Establishing the Most Important Clinical Skills for New Graduate Veterinarians by Comparing Published Lists with Regional Stakeholder Expectations: A Bangladesh Experience.

Veterinary clinical skills training is evolving rapidly around the world and there is increasing interest in Bangladesh in opening clinical skills laboratories and using models in teaching. The first clinical skills laboratory was opened at Chattogram Veterinary and Animal Sciences University in 2019. The current study aimed to identify the most important clinical skills for veterinarians in Bangladesh to inform the further development of clinical skills laboratories and ensure resources are deployed effectively and efficiently. Lists of clinical skills were collated from the literature, national and international accreditation standards, and regional syllabi. The list was refined through local consultation, focused on farm and pet animals, and was disseminated via an online survey to veterinarians and final-year students who were asked to rate the level of importance of each skill for a new graduate. The survey was completed by 215 veterinarians and 115 students. A ranked list was generated with injection techniques, animal handling, clinical examination, and basic surgical skills among the most important. Some techniques requiring specific equipment and some advanced surgical procedures were considered less important. As a result of the study, the most important clinical skills for a new graduate in Bangladesh have been identified for the first time. The results will inform the development of models, the use of clinical skills laboratories, and the design of clinical skills courses for veterinary training. Our approach of drawing upon existing lists followed by local stakeholders consultation is recommended to others to ensure clinical skills teaching is regionally relevant.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Hydroxyapatite reinforced inherent RGD containing silk fibroin composite scaffolds: Promising platform for bone tissue engineering.

Replacement and repair of ectopic bone defects and traumatized bone tissues are done using porous scaffolds and composites. The prerequisites for such scaffolds include high mechanical strength, osseoconductivity and cytocompatibility. The present work is designed to address such requirements by fabricating a reinforced cytocompatible scaffold. Biocompatible silk protein fibroin collected from tropical non-mulberry tasar silkworm (Antheraea mylitta) is used to fabricate fibroin-hydroxyapatite (HAp) nanocomposite particles using chemical precipitation method. In situ reinforcement of fibroin-HAp nanocomposite and external deposition of HAp particles on fibroin scaffold is carried out for comparative evaluations of bio-physical and biochemical characteristics. HAp deposited fibroin scaffolds provide greater mechanical strength and cytocompatibility, when compared with fibroin-HAp nanoparticles reinforced fibroin scaffolds. Minimal immune responses of both types of composite scaffolds are observed using osteoblast-macrophage co-culture model. Nanocomposite reinforced fibroin scaffold can be tailored further to accommodate different requirements depending on bone type or bone regeneration period.

Silk fibroin nanoparticles support in vitro sustained antibiotic release and osteogenesis on titanium surface.

UNLABELLED: Increasing amounts of metal-based implants are used for orthopedic or dental surgeries throughout the world. Still several implant-related problems such as inflammation, loosening and bacterial infection are prevalent. These problems stem from the immediate microbial contamination and failure of initial osteoblast adhesion. Additionally, bacterial infections can cause serious and life-threatening conditions such as osteomyelitis. Here, antibiotic (gentamicin)-loaded silk protein fibroin (non-mulberry silkworm, Antheraea mylitta) nanoparticles are fabricated and deposited over the titanium surface to achieve sustained drug release in vitro and to alter the surface nano-roughness. Based on the altered surface topography, chemistry and antibacterial activity, we conclude that the nanoparticle-deposited surfaces are superior for osteoblast adhesion, proliferation and differentiation in comparison to bare Ti. This method can be utilized as a cost-effective approach in implant modification. FROM THE CLINICAL EDITOR: Titanium-based implants are commonly used in the field of orthopedics or dentistry. Surface modification of an implant is vital to ensure osseointegration. In this article, the author investigated the use of silk protein fibroins for metal surface modification and also for drug delivery against bacteria. The encouraging data should provide a new method in terms of nanotechnology in the respective clinical fields.

CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics.

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

Cytotoxicity and sustained release of modified divinylsulfone from silk based 3D construct.

Monovinylsulfones have been extensively studied for its biological activities but modified divinylsulfones (mDVS2) were largely neglected due to the non-availability of appropriate synthetic routes. The present report describes the potential of a unique derivative of divinylsulfone as a remedial molecule. The mDVS2, available in reasonably large amount through an easy synthesis route, incites necrosis in invasive and non-invasive breast cancer cells in a time and concentration dependent manner. This molecule is further used to fabricate mDVS2 embedded silk based 3D scaffolds in order to achieve sustained release. The entrapped molecules retain their activity over time, as 100% cell death is observed within 7 days. The findings demonstrate the cytotoxic property of mDVS and highlight the importance of under utilized mDVSs as potential therapeutic agents.

