The marine environment is known for its vast diversity of the microbial population; however, less explored for bioactive compounds. In this study, an AMP produced by a new marine isolate, Vibrio proteolyticus MT110, showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. The AMP was purified to homogeneity using ethyl acetate extraction followed by RP-HPLC, and LC-MS analysis showed its molecular weight as 980 Da. The MIC of AMP (peptide-MT110) was obtained in the 7.81-31.25 µg/mL range against different indicator strains. Peptide-MT110 showed stability of its antimicrobial activity at 15-121 °C and pH 4-10 and in the presence of various hydrolytic enzymes. The peaks at 1536 cm-1 and 1712 cm-1 wavenumbers in FTIR spectra confirmed the peptidic nature of AMP, and its amino acid analysis confirmed the presence of tyrosine and isoleucine. The antibacterial activity of peptide-MT110 is confirmed by PI assay and TEM. The optimization of peptide-MT110 production using statistical methods resulted in a 2.64-fold higher production. The physicochemical properties and stability in wide pH and temperature ranges showed the potential of peptide-MT110 for its development as a drug candidate. This is believed to be the first report on an AMP from Vibrio proteolyticus.

Early detection of brain tumor has an important role in further developing therapeutic outcomes, and hence functioning in endurance tolerance. Physically evaluating the various reversion imaging (magnetic resonance imaging [MRI]) images that are regularly distributed at the center is a problematic cycle. Along these lines, there is a significant need for PC-assisted strategies with improved accuracy for early detection of cancer. PC-backed brain cancer detection from MR images including growth location, division, and order processes. In recent years, many inquiries have turned to zero in traditional or outdated AI procedures for brain development findings. Presently, there has been an interest in using in-depth learning strategies to detect cerebral growths with an excellent accuracy and heart rate. This review presents a far-reaching audit of traditional AI strategies and in-depth study methods for diagnosing brain cancer. This research paper distinguishes three main benefits i.e. exhibition, estimation and measurements of brain tumour detection.

ABSTRACTThe present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcatwere 9.49×10-3M, 25.13 IUmL-1min-1, and 3.01×103s-1, respectively. The CD spectra showed that PrASNase consists of 30.9% α-helix and 69.1% other structures in its native form. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Different from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37°C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50of 7.4 µg.mL-1and 5.6 µg.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrates the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.HIGHLIGHTSA new L-asparaginase (PrASNase) was structurally and functionally characterized.SAXS revealed PrASNase is functionally active in monomeric form and oligomerizes with temperature rise.Monomeric PrASNase showed an IC50value of 7.4 and 5.6 µg mL-1against WIL2-S and TF-1.28 cells.Cytotoxicity of PrASNase against leukemic cell lines showed its potential as a biotherapeutic.GRAPHICAL ABSTRACT

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23H T showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095 T , 7.8% from Debaryomyces udenii JCM 7855 T , and Debaryomyces coudertii JCM 2387 T in the D1/D2 domain. In the ITS region sequences, strain CIG-23H T showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095 T and Debaryomyces coudertii JCM 2387 T . In 18S rRNA gene sequence, the strain CIG-23H T showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23H T can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23H T is a novel species of the genus Debaryomyces , and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297 T , and the isotypes are MTCC 12914 T and KCTC 37024 T . The MycoBank number of Debaryomyces apis f.a., sp. nov . is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Purpose A person's healthy activities are determined by the state of his or her brain. The brain is in charge of all of a person's activities. If a small abnormality develops in the brain, it will have a negative impact on the person regardless of whether the other organs are in good condition. As a result, early detection of any abnormal growth in the brain is essential. Methods In this work, the authors have utilized data pre-processing using discrete wavelet transform (DWT) and segmentation, whereas, for detection, an ensemble learning technique is proposed. DWT and segmentation help in increasing the dataset size that is used to train the deep learning model. Segmentation using supervised Auto-encoder (AE) is used for data enhancement to strengthen the training process. The original data, outputs of DWT, and segmented images are utilized for the training of the ensemble model designed with three parallel-connected convolutional neural networks (CNNs). Results The detection results obtained from the ensemble of these recurrent models are then passed through the Multilayer Perceptron (MLP) for final detection. Kaggle brain MRI image dataset is used to complete the proposed method. Test accuracy, F1-score, precision, sensitivity, and specificity provided by this method are 98.08%, 0.9836, 1.0000, 0.9677, and 1.0000 respectively. In comparison to state-of-the-art models, the proposed model produces competitive outcomes. Conclusion In time detection of the tumor may lead to the survival of the patient. Automatic and accurate detection is another perspective of this field. For this purpose, we have proposed a deep ensemble model with wavelet features. The ensemble model provides increased performance in comparison to single models due to the parallel training.

In this work, the authors have applied image inpainting on MRI images of the brain to highlight the tumors present in the image. These highlighted images are used for the training of the ensemble model. Three convolutional neural networks (CNNs) are used as the base classifier and their outputs are fed to a Multilayer Perceptron (MLP) for further training and final classification. Classification is done to check whether the brain is having a tumor or it is healthy using a data set that is available at Kaggle for open access. The proposed method provided 100% and 98.33% training and testing accuracies that show the effectiveness of applying inpainting on the data set images.

Image segmentation is an essential technique of brain tumor MRI image processing for automated diagnosis of an image by partitioning it into distinct regions referred to as a set of pixels. The classification of the tumor affected and non-tumor becomes an arduous task for radiologists. This paper presents a novel image enhancement based on the SCA (Sine Cosine Algorithm) optimization technique for the improvement of image quality. The improved FLICM (Fuzzy Local Information C Means) segmentation technique is proposed to detect the affected regions of brain tumor from the MRI brain tumor images and reduction of noise from the MRI images by introducing a fuzzy factor to the objective function. The SCA weight-optimized Wavelet-Extreme Learning Machine (SCA-WELM) model is also proposed for the classification of benign tumors and malignant tumors from MRI brain images. In the first instance, the enhanced images are undergone improved FLICM Segmentation. In the second phase, the segmented images are utilized for feature extraction. The GLCM feature extraction technique is considered for feature extraction. The extracted features are aligned as input to the SCA-WELM model for the classification of benign and malignant tumors. The following dataset (Dataset-255) is considered for evaluating the proposed classification approach. An accuracy of 99.12% is achieved by the improved FLICM segmentation technique. The classification performance of the SCA-WELM is measured by sensitivity, specificity, accuracy, and computational time and achieved 0.98, 0.99, 99.21%, and 97.2576 seconds respectively. The comparison results of SVM (Support Vector Machine), ELM, SCA-ELM, and proposed SCA-WELM models are presented to show the robustness of the proposed SCA-WELM classification model.

Epidermal growth factor receptor (EGFR) is the primary target for the treatment of colorectal cancer, the third most diagnosed cancer worldwide. In recent years, regulatory changes have facilitated the approval of biosimilars aimed to bring more access to biologics to patients. However, it has also expended the requirements of non-clinical characterisation data using state-of-the-art and orthogonal methodologies to demonstrate similarity between proposed biologic and its reference medicinal product (RMP). The current study was aimed to develop a stable CHO-S cell line producing panitumumab biosimilar candidate, P-mAb, a fully human IgG2 anti-EGFR monoclonal antibody and assess its physicochemical and functional similarity with RMP, Vectibix. The single-cell clone from stably transfected CHO-S cell pools was used for the production of P-mAb. This was followed by purification and comparative physicochemical and biological characterisation of P-mAb and RMP using SDS-PAGE, LC/MS, MALDI, MS/MS, CD spectrometry, DSF, SAXS, ITF, MTT assay and binding affinity. SAXS and MST assays are being used for first time in biosimilarity analysis of therapeutic monoclonal antibody. The results of structural and functional analysis of anti-EGFR P-mAb, produced by stable CHO-S cell line revealed high similarity between P-mAb and RMP, vectibix, thus providing the scientific basis of its potential for therapeutic applications.

L-asparaginase has been widely accepted as a standard anticancer drug for acute lymphoblastic leukaemia (ALL). Presently in L-asparaginase biotherapeutic applications, the main focus is developing new L-asparaginase with minimal or without any glutaminase activity to reduce the associated adverse drug reactions. In this study, Bacillus flexus strain SS (NCBI GenBank Accession Number MN420983) was identified as a promising producer of L-asparaginase. L-asparaginase production was optimized by response surface methodology statistical bioprocess modelling, and enzyme yield of 25.08 IU/mL was reached at the bioreactor scale. The purification of L-asparaginase included ammonium sulfate precipitation, ion-exchange chromatography and size exclusion chromatography yielding 5.27-fold purification. The SDS-PAGE and CE-SDS revealed the monomeric L-asparaginase with molecular weight of 33 kDa. The purified enzyme was highly specific to substrate L-Asn and free from glutaminase activity. The anticancer activity of purified L-asparaginase was found specific against tumor cell lines SKBR3 (IC 50 = 0.8 µg/mL), WIL2-S (IC 50 = 16.2 µg/mL), and TF-1 (IC 50 = 47 µg/mL), but not against negative control HUVEC cells. Therefore, L-asparaginase from B. flexus SS with no glutaminase activity could be a potential new candidate for anticancer drug for ALL with reduced adverse effects.

The K. marxianus has emerged as a potential yeast strain for various biotechnological applications. However, the limited number of available genetic tools has hindered the widespread usage of this yeast. In the current study we have expanded the molecular tool box by identifying novel sets of promoters and terminators for increased recombinant protein expression in K. marxianus. The previously available transcriptomic data were analyzed to identify top 10 promoters of highest gene expression activity. We further characterized and compared strength of these identified promoters using eGFP as a reporter protein, at different temperatures and carbon sources. To examine the regulatory region driving protein expression, serially truncated shorter versions of two selected strong promoters were designed, and examined for their ability to drive eGFP protein expression. The activities of these two promoters were further enhanced using different combinations of native transcription terminators of K. marxianus. We further utilized the identified DNA cassette encoding strong promoter in metabolic engineering of K. marxianus for enhanced β-galactosidase activity. The present study thus provides novel sets of promoters and terminators as well as engineered K. marxianus strain for its wider utility in applications requiring lactose degradation such as in cheese whey and milk.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC3 (5) and H2DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5–10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2–16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC4 (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.

In the current study, the production of novel glutaminase free l -asparaginase from a new microbial source ( Pseudomonas resinovorans IGS-131) is reported. Optimization of l -asparaginase production using conventional and statistical optimization techniques resulted in an enzyme yield of 37.63 IU/mL, which was 3.45-fold higher than the initial enzyme activity (i.e., 10.91 IU/mL). l -Asparaginase production from P . resinovorans IGS-131 was successfully carried out at the bioreactor level and investigations on the effect of agitation rates showed a maximum asparaginase yield of 38.88 IU/mL after 24 h fermentation at 400 rpm. The l -asparaginase gene from this source, showing 78% identity with a reported sequence in GenBank, was expressed in Escherichia coli rosetta DE3. The molecular weight of the recombinant protein was determined as 35.6 kDa. Downstream processing of recombinant l -asparaginase resulted in a purified protein concentration of 62.53 mg/L, which showed good free radical scavenging activity of 62%. The current findings provide promising results for a process of l -asparaginase production from P . resinovorans IGS-131. Furthermore, the recombinant production of this enzyme could help in avoiding the complexity of down streaming processes associated with the purification of this enzyme from wild-type organisms.

Antimicrobial resistance (AMR) is a global public health threat in which microorganisms have generated the capacity to tolerate or withstand the impact of antimicrobials. In recent years, studies of antimicrobial peptides (AMPs), biologically active oligopeptides having bioactivity against broad spectrum of microorganism (from viruses to protozoans), have opened a new field of research and widened the application of AMPs which could also be a potential alternative to conventional antibiotics to combat the multiple drug resistant (MDR) pathogens in future. AMPs produced by bacteria are of two types (i) ribosomally synthesized i.e., bacteriocins, (ii) non-ribosomally synthesized. The non-ribosomally synthesized AMPs kill the bacteria by damaging its membrane, which leads to cell death. The ribosomally synthesised bacteriocins are mostly produced by gram-positive and gram-negative bacteria and have antimicrobial activities against similar or closely related bacterial strain(s). This chapter summarizes the current information on classification, mode of action and potential applications of AMPs including those in food and pharmaceutical industries.

Erythritol belongs to the category of sugar alcohols/polyols and consists of four carbon sugar alcohols. Erythritol occurs naturally in many food items such as grapes, wine, beer, sake, soy sauce, peers, mushrooms, and watermelon, etc. Among different sugar alcohols, erythritol is non-caloric and non-fermentable polyol, which makes it as an attractive sugar substitute. It has a small molecular size, and because of this reason, it is mostly absorbed in the small intestine. As it is not metabolized in the body, it does not show any glycaemic and insulinaemic effect. These properties make erythritol an essential sweetener for those people who want to reduce their blood sugar level. Presently, erythritol is used in baked products, confectionery, beverages, manufacturing of chewing gums, etc., and it has potential to be used as a sugar additive in larger global demand. Herein, this chapter focuses on potential applications of erythritol as a sugar substrate in the current global scenario.

l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB)1 dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient.

A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 101CFUmL−1, with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.

This chapter aims to survey the current status of nanotechnology with particular emphasis to carbon nanomaterials (CNMs) in electrochemical biosensors. CNMs have merged as an excellent sensing platform. These materials have been prospected as promising carriers or support for enzyme immobilization. The CNMs-based enzyme nanobiosensors are notable for the high surface area, which allow many simultaneous detection events. This chapter has been started with CNMs such as carbon nanotubes, graphene, nanodiamonds, fullerenes, carbon nanoonion, carbon nanohorns, carbon nanofibers, and carbon nanodot and their composite for electrochemical sensing of glucose, dopamine, cholesterol, creatinine, bilirubin, ascorbic acid, xanthine, hypoxanthine, uric acid, and amino acid. CNMs-based composites have been fabricated, and their novel properties are being gradually discovered, and their applications have also greatly advanced the performance of biosensors. CNMs-based nanobiosensors have generated a great deal of excitement due to their ability to detect a wide range of materials at incredibly small concentrations.