Search results for: fusion proteins

Authors: Qinghua Xing, Xinyi Tao, Haisheng Wang, Baisuo Zhao

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The first true anaerobic, halophilic alkali thermophile, Natranaerobius thermophilus DSM 18059T, serves as a valuable model for studying cellular adaptations to saline, alkaline and thermal extremes. To uncover the adaptive strategies employed by N. thermophilus in coping with these challenges, we conducted a comprehensive iTRAQ-based quantitative proteomic analysis under different conditions of salinity (3.5 M vs. 2.5 M Na+), pH (pH 9.6 vs. pH 8.6), and temperature (52°C vs. 42°C). The increased intracellular accumulation of glycine betaine, through both synthesis and transport, plays a critical role in N. thermophilus' adaptation to these combined stresses. Under all three stress conditions, the up-regulation of Trk family proteins responsible for K+ transport is observed. Intracellular K+ concentration rises in response to salt and pH levels. Multiple types of Na+/H+ antiporter (NhaC family, Mrp family and CPA family) and a diverse range of FOF1-ATP synthase are identified as vital components for maintaining ionic balance under different stress conditions. Importantly, proteins involved in amino acid metabolism, carbohydrate metabolism, ABC transporters, signaling and chemotaxis, as well as biological macromolecule repair and protection, exhibited significant up-regulation in response to these extreme conditions. These metabolic pathways emerge as critical factors in N. thermophilus' adaptation mechanisms under extreme environmental stress. To validate the proteomic data, ddPCR analysis confirmed changes in mRNA expression, thereby corroborating the up-regulation and down-regulation patterns of 19 co-up-regulated and 36 key proteins under saline, alkaline and thermal stresses. This research enriches our understanding of the complex regulatory systems that enable polyextremophiles to survive in combined extreme conditions.

Keywords: polyextremophiles, natranaerobius thermophilus, saline- alkaline- thermal stresses, combined extremes

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Authors: Ximena Calle, Plinio Cantero-López, Felipe Muñoz-Córdova, Mayarling-Francisca Troncoso, Sergio Lavandero, Valentina Parra

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MUL1, a mitochondrial E3 ubiquitin ligase anchored to the outer mitochondrial membrane, is highly expressed in the heart. MUL1 is involved in multiple biological pathways associated with mitochondrial dynamics. Increased MUL1 affects the balance between fission and fusion, affecting mitochondrial function, which plays a crucial role in myocardial function. Therefore, it is interesting to evaluate the effect of cardiac-specific overexpression of MUL1 on myocardial function. Aim: To determine heart functionality in a mouse model with cardio-specific overexpression MUL1 protein. Methods and Results: Male C57BL/Tg transgenic mice with cardiomyocyte-specific overexpression of MUL1 (n=10) and control (n=4) were evaluated at 12, 27, and 35 weeks of age. Glucose tolerance curve determination was performed after a 6-hours fast to assess metabolic capacity, treadmill test, and systolic, and diastolic pressure was evaluated by the mouse tail-cuff blood pressure system equipment. The result showed no glucose tolerance curve, and the treadmill test demonstrated no significant changes between groups. However, substantial changes in diastolic function were observed by ultrasound and determination of cardiac hypertrophy proteins by western blot. Conclusions: Cardio-specific overexpression of MUL1 in mice without any treatment affects diastolic cardiac function, thus showing the important role contributed by MUL1 in the heart. Future research should evaluate the effect of cardiomyocyte-specific overexpression of MUL1 in pathological conditions such as a high-fat diet is one of the main risk factors for cardiovascular disease.

Keywords: diastolic dysfunction, hypertrophy cardiac, mitochondrial E3 ubiquitin ligase 1, MUL1

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Authors: Tobias Schmithuesen, Johannes Henrich Schleifenbaum

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The additive manufacturing process laser powder bed fusion offers many advantages over conventional manufacturing processes. For example, almost any complex part can be produced, such as topologically optimized lightweight parts, which would be inconceivable with conventional manufacturing processes. A major challenge posed by the LPBF process, however, is, in most cases, the need to use and remove support structures on critically inclined part surfaces (α < 45 ° regarding substrate plate). These are mainly used for dimensionally accurate mapping of part contours and to reduce distortion by absorbing process-related internal stresses. Furthermore, they serve to transfer the process heat to the substrate plate and are, therefore, indispensable for the LPBF process. A major challenge for the economical use of the LPBF process in industrial process chains is currently still the high manual effort involved in removing support structures. According to the state of the art (SoA), the parts are usually treated by simple hand tools (e.g., pliers, chisels) or by machining (e.g., milling, turning). New automatable approaches are the removal of support structures by means of wet chemical ablation and thermal deburring. According to the state of the art, the support structures are essentially adapted to the LPBF process and not to potential post-processing steps. The aim of this study is the determination of support structure designs that are adapted to the mentioned post-processing approaches. In the first step, the essential boundary conditions for complete removal by means of the respective approaches are identified. Afterward, a representative demonstrator part with various macroscopic support structure designs will be LPBF-manufactured and tested with regard to a complete powder and support removability. Finally, based on the results, potentially suitable support structure designs for the respective approaches will be derived. The investigations are carried out on the example of the aluminum alloy AlSi10Mg.

Keywords: additive manufacturing, laser powder bed fusion, laser beam melting, selective laser melting, post processing, tool-free, wet chemical ablation, thermal deburring, aluminum alloy, AlSi10Mg

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Authors: Ieva Bruzauskaite, Jovile Raudoniute, Karina Poliakovaite, Danguole Zabulyte, Daiva Bironaite, Ruta Aldonyte

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Introduction: Air pollution is a growing global problem which has been shown to be responsible for various adverse health outcomes. Immunotoxicity, such as dysregulated inflammation, has been proposed as one of the main mechanisms in air pollution-associated diseases. Chronic obstructive pulmonary disease (COPD) is among major morbidity and mortality causes worldwide and is characterized by persistent airflow limitation caused by the small airways disease (obstructive bronchiolitis) and irreversible parenchymal destruction (emphysema). Exact pathways explaining the air pollution induced and mediated disease states are still not clear. However, modern societies understand dangers of polluted air, seek to mitigate such effects and are in need for reliable biomarkers of air pollution. We hypothesise that post-translational modifications of structural proteins, e.g. citrullination, might be a good candidate biomarker. Thus, we have designed this study, where mice were exposed to diesel exhaust and the ongoing protein modifications and inflammation in lungs and other tissues were assessed. Materials And Methods: To assess the effects of diesel exhaust a in vivo study was designed. Mice (n=10) were subjected to everyday 2-hour exposure to diesel exhaust for 14 days. Control mice were treated the same way without diesel exhaust. The effects within lung and other tissues were assessed by immunohistochemistry of formalin-fixed and paraffin-embedded tissues. Levels of inflammation and citrullination related markers were investigated. Levels of parenchymal damage were also measured. Results: In vivo study corroborates our own data from in vitro and reveals diesel exhaust initiated inflammatory shift and modulation of lung peptidyl arginine deiminase 4 (PAD4), citrullination associated enzyme, levels. In addition, high levels of citrulline were observed in exposed lung tissue sections co-localising with increased parenchymal destruction. Conclusions: Subacute exposure to diesel exhaust renders mice lungs inflammatory and modifies certain structural proteins. Such structural changes of proteins may pave a pathways to lost/gain function of affected molecules and also propagate autoimmune processes within the lung and systemically.

Keywords: air pollution, citrullination, in vivo, lungs

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Authors: Sayyed Mostafa Ahmadi, Neda Raeesi

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Background and Objectives: Cervical spondylosis is a common problem that affects the adult spine and is the most common cause of radiculopathy and myelopathy in older patients. Anterior discectomy and fusion is a well-known technique in degenerative cervical disc disease. However, one of the late undesirable complications is adjacent disc degeneration, which affects about 91% of patients in ten years. Many factors can be effective in causing this complication, but some are still debatable. Discovering these risk factors and eliminating them can improve the quality of life. Methods: This is a retrospective cohort study. All patients who underwent anterior discectomy and fusion surgery in the neurosurgery ward of Imam Khomeini Hospital between 2013 and 2016 were evaluated. Their demographic information was collected. All patients were visited and examined for radiculopathy, myelopathy, and muscular force. At the same visit, all patients were asked to have a facelift, and neck profile, as well as a neck MRI(General Tesla 3). Preoperative graphs were used to measure the diameter of the cervical canal(Pavlov ratio) and to evaluate sagittal alignment(Cobb Angle). Preoperative MRI of patients was reviewed for anterior and posterior longitudinal ligament calcification. Result: In this study, 57 patients were studied. The mean age of patients was 50.63 years, and 49.1% were male. Only 3.5% of patients had anterior and posterior longitudinal ligament calcification. Symptomatic ASD was observed in 26.6%. The X-rays and MRIs showed evidence of 80.7% radiological ASD. Among patients who underwent one-level surgery, 20% had symptomatic ASD, but among patients who underwent two-level surgery, the rate of ASD was 50%.In other words, the higher the number of surfaces that are operated and fused, the higher the probability of symptomatic ASD(P-value <0.05). The X-rays and MRIs showed 80.7% of radiological ASD. Among patients who underwent surgery at one level, 78% had radiological ASD, and this number was 92% among patients who underwent two-level surgery(P-value> 0.05). Demographic variables such as age, sex, height, weight, and BMI did not have a significant effect on the incidence of radiological ASD(P-value> 0.05), but sex and height were two influential factors on symptomatic ASD(P-value <0.05). Other related variables such as family history, smoking and exercise also have no significant effect(P-value> 0.05). Radiographic variables such as Pavlov ratio and sagittal alignment were also unaffected by the incidence of radiological and symptomatic ASD(P-value> 0.05). The number of surgical surfaces and the incidence of anterior and posterior longitudinal ligament calcification before surgery also had no statistically significant effect(P-value> 0.05). In the study of the ability of the neck to move in different directions, none of these variables are statistically significant in the two groups with radiological and symptomatic ASD and the non-affected group(P-value> 0.05). Conclusion: According to the findings of this study, this disease is considered to be a multifactorial disease. The incidence of radiological ASD is much higher than symptomatic ASD (80.7% vs. 26.3%) and sex, height and number of fused surfaces are the only factors influencing the incidence of symptomatic ASD and no variable influences radiological ASD.

Keywords: risk factors, anterior neck disectomy and fusion, adjucent segment degeneration, complication

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Authors: Rui Liu, Pengyu Cui, Nan Jiang

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At present, there is much research on the application of centralized management and cross-integration application of basic geographic information. However, the idea of information integration and sharing between land, sea, and air navigation targets is not deeply applied into the research of navigation information service, especially in the information expression. Targeting at this problem, the paper carries out works about the expression pattern of navigation electronic map for ground vehicles under air and land united navigation mechanism. At first, with the support from multi-source information fusion of GIS vector data, RS data, GPS data, etc., an air and land united information expression pattern is designed aiming at specific navigation task of emergency rescue in the earthquake. And then, the characteristics and specifications of the united expression of air and land navigation information under the constraints of map load are summarized and transferred into expression rules in the rule bank. At last, the related navigation experiment is implemented to evaluate the effect of the expression pattern. The experiment selects evaluation factors of the navigation task accomplishment time and the navigation error rate as the main index, and make comparisons with the traditional single information expression pattern. To sum up, the research improved the theory of navigation electronic map and laid a certain foundation for the design and realization of united navigation system in the aspect of real-time navigation information delivery.

Keywords: navigation electronic map, united navigation, multi-element expression pattern, multi-source information fusion

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Authors: Rauan Sakenov, Peter Bukovics, Peter Gaszler, Veronika Tokacs-Kollar, Beata Bugyi

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FH2 (formin homology-2) domains of several proteins, collectively known as formins, including DAAM, DAAM1 and mDia1, promote G-actin nucleation and elongation. FH2 domains of these formins exist as oligomers. Chain dimerization by ring structure formation serves as a structural basis for actin polymerization function of FH2 domain. Proper single chain configuration and specific interactions between its various regions are necessary for individual chains to form a dimer functional in G-actin nucleation and elongation. FH1 and WH2 domain-containing formins were shown to behave as intrinsically disordered proteins. Thus, the aim of this research was to study structural dynamics of FH2 domain of DAAM. To investigate structural features of FH2 domain of DAAM, molecular dynamics simulation of chain A of FH2 domain of DAAM solvated in water box in 50 mM NaCl was conducted at temperatures from 293.15 to 353.15K, with VMD 1.9.2, NAMD 2.14 and Amber Tools 21 using 2z6e and 1v9d PDB structures of DAAM was obtained on I-TASSER webserver. Calcium and ATP bound G-actin 3hbt PDB structure was used as a reference protein with well-described structural dynamics of denaturation. Topology and parameter information of CHARMM 2012 additive all-atom force fields for proteins, carbohydrate derivatives, water and ions were used in NAMD 2.14 and ff19SB force field for proteins in Amber Tools 21. The systems were energy minimized for the first 1000 steps, equilibrated and produced in NPT ensemble for 1ns using stochastic Langevin dynamics and the particle mesh Ewald method. Our root-mean square deviation (RMSD) analysis of molecular dynamics of chain A of FH2 domains of DAAM revealed similar insignificant changes of total molecular average RMSD values of FH2 domain of these formins at temperatures from 293.15 to 353.15K. In contrast, total molecular average RMSD values of G-actin showed considerable increase at 328K, which corresponds to the denaturation of G-actin molecule at this temperature and its transition from native, ordered, to denatured, disordered, state which is well-described in the literature. RMSD values of lasso and tail regions of chain A of FH2 domain of DAAM exhibited higher than total molecular average RMSD at temperatures from 293.15 to 353.15K. These regions are functional in intra- and interchain interactions and contain highly conserved tryptophan residues of lasso region, highly conserved GNYMN sequence of post region and amino acids of the shell of hydrophobic pocket of the salt bridge between Arg171 and Asp321, which are important for structural stability and ordered state of FH2 domain of DAAM and its functions in FH2 domain dimerization. In conclusion, higher than total molecular average RMSD values of lasso and post regions of chain A of FH2 domain of DAAM may explain disordered state of FH2 domain of DAAM at temperatures from 293.15 to 353.15K. Finally, absence of marked transition, in terms of significant changes in average molecular RMSD values between native and denatured states of FH2 domain of DAAM at temperatures from 293.15 to 353.15K, can make it possible to attribute these formins to the group of intrinsically disordered proteins rather than to the group of intrinsically ordered proteins such as G-actin.

Keywords: FH2 domain, DAAM, formins, molecular modelling, computational biophysics

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Hantian Wu, Bo Huang, Yuan Zeng

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Divisions between wealthy and poor, private and public landscapes are propagated by the increasing economic inequality of cities. While these are the spatial reflections of larger social issues and problems, urban design can at least employ spatial techniques that promote more inclusive rather than exclusive, overlapping rather than segregated, interlinked rather than disconnected landscapes. Indeed, the type of edge or border between urban landscapes plays a critical role in the way the environment is perceived. China experiences rapid urbanization, which poses unpredictable environmental challenges. The urban green cover and water body are under changes, which highly relevant to resident wealth and happiness. However, very limited knowledge and data on their rapid changes are available. In this regard, enhancing the monitoring of urban landscape with high-frequency method, evaluating and estimating the impacts of the urban landscape changes, and understating the driving forces of urban landscape changes can be a significant contribution for urban planning and studying. High-resolution remote sensing data has been widely applied to urban management in China. The map of urban land use map for the entire China of 2018 with 10 meters resolution has been published. However, this research focuses on the large-scale and high-resolution remote sensing land use but does not precisely focus on the seasonal change of urban covers. High-resolution remote sensing data has a long-operation cycle (e.g., Landsat 8 required 16 days for the same location), which is unable to satisfy the requirement of monitoring urban-landscape changes. On the other hand, aerial-remote or unmanned aerial vehicle (UAV) sensing are limited by the aviation-regulation and cost was hardly widely applied in the mega-cities. Moreover, those data are limited by the climate and weather conditions (e.g., cloud, fog), and those problems make capturing spatial and temporal dynamics is always a challenge for the remote sensing community. Particularly, during the rainy season, no data are available even for Sentinel Satellite data with 5 days interval. Many natural events and/or human activities drive the changes of urban covers. In this case, enhancing the monitoring of urban landscape with high-frequency method, evaluating and estimating the impacts of the urban landscape changes, and understanding the mechanism of urban landscape changes can be a significant contribution for urban planning and studying. This project aims to use the high spatiotemporal fusion of remote sensing data to create short-cycle, high-resolution remote sensing data sets for exploring the high-frequently urban cover changes. This research will enhance the long-term monitoring applicability of high spatiotemporal fusion of remote sensing data for the urban landscape for optimizing the urban management of landscape border to promoting the inclusive of the urban landscape to all communities.

Keywords: urban land cover changes, remote sensing, high spatiotemporal fusion, urban management

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Authors: Matthew Yeager, Christopher Willy, John Bischoff

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The conceptualization and design phases of a system lifecycle consume a significant amount of the lifecycle budget in the form of direct tasking and capital, as well as the implicit costs associated with unforeseeable design errors that are only realized during downstream phases. Ad hoc or iterative approaches to generating system requirements oftentimes fail to consider the full array of feasible systems or product designs for a variety of reasons, including, but not limited to: initial conceptualization that oftentimes incorporates a priori or legacy features; the inability to capture, communicate and accommodate stakeholder preferences; inadequate technical designs and/or feasibility studies; and locally-, but not globally-, optimized subsystems and components. These design pitfalls can beget unanticipated developmental or system alterations with added costs, risks and support activities, heightening the risk for suboptimal system performance, premature obsolescence or forgone development. Supported by rapid advances in learning algorithms and hardware technology, sensors and sensor systems have become commonplace in both commercial and industrial products. The evolving array of hardware components (i.e. sensors, CPUs, modular / auxiliary access, etc…) as well as recognition, data fusion and communication protocols have all become increasingly complex and critical for design engineers during both concpetualization and implementation. This work seeks to develop and utilize a non-deterministic approach for sensor system design within the multi-attribute tradespace exploration (MATE) paradigm, a technique that incorporates decision theory into model-based techniques in order to explore complex design environments and discover better system designs. Developed to address the inherent design constraints in complex aerospace systems, MATE techniques enable project engineers to examine all viable system designs, assess attribute utility and system performance, and better align with stakeholder requirements. Whereas such previous work has been focused on aerospace systems and conducted in a deterministic fashion, this study addresses a wider array of system design elements by incorporating both traditional tradespace elements (e.g. hardware components) as well as popular multi-sensor data fusion models and techniques. Furthermore, statistical performance features to this model-based MATE approach will enable non-deterministic techniques for various commercial systems that range in application, complexity and system behavior, demonstrating a significant utility within the realm of formal systems decision-making.

Keywords: multi-attribute tradespace exploration, data fusion, sensors, systems engineering, system design

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Authors: Valencia Fernandes, Dharmendra Kumar Khatri, Shashi Bala Singh

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Background and Aim: Epigenetics are the inaudible signatures of several pathological processes in the brain. This study understands the influence of DNA methylation, a major epigenetic modification, in the prefrontal cortex and hippocampus of the diabetic brain and its notable effect on the cellular chaperones and synaptic proteins. Method: Chronic high fat diet and STZ-induced diabetic mice were studied for cognitive dysfunction, and global DNA methylation, as well as DNA methyltransferase (DNMT) activity, were assessed. Further, the cellular chaperones and synaptic proteins were examined using DNMT inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC)-via intracerebroventricular injection. Moreover, % methylation of these synaptic proteins were also studied so as to correlate its epigenetic involvement. Computationally, its interaction with the DNMT enzyme were also studied using bioinformatic tools. Histological studies for morphological alterations and neuronal degeneration were also studied. Neurogenesis, a characteristic marker for new learning and memory formation, was also assessed via the BrdU staining. Finally, the most important behavioral studies, including the Morris water maze, Y maze, passive avoidance, and Novel object recognition test, were performed to study its cognitive functions. Results: Altered global DNA methylation and increased levels of DNMTs within the nucleus were confirmed in the cortex and hippocampus of the diseased mice, suggesting hypermethylation at a genetic level. Treatment with AzadC, a global DNA demethylating agent, ameliorated the protein and gene expression of the cellular chaperones and synaptic fidelity. Furthermore, the methylation analysis profile showed hypermethylation of the hsf1 protein, a master regulator for chaperones and thus, confirmed the epigenetic involvement in the diseased brain. Morphological improvements and decreased neurodegeneration, along with enhanced neurogenesis in the treatment group, suggest that epigenetic modulations do participate in learning and memory. This is supported by the improved behavioral test battery seen in the treatment group. Conclusion: DNA methylation could possibly accord in dysregulating the memory-associated proteins at chronic stages in type 2 diabetes. This could suggest a substantial contribution to the underlying pathophysiology of several metabolic syndromes like insulin resistance, obesity and also participate in transitioning this damage centrally, such as cognitive dysfunction.

Keywords: epigenetics, cognition, chaperones, DNA methylation

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Authors: Mayada Mazher, Ahmed Moustafa, Ahmed Abdellatif

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Background: Autophagy is a eukaryotic, highly conserved catabolic process implicated in many pathophysiologies such as wound healing. Autophagy-associated genes serve as a scaffolding platform for signal transduction of macrophage polarization during the inflammatory phase of wound healing and tissue repair process. In the current study, we report a model for the interplay between autophagy-associated genes and macrophages polarization associated genes. Methods: In silico analysis was performed on 249 autophagy-related genes retrieved from the public autophagy database and gene expression data retrieved from Gene Expression Omnibus (GEO); GSE81922 and GSE69607 microarray data macrophages polarization 199 DEGS. An integrated protein-protein interaction network was constructed for autophagy and macrophage gene sets. The gene sets were then used for GO terms pathway enrichment analysis. Common transcription factors for autophagy and macrophages' polarization were identified. Finally, microRNAs enriched in both autophagy and macrophages were predicated. Results: In silico prediction of common transcription factors in DEGs macrophages and autophagy gene sets revealed a new role for the transcription factors, HOMEZ, GABPA, ELK1 and REL, that commonly regulate macrophages associated genes: IL6,IL1M, IL1B, NOS1, SOC3 and autophagy-related genes: Atg12, Rictor, Rb1cc1, Gaparab1, Atg16l1. Conclusions: Autophagy and macrophages' polarization are interdependent cellular processes, and both autophagy-related proteins and macrophages' polarization related proteins coordinate in tissue remodelling via transcription factors and microRNAs regulatory network. The current work highlights a potential new role for transcription factors HOMEZ, GABPA, ELK1 and REL in wound healing.

Keywords: autophagy related proteins, integrated network analysis, macrophages polarization M1 and M2, tissue remodelling

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: N. R. Prasannakumar, M. N. Maruthi

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The whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae) is a highly polyphagous pest reported to infest over 600 plant hosts globally. About 42 genetic groups/cryptic species of B. tabaci exist in the world on different hosts. The species have variable behaviour with respect to feeding, development and transmission of viral diseases. Feeding on diverse host plants affect both whitefly development and the population of the endosymbionts harboured by the insects. Due to changes in the level of endosymbionts, the virus transmission efficiency by the vector also gets affected. We investigated these interactions on five host plants – egg plant, tomato, beans, okra and cotton - using a single whitefly species Asia 1 infected with three different bacteria Portiera, Wolbachia and Arsenophonus. The Asia 1 transmits the Tomato leaf curl Bangalore virus (ToLCBV) effectively and thus was used in the interaction studies. We found a significant impact of hosts on whitefly growth and development; eggplant was most favourable host, while okra and tomato were least favourable. Among the endosymbiotic bacteria, the titre of Wolbachia was significantly affected by feeding of B. tabaci on different host plants whereas Arsenophonus and Portiera were unaffected. When whitefly fed on ToLCBV-infected tomato plants, the Arsenophonus population was significantly increased, indicating its previously confirmed role in ToLCBV transmission. Further, screening of total proteins of B. tabaci Asia 1 genetic group interacting with ToLCBV coat protein was carried out using Y2H system. Some of the proteins found to be interacting with ToLCBV CP were HSPs 70kDa, GroEL, nucleoproteins, vitellogenins, apolipophorins, lachesins, enolase. The reported protein thus would be the potential targets for novel whitefly control strategies such as RNAi or novel insecticide target sites for sustainable whitefly management after confirmation of genuine proteins.

Keywords: cDNA, whitefly, ToLCBV, endosymbionts, Y2H

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Authors: Chien Y. Lin, Jung Y. Huang, Leu-Wei Lo

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A growing body of data reveals that the membrane cholesterol molecules can alter the signaling pathways of living cells. However, the understanding about how membrane cholesterol modulates receptor proteins is still lacking. Single-molecule tracking can effectively probe into the microscopic environments and thermal fluctuations of receptor proteins in a living cell. In this study we applies single-molecule optical tracking on ligand-induced dimerization process of EGFRs in the plasma membranes of two cancer cell lines (HeLa and A431) and one normal endothelial cell line (MCF12A). We tracked individual EGFR and dual receptors, diffusing in a correlated manner in the plasma membranes of live cells. We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to help extracting important information from single-molecule trajectories. From the study, we discovered that ligand-bound EGFRs move from non-raft areas into lipid raft domains. This ligand-induced motion is a common behavior in both cancer and normal cells. By manipulating the total amount of membrane cholesterol with methyl-β-cyclodextrin and the local concentration of membrane cholesterol with nystatin, we further found that the amount of cholesterol can affect the stability of EGFR dimers. The EGFR dimers in the plasma membrane of normal cells are more sensitive to the local concentration changes of cholesterol than EGFR dimers in the cancer cells. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

Keywords: membrane proteins, single-molecule tracking, Cahn-Hilliard equation, EGFR dimers

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Authors: Harshit Agrawal, Salman Muhammad

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Degradation to reinforced concrete (RC), primarily due to corrosion of embedded reinforcement, has been a major cause of concern worldwide. Among several ways to control corrosion, the use of coated reinforcement has gained significant interest in field applications. However, the choice of proper coating material and the effect of damage over coating are yet to be addressed for effective application of coated reinforcements. The present study aims to investigate and compare the performance of three different types of coated reinforcements —Fusion-Bonded Epoxy Coating (FBEC), Cement Polymer Composite Coating (CPCC), and Dual Zinc-Epoxy Coating (DZEC) —in concrete structures. The aim is to assess their corrosion resistance, durability, and overall effectiveness as coated reinforcement materials both in undamaged and simulated damaged conditions. Through accelerated corrosion tests, electrochemical analysis, and exposure to aggressive marine environments, the study evaluates the long-term performance of each coating system. This research serves as a crucial guide for engineers and construction professionals in selecting the most suitable corrosion protection for reinforced concrete, thereby enhancing the durability and sustainability of infrastructure.

Keywords: corrosion, reinforced concrete, coated reinforcement, seawater exposure, electrochemical analysis, service life, corrosion prevention

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Authors: Mohammadjavad Sotoudeheian

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Heart failure (HF) prevalence, as a global cardiovascular problem, is increasing gradually. A variety of molecular mechanisms contribute to HF. Proteins involved in cardiac contractility regulation, such as ion channels and calcium handling proteins, are altered. Additionally, epigenetic modifications and gene expression can lead to altered cardiac function. Moreover, inflammation and oxidative stress contribute to HF. The progression of HF can be attributed to mitochondrial dysfunction that impairs energy production and increases apoptosis. Molecular mechanisms such as these contribute to the development of cardiomyocyte defects and HF and can be therapeutically targeted. The heart's contractile function is controlled by cardiomyocytes. Calpain, and its related molecules, including Bax, VEGF, and AMPK, are among the proteins involved in regulating cardiomyocyte function. Apoptosis is facilitated by Bax. Cardiomyocyte apoptosis is regulated by this protein. Furthermore, cardiomyocyte survival, contractility, wound healing, and proliferation are all regulated by VEGF, which is produced by cardiomyocytes during inflammation and cytokine stress. Cardiomyocyte proliferation and survival are also influenced by AMPK, an enzyme that plays an active role in energy metabolism. They all play key roles in apoptosis, angiogenesis, hypertrophy, and metabolism during myocardial inflammation. The role of calpains has been linked to several molecular pathways. The calpain pathway plays an important role in signal transduction and apoptosis, as well as autophagy, endocytosis, and exocytosis. Cell death and survival are regulated by these calcium-dependent cysteine proteases that cleave proteins. As a result, protein fragments can be used for various cellular functions. By cleaving adhesion and motility proteins, calcium proteins also contribute to cell migration. HF may be brought about by calpain-mediated pathways. Many physiological processes are mediated by the calpain molecular pathways. Signal transduction, cell death, and cell migration are all regulated by these molecular pathways. Calpain is activated by calcium binding to calmodulin. In the presence of calcium, calmodulin activates calpain. Calpains are stimulated by calcium, which increases matrix metalloproteinases (MMPs). In order to develop novel treatments for these diseases, we must understand how this pathway works. A variety of myocardial remodeling processes involve calpains, including remodeling of the extracellular matrix and hypertrophy of cardiomyocytes. Calpains also play a role in maintaining cardiac homeostasis through apoptosis and autophagy. The development of HF may be in part due to calpain-mediated pathways promoting cardiomyocyte death. Numerous studies have suggested the importance of the Ca2+ -dependent protease calpain in cardiac physiology and pathology. Therefore, it is important to consider this pathway to develop and test therapeutic options in humans that targets calpain in HF. Apoptosis, autophagy, endocytosis, exocytosis, signal transduction, and disease progression all involve calpain molecular pathways. Therefore, it is conceivable that calpain inhibitors might have therapeutic potential as they have been investigated in preclinical models of several conditions in which the enzyme has been implicated that might be treated with them. Ca 2+ - dependent proteases and calpains contribute to adverse ventricular remodeling and HF in multiple experimental models. In this manuscript, we will discuss the calpain molecular pathway's important roles in HF development.

Keywords: calpain, heart failure, autophagy, apoptosis, cardiomyocyte

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Authors: Manar Makhoul, Buthainah Alsalamah, Salam Lawand, Hassan Azzam

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The major storage proteins in endosperm of 33 cultivated and wild barley genotypes (H.vulgare, H. spontaneum, H. bulbosum, H. murinum, H. marinum) were analyzed to demonstrate the variation in the hordein polypeptides encoded by multigene families in grains. The SDS-PAGE revealed 13 and 17 alleles at the Hor1 and the Hor2 loci respectively, with frequencies from 0.83 to 14 and 0.56 to 13.41% respectively, while seven alleles at the Hor3 locus with frequencies from 3.63 to 30.91% were recognized. The phylogenetic analysis indicated to relevance of the polymorphism in hordein patterns as successful tool in identifying the individual genotypes and discriminating the species according to genome type. We also reported in this research complete nucleotide sequence B-hordein genes of seven wild and cultivated barley genotypes. A 152bp upstream sequence of B-hordein promoter contained a TATA box, CATC box, AAAG motif, N-motif and E-motif. In silico analysis of B-Hordein sequences demonstrated that the coding regions were not interrupted by any intron, and included the complete ORF which varied between 882 and 906 bp, and encoded mature proteins with 293-301 residues characterized by high contents of glutamine (29%), and proline (18%). Comparison of the predicted polypeptide sequences with the published ones suggested that all S-rich prolamins genes are descended from common ancestor. The sequence started at N-terminal with a signal peptide, and then followed directly by two domains; a repetitive one based on the repetition of the repeat unit PQQPFPQQ and C-terminal domain. Also, it was found that positions of the eight cysteine residues were highly conserved in all the B-hordein sequences, but Hordeum bulbosum had additional unpaired one. The phylogenetic tree of B-hordein polypeptide separated the genotypes in distinct seven subgroups. In general, the high homology between B-hordeins and LMW glutenin subunits suggests similar bread-making influences for these B-hordeins.

Keywords: hordeum, phylogenetic tree, sequencing, storage protein

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Authors: Majid Afkhami, Maryam Ehsanpour

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The present study provides information about the nature of adverse effects on fish and the ecological impact that polycyclic aromatic hydrocarbons (PAHs) pollutant are having in the northern coast of Hormuz Strait. The glucose and cholesterol levels were higher in fish from the St3 than in Walton's mudskipper from other stations however St3 samples had lowest total proteins levels. There was a significant positive correlation between glucose and cholesterol with PAHs concentrations in sediment and tissue samples (P<0.05). However, total proteins had adverse significant correlation with PAHs concentrations (P>0.05). The adverse correlation was seen between length and body weight of fish samples with PAHs concentrations. According to the results of this study, the monitoring of contaminants bioaccumulation in the northern part of Hormuz Strait is necessary, because this will give an indication of the temporal and spatial extent of the process, as well as an assessment of the potential impact on aquatic organisms health.

Keywords: PAHs, blood metabolic factors, Periophthalmus waltoni, Hormuz Strait

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Authors: Gayathri Devi V, Aravamudan Kannan, Amit Sircar

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In the inner fuel cycle system of a nuclear fusion reactor, the Hydrogen Isotopes Removal System (HIRS) plays a pivoted role. It enables the effective extraction of the hydrogen isotopes from the breeder purge gas which helps to maintain the tritium breeding ratio and sustain the fusion reaction. One of the components of HIRS, Cryogenic Molecular Sieve Bed (CMSB) columns with zeolites adsorbents are considered for the physisorption of hydrogen isotopes at 1 bar and 77 K. Even though zeolites have good thermal stability and reduced activation properties making them ideal for use in nuclear reactor applications, their modest capacity for hydrogen isotopes adsorption is a cause of concern. In order to enhance the adsorbent capacity in an informed manner, it is helpful to understand the adsorption phenomena at the quantum electronic structure level. Physicochemical modifications of the adsorbent material enhances the adsorption capacity through the incorporation of active sites. This may be accomplished through the incorporation of suitable metal ions in the zeolite framework. In this work, molecular hydrogen isotopes adsorption on the active sites of functionalized zeolites are investigated in detail using Density Functional Theory (DFT) study. This involves the utilization of hybrid Generalized Gradient Approximation (GGA) with dispersion correction to account for the exchange and correlation functional of DFT. The electronic energies, adsorption enthalpy, adsorption free energy, Highest Occupied Molecular Orbital (HOMO), Lowest Unoccupied Molecular Orbital (LUMO) energies are computed on the stable 8T zeolite clusters as well as the periodic structure functionalized with different active sites. The characteristics of the dihydrogen bond with the active metal sites and the isotopic effects are also studied in detail. Validation studies with DFT will also be presented for adsorption of hydrogen on metal ion functionalized zeolites. The ab-inito screening analysis gave insights regarding the mechanism of hydrogen interaction with the zeolites under study and also the effect of the metal ion on adsorption. This detailed study provides guidelines for selection of the appropriate metal ions that may be incorporated in the zeolites framework for effective adsorption of hydrogen isotopes in the HIRS.

Keywords: adsorption enthalpy, functionalized zeolites, hydrogen isotopes, nuclear fusion, physisorption

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Authors: Sahila Chopra, Hemdeep, Arshdeep Kaur, Raj K. Gupta

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In recent times, we noticed an interesting and important role of non-coplanar degree-of-freedom (Φ = 00) in heavy ion reactions. Using the dynamical cluster-decay model (DCM) with Φ degree-of-freedom included, we have studied three compound systems 246Bk∗, 164Yb∗ and 105Ag∗. Here, within the DCM with pocket formula for nuclear proximity potential, we look for the effects of including compact, non-coplanar configurations (Φc = 00) on the non-compound nucleus (nCN) contribution in total fusion cross section σfus. For 246Bk∗, formed in 11B+235U and 14N+232Th reaction channels, the DCM with coplanar nuclei (Φc = 00) shows an nCN contribution for 11B+235U channel, but none for 14N+232Th channel, which on including Φ gives both reaction channels as pure compound nucleus decays. In the case of 164Yb∗, formed in 64Ni+100Mo, the small nCN effects for Φ=00 are reduced to almost zero for Φ = 00. Interestingly, however, 105Ag∗ for Φ = 00 shows a small nCN contribution, which gets strongly enhanced for Φ = 00, such that the characteristic property of PCN presents a change of behaviour, like that of a strongly fissioning superheavy element to a weakly fissioning nucleus; note that 105Ag∗ is a weakly fissioning nucleus and Psurv behaves like one for a weakly fissioning nucleus for both Φ = 00 and Φ = 00. Apparently, Φ is presenting itself like a good degree-of-freedom in the DCM.

Keywords: dynamical cluster-decay model, fusion cross sections, non-compound nucleus effects, non-coplanarity

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Authors: Jinan Fiaidhi, Sabah Mohammed

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Diagnosing cataract severity is an important factor in deciding to undertake surgery. It is usually conducted by an ophthalmologist or through taking a variety of fundus photography that needs to be examined by the ophthalmologist. This paper carries out an investigation using a Siamese neural net that can be trained with small anchor samples to score cataract severity. The model used in this paper is based on a triplet loss function that takes the ophthalmologist best experience in rating positive and negative anchors to a specific cataract scaling system. This approach that takes the heuristics of the ophthalmologist is generally called the thick data approach, which is a kind of machine learning approach that learn from a few shots. Clinical Relevance: The lens of the eye is mostly made up of water and proteins. A cataract occurs when these proteins at the eye lens start to clump together and block lights causing impair vision. This research aims at employing thick data machine learning techniques to rate the severity of the cataract using Siamese neural network.

Keywords: thick data analytics, siamese neural network, triplet-loss model, few shot learning

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Authors: Hiba Naccache, Rima Hleiss

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Additive manufacturing AM or 3D printing is one of the pillars of Industry 4.0. Compared to traditional manufacturing, AM provides a prototype before production in order to optimize the design and avoid the stock market and uses strictly necessary material which can be recyclable, for the benefit of leaning towards local production, saving money, time and resources. Different types of AM exist and it has a broad range of applications across several industries like aerospace, automotive, medicine, education and else. The Laser Powder Bed Fusion (LPBF) is a metal AM technique that uses a laser to liquefy metal powder, layer by layer, to build a three-dimensional (3D) object. In industry 4.0 and aligned with the numbers 9 (Industry, Innovation and Infrastructure) and 12 (Responsible Production and Consumption) of the Sustainable Development Goals of the UNESCO 2030 Agenda, the AM’s manufacturers committed to minimizing the environmental impact by being sustainable in every production. The LPBF has several environmental advantages, like reduced waste production, lower energy consumption, and greater flexibility in creating components with lightweight and complex geometries. However, LPBF also have environmental drawbacks, like energy consumption, gas consumption and emissions. It is critical to recognize the environmental impacts of LPBF in order to mitigate them. To increase awareness and promote sustainable practices regarding LPBF, the researchers use the Elaboration Likelihood Model (ELM) theory where people from multiple universities in France and Qatar process information in two ways: peripherally and centrally. The peripheral campaigns use superficial cues to get attention, and the central campaigns provide clear and concise information. The authors created a seminar including a video showing LPBF production and a website with educational resources. The data is collected using questionnaire to test attitude about the public awareness before and after the seminar. The results reflected a great shift on the awareness toward LPBF and its impact on the environment. With no presence of similar research, to our best knowledge, this study will add to the literature on the sustainability of the LPBF production technique.

Keywords: additive manufacturing, laser powder bed fusion, elaboration likelihood model theory, sustainable development goals, education-awareness, France, Qatar, specific energy consumption, environmental impact, lightweight components

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Authors: Alexander A. Tokmakov

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Cell-free protein synthesis is widely used to synthesize recombinant proteins. It allows genome-scale expression of various polypeptides under strictly controlled uniform conditions. However, only a minor fraction of all proteins can be successfully expressed in the systems of protein synthesis that are currently used. The factors determining expression success are poorly understood. At present, the vast volume of data is accumulated in cell-free expression databases. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with successful cell-free expression. Here, we describe an approach aimed at identification of multiple physicochemical and structural properties of amino acid sequences associated with protein solubility and aggregation and highlight major correlations obtained using this approach. The developed method includes: categorical assessment of the protein expression data, calculation and prediction of multiple properties of expressed amino acid sequences, correlation of the individual properties with the expression scores, and evaluation of statistical significance of the observed correlations. Using this approach, we revealed a number of statistically significant correlations between calculated and predicted features of protein sequences and their amenability to cell-free expression. It was found that some of the features, such as protein pI, hydrophobicity, presence of signal sequences, etc., are mostly related to protein solubility, whereas the others, such as protein length, number of disulfide bonds, content of secondary structure, etc., affect mainly the expression propensity. We also demonstrated that amenability of polypeptide sequences to cell-free expression correlates with the presence of multiple sites of post-translational modifications. The correlations revealed in this study provide a plethora of important insights into protein folding and rationalization of protein production. The developed bioinformatics approach can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

Keywords: bioinformatics analysis, cell-free protein synthesis, expression success, optimization, recombinant proteins

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Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus

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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.

Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor

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Authors: Manuel A. Alonso-Tarajano, Roberto Mosca, Patrick Aloy

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Protein kinases participate in a myriad of cellular processes of major biomedical interest. The in vivo substrate specificity of these enzymes is a process determined by several factors, and despite several years of research on the topic, is still far from being totally understood. In the present work, we have quantified the contributions to the kinase substrate specificity of i) the phosphorylation sites and their surrounding residues in the sequence and of ii) the association of kinases to adaptor or scaffold proteins. We have used position-specific scoring matrices (PSSMs), to represent the stretches of sequences phosphorylated by 93 families of kinases. We have found negative correlations between the number of sequences from which a PSSM is generated and the statistical significance and the performance of that PSSM. Using a subset of 22 statistically significant PSSMs, we have identified specificity determinant residues (SDRs) for 86% of the corresponding kinase families. Our results suggest that different SDRs can function as positive or negative elements of substrate recognition by the different families of kinases. Additionally, we have found that human proteins with known function as adaptors or scaffolds (kAS) tend to interact with a significantly large fraction of the substrates of the kinases to which they associate. Based on this characteristic we have identified a set of 279 potential adaptors/scaffolds (pAS) for human kinases, which is enriched in Pfam domains and functional terms tightly related to the proposed function. Moreover, our results show that for 74.6% of the kinase– pAS association found, the pAS colocalize with the substrates of the kinases they are associated to. Finally, we have found evidence suggesting that the association of kinases to adaptors and scaffolds, may contribute significantly to diminish the in vivo substrate crossed- specificity of protein kinases. In general, our results indicate the relevance of several SDRs for both the positive and negative selection of phosphorylation sites by kinase families and also suggest that the association of kinases to pAS proteins may be an important factor for the localization of the enzymes with their set of substrates.

Keywords: kinase, phosphorylation, substrate specificity, adaptors, scaffolds, cellular colocalization

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Authors: Karim Kheloufi, El Hachemi Amara

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In the present study, a three-dimensional transient numerical model was developed to study the temperature field and cutting kerf shape during laser fusion cutting. The finite volume model has been constructed, based on the Navier–Stokes equations and energy conservation equation for the description of momentum and heat transport phenomena, and the Volume of Fluid (VOF) method for free surface tracking. The Fresnel absorption model is used to handle the absorption of the incident wave by the surface of the liquid metal and the enthalpy-porosity technique is employed to account for the latent heat during melting and solidification of the material. To model the physical phenomena occurring at the liquid film/gas interface, including momentum/heat transfer, a new approach is proposed which consists of treating friction force, pressure force applied by the gas jet and the heat absorbed by the cutting front surface as source terms incorporated into the governing equations. All these physics are coupled and solved simultaneously in Fluent CFD®. The main objective of using a transient phase change model in the current case is to simulate the dynamics and geometry of a growing laser-cutting generated kerf until it becomes fully developed. The model is used to investigate the effect of some process parameters on temperature fields and the formed kerf geometry.

Keywords: laser cutting, numerical simulation, heat transfer, fluid flow

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Authors: Razieh Karimi Aghcheh, Christian Kubicek, Joseph Strauss, Gerhard Braus

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Filamentous fungi are of considerable economic and social significance for human health, nutrition and in white biotechnology. These organisms are dominant producers of a range of primary metabolites such as citric acid, microbial lipids (biodiesel) and higher unsaturated fatty acids (HUFAs). In particular, they produce also important but structurally complex secondary metabolites with enormous therapeutic applications in pharmaceutical industry, for example: cephalosporin, penicillin, taxol, zeranol and ergot alkaloids. Several fungal secondary metabolites, which are significantly relevant to human health do not only include antibiotics, but also e.g. lovastatin, a well-known antihypercholesterolemic agent produced by Aspergillus. terreus, or aflatoxin, a carcinogen produced by A. flavus. In addition to their roles for human health and agriculture, some fungi are industrially and commercially important: Species of the ascomycete genus Hypocrea spp. (teleomorph of Trichoderma) have been demonstrated as efficient producer of highly active cellulolytic enzymes. This trait makes them effective in disrupting and depolymerization of lignocellulosic materials and thus applicable tools in number of biotechnological areas as diverse as clothes-washing detergent, animal feed, and pulp and fuel productions. Fungal LaeA/LAE1 (Loss of aflR Expression A) homologs their gene products act at the interphase between secondary metabolisms, cellulase production and development. Lack of the corresponding genes results in significant physiological changes including loss of secondary metabolite and lignocellulose degrading enzymes production. At the molecular level, the encoded proteins are presumably methyltransferases or demethylases which act directly or indirectly at heterochromatin and interact with velvet domain proteins. Velvet proteins bind to DNA and affect expression of secondary metabolites (SMs) genes and cellulases. The dynamic interplay between LaeA/LAE1, velvet proteins and additional interaction partners is the key for an understanding of the coordination of metabolic and morphological functions of fungi and is required for a biotechnological control of the formation of desired bioactive products. Aspergilli and Trichoderma represent different biotechnologically significant species with significant differences in the LaeA/LAE1-Velvet protein machinery and their target proteins. We, therefore, performed a comparative study of the interaction partners of this machinery and the dynamics of the various protein-protein interactions using our robust proteomic and mass spectrometry techniques. This enhances our knowledge about the fungal coordination of secondary metabolism, cellulase production and development and thereby will certainly improve recombinant fungal strain construction for the production of industrial secondary metabolite or lignocellulose hydrolytic enzymes.

Keywords: cellulases, LaeA/1, proteomics, secondary metabolites

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Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Huawei Bai, Jianguo Yao, Fellow, IEEE

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Atrial fibrillation (AF) is the most common form of heart arrhythmia and is closely associated with mortality and morbidity in heart failure, stroke, and coronary artery disease. The development of single spot optical sensors enables widespread photoplethysmography (PPG) screening, especially for AF, since it represents a more convenient and noninvasive approach. To our knowledge, most existing studies based on public and unbalanced datasets can barely handle the multiple noises sources in the real world and, also, lack interpretability. In this paper, we construct a large- scale PPG dataset using measurements collected from PPG wrist- watch devices worn by volunteers and propose an attention-based two-stream heterogeneous neural network (TSHNN). The first stream is a hybrid neural network consisting of a three-layer one-dimensional convolutional neural network (1D-CNN) and two-layer attention- based bidirectional long short-term memory (Bi-LSTM) network to learn representations from temporally sampled signals. The second stream extracts latent representations from the PPG time-frequency spectrogram using a five-layer CNN. The outputs from both streams are fed into a fusion layer for the outcome. Visualization of the attention weights learned demonstrates the effectiveness of the attention mechanism against noise. The experimental results show that the TSHNN outperforms all the competitive baseline approaches and with 98.09% accuracy, achieves state-of-the-art performance.

Keywords: PPG wearables, atrial fibrillation, feature fusion, attention mechanism, hyber network

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