Impact of Chemotherapeutic Stress Depends on The Nature of Breast Cancer Spheroid and Induce Behavioral Plasticity to Resistant Population.

Cellular or tumor dormancy, identified recently as one of the main reasons behind post-therapy recurrence, can be caused by diverse reasons. Chemotherapy has recently been recognized as one of such reasons. However, in-depth studies of chemotherapy-induced dormancy are lacking due to the absence of an in vitro human-relevant model tailor-made for such a scenario. This report utilized multicellular breast cancer spheroid to create a primary platform for establishing a chemotherapy-induced dormancy model. It is observed that extreme chemotherapeutic stress affects invasive and non-invasive spheroids differently. Non-invasive spheroids exhibit more resilience and maintain viability and migrational ability, while invasive spheroids display heightened susceptibility and improved tumorigenic capacity. Heterogenous spheroids exhibit increased tumorigenic capacity while show minimal survival ability. Further probing of chemotherapeutically dormant spheroids is needed to understand the molecular mechanism and identify dormancy-related markers to achieve therapeutic success in the future.

Mitochondria: A source of potential biomarkers for non-communicable diseases.

Mitochondria, as an endosymbiont of eukaryotic cells, controls multiple cellular activities, including respiration, reactive oxygen species production, fatty acid synthesis, and death. Though the majority of functional mitochondrial proteins are translated through a nucleus-controlled process, very few of them (∼10%) are translated within mitochondria through their own machinery. Germline and somatic mutations in mitochondrial and nuclear DNA significantly impact mitochondrial homeostasis and function. Such modifications disturbing mitochondrial biogenesis, metabolism, or mitophagy eventually resulted in cellular pathophysiology. In this chapter, we discussed the impact of mitochondria and its dysfunction on several non-communicable diseases like cancer, diabetes, neurodegenerative, and cardiovascular problems. Mitochondrial dysfunction and its outcome could be screened by currently available omics-based techniques, flow cytometry, and high-resolution imaging. Such characterization could be evaluated as potential biomarkers to assess the disease burden and prognosis.

Biomarkers of mitochondrial origin: a futuristic cancer diagnostic.

Cancer is a highly fatal disease without effective early-stage diagnosis and proper treatment. Along with the oncoproteins and oncometabolites, several organelles from cancerous cells are also emerging as potential biomarkers. Mitochondria isolated from cancer cells are one such biomarker candidates. Cancerous mitochondria exhibit different profiles compared with normal ones in morphology, genomic, transcriptomic, proteomic and metabolic landscape. Here, the possibilities of exploring such characteristics as potential biomarkers through single-cell omics and Artificial Intelligence (AI) are discussed. Furthermore, the prospects of exploiting the biomarker-based diagnosis and its futuristic utilization through circulatory tumor cell technology are analyzed. A successful alliance of circulatory tumor cell isolation protocols and a single-cell omics platform can emerge as a next-generation diagnosis and personalized treatment procedure.

Comparing mechano-transduction in fibroblasts deficient of focal adhesion proteins.

Mechano-transduction was studied in wildtype and focal adhesion (FA) protein-deficient mouse embryonic fibroblasts (MEFs). Using a cell stretcher, we determined the effect of stretch on cell morphology, apoptosis, and phosphorylation of ERK(1/2). After 20% cyclic, uniaxial stretch, FA-deficient MEFs showed morphological changes and levels of apoptosis of the order: focal adhesion kinase>p130Cas>vinculin compared to wildtype cells. ERK(1/2) phosphorylation peaked in wildtype cells at around 10 min, and in all FA-deficient cells at around 5 min. The relative change in strain energy of FA-deficient cells compared to wildtype cells was of the order: vinculin>FAK>p130Cas. Taken together, FAK and p130Cas are more important in the stretch-mediated downstream signaling and cell survival pathway, while vinculin is more critical in maintaining cell contractility.

Nonmulberry silk proteins: multipurpose ingredient in bio-functional assembly.

