns exhibited by data. Of particular interest is the assessment of conditional attributes (i.e., the selected indicators), and the information connecting them to sustainability, as a decision attribute. The most important result is rule generation, specifically, decision rules that are able to suggest tools for policy makers at different levels

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab