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Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-d-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70 to 80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80°C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α) 8 -barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Low back pain is an important cause of disability. The aim of treatment is to reduce the pain and minimize its consequences. Management includes counseling and reassurance, use of analgesics, non-pharmacological therapies, and regular review., Method: An open-label, randomized, and parallel-group clinical trial was conducted in which Group A patients received Hijama bila Shart (dry cupping) while group B patients received Bukhoor (medicated steam application); both therapies were given for 5 min in 3 sittings (day 1, 4 and 8)., Result: Oswestry LBP Total Scores in Group A decreased from 20.70 ± 0.49 to 6.77 ± 0.37, while in Group B it decreased from 20.07 ± 3.63 to 5.83 ± 2.36. The pain intensity also decreased from 3.10 ± 0.55 to 1.57 ± 0.57 in group A, while from 2.97 ± 0.61 to 1.30 ± 0.75 in group B. Improvement in disability was also found statistically significant. On comparing the improvement in disability between the group, the difference was significant (p < 0.05), and improvement in group A was a little better as compared to group B., Discussion: The effect of dry cupping may be due to increased blood circulation; lymphatic channelization and dispersion of inflammatory chemicals from the site of pain. The effect of medicated steam application may be due to the anti-inflammatory and muscle relaxing effects of steam and heat of the steam itself; the drugs used for steam application are reported to reduce inflammation on local application., Conclusion: Dry cupping and Medicated Steam application both are beneficial, safe, and cost effective treatment modalities for the Low back pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

It is no surprise that nature has provided us with many therapeutic compounds for thousands of years. Throughout history, plants and their derivatives have been used orally and locally to treat most of the diseases. In addition to providing a wealth of health benefits, Azadirachta indica stands out among all of them as one of the most universal and prolific trees. Since antiquity, this medicinal plant has been used in the Unani System of Medicine as well as in other traditional systems of medicine (Ayurveda, Homeopathic, Chinese, and European "Materia Medica") to treat a wide range of diseases. Almost every part of the tree can be used as medicine, including its leaves, flowers, seeds, fruits, roots, and bark, both locally and systemically after being properly processed in pharmaceutical industries. Apart from treating various skin ailments like dermatitis, eczema, acne, and fungal and bacterial infections, it also exhibits anti-malarial, anticancer, antifertility, antioxidant, antidiabetic, anti-inflammatory, hepatoprotective, neuroprotective, and wound healing properties. In modern times, it is also used in cosmetics, toiletries, and pharmaceutical products. The purpose of this review is to gather all the information available about Azadirachta indica, including its distribution, botanical description, commercial use, and medicinal properties.

Multi-drug resistant (MDR) E. coli constitute a major public health burden globally, reaching the highest prevalence in the global south yet frequently flowing with travellers to other regions. However, our comprehension of the entire genetic diversity of E. coli colonising local populations remains limited. We quantified this diversity, its associated antimicrobial resistance (AMR), and assessed the impact of antibiotic use by recruiting 494 outpatients and 423 community dwellers in the Punjab province, Pakistan. Rectal swab and stool samples were cultured on CLED agar and DNA extracted from plate sweeps was sequenced en masse to capture both the genetic and AMR diversity of E. coli. We assembled 5,247 E. coli genomes from 1,411 samples, displaying marked genetic diversity in gut colonisation. Compared with high income countries, the Punjabi population generally showed a markedly different distribution of genetic lineages and AMR determinants, while use of antibiotics elevated the prevalence of well-known globally circulating MDR clinical strains. These findings implicate that longitudinal multi-regional genomics-based surveillance of both colonisation and infections is a prerequisite for developing mechanistic understanding of the interplay between ecology and evolution in the maintenance and dissemination of (MDR) E. coli., (© 2024. The Author(s).)

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The K m value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding V max value was 25.00 ± 0.67 μmol min -1 mg -1 in the presence of 2 mM CaCl 2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (μmol min -1  mg -1 ) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Amylases are biologically active enzymes that can hydrolyze starch to produce dextrin, glucose, maltose, and oligosaccharides. The amylases contribute approximately 30% to the global industrial enzyme market. The globally produced amylases are widely used in textile, biofuel, starch processing, food, bioremediation of environmental pollutants, pulp, and paper, clinical, and fermentation industries. The purpose of this review article is to summarize recent trends and aspects of α-amylases, classification, microbial production sources, biosynthesis and production methods, and its broad-spectrum applications for industrial purposes, which will depict the latest trends in α-amylases production. In the present article, we have comprehensively compared the biodiversity of α-amylases in different model organisms ranging from archaea to eukaryotes using in silico structural analysis tools. The detailed comparative analysis: regarding their structure, function, cofactor, signal peptide, and catalytic domain along with their catalytic residues of α-amylases in 16 model organisms were discussed in this paper. The comparative studies on alpha (α) amylases' secondary and tertiary structures, multiple sequence alignment, transmembrane helices, physiochemical properties, and their phylogenetic analysis in model organisms were briefly studied. This review has documented the recent trends and future perspectives of industrially important novel thermophilic α-amylases. In conclusion, this review sheds light on the current understanding and prospects of α-amylase research, highlighting its importance as a versatile enzyme with numerous applications and emphasizing the need for further exploration and innovation in this field., (© 2023 Wiley Periodicals LLC.)

TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5-7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6-0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications., Competing Interests: Competing interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Melasma is a chronic, acquired, symmetrical hyper melanosis of skin, characterized by irregular light to dark brown patches on sun-exposed areas, with a significant effect on psychological health; melasma is termed as Kalf in Unani medicine. Conventional treatments have transitory results and often carry adverse effects like skin irritation, scarring, etc. This study was planned to evaluate the safety and efficacy of a Unani pharmacopoeial formulation Tila-e-Kalf, comprising of lentil ( Lens culinaris ), bitter almond ( Prunus amygdalus ), and fig ( Ficus carica ), and to compare its efficacy with standard drug hydroquinone in patients of melasma., Materials and Methods: This was an 8-week open-label, standard controlled, randomized clinical study conducted on patients of epidermal melasma. The test group received Tila-e-Kalf while the control group received hydroquinone 4% cream for local application once daily. Efficacy was assessed by MASI (Melasma Area Severity Index), DLQI (Dermatology Life Quality Index), and PGA (Physician Global Assessment) and colored photographs., Results: Mean MASI score decreased from10.65±0.85 to 7.07±0.74 in the test group (p<0.0001) and from 11.28±1.24 to 7.76±0.9 (p<0.0001) the in control group. Similar improvement was noticed in other parameters also. A large number of patients in the control group reported mild burning, itching, dryness, and skin rashes, while only one patient in the test group reported mild itching., Conclusion: Tila-e-Kalf as a topical depigmenting agent was found equally effective with better tolerability and safety as compared to hydroquinone., Competing Interests: The authors have declared that there is no conflict of interest.