The emerging field of tissue engineering and regenerative medicines utilising artificial polymers is facing many problems. Despite having mechanical stability, non-toxicity and biodegradability, most of them lack cytocompatibility and biocompatibility. Natural polymers (such as collagen, hyaluronic acid, fibrin, fibroin, and others), including blends, are introduced to the field to solve some of the relevant issues. Another natural biopolymer: silkworm silk gained special attention primarily due to its specific biophysical, biochemical, and material properties, worldwide availability, and cost-effectiveness. Silk proteins, namely fibroin and sericin extracted from domesticated mulberry silkwormBombyx mori, are studied extensively in the last few decades for tissue engineering. Wild nonmulberry silkworm species, originated from India and other parts of the world, also produce silk proteins with variations in their nature and properties. Among the nonmulberry silkworm species,Antheraea mylitta(Indian Tropical Tasar),A. assamensis/A. assama(Indian Muga), andSamia ricini/Philosamia ricini(Indian Eri), along withA. pernyi(Chinese temperate Oak Tasar/Tussah) andA. yamamai(Japanese Oak Tasar) exhibit inherent tripeptide motifs of arginyl glycyl aspartic acid in their fibroin amino acid sequences, which support their candidacy as the potential biomaterials. Similarly, sericin isolated from such wild species delivers unique properties and is used as anti-apoptotic and growth-inducing factors in regenerative medicines. Other characteristics such as biodegradability, biocompatibility, and non-inflammatory nature make it suitable for tissue engineering and regenerative medicine based applications. A diverse range of matrices, including but not limited to nano-micro scale structures, nanofibres, thin films, hydrogels, and porous scaffolds, are prepared from the silk proteins (fibroins and sericins) for biomedical and tissue engineering research. This review aims to represent the progress made in medical and non-medical applications in the last couple of years and depict the present status of the investigations on Indian nonmulberry silk-based matrices as a particular reference due to its remarkable potentiality of regeneration of different types of tissues. It also discusses the future perspective in tissue engineering and regenerative medicines in the context of developing cutting-edge techniques such as 3D printing/bioprinting, microfluidics, organ-on-a-chip, and other electronics, optical and thermal property-based applications.

Size-selective separation of macromolecules by nanochannel titania membrane with self-cleaning (declogging) ability.

We report on a simple and self-organizing process for the fabrication of TiO(2) nanochannel membranes with a channel width of 8-10 nm that can be used for size selective separation of macromolecules (proteins). The membrane, consisting of self-aligned oxide channels, is formed by complete anodization of a thin Ti foil under specific electrochemical conditions in a glycerol-phosphate electrolyte. Due to self-cleaning properties of TiO(2), clogged membranes (for example due to extended use) can easily be fully reopened and thus are reusable. As the TiO(2) after anodic formation directly contains anatase crystallites (the most photoactive TiO(2) crystal form) no thermal treatment of the membrane is required (avoiding the danger of thermally induced cracking).

First synthesis and antiprotozoal activities of divinyl sulfone-modified carbohydrates.

Divinyl sulfone-modified carbohydrates have been synthesized for the first time by reacting easily available carbohydrate epoxides with thioethanol in a regiospecific fashion. One of the modified divinyl sulfones initiated significant cell death in Entamoeba species and the influence of the anomeric configurations on the biological activities of these sugar-derived divinyl sulfones has been highlighted. The most active compound in this series was found to be devoid of any toxicity.

Tumor-macrophage crosstalk: how to listen.

The tumor microenvironment contains many cellular components influencing tumor behaviors, such as metastasis, angiogenesis and chemo-resistance. Tumor-associated macrophages (TAMs) are one of such components that can also manipulate the overall prognosis and patient survival. Analysis of tumor-macrophage crosstalk is crucial as tumor cells can polarize circulatory monocytes into TAMs. Such trans-polarization of macrophages support tumor mediated evasion and suppression of immune response. Additionally, such TAMs significantly influence tumor growth and proliferation, making them a potential candidate for precision therapeutics. However, the failure of macrophage-dependent therapies at clinical trials emphasizes the fault in current perception and research modality. This review discussed this field's progress regarding emerging model systems with a focused view on the in vitro platforms. The inadequacy of currently available models and their implications on existing studies also analyzed. The need for a conceptual and experimental leap toward a human-relevant in vitro custom-built platform for studying tumor-macrophage crosstalk is acknowledged.

Entamoeba invadens: cloning and molecular characterization of chitinases.

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.

Inherent aggressive character of invasive and non-invasive cells dictates the in vitro migration pattern of multicellular spheroid.

Cellular migration, a process relevant to metastasis, is mostly studied in the conventional 2D condition. However, cells cultured in the 3D condition assumed to mimic the in vivo conditions better. The current study is designed to compare an invasive and non-invasive adenocarcinoma cell with an invasive fibrosarcoma cell to understand the migration pattern of the multicellular spheroid. It is observed that conventional haplotaxis, chemotactic and pseudo-3D migration assay cannot distinguish between the invasive and non-invasive cells conclusively under 2D condition. Invasive spheroids migrate rapidly in sprouting assay in comparison to non-invasive spheroids. Effects of cytochalasin B, marimastat and blebbistatin are tested to determine the influence of different migration modality namely actin polymerization, matrix metalloprotease and acto-myosin in both culture conditions. Altered mRNA profile of cellular migration related genes (FAK, Talin, Paxillin, p130cas and Vinculin) is observed between 2D and 3D condition followed by the changed expression of matrix metallo proteases. A distinct difference is observed in distribution and formation of focal adhesion complex under these culture conditions. This study demonstrates the efficacy of multicellular spheroids in identifying the intrinsic aggressive behavior of different cell lines as a proof of concept and recognizes the potential of spheroids as a migration model.

Target specific delivery of anticancer drug in silk fibroin based 3D distribution model of bone-breast cancer cells.

To avoid the indiscriminating action of anticancer drugs, the cancer cell specific targeting of drug molecule becomes a preferred choice for the treatment. The successful screening of the drug molecules in 2D culture system requires further validation. The failure of target specific drug in animal model raises the issue of creating a platform in between the in vitro (2D) and in vivo animal testing. The metastatic breast cancer cells migrate and settle at different sites such as bone tissue. This work evaluates the in vitro 3D model of the breast cancer and bone cells to understand the cellular interactions in the presence of a targeted anticancer drug delivery system. The silk fibroin based cytocompatible 3D scaffold is used as in vitro 3D distribution model. Human breast adenocarcinoma and osteoblast like cells are cocultured to evaluate the efficiency of doxorubicin loaded folic acid conjugated silk fibroin nanoparticle as drug delivery system. Decreasing population of the cancer cells, which lower the levels of vascular endothelial growth factors, glucose consumption, and lactate production are observed in the drug treated coculture constructs. The drug treated constructs do not show any major impact on bone mineralization. The diminished expression of osteogenic markers such as osteocalcein and alkaline phosphatase are recorded. The result indicates that this type of silk based 3D in vitro coculture model may be utilized as a bridge between the traditional 2D and animal model system to evaluate the new drug molecule (s) or to reassay the known drug molecules or to develop target specific drug in cancer research.

Non-mulberry silk fibroin influence osteogenesis and osteoblast-macrophage cross talk on titanium based surface.

The titanium and its alloys are used as orthopedic dental implants due to their mechanical and bio-inert properties. The bare metal implants are not the ultimate answer for better osteogenesis and implant integration. Physical and chemical modifications are carried out to achieve the goal of improved adhesion and differentiation of the osteoblast. In this work, the silk fibroins from both mulberry and non-mulberry sources are used for surface modification. Silk fibroins are immobilized on titanium surface to facilitate the initial cell adhesion followed by improved cell spreading and better mineralization in order to achieve enhanced osseointegration. The immunological responses along with the effect of cytokines on osteoblast adhesion and function are investigated. The non-mulberry fibroin performs better in the context of the cell adherence and differentiation, which lead to better mineralization. The results indicate that the silk fibroin from non-mulberry source can be used for better osteogenesis on orthopedic implants.

Modified dextran cross-linked electrospun gelatin nanofibres for biomedical applications.

Electrospun gelatin nanofibres attract attention of bioengineering arena because of its excellent biocompatibility and structural resemblance with native extracellular matrix. In this study, we have developed gelatin nanofibres using an innovative cross-linking approach to minimize cytotoxic effects. Gelatin was dissolved in water:acetic acid (8:2, v/v) solution and electrospun to form nanofibres with diameter in the range of 156 ± 30 nm. The nanofibres were cross-linked with a modified polysaccharide, namely, dextran aldehyde (DA). Cross-linking with DA could be achieved without compromising the fibrous architecture. DA cross-linked gelatin nanofibres maintained the fibrous morphology in aqueous medium. These mats exhibit improved mechanical properties and gradual degradation behaviour. The nanofibres were evaluated for cytotoxicity, cell adhesion, viability, morphology and proliferation using L-929 fibroblast cells. The results confirmed that DA cross-linked mats were non cytotoxic towards L-929 cells with good cell adhesion, spreading and proliferation.

Silk protein lithography as a route to fabricate sericin microarchitectures.

Photolithographic fabrication via a "silk sericin photoresist" is used to form precise protein microstructures directly and rapidly on a variety of substrates. High-resolution and fidelity architectures in two and three dimensions with line widths down to 1 µm are formed. Photo-crosslinked protein structures provide structural iridescence and guide cell adhesion with precise spatial control.