Search results for: diagnostic enzymes

Authors: Swati Kapoor, Rajeev Upreti, Monica Mahajan, Abhaya Indrayan, Dinesh Srivastava

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Enteric Fever is caused by gram negative bacilli Salmonella typhi and paratyphi. It is associated with high morbidity and mortality worldwide. Timely initiation of treatment is a crucial step for prevention of any complications. Cultures of body fluids are diagnostic, but not always conclusive or practically feasible in most centers. Moreover, the results of cultures delay the treatment initiation. Serological tests lack diagnostic value. The blood counts can offer a promising option in diagnosis. A retrospective study to find out the relevance of leucopenia and eosinopenia was conducted on 203 culture proven enteric fever patients and 159 culture proven non-enteric fever patients in a tertiary care hospital in New Delhi. The patient details were retrieved from the electronic medical records section of the hospital. Absolute eosinopenia was considered as absolute eosinophil count (AEC) of less than 40/mm³ (normal level: 40-400/mm³) using LH-750 Beckman Coulter Automated machine. Leucopoenia was defined as total leucocyte count (TLC) of less than 4 X 10⁹/l. Blood cultures were done using BacT/ALERT FA plus automated blood culture system before first antibiotic dose was given. Case and control groups were compared using Pearson Chi square test. It was observed that absolute eosinophil count (AEC) of 0-19/mm³ was a significant finding (p < 0.001) in enteric fever patients, whereas leucopenia was not a significant finding (p=0.096). Using Receiving Operating Characteristic (ROC) curves, it was observed that patients with both AEC < 14/mm³ and TCL < 8 x 10⁹/l had 95.6% chance of being diagnosed as enteric fever and only 4.4% chance of being diagnosed as non-enteric fever. This result was highly significant with p < 0.001. This is a very useful association of AEC and TLC found in enteric fever patients of this study which can be used for the early initiation of treatment in clinically suspected enteric fever patients.

Keywords: absolute eosinopenia, absolute eosinophil count, enteric fever, leucopenia, total leucocyte count

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Authors: Onur Ercan, Buket Askin, Erdogan Kucukoner

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Blueberry (Vaccinium corymbosum, bluegold) has become popular beverage due to their nutritional values such as vitamins, minerals, and antioxidants. In the study, the effects of pressing, mashing, enzymatic treatment, and pasteurization on anthocyanins, colour, and polyphenolics of blueberry juice (BJ) were studied. The blueberry juice (BJ) was produced with two different methods that direct juice extraction (DJE) and mash treatment process (MTP) were applied. After crude blueberry juice (CBJ) production, the samples were first treated with commercial enzymes [Novoferm-61 (Novozymes A/S) (2–10 mL/L)], to break down the hydrocolloid polysaccharides, mainly pectin and starch. The enzymes were added at various concentrations. The highest transmittance (%) was obtained for Novoferm-61 at a concentration of 2 mL/L was 66.53%. After enzymatic treatment, clarification trials were applied to the enzymatically treated BJs with adding various amounts of bentonite (10%, w/v), gelatin (1%, w/v) and kiselsol (15%, v/v). The turbidities of the clarified samples were then determined. However, there was no significant differences between transmittances (%) for samples. For that, only enzymatic treatment was applied to the blueberry juice processing (DDBJ, depectinized direct blueberry juice). Based on initial pressing process made to evaluate press function, it was determined that pressing fresh blueberries with no other processing did not render adequate juice due to lack of liquefaction. Therefore, the blueberries were mash into small pieces (3 mm) and then enzymatic treatments and clarification trials were performed. Finally, both BJ samples were pasteurized. Compositional analyses, colour properties, polyphenols and antioxidant properties were compared. Enzymatic treatment caused significant reductions in ACN content (30%) in Direct Blueberry Juice Processing (DBJ), while there was a significant increasing in Mash Treatment Processing (MTP). Overall anthocyanin levels were higher intreated samples after each processing step in MTP samples, but polyphenolic levels were slightly higher for both processes (DBJ and MTP). There was a reduction for ACNs and polyphenolics only after pasteurization. It has a result that the methods for tried to blueberry juice is suitable into obtain fresh juice. In addition, we examined fruit juice during processing stages; anthocyanin, phenolic substance content and antioxidant activity are higher, and yield is higher in fruit juice compared to DBJ method in MTP method, the MTP method should be preferred in processing juice of blueberry into fruit juice.

Keywords: anthocyanins, blueberry, depectinization, polyphenols

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Authors: Yanling Li, Linying Ji, Zita Oravecz, Timothy R. Brick, Michael D. Hunter, Sy-Miin Chow

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Assessing several individuals intensively over time yields intensive longitudinal data (ILD). Even though ILD provide rich information, they also bring other data analytic challenges. One of these is the increased occurrence of missingness with increased study length, possibly under non-ignorable missingness scenarios. Multiple imputation (MI) handles missing data by creating several imputed data sets, and pooling the estimation results across imputed data sets to yield final estimates for inferential purposes. In this article, we introduce dynr.mi(), a function in the R package, Dynamic Modeling in R (dynr). The package dynr provides a suite of fast and accessible functions for estimating and visualizing the results from fitting linear and nonlinear dynamic systems models in discrete as well as continuous time. By integrating the estimation functions in dynr and the MI procedures available from the R package, Multivariate Imputation by Chained Equations (MICE), the dynr.mi() routine is designed to handle possibly non-ignorable missingness in the dependent variables and/or covariates in a user-specified dynamic systems model via MI, with convergence diagnostic check. We utilized dynr.mi() to examine, in the context of a vector autoregressive model, the relationships among individuals’ ambulatory physiological measures, and self-report affect valence and arousal. The results from MI were compared to those from listwise deletion of entries with missingness in the covariates. When we determined the number of iterations based on the convergence diagnostics available from dynr.mi(), differences in the statistical significance of the covariate parameters were observed between the listwise deletion and MI approaches. These results underscore the importance of considering diagnostic information in the implementation of MI procedures.

Keywords: dynamic modeling, missing data, mobility, multiple imputation

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Authors: T. Urushadze, R. Khvedelidze, L. Kutateladze, M. Jobava, T. Burduli, T. Alexidze

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There is an increasing interest in reliable, wasteless, ecologically friendly technologies. About 40% of enzymes produced all over the world are used for production of syrups with high concentration of glucose-fructose. One of such technologies complies obtaining fermentable sugar glucose from raw materials containing starch by means of amylases. In modern alcohol-producing factories this process is running in two steps, involving two enzymes of different origin: bacterial α-amylase and fungal glucoamylase, as generally fungal amylases are less thermostable as compared to bacterial amylases. Selection of stable and operable at 700С and higher temperatures enzyme preparation with both α- and glucoamylase activities will allow conducting this process in one step. S. Durmishidze Institute of Biochemistry and Biotechnology owns unique collection of mycelial fungi, isolated from different ecological niches of Caucasus. As a result of screening our collection 39 strains poducing amylases were revealed. Most of them belong to the genus Aspergillus. Optimum temperatures of action of selected amylases from three producers were estableshed to be within the range 67-80°C. A. niger B-6 showed higher α-amylase activity at 67°C, and glucoamylase activity at 62°C, A. niger 6-12 showed higher α-amylase activity at 72°C, and glucoamylase activity at 65°C, Aspergillus niger p8-3 showed higher activities at 82°C and 70°C, for α-amylase and glucoamylase activities, respectively. Exhaustive hydrolysis process of starch solutions of different concentrations (3, 5, 15, and 30 %) with cultural liquid and technical preparation of Aspergillus niger p8-3 enzyme was studied. In case of low concentrations exhaustive hydrolysis of starch lasts 40–60 minutes, in case of high concentrations hydrolysis takes longer time. 98, 6% yield of glucose can be reached at incubation during 12 hours with enzyme cultural liquid and 8 hours incubation with technical preparation of the enzyme at gradual increase of temperature from 50°C to 82°C during the first 20 minutes and further decrease of temperature to 70°C. Temperature setting for high yield of glucose and high hydrolysis (pasteurizing), optimal for activity of these strains is the prerequisite to be able to carry out hydrolysis of starch to glucose in one step, and consequently, using one strain, what will be economically justified.

Keywords: amylase, glucose hydrolisis, stability, starch

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Authors: Muhammad Suhail Ibrahim, Ahmad Mujtaba

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Background: The accuracy of the most frequently used tests for diagnosing Helicobacter pylori is always under consideration in clinical settings. A reliable diagnosis is crucial to confirm the success of therapy. Objective: The aim of this research was to study the isolation frequency of H. pylori from patients compatible with gastritis or gastric ulcer and to compare some feasible non-invasive and invasive methods for the diagnosis of infection. Materials and Methods: Ninety-six gastric biopsy and blood samples were obtained with various gastroduodenal symptoms after obtaining informed consent. The biopsies were analyzed and compared using the culture, microscopic examination, histopathology, Rapid urease RUT), serology, biochemical, antibiotic susceptibility test and molecular method. Results: A number of 40 (41.67%) were considered H. pylori positive in both histopathology and RUT. On the other hand, 46 patients were positive against anti IgA and IgG by ELISA. Eighteen biopsies were positive according to the culture test. This was further confirmed by endoscopic examination, urease, catalase and oxidase tests. A high percentage of resistance to polymyxin B, amoxicillin, and kanamycin was observed (100, 88.89, and 77.78%, respectively). A gene (Cag A) was also detected by using molecular technique which appeared positive in 16 patients. The sensitivity/specificity (%) of diagnostic method was 95/77 for histology, 100/83.5 for rapid urease, 85.7/90 for gram staining, 100/66.6 for IgG serology, 100/79.5 for IgA serology, 100/75.0 for PCR, 100/79.04 for combination of RUT and IgG serology and 100/92.4 for combination of RUT, gram staining and IgG serology. Conclusion: In view of the result obtained, PCR appeared to be the most reliable test. However, higher sensitivity and specificity were also recorded for other tests. So, for more accurate results, it is advisable not to rely solely on a single method for detection.

Keywords: helicobacter pylori, isolation, detection, culture, urease, polymerase chain reaction, antibiotic susceptibility test, dyspeptic patients

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Authors: Chengcao Sun, Cuili Yang, Shujun Li, Ruilin Xue, Liang Wang, Yongyong Xi, Dejia Li

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Backgrounds: Sulforaphane, one of the most important isothiocyanates in the human diet, is known to have chemopreventive and antioxidant activities in different tissues via activation of NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of Sulforaphane on Duchenne muscular dystrophy (DMD). Methods: 4-week-old mdx mice were treated with SFN by gavage (2 mg/kg body weight per day) for 8 weeks. Blood was collected from eye socket every week, and tibial anterior, extensor digitorum longus, gastrocnemius, soleus, triceps brachii muscles and heart samples were collected after 8-week gavage. Force measurements and mice exercise capacity assays were detected. GSH/GSSG ratio, TBARS, CK and LDH levels were analyzed by spectrophotometric methods. H&E staining was used to analyze histological and morphometric of skeletal muscles of mdx mice, and Evas blue dye staining was made to detect sarcolemmal integrity of mdx mice. Further, the role of Sulforaphane on Nrf2/ARE signaling pathway was analyzed by ELISA, western blot and qRT-PCR. Results: Our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NQO1 and HO-1 with Nrf2 dependent manner. SFN significantly increased skeletal muscle mass, muscle force (~30%), running distance (~20%) and GSH/GSSG ratio (~3.2 folds) of mdx mice, and decreased the activities of plasma creatine phosphokinase (CK) (~45%) and lactate dehydrogenase (LDH) (~40%), gastrocnemius hypertrophy (~25%), myocardial hypertrophy (~20%) and MDA levels (~60%). Further, SFN treatment also reduced the central nucleation (~40%), fiber size variability, inflammation and improved the sarcolemmal integrity of mdx mice. Conclusions: Collectively, these results show that SFN can improve muscle function, pathology and protect dystrophic muscle from oxidative damage in mdx mice through Nrf2 signaling pathway, which indicate Nrf2 may have clinical implications for the treatment of patients with muscular dystrophy.

Keywords: sulforaphane, duchenne muscular dystrophy, Nrf2, oxidative stress

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Authors: Andrew White, Abigail Lowe, Kory Watkins, Hamed Akhlaghi, Nicole Winter

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The timing and appropriateness of diagnostic imaging are critical to the evaluation and management of traumatic injuries. Within the subclass of trauma patients, the prevalence of c-spine injury is less than 4%. However, the incidence of delayed diagnosis within this cohort has been documented as up to 20%, with inadequate radiological examination most cited issue. In order to assess those in which c-spine injury cannot be fully excluded based on clinical examination alone and, therefore, should undergo diagnostic imaging, a set of criteria is used to provide clinical guidance. The NEXUS (National Emergency X-Radiography Utilisation Study) criteria is a validated clinical decision-making tool used to facilitate selective c-spine radiography. The criteria allow clinicians to determine whether cervical spine imaging can be safely avoided in appropriate patients. The NEXUS criteria are widely used within the Emergency Department setting given their ease of use and relatively straightforward application and are used in the Victorian State Trauma System’s guidelines. This audit utilized retrospective data collection to examine the concordance of c-spine imaging in trauma patients to that of the NEXUS criteria and assess compliance with state guidance on diagnostic imaging in trauma. Of the 183 patients that presented with trauma to the head, neck, or face (244 excluded due to incorrect triage), 98 did not undergo imaging of the c-spine. Out of those 98, 44% fulfilled at least one of the NEXUS criteria, meaning the c-spine could not be clinically cleared as per the current guidelines. The criterion most met was intoxication, comprising 42% (18 of 43), with midline spinal tenderness (or absence of documentation of this) the second most common with 23% (10 of 43). Intoxication being the most met criteria is significant but not unexpected given the cohort of patients seen at St Vincent’s and within many emergency departments in general. Given these patients will always meet NEXUS criteria, an element of clinical judgment is likely needed, or concurrent use of the Canadian C-Spine Rules to exclude the need for imaging. Midline tenderness as a met criterion was often in the context of poor or absent documentation relating to this, emphasizing the importance of clear and accurate assessments. The distracting injury was identified in 7 out of the 43 patients; however, only one of these patients exhibited a thoracic injury (T11 compression fracture), with the remainder comprising injuries to the extremities – some studies suggest that C-spine imaging may not be required in the evaluable blunt trauma patient despite distracting injuries in any body regions that do not involve the upper chest. This emphasises the need for standardised definitions for distracting injury, at least at a departmental/regional level. The data highlights the currently poor application of the NEXUS guidelines, with likely common themes throughout emergency departments, highlighting the need for further education regarding implementation and potential refinement/clarification of criteria. Of note, there appeared to be no significant differences between levels of experience with respect to inappropriately clearing the c-spine clinically with respect to the guidelines.

Keywords: imaging, guidelines, emergency medicine, audit

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Authors: Kumari Rekha, Mathur K Dhananjay, Maheshwari Deepanshu, Nautiyal Nidhi, Shubham Smriti, Laal Deepika, Sinha Swati, Kumar Anupam, Biswas Subhrajit, Shiv Kumar Sarin

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Background: Mesenchymal stem cells are multipotent stem cells derived from mesoderm and are used for therapeutic purposes because of their self-renewal, homing capacity, Immunomodulatory capability, low immunogenicity and mitochondrial transfer signaling. MSCs have the ability to regulate the mechanism of both innate as well as adaptive immune responses through the modulation of cellular response and the secretion of inflammatory mediators. Different sources of MSC are UC MSC, BM MSC, Dental Pulp, and Adipose MSC. The most frequent source used is umbilical cord tissue due to its being easily available and free of limitations of collection procedures from respective hospitals. The immunosuppressive role of MSCs is particularly interesting for clinical use since it confers resistance to rejection by the host immune response. Methodology: In this study, T helper cells (TH4), Cytotoxic T cells (CD-8), immunoregulatory cells (CD25 +FOXP3+) are compared from isolated MSC from three different methods, UC Dissociation Kit (Miltenyi), Explant Culture and Collagenase Type-IV. To check the immunomodulatory property, these MSCs were seeded with PBMC(Coculture) in CD3 coated 24 well plates. Cd28 antibody was added in coculture for six days. The coculture was analyzed in FACS Verse flow cytometry. Results: From flow cytometry analysis of coculture, it found that All over T helper cells (CD4+) number p<0.0264 increases in (All Enzymes) MSC rather than explant MSC(p>0.0895) as compared to Collagenase(p>0.7889) in a coculture of Activated T cell and Mesenchymal Stem Cell. Similar T reg cells (CD25+, FOXP3+) expression p<0.0234increases in All Enzymes), decreases in Explant and Collagenase. Experiments have shown that MSCs can also directly prevent the cytotoxic activity of CD8 lymphocytes mainly by blocking their proliferation rather than by inhibiting the cytotoxic effect. And promoting the t-reg cells, which helps in the mediation of immune response in various diseases. Conclusion: MSC suppress Cytotoxic CD8 T cell and Enhance immunoregulatory T reg (CD4+, CD25+, FOXP3+) Cell expression. Thus, MSC maintains a proper balance(ratio) between CD4 T cells and Cytotoxic CD8 T cells.

Keywords: MSC, disease, T cell, T regulatory

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Authors: Sally Ibrahim, Mohamed Hedia, Mohamed Taqi, Mohamed Derbala, Karima Mahmoud, Youssef Ahmed, Sayed Ismail, Mohamed El-Belely

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The identification and quantification of serum microRNA (miRNA) from mares with endometritis might serve as useful and implementable clinical biomarkers for the early diagnosis of endometiritis. Aims of the current study were (I) to study the expression pattern of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205, and (II) to determine the levels of interleukin 6 (IL-6), prostaglandins (PGF₂α and PGE₂), in the serum of Arabian mares with healthy and abnormal uterine status (endometritis). This study was conducted on 80 Arabian mares (4-14 years old). Mares were divided into 48 sub-fertile mares suspected of endometritis and 32 fertile at stud farms. The criteria for mares to be enrolled in the endometritis group were that they had been bred three or more times unsuccessfully in the breeding season or had a history of more than one year of reproductive failure. In addition, two or more of the following criteria on a checklist were present: abnormal clinical findings, transrectal ultrasonographic uterine examination showed abnormal fluid in the uterus (echogenic or ≥2 cm in diameter), positive endometrial cytology; and bacterial and/or fungal growth. Serum samples were collected for measuring IL-6, PGF₂α, and PGE₂ concentrations, as well as serum miRNA isolation and quantitative real-time PCR. Serum concentrations of IL-6, PGE₂, and PGF₂α were higher (P ≤ 0.001) in mares with endometritis compared to the control healthy ones. The expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 increased (P≤0.001) in mares with endometritis compared to the control ones. To the best of our knowledge, this is the first study that revealed that serum miRNA and serum inflammatory mediators (IL-6, PGE₂, and PGF₂α) could be used as non-invasive gold standard biomarkers, and therefore might be served as an important additional diagnostic tool for endometritis in Arabian mares. Moreover, estimation of the serum concentrations of serum miRNA, IL-6, PGE₂, and PGF₂α is a promising recommended tool during the breeding soundness examination in mares.

Keywords: Arabian Mares, endometritis, inflammatory mediators, serum miRNA

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Authors: Basant Singh Sikarwar, Mukesh Roy, Ayush Goyal, Priya Ranjan

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This work combines two bodies of knowledge which includes biomedical basis of blood stain formation and fluid communities’ wisdom that such formation of blood stain depends heavily on physical properties. Moreover biomedical research tells that different patterns in stains of blood are robust indicator of blood donor’s health or lack thereof. Based on these valuable insights an innovative screening tool is proposed which can act as an aide in the diagnosis of diseases such Anemia, Hyperlipidaemia, Tuberculosis, Blood cancer, Leukemia, Malaria etc., with enhanced confidence in the proposed analysis. To realize this powerful technique, simple, robust and low-cost micro-fluidic devices, a micro-capillary viscometer and a pendant drop tensiometer are designed and proposed to be fabricated to measure the viscosity, surface tension and wettability of various blood samples. Once prognosis and diagnosis data has been generated, automated linear and nonlinear classifiers have been applied into the automated reasoning and presentation of results. A support vector machine (SVM) classifies data on a linear fashion. Discriminant analysis and nonlinear embedding’s are coupled with nonlinear manifold detection in data and detected decisions are made accordingly. In this way, physical properties can be used, using linear and non-linear classification techniques, for screening of various diseases in humans and cattle. Experiments are carried out to validate the physical properties measurement devices. This framework can be further developed towards a real life portable disease screening cum diagnostics tool. Small-scale production of screening cum diagnostic devices is proposed to carry out independent test.

Keywords: blood, physical properties, diagnostic, nonlinear, classifier, device, surface tension, viscosity, wettability

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Authors: Z. Abdessemed, N. Messaâdia, M. Houhamdi

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The PAH (Polycyclic Aromatic Hydrocarbons) represent a persistent source of pollution for oil field soils. Their degradation, essentially dominated by the aerobic bacterial and fungal flora, exhibits certain aspects for remediation of these soils microbial oxygenases have, as their substrates, a large range of PAH. The variety and the performance of these enzymes allow the initiation of the biodegradation of any PAH through many different metabolic pathways. These pathways are very important for the recycling of the PAH in the biosphere, where substances supposed indigestible by living organisms are rapidly transformed into simples compounds, directly assimilated by the intermediate metabolism of other microorganisms.

Keywords: polycyclic aromatic hydrocarbons, microbial oxygenases, biodegradation, metabolic pathways

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Authors: Nacera Yassa, Abdelmalek Saidoune, Ghania Ouadfel, Hamza Houassine

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The rapid advancement in electrical technologies has underscored the increasing importance of multiphase machines across various industrial sectors. These machines offer significant advantages in terms of efficiency, compactness, and reliability compared to their single-phase counterparts. However, early detection and diagnosis of electrical faults remain critical challenges to ensure the durability and safety of these complex systems. This paper presents an advanced analytical model for multiphase machines, with a particular focus on dual stator asynchronous machines. The primary objective is to develop a robust diagnostic tool capable of effectively detecting and locating electrical faults in these machines, including short circuits, winding faults, and voltage imbalances. The proposed methodology relies on an analytical approach combining electrical machine theory, modeling of magnetic and electrical circuits, and advanced signal analysis techniques. By employing detailed analytical equations, the developed model accurately simulates the behavior of multiphase machines in the presence of electrical faults. The effectiveness of the proposed model is demonstrated through a series of case studies and numerical simulations. In particular, special attention is given to analyzing the dynamic behavior of machines under different types of faults, as well as optimizing diagnostic and recovery strategies. The obtained results pave the way for new advancements in the field of multiphase machine diagnostics, with potential applications in various sectors such as automotive, aerospace, and renewable energies. By providing precise and reliable tools for early fault detection, this research contributes to improving the reliability and durability of complex electrical systems while reducing maintenance and operation costs.

Keywords: faults, diagnosis, modelling, multiphase machine

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Authors: Mualla McManus, Jenna Luche Thaye

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World Health Organisation International Classification of Disease codes were created to define disease including infections in order to guide and educate diagnosticians. Most infectious diseases such as syphilis are clearly defined by their ICD 10 codes and aid/help to educate the clinicians in syphilis diagnosis and treatment globally. However, current ICD 10 codes for relapsing fever Borreliosis and Lyme disease are less clearly defined and can impede appropriate diagnosis especially if the clinician is not familiar with the symptoms of these infectious diseases. This is despite substantial number of scientific articles published in peer-reviewed journals about relapsing fever and Lyme disease. In the USA there are estimated 380,000 people annually contacting Lyme disease, more cases than breast cancer and 6x HIV/AIDS cases. This represents estimated 0.09% of the USA population. If extrapolated to the global population (7billion), 0.09% equates to 63 million people contracting relapsing fever or Lyme disease. In many regions, the rate of contracting some form of infection from tick bite may be even higher. Without accurate and appropriate diagnostic codes, physicians are impeded in their ability to properly care for their patients, leaving those patients invisible and marginalized within the medical system and to those guiding public policy. This results in great personal hardship, pain, disability, and expense. This unnecessarily burdens health care systems, governments, families, and society as a whole. With accurate diagnostic codes in place, robust data can guide medical and public health research, health policy, track mortality and save health care dollars. Better defined ICD codes are the way forward in educating the diagnosticians about relapsing fever and Lyme diseases.

Keywords: WHO ICD codes, relapsing fever, Lyme diseases, World Health Organisation

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Authors: Sunil Mittal, Sukhpreet Singh, Hardeep Kaur

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A simple voltammetric technique for on-line analysis of arsenite [As (III)] is reported. Owing to the affinity of As (III) with thiol group of proteins and enzymes, cysteine has been employed as reducing agent. The reduction study of As(III)-cysteine complex on indium tin oxide (ITO) electrode has been explored. The experimental parameters such as scan rate, cysteine concentration, pH etc. were optimized to achieve As (III) determination. The developed method provided dynamic linear range of detection from 0.1 to 1 mM with a detection limit of 0.1 mM. The method is applicable to environmental monitoring of As (III) from highly contaminated sources such as industrial effluents, wastewater sludge etc.

Keywords: arsenite, cysteine, linear sweep voltammetry, reduction

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Authors: Timea Harmath-Tánczos

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Drug-resistant epilepsy (DRE) is defined as the persistence of seizures despite at least two syndrome-adapted antiseizure drugs (ASD) used at efficacious daily doses. About a third of patients with epilepsy suffer from drug resistance. Cognitive assessment has a crucial role in the diagnosis and clinical management of epilepsy. Previous studies have addressed the clinical targets and indications for measuring neuropsychological functions; best to our knowledge, no studies have examined it in a Hungarian therapy-resistant population. To fill this gap, we investigated the Hungarian diagnostic protocol between 18 and 65 years of age. This study aimed to describe and analyze neuropsychological functions in patients with drug-resistant epilepsy and identify factors associated with neuropsychology deficits. We perform a prospective case-control study comparing neuropsychological performances in 50 adult patients and 50 healthy individuals between March 2023 and July 2023. Neuropsychological functions were examined in both patients and controls using a full set of specific tests (general performance level, motor functions, attention, executive facts., verbal and visual memory, language, and visual-spatial functions). Potential risk factors for neuropsychological deficit were assessed in the patient group using a multivariate analysis. The two groups did not differ in age, sex, dominant hand and level of education. Compared with the control group, patients with drug-resistant epilepsy showed worse performance on motor functions and visuospatial memory, sustained attention, inhibition and verbal memory. Neuropsychological deficits could therefore be systematically detected in patients with drug-resistant epilepsy in order to provide neuropsychological therapy and improve quality of life. The analysis of the classical and complex indices of the special neuropsychological tasks presented in the presentation can help in the investigation of normal and disrupted memory and executive functions in the DRE.

Keywords: drug-resistant epilepsy, Hungarian diagnostic protocol, memory, executive functions, cognitive neuropsychology

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Authors: Zohreh Bayat, Dariush Minai-Tehrani

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Lipases constitute one of the most important groups of industrial enzymes that catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids. Muscarinic antagonist relieves smooth muscle spasm of the gastrointestinal tract and effect on the cardiovascular system. In this research, the effect of a muscarinic antagonist on the lipase activity of Pseudomonas aeruginosa was studied. Lineweaver–Burk plot showed that the drug inhibited the enzyme by competitive inhibition. The IC50 value (60 uM) and Ki (30 uM) of the drug revealed the drug bound to the enzyme with high affinity. Determination of enzyme activity in various pH and temperature showed that the maximum activity of lipase was at pH 8 and 60°C both in presence and absence of the drug.

Keywords: bacteria, inhibition, kinetics, lipase

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Authors: H. M. El-Rafie, A. H. Abou Zeid, R. S. Mohammed, A. A. Sleem

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Acrocarpus is a genus of flowering plants in the legume family Fabaceae which considered as a large and economically important family. This study aimed to investigate the phytoconstituents of the petroleum ether extract (PEE) of Acrocarpus fraxinofolius bark by Gas chromatography coupled with mass spectrometry (GC/MS) analysis of its fractions (fatty acid and unsaponifiable matter). Concerning this, identification of 52 compounds constituting 97.03 % of the total composition of the unsaponifiable matter fraction. Cycloeucalenol was found to be the major compound representing 32.52% followed by 4a, 14a-dimethyl-A8~24(28)-ergostadien (26.50%) and ß-sitosterol(13.74%), furthermore Gas liquid chromatography (GLC) analysis of the sterol fraction revealed the identification of cholesterol (7.22 %), campesterol (13.30 %), stigmasterol (10.00 %) and β - sitosterol (69.48 %). Meanwhile, the identification of 33 fatty acids representing 90.71% of the total fatty acid constituents. Methyl-9,12-octadecadienoate (40.39%) followed by methyl hexadecanoate (23.64%) were found to be the major compounds. On the other hand, column chromatography and Thin layer chromatography (TLC) fractionation of PEE separate the triterpenoid: 21β-hydroxylup-20(29)-en-3-one and β- amyrin which were structurally identified by spectroscopic analysis (NMR, MS and IR). PEE has been biologically evaluated for 1: management of diabetes in alloxan induced diabetic rats 2: cytotoxic activity against four human tumor cell lines (Cervix carcinoma cell line[HELA], Breast carcinoma cell line [MCF7], Liver carcinoma cell line[HEPG2] and Colon carcinoma cell line[HCT-116] 3: hepatoprotective activity against CCl4-induced hepatotoxicity in rats and the activity was studied by assaying the serum marker enzymes like AST, ALT, and ALP. Concerning this, the anti-diabetic activity exhibited by 100mg of PEE extract was 74.38% relative to metformin (100% potency). It also showed a significant anti-proliferative activity against MCF-7 (IC50= 2.35µg), Hela(IC50=3.85µg) and HEPG-2 (IC50= 9.54µg) compared with Doxorubicin as reference drug. The hepatoprotective activity was evidenced by significant decrease in liver function enzymes, i.e. AST, ALT and ALP by (29.18%, 28.26%, and 34.11%, respectively using silymarin as the reference drug, compared to their concentration levels in an untreated group with liver damage induced by CCl₄. This study was performed for the first time on the bark of this species.

Keywords: Acrocarpus fraxinofolius, antidiabetic, cytotoxic, hepatoprotective

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Authors: Shanza Akram, Sameen Toor, Heba Harb Abu Alkass, Zainab Abdulsalam Altaha, Sara Taha Abdulla, Saleem Imran

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Acute pulmonary embolism (PE) is a common disease and can be fatal. The clinical presentation is variable and nonspecific, making accurate diagnosis difficult. Testing patients with suspected acute PE has increased dramatically. However, the overuse of some tests, particularly CT and D-dimer measurement, may not improve care while potentially leading to patient harm and unnecessary expense. CTPA is the investigation of choice for PE. Its easy availability, accuracy and ability to provide alternative diagnosis has lowered the threshold for performing it, resulting in its overuse. Guidelines have recommended the use of clinical pretest probability tools such as ‘Wells score’ to assess risk of suspected PE. Unfortunately, implementation of guidelines in clinical practice is inconsistent. This has led to low risk patients being subjected to unnecessary imaging, exposure to radiation and possible contrast related complications. Aim: To study the diagnostic yield of CT PA, clinical pretest probability of patients according to wells score and to determine whether or not there was an overuse of CTPA in our service. Methods: CT scans done on patients with suspected P.E in our hospital from 1st January 2014 to 31st December 2014 were retrospectively reviewed. Medical records were reviewed to study demographics, clinical presentation, final diagnosis, and to establish if Wells score and D-Dimer were used correctly in predicting the probability of PE and the need for subsequent CTPA. Results: 100 patients (51male) underwent CT PA in the time period. Mean age was 57 years (24-91 years). Majority of patients presented with shortness of breath (52%). Other presenting symptoms included chest pain 34%, palpitations 6%, collapse 5% and haemoptysis 5%. D Dimer test was done in 69%. Overall Wells score was low (<2) in 28 %, moderate (>2 - < 6) in 47% and high (> 6) in 15% of patients. Wells score was documented in medical notes of only 20% patients. PE was confirmed in 12% (8 male) patients. 4 had bilateral PE’s. In high-risk group (Wells > 6) (n=15), there were 5 diagnosed PEs. In moderate risk group (Wells >2 - < 6) (n=47), there were 6 and in low risk group (Wells <2) (n=28), one case of PE was confirmed. CT scans negative for PE showed pleural effusion in 30, Consolidation in 20, atelactasis in 15 and pulmonary nodule in 4 patients. 31 scans were completely normal. Conclusion: Yield of CT for pulmonary embolism was low in our cohort at 12%. A significant number of our patients who underwent CT PA had low Wells score. This suggests that CT PA is over utilized in our institution. Wells score was poorly documented in medical notes. CT-PA was able to detect alternative pulmonary abnormalities explaining the patient's clinical presentation. CT-PA requires concomitant pretest clinical probability assessment to be an effective diagnostic tool for confirming or excluding PE. . Clinicians should use validated clinical prediction rules to estimate pretest probability in patients in whom acute PE is being considered. Combining Wells scores with clinical and laboratory assessment may reduce the need for CTPA.

Keywords: CT PA, D dimer, pulmonary embolism, wells score

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Authors: Angelis P. Barlampas

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Purpose of Learning Objective: This case depicts the need for cooperation between the emergency department and the radiologist to achieve the best diagnostic result for the patient. The clinical picture must correlate well with the radiology report and when it does not, this is not necessarily someone’s fault. Careful interpretation and good knowledge of the limitations, advantages and disadvantages of each imaging procedure are essential for the final diagnostic goal. Methods or Background: A patient was brought to the emergency department by their relatives. He was suddenly confused and his mental status was altered. He hadn't any history of mental illness and was otherwise healthy. A computing tomography scan without contrast was done, but it was unremarkable. Because of high clinical suspicion of probable neurologic disease, he was admitted to the hospital. Results or Findings: Another T was done after 48 hours. It showed a hypodense region in both thalamic areas. Taking into account that the first CT was normal, but the initial clinical picture of the patient was alerting of something wrong, the repetitive CT exam is highly suggestive of a probable diagnosis of bilateral thalamic infractions. Differential diagnosis: Primary bilateral thalamic glioma, Wernicke encephalopathy, osmotic myelinolysis, Fabry disease, Wilson disease, Leigh disease, West Nile encephalitis, Greutzfeldt Jacob disease, top of the basilar syndrome, deep venous thrombosis, mild to moderate cerebral hypotension, posterior reversible encephalopathy syndrome, Neurofibromatosis type 1. Conclusion: As is the case of limitations for any imaging procedure, the same applies to CT. The acute ischemic attack can not depict on CT. A period of 24 to 48 hours has to elapse before any abnormality can be seen. So, despite the fact that there are no obvious findings of an ischemic episode, like paresis or imiparesis, one must be careful not to attribute the patient’s clinical signs to other conditions, such as toxic effects, metabolic disorders, psychiatric symptoms, etc. Further investigation with MRI or at least a repeated CT must be done.

Keywords: CNS, CT, thalamus, emergency department

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Authors: Naglaa A. Abd Elkader, Haithem A. Farghali

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The present study carried out on fifteen clinical cases of different species of dogs which admitted to surgical clinic of veterinary medicine with different symptoms (Acute vomiting, hematemesis and anorexia). There was diagnostic march which including plain radiograph and endoscopic examination. Treatment was including surgical interference and endoscopic retrieval followed by medicinal treatment. This study was aimed the detection of different foreign bodies by the most suitable method according to the type of the foreign bodies.

Keywords: stomach, endoscopy, foreign bodies, dogs

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Authors: Dipti Chand, Riya Saboo

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OBJECTIVES: Early and correct diagnosis may present a significant clinical challenge in diagnosis of patients presenting to Emergency Department with Acute Dyspnoea. The common cause of acute dyspnoea and respiratory distress in Emergency Department are Decompensated Heart Failure (HF), Chronic Obstructive Pulmonary Disease (COPD), Asthma, Pneumonia, Acute Respiratory Distress Syndrome (ARDS), Pulmonary Embolism (PE), and other causes like anaemia. The aim of the study was to measure NT-pro Brain Natriuretic Peptide (BNP) and exhaled End-Tidal Carbon dioxide (ETCO2) in patients presenting with dyspnoea. MATERIAL AND METHODS: This prospective, cross-sectional and observational study was performed at the Government Medical College and Hospital, Nagpur, between October 2019 and October 2021 in patients admitted to the Medicine Intensive Care Unit. Three groups of patients were compared: (1) HFrelated acute dyspnoea group (n = 52), (2) pulmonary (COPD/PE)-related acute dyspnoea group (n = 31) and (3) sepsis with ARDS-related dyspnoea group (n = 13). All patients underwent initial clinical examination with a recording of initial vital parameters along with on-admission ETCO2 measurement, NT-proBNP testing, arterial blood gas analysis, lung ultrasound examination, 2D echocardiography, chest X-rays, and other relevant diagnostic laboratory testing. RESULTS: 96 patients were included in the study. Median NT-proBNP was found to be high for the Heart Failure group (11,480 pg/ml), followed by the sepsis group (780 pg/ml), and pulmonary group had an Nt ProBNP of 231 pg/ml. The mean ETCO2 value was maximum in the pulmonary group (48.610 mmHg) followed by Heart Failure (31.51 mmHg) and the sepsis group (19.46 mmHg). The results were found to be statistically significant (P < 0.05). CONCLUSION: NT-proBNP has high diagnostic accuracy in differentiating acute HF-related dyspnoea from pulmonary (COPD and ARDS)-related acute dyspnoea. The higher levels of ETCO2 help in diagnosing patients with COPD.

Keywords: NT PRO BNP, ETCO2, dyspnoea, lung USG

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Authors: Vivek Kumar Gupta, Kripi Vohra, Monika Gupta

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Pulses have been a vital ingredient of the balanced human diet in India. Lentil (Lens culinaris Medikus or Lens esculenta Moench.) is a common legume known since biblical times. Lentil seeds, with or without hulls, are cooked as dhal and this has been the main dish for millennia in the South Asian region. Oxidative stress can damage lipids, proteins, enzymes, carbohydrates and DNA in cells and tissues, resulting in membrane damage, fragmentation or random cross linking of molecules like DNA, enzymes and structural proteins and even lead to cell death induced by DNA fragmentation and lipid peroxidation. These consequences of oxidative stress construct the molecular basis in the development of cancer, neurodegenerative disorders, cardiovascular diseases, diabetes and autoimmune. The aim of the present work is to assess the antioxidant potential of the peteroleum ether, acetone, methanol and water extract of the Lens esculenta seeds. In vitro antioxidant assessment of the extracts was carried out using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging activity, reducing power assay. The quantitative estimation of total phenolic content, total flavonoid content in extracts and in plant material, total saponin content, total alkaloid content, crude fibre content, total volatile content, fat content and mucilage content in drug material was also carried out. Though all the extracts exhibited dose dependent reducing power activity the acetone extract was found to possess significant hydrogen donating ability in DPPH (45.83%-93.13%) and hydroxyl radical scavenging system (28.7%-46.41%) than the peteroleum ether, methanol and water extracts. Total phenolic content in the acetone and methanol extract was found to be 608 and 188 mg gallic acid equivalent of phenol/g of sample respectively. Total flavonoid content of acetone and methanol extract was found to be 128 and 30.6 mg quercetin equivalent/g of sample respectively. It is evident that acetone extract of Lentil seeds possess high levels of polyphenolics and flavonoids that could be utilized as antioxidants and neutraceuticals.

Keywords: antioxidant, flavanoids, Lens esculenta, polyphenols

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Authors: Shringika Soni, Utkarsh Jain, Nidhi Chauhan

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Aptamers are recently discovered ~80-100 bp long artificial oligonucleotides that not only demonstrated their applications in therapeutics; it is tremendously used in diagnostic and sensing application to detect different biomarkers and drugs. Synthesizing aptamers for proteins or genomic template is comparatively feasible in laboratory, but drugs or other chemical target based aptamers require major specification and proper optimization and validation. One has to optimize all selection, amplification, and characterization steps of the end product, which is extremely time-consuming. Therefore, we performed asymmetric PCR (polymerase chain reaction) for random oligonucleotides pool synthesis, and further use them in Systematic evolution of ligands by exponential enrichment (SELEX) for anti-psychotic drugs based aptamers synthesis. Anti-psychotic drugs are major tranquilizers to control psychosis for proper cognitive functions. Though their low medical use, their misuse may lead to severe medical condition as addiction and can promote crime in social and economical impact. In this work, we have approached the in-vitro SELEX method for ssDNA synthesis for anti-psychotic drugs (in this case ‘target’) based aptamer synthesis. The study was performed in three stages, where first stage included synthesis of random oligonucleotides pool via asymmetric PCR where end product was analyzed with electrophoresis and purified for further stages. The purified oligonucleotide pool was incubated in SELEX buffer, and further partition was performed in the next stage to obtain target specific aptamers. The isolated oligonucleotides are characterized and quantified after each round of partition, and significant results were obtained. After the repetitive partition and amplification steps of target-specific oligonucleotides, final stage included sequencing of end product. We can confirm the specific sequence for anti-psychoactive drugs, which will be further used in diagnostic application in clinical and forensic set-up.

Keywords: anti-psychotic drugs, aptamer, biosensor, ssDNA, SELEX

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Authors: Vadanasundari Vedarethinam, Kun Qian

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The metabolic analysis is more distal over proteomics and genomics engaging in clinics and needs rationally distinct techniques, designed materials, and device for clinical diagnosis. Conventional techniques such as spectroscopic techniques, biochemical analyzers, and electrochemical have been used for metabolic diagnosis. Currently, there are four major challenges including (I) long-term process in sample pretreatment; (II) difficulties in direct metabolic analysis of biosamples due to complexity (III) low molecular weight metabolite detection with accuracy and (IV) construction of diagnostic tools by materials and device-based platforms for real case application in biomedical applications. Development of chips with nanomaterial is promising to address these critical issues. Mass spectroscopy (MS) has displayed high sensitivity and accuracy, throughput, reproducibility, and resolution for molecular analysis. Particularly laser desorption/ ionization mass spectrometry (LDI MS) combined with devices affords desirable speed for mass measurement in seconds and high sensitivity with low cost towards large scale uses. We developed a plasmonic chip for clinical metabolic fingerprinting as a hot carrier in LDI MS by series of chips with gold nanoshells on the surface through controlled particle synthesis, dip-coating, and gold sputtering for mass production. We integrated the optimized chip with microarrays for laboratory automation and nanoscaled experiments, which afforded direct high-performance metabolic fingerprinting by LDI MS using 500 nL of serum, urine, cerebrospinal fluids (CSF) and exosomes. Further, we demonstrated on-chip direct in-vitro metabolic diagnosis of early-stage lung cancer patients using serum and exosomes without any pretreatment or purifications. To our best knowledge, this work initiates a bionanotechnology based platform for advanced metabolic analysis toward large-scale diagnostic use.

Keywords: plasmonic chip, metabolic fingerprinting, LDI MS, in-vitro diagnostics

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Authors: Pao-Huei Chen, Shu-Mei Lin, Yih-Ming Weng, Zer-Ran Yu, Be-Jen Wang

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Pancreatic α-amylase and intestinal α-glucosidase are the critical enzymes for the breakdown of complex carbohydrates into di- or mono-saccharide, which play an important role in modulating postprandial blood sugars. Angiotensin converting enzyme (ACE) converts inactive angiotensin-I into active angiotensin-II, which subsequently increase blood pressure through triggering vasoconstriction and aldosterone secretion. Thus, inhibition of carbohydrate-digestion enzymes and ACE will help the management of blood glucose and blood pressure, respectively. Studies showed Pleurotus citrinopileatus (PC), an edible mushroom and commonly cultured in oriental countries, exerted anticancer, immune improving, antioxidative, hypoglycemic and hypolipidemic effects. Previous studies also showed various molecular weights (MW) fractioned from extracts may affect biological activities due to varying contents of bioactive components. Thus, the objective of this study is to investigate the in vitro antioxidant, hypoglycemic and hypotenstive effects and distribution of active compounds of various MWs of cold water extract from P. citrinopileatus (CWEPC). CWEPC was fractioned into four various MW fractions, PC-I (＜1 kDa), PC-II (1-3.5 kDa), PC-III (3.5-10 kDa), and PC-IV (＞10 kDa), using an ultrafiltration system. The physiological activities, including antioxidant activities, the inhibition capabilities of pancreatic α-amylase, intestinal α-glucosidase, and hypertension-linked ACE, and the active components, including polysaccharides, protein, and phenolic contents, of CWEPC and four fractions were determined. The results showed that fractions with lower MW exerted a higher antioxidant activity (p<0.05), which was positively correlated to the levels of total phenols. In contrast, the inhibition effects on the activities of α-amylase, α-glucosidase, and ACE of PC-IV fraction were significantly higher than CWEPC and the other three low MW fractions (< 10 kDa), which was more related to protein contents. The inhibition capability of CWEPC and PC-IV on α-amylase activity was 1/13.4 to 1/2.7 relative to that of acarbose (positive control), respectively. However, the inhibitory ability of PC-IV on α-glucosidase (IC50 = 0.5 mg/mL) was significantly higher than acarbose (IC50 = 1.7 mg/mL). Kinetic data revealed that PC-IV fraction followed a non-competitive inhibition on α-glucosidase activity. In conclusion, the distribution of various bioactive components contribute to the functions of different MW fractions on oxidative stress prevention, and blood pressure and glucose modulation.

Keywords: α-Amylase, angiotensin converting enzyme, α-Glucosidase, Pleurotus citrinopileatus

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Authors: J. M. Cruz, X. Vecıno, L. Rodrıguez-López, J. M. Dominguez, A. B. Moldes

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The cosmetic and personal care industry are the fields where biosurfactants could have more possibilities of success because in this kind of products the replacement of synthetic detergents by natural surfactants will provide an additional added value to the product, at the same time that the harmful effects produced by some synthetic surfactants could be avoided or reduced. Therefore, nowadays, consumers are disposed to pay and additional cost if they obtain more natural products. In this work we provide data about the potential of biosurfactants in the cosmetic and personal care industry. Biosurfactants from corn steep liquor, that is a fermented and condensed stream, have showed good surface-active properties, reducing substantially the surface tension of water. The bacteria that usually growth in corn steep liquor comprises Lactobacillus species, generally recognize as safe. The biosurfactant extracted from CSL consists of a lipopeptide, composed by fatty acids, which can reduce the surface tension of water in more than 30 units. It is a yellow and viscous liquid with a density of 1.053 mg/mL and pH=4. By these properties, they could be introduced in the formulation of cosmetic creams, hair conditioners or shampoos. Moreover this biosurfactant extracted from corn steep liquor, have showed a potent antimicrobial effect on different strains of Streptococcus. Some species of Streptococcus are commonly found weakly living in the human respiratory and genitourinary systems, producing several diseases in humans, including skin diseases. For instance, Streptococcus pyogenes produces many toxins and enzymes that help to stabilize skin infections; probably biosurfactants from corn steep liquor can inhibit the mechanisms of the S. pyogenes enzymes. S. pyogenes is an important cause of pharyngitis, impetigo, cellulitis and necrotizing fasciitis. In this work it was observed that 50 mg/L of biosurfactant extract obtained from corn steep liquor is able to inhibit more than 50% the growth of S. pyogenes. Thus, cosmetic and personal care products, formulated with biosurfactants from corn steep liquor, could have prebiotic properties. The natural biosurfactant presented in this work and obtained from corn milling industry streams, have showed a high potential to provide an interesting and sustainable alternative to those, antibacterial and surfactant ingredients used in cosmetic and personal care manufacture, obtained by chemical synthesis, which can cause irritation, and often only show short time effects.

Keywords: antimicrobial activity, biosurfactants, cosmetic, personal care

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Authors: Kira Rysakova, Vitaly Novikov

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An improved method of obtaining enzymatic collagen hydrolysate from the tissues of marine hydrobionts is proposed, which allows to obtain hydrolysate without pre-isolation of pure collagen. The method can be used to isolate enzymatic collagen hydrolysate from the waste of industrial processing of Red King crab and non-traditional objects - marine holothurias. Comparative analysis of collagen hydrolysates has shown the possibility of their use in a number of nutrient media, but this requires additional optimization of their composition and biological tests on wide sets of test strains of microorganisms.

Keywords: collagen hydrolysate, marine hydrobionts, red king crab, marine holothurias, enzymes, exclusive HPLC

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Authors: Supriya Chatla, Anjana Male, Srikala Kamireddy

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L-asparaginase (E.C.3.5.1.1) is an anti-cancer enzyme that has been purified and characterized for decades to study and evaluate its anti-carcinogenic activity against Hodgkin’s lymphoma. The present investigation deals with screening, isolation and optimization of L-asparaginase giving fungal strain of soil samples from different areas of AP, India. L-Aspariginase activity was estimated on the basis of the pink color surrounding the growing colony. A total of 132 colonies were screened and isolated from different samples. Based on the zone diameter, L-asparaginase activity is determined, L- asparaginase activity is optimized at 28oc and Immobilized Aspariginase had more potency than the free enzymes.

Keywords: aspariginase, anticancer enzyme, Isolation, optimization

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Authors: Mona G. Alharbi

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A new family of methionine-sulfoxide reductase (Msr) was recently discovered and was named free methionine sulfoxide reductase (fRMsr). This family includes enzymes with a reductase activity toward the free R isomer of a methionine sulfoxide substrate. The fRMsrs have a GAF domain topology, a domain, which was previously identified as having in some cases a cyclic nucleotide phosphodiesterase activity. The classification of fRMsrs as GAF domains revealed a new function can be added to the GAF domain family. Interestingly the four members identified in the fRMsr family share the GAF domain structure and the presence of three conserved cysteines in the active site with free R methionine sulfoxide substrate specificity. This thesis presents the crystal structures of reduced, free Met-SO substrate-bound and MES-bound forms of a new fRMsr from Burkholderia pseudomallei (BPSL2418). BPSL2418 was cloned, overexpressed and purified to enable protein crystallization. The crystallization trials for reduced, Met-SO-bound and MES-bound forms of BPSL2418 were prepared and reasonable crystals of each form were produced. The crystal structures of BPSL2418MES, BPSL2418Met-SO and BPSL2418Reduced were solved at 1.18, 1.4 and 2.0Å, respectively by molecular replacement. The BPSL2418MES crystal belongs to space group P 21 21 21 while BPSL2418Met-SO and BPSL2418Reduced crystals belong to space group P 1 21 1. All three forms share the GAF domain structure of six antiparallel β-strands and four α-helices with connecting loops. The antiparallel β-strands (β1, β2, β5 and β6) are located in the center of the BPSL2418 structure flanked on one side by a three α-helices (α1, α2 and α4) and on the other side by a (loop1, β3, loop2, α3, β4 loop4) unit where loop4 forms a capping flap and covers the active site. The structural comparison of the three forms of BPSL2418 indicates that the catalytically important cysteine is CYS109, where the resolving cysteine is CYS75, which forms a disulfide bond with CYS109. They also suggest that the third conserved cysteine in the active site, CYS85, which is located in α3, is a non-essential cysteine for the catalytic function but it may play a role in the binding of the substrate. The structural comparison of the three forms reveals that conformational changes appear in the active site particularly involving loop4 and CYS109 during catalysis. The 3D structure of BPSL2418 shows strong structure similarity to fRMsrs enzymes, which further suggests that BPSL2418 acts as a free Met-R-SO reductase and shares the catalytic mechanism of fRMsr family.

Keywords: Burkholderia pseudomallei, GAF domain protein, methionine sulfoxide reductase, protein crystallization

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Authors: Minu Bharati, Priyanka Rai, Satarupa Dutta, Dhiraj Saha

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Culex quinquefasciatus Say, is a mosquito of immense public health concern due to its role in transmission of filariasis, which is an endemic disease in 20 states and union territories of India, putting about 600 million people at the risk of infection. The main strategies to control filaria in India include anti-larval measures in urban areas, Indoor Residual Spray (IRS) in rural areas and mass diethylcarbamazine citrate (DEC) administration. Larval destruction measures and IRS are done with the use of insecticides. In this study, Susceptibility/ Resistance to insecticides were assessed in Culex quinquefasciatus mosquitoes collected from eight densely populated areas of Siliguri subdivision, which has a high rate of filarial infection. To unveil the insecticide susceptibility status of Culex quinquefasciatus, bioassays were performed on field-caught mosquitoes against two major groups of insecticides, i.e. Synthetic Pyrethroids (SPs): 0.05% deltamethrin and 0.05% lambda-cyhalothrin and Organophosphates (OPs): 5% malathion and temephos using World Health Organisation (WHO) discriminating doses. The knockdown rates and knockdown times (KDT50) were also noted against deltamethrin, lambda-cyhalothrin and malathion. Also, activities of major detoxifying enzymes, i.e. α-carboxylesterases, β-carboxylesterases and cytochrome P450 (CYP450) monooxygenases were determined to find the involvement of biochemical mechanisms in resistance phenomenon (if any). The results obtained showed that, majority of the mosquito populations were moderately to severely resistant against both the SPs and one OP, i.e. temephos. Whereas, most of the populations showed 100% susceptibility to malathion. The knockdown rates and KDT50 in response to above-mentioned insecticides showed significant variation among different populations. Variability in activities of carboxylesterases and CYP450 monooxygenases were also observed with hints of their involvement in contribution towards insecticide resistance in some of the tested populations. It may be concluded that, Culex quinquefasciatus has started developing resistance against deltamethrin, lambda-cyhalothrin and temephos in Siliguri subdivision. Malathion seems to hold the greatest potentiality for control of these mosquitoes in this area as revealed through this study. Adoption of Integrated mosquito management (IMM) strategy should be the prime objective of the concerned authorities to delimit the insecticide resistance phenomenon and filariasis infections.

Keywords: Culex quinquefasciatus, detoxifying enzymes, insecticide resistance, knockdown rate

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