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 U μmol -1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90 °C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60 °C for 1 h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The component of human life that has been most significantly altered by the COVID-19 epidemic is travel. Due to the upheaval produced by the pandemic breakout, countries are becoming increasingly avaricious and are scrambling to stockpile vaccines. The world has been locked down to reduce/control the pandemic outbreak, driving countries to shut their doors to other people from countries. The recent pandemic has had a short-term, positive effect on the environment, but travel restrictions have caused problems for the common person and are expected to deteriorate more soon, necessitating longer quarantines, vaccination requirements, vaccine passports, and immunization certificates required by countries for safe travel. Thus, this study has three objectives. First, we investigate the impact of COVID-19 on travel and the environment, as well as the role that tourists play in the transmission of the virus. Second, we examine how countries are handling COVID-19 vaccines. Finally, we pinpoint differences in vaccination coverage., (© The Author(s), under exclusive licence to Springer Nature B.V. 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Non availability of effective anti-TB vaccine impedes TB control which remains a crucial global health issue. A fusion molecule based on immunogenic antigens specific to different growth phases of Mycobacterium tuberculosis can enhance T-cell responses required for developing a potent vaccine. In this study, six antigens including EspC, TB10.4, HspX, PPE57, CFP21 and Rv1352 were selected for constructing EspC-TB10.4 (bifu25), TnCFP21-Rv1352 (bifu29), HspX-EspC-TB10.4 (trifu37), HspX-TnCFP21-Rv1352 (trifu44) and HspX-EspC-TB10.4-PPE57 (tetrafu56) fusion proteins. Th1-cell epitopes of EspC, PPE57 and Rv1352 antigens were predicted for the first time using different in silico tools. The fusion molecule tetrafu56, which consisted of antigens from both the replicating and the dormant stages of Mtb, induced a release of 397 pg/mL of IFN-γ from PBMCs of the active TB patients. This response was comparable to the response obtained with cocktail of the component antigens (396 pg/mL) as well as to the total of the responses obtained separately for each of its component antigens (388 pg/mL). However, PBMCs from healthy samples in response to tetrafu56 showed IFN-γ release of only 26.0 pg/mL Thus a previous exposure of PBMCs to Mtb antigens in TB plasma samples resulted in 15-fold increase in IFN-γ response to tetrafu56 as compared to the PBMCs from the healthy controls. Hence, most of the T-cell epitopes of the individual antigens seem to be available for T-cell interactions in the form of the fusion. Further investigation in animal models should substantiate the immune efficacy of the fusion molecule. Thus, the fusion tetrafu56 seems to be a potential candidate for developing an effective multistage vaccine against TB., Competing Interests: The authors declare that there is no conflict of interest regarding this manuscript.

Climate change has increasingly been recognised and associated with consumer behaviour: Practitioners are developing their strategies to reduce environmental degradation while increasing the management of sustainable consumption; it needs to better understand consumer attitudes and eco-friendly factors about the issue. Therefore, the current study focused to understand the effects of pro-environmental factors on individuals' environmental attitudes (purchase behaviour towards products with recycled packaging) through the lens of theory of planned behaviour in a cross-cultural setting. Moreover, present research focuses on the moderating role that religiosity plays in causal pathways between certain determinants (attitude, subjective norms, and perceived behavioural control) and intentions in this context. A multi-wave time-lagged research design was employed in this study, and university students from two developing countries were surveyed ( N  = 324, 266). The findings revealed pronounced similarities between the two examined countries. Overwhelmingly, pro-environmental factors examined (environmental values, environmental knowledge, and environmental concern) were found to be positively related to attitude formation. Further results showed that attitude and subjective norms are significant predictors of the intention to purchase products with recycled packaging. Moreover, with the exception of perceived behavioural control, religiosity moderates the relationships between all the determinants of TPB and intention to purchase recycled packaged products. Present study offers insightful implications to management of these emerging and/or similar cultural markets regarding customer value for green products. Using TPB, present study broadened and deepen extant stream of literature on consumption of recycled packaged products in two highly emerging markets; Pakistan and Malaysia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Husnain, Usman, Akhtar, Ali, Khan, Abbas, Ismail, Rehman and Akram.)

Tuberculosis (TB) stays a major cause of death globally after COVID-19 and HIV. An early diagnosis to control TB effectively, needs a fast reliable diagnostic method with high sensitivity. Serodiagnosis involving polyclonal antibodies detection against an antigen of Mycobacterium tuberculosis (Mtb) in serum samples can be instrumental. In our study, Rv3874 and Rv3875 antigens were cloned, expressed, and purified individually and as a chimeric construct in Escherichia coli BL21. Enzyme-Linked Immunosorbent Assay (ELISA) based findings revealed that the Rv3874-Rv3875 chimeric construct was two-fold more sensitive (59.7%) than the individual sensitivities of Rv3874 (28.4%) and Rv3875 (24.9%) for 201 serum TB positive samples. Furthermore, the fusion construct was a little more sensitive (60.4%) for male subjects than that for females (58.8%). Lastly, our preliminary findings, molecular insights of secondary structure, and statistical and in silico analysis of each construct also advocate that CEP can be considered a better immunodiagnostic tool in addition to previously reported EC skin test., (Copyright © 2022. Published by Elsevier Ltd.)

Utilizing the conservation of resources theory, this study investigates serial mediation of facades of conformity and depression between exploitative leadership and absenteeism. A total of 211 education sector employees using the convenient sampling technique took part in the survey with data collected in a time-lagged research design. Findings of the study reveal that facades of conformity and depression mediate the independent paths and play a serial mediating role between EL and absenteeism path. This study suggests that EL works as a workplace stressor, under which employees try to protect their valuable resources from further loss in the form of facades of conformity, in doing so, it leads to depression; thus, employees ultimately use absenteeism as an active coping strategy to cope with workplace stressors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SA declared a shared affiliation with the authors MWA and MSS to the handling editor at the time of review., (Copyright © 2022 Akhtar, Huo, Syed, Safdar, Rasool, Husnain, Awais and Sajjad.)

Using social information processing theory, our study investigates the effect of responsible leadership on employee career success via work engagement. The model also examines whether self-enhancement motives moderate the aforementioned mediating linkages. In three waves, data were collected from employees in the education sector. Macro PROCESS was used to assess the hypotheses. According to the findings, responsible leadership boosts employee work engagement, which leads to career success. The results also suggest that responsible leadership has a stronger positive effect on work engagement among individuals high on self-enhancement motives. There is no evidence in the educational literature about the underlying process through which a responsible leadership impacts employee success. Our research addresses this gap by suggesting work engagement as a mediator of the effect of responsible leadership on individuals' career success at various degrees of self-enhancement motives., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Yang and Akhtar.)

Emergencies and corruption go hand in hand in times of crisis. We are currently living in a pandemic phase, and corruption is even more damaging during these times of crisis that the world is experiencing with COVID-19. Vaccination is the only survival option that we have. The development of a nation will soon be measured by the criteria of who owns more vaccines. This study has four objectives. The first is to explore the most recent relevant literature. Moreover, we also investigate the unique trilogy of corruption, the environment, and the COVID-19 pandemic. The second is to identify adequate channels for distributing the COVID-19 vaccines. The vaccines should be dispersed based on the categories of age, gender, ethnicity, profession, and health conditions. Third, we explored the factors that are causing corruption in the distribution of the COVID-19 vaccines. Our findings show that unequal distribution, theft and black markets, weaponization of vaccines, logistical challenges, and substandard and falsified vaccines are the factors that potentially lead to corruption. The fourth objective is to investigate solutions for mitigating corruption. We revealed that blockchain, awareness, well-planned distribution channels, and prioritization of vulnerable groups are the steps that could effectively reduce corruption., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Oral squamous cell carcinoma (OSCC) is a main cause of oral cancer mortality and morbidity in central south Asia. To improve the clinical outcome of OSCC patients, detection markers are needed, which are preferably non-invasive and thus independent of a tissue biopsy., Methods: In the present study, we aimed to identify robust candidate protein biomarkers for non-invasive OSCC diagnosis. To this end, we measured the global protein profiles of OSCC tissue lysates to matched normal adjacent mucosa samples (n = 14) and the secretomes of nine HNSCC cell lines using LC-MS/MS-based proteomics., Results: A total of 5123 tissue proteins were identified, of which 205 were robustly up- regulated (p-value < 0.01, fold change > + 2) in OSCC-tissues compared to normal adjacent tissues. The biological process "Secretion" was highly enriched in this set of proteins. Other upregulated biological pathways included "Unfolded Protein Response", "Spliceosomal complex assembly", "Protein localization to endosome" and "Interferon Gamma Response". Transcription factor analysis implicated Creb3L1, ESRRA, YY, ELF2, STAT1 and XBP as potential regulators. Of the 205 upregulated tissue proteins, 132 were identified in the cancer cell line secretomes, underscoring their potential use as non-invasive biofluid markers. To further prioritize our candidate markers for non-invasive OSCC detection, we integrated our data with public biofluid datasets including OSCC saliva, yielding 25 candidate markers for further study., Conclusions: We identified several key proteins and processes that are associated with OSCC tissues, underscoring the importance of altered secretion. Cancer-associated OSCC secretome proteins present in saliva have potential to be used as novel non-invasive biomarkers for the diagnosis of OSCC., (© 2022. The Author(s).)

The effect of multiwall carbon nanotubes (MWCNTs) and magnesium oxide (MgO) on the thermal conductivity of MWCNTs and MgO-reinforced silicone rubber was studied. The increment of thermal conductivity was found to be linear with respect to increased loading of MgO. In order to improve the thermal transportation of phonons 0.3 wt % and 0.5 wt % of MWCNTs were added as filler to MgO-reinforced silicone rubber. The MWCNTs were functionalized by hydrogen peroxide (H 2 O 2 ) to activate organic groups onto the surface of MWCNTs. These functional groups improved the compatibility and adhesion and act as bridging agents between MWCNTs and silicone elastomer, resulting in the formation of active conductive pathways between MgO and MWCNTs in the silicone elastomer. The surface functionalization was confirmed with XRD and FTIR spectroscopy. Raman spectroscopy confirms the pristine structure of MWCNTs after oxidation with H 2 O 2 . The thermal conductivity is improved to 1 W/m·K with the addition of 20 vol% with 0.5 wt % of MWCNTs, which is an ~8-fold increment in comparison to neat elastomer. Improved thermal conductive properties of MgO-MWCNTs elastomer composite will be a potential replacement for conventional thermal interface materials.

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2 -ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future., (© 2021. The Author(s).)

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population., Competing Interests: No authors have competing interests.

Mungbean yellow mosaic virus (MYMV) is an important constraint in successful production of mungbean (Vigna radiata L.) in many countries, including Pakistan. The MYMV spreads by insect vector whitefly (Bemisia tabaci Gennadius). The use of resistant cultivars is the most effective management tactics for MYMV. Twenty mungbean varieties/lines were screened against insect vector of MYMV under field condition in the current study. Resistance levels for varieties/lines were assessed through visual scoring of typical disease symptoms. Furthermore, the impacts of two insecticides 'Imidacloprid' and 'Thiamethoxam' and two plant extracts, i.e., neem (Azadirachta indica), and Eucalyptus (Eucalyptus camaldulensis) were tested on the suppression of whitefly. Field screening indicated that none of the tested varieties/lines proved immune/highly resistant, while significant variations were recorded among varieties/lines for resistance level. All varieties/lines were systemically infected with MYMV. The varieties 'AARI-2006' and 'Mung-14043' were considered as resistant to MYMV based on visual symptoms and the lowest vector population. These varieties were followed by 'NM-2006' and 'NL-31', which proved as moderately resistant to MYMV. All remaining varieties/lines were grouped as moderately to highly susceptible to MYMV based on visual symptoms' scoring. These results revealed that existing mungbean germplasm do not possess high resistance level MYMV. However, the lines showing higher resistance in the current study must be exploited in breeding programs for the development of resistant mungbean varieties/lines against MYMV. Imidacloprid proved as the most effective insecticide at all concentrations to manage whitefly population. Therefore, use of the varieties with higher resistance level and spraying Imidacloprid could lower the incidence of MYMV., Competing Interests: The authors have declared that no competing interests exist.

Food production and waste recycling are the two major issues faced globally with rapidly increasing population. Recycling organic wastes to crop amendments could be a possible solution to these issues. Earthworms transfer organic waste to compost, which is used to grow crops and increase crop productivity. This study assessed the impact of vermicompost produced from the residues of six desert plant species, i.e., (Ziziphus mauritiana, Aerva javanica, Calligonum comosum, Sacchrum benghalens, Calligonum polygonoides and Prosopis cineraria) combined with farmyard manure (5 t ha-1) on growth, yield and photosynthetic activity of maize crop. Earthworm species Eisenia fetida (Savigny, 1826) was used to prepare vermicomposting of all tested plant species. The desert species were collected from natural habitats, chopped, dried, mixed with FYM and then earthworms were released to prepare the vermicompost. The earthworms were excluded twenty days after release and resultant was considered as compost and used in the experiment. Results revealed that application of P. cineraria vermicompost resulted in the highest plant height (75.33 cm), stem diameter (22.66 mm), cob length (17.66 cm), number of grains/cob (374.67), 1000-grain weight (260.41 g) and grains yield (3.20 t/ha). Application of P. cineraria vermicompost resulted in the highest uptake of macronutrients, i.e., N (91.01%), P (22.07%), K (80.41%), micronutrients, i.e., Fe (19.07 ppm), Zn (40.05 ppm), and phenolic contents (150). Application of P. cineraria vermicompost also resulted in the highest quantum photosynthetic yield (0.42 mole C/mole of photon), chlorophyll florescence (355.18 moles of photon m-2s-1) and electron transport rate (310.18 micro mole m-2s-1). It is concluded that vermicomposting has the potential to improve growth and yield of maize crop. Particularly, application of vermicompost obtained from P. cineraria can be used to improve the growth and yield of maize crop. Nonetheless, field trials are necessary for a wide scale recommendation., Competing Interests: The authors have declared that no competing interests exist.

Tuberculosis vaccines capable of reducing disease worldwide have proven difficult to develop. BCG is effective in limiting childhood disease, but adult TB is still a major public health issue. Development of new vaccines requires identification of antigens that are both spatially and temporally available throughout infection, and immune responses to which reduce bacterial burden without increasing pathologic outcomes. Subunit vaccines containing antigen require adjuvants to drive appropriate long-lived responses. We generated a triple-antigen fusion containing the virulence-associated EsxN (Rv1793), the PPE42 (Rv2608), and the latency associated Rv2628 to investigate the balance between bacterial reduction and weight loss in an animal model of aerosol infection. We found that in both a low pattern recognition receptor (PRR) engaging adjuvant and a high PRR-engaging adjuvant (MPL/TDM/DDA) the triple-antigen fusion could reduce the bacterial burden, but also induced weight loss in the mice upon aerosol infection. The weight loss was associated with an imbalance between TNFα and IL-17 transcription in the lung upon challenge. These data indicate the need to assess both protective and pathogenic responses when investigating subunit vaccine activity.

Ovarian cancer (OC) is the most lethal among female reproductive system malignancies. Depending upon the stage at presentation, the five year survival ratio varies from ∼92 to ∼30%. The role of biomarkers in early cancer diagnosis, including OC, is well understood. In our previous study, through an initial screening, we have analyzed eleven proteins that exhibited differential expression in OC using two-dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometric (MALDI-TOF MS) analysis. In continuation of our previous study, the present work describes analysis of twenty more proteins that showed aberrant expression in OC. Among these, six showed consistent significant deregulation in the OC false discovery rate [FDR ≤ 0.05]. Upon MS analysis, they were identified as vimentin, tubulin beta 2C chain, tubulin alpha 1C chain, actin cytoplasmic 2, apolipoprotein A-I, and collagen alpha 2(VI) chain [peptide mass fingerprint (PMF) score ≥ 79]. One of the differentially regulated proteins, tubulin beta 2C chain, was found to be significantly (fold change, 2.5) enhanced in OC. Verification by western blot and enzyme-linked immunosorbent assay (ELISA) demonstrated that the tubulin beta 2C chain may serve as a valuable marker for OC (ANOVA p < 0.0001). The assessment of the likely association of TBB2C with OC in a larger population will not only help in developing clinically useful biomarkers in the future but also improve our understanding of the progression of OC disease., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca 2+ -dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential. SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD., (Copyright © 2021 the authors.)

Tuberculosis (TB) is a global health problem, being prevalent in the developing countries. A rapid, reliable and cost effective diagnostic method would help in controlling TB in the endemic populations. Development of suitable fusion molecules detecting multiple antibodies produced against Mycobacterium tuberculosis antigens would enhance sensitivity of serodiagnostic assays. In this study, EspC, CFP7 and PPE57 antigens of M. tuberculosis were selected for constructing fusion molecules after prediction of B-cell epitopes using in silico tools. Fusion proteins EspC-CFP7, HspX-EspC-CFP7 and HspX-EspC-CFP7-PPE57 were expressed in E.coli (BL21). The serodiagnostic potential of the individual antigens and their fusions was analyzed by screening 230 plasma samples of pulmonary TB patients. The single antigens HspX, EspC, CFP7, PPE57 showed sensitivities of 30%, 31%, 22% and 35%, respectively. The fusion protein EspC-CFP7 showed sensitivity of 43%. Linking of HspX antigen to the N-terminus of EspC-CFP7 fusion molecule increased sensitivity to 58%, while joining PPE57 antigen to the C-terminus of HspX-EspC-CFP7 increased sensitivity to 69%. The fusion protein HspX-EspC-CFP7-PPE57 seems to be a promising molecule for use in the development of fusions with higher sensitivity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment., Competing Interests: The authors have declared that no competing interests exist.

Using the assumptions of Sternberg (2003) Duplex Theory of Hate, the present study reveals the combined effects of similar competitor offer and narcissistic personality on brand equity through the underlying mechanism of brand hate. Specifically, we hypothesize that brand hate mediates the relationship between similar competitor offer and brand equity. Moreover, we propose that similar competitor offer and brand hate relationship are stronger for narcissistic individuals. By employing a multi-wave time-lagged research design, we collected data from a sample of ( N = 338) dairy product consumers in Pakistan. The findings of moderated-mediation regression analyses indicate that (a) Brand hate mediates the relationship between similar competitor offer and brand equity; and (b) Narcissistic personality moderates a similar competitor offer and brand hate relationship such that a high similar competitor offer led to greater brand hate when narcissism was high. Furthermore, conditional indirect effects reveal that brand hate mediates the relationship between similar competitor offer and brand equity only with individuals exhibiting narcissistic personality traits. The current study offers great insights to managers that by managing similar competitor offer, they can manage the development of brand hate, which can subsequently effect brand equity. Moreover, by profiling customers on the basis of their personalities, marketing managers can effectively invest only in customers with positive tendencies. The current study is unique in that it highlights new avenues in existing research by extending the nascent domain of brand hate in consumer-brand relationships., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Husnain, Wang, Poulova, Syed, Akbar, Akhtar, Akbar and Usman.)

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at G o /G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications. KEY POINTS: • Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli. • Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media. • Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL. • Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features. Graphical abstract.

Tuberculosis (TB) is amongst the deadliest diseases worldwide. For effective control of TB a rapid, reliable and sensitive method for its diagnosis is essential. Serodiagnosis detecting multiple antibodies against antigens of Mycobacterium tuberculosis (Mtb) in blood samples could prove beneficial. Based on the epitope position in the molecule, two truncated variants of Rv1984c, i.e., Tn1Rv1984c and Tn2Rv1984c were expressed in Escherichia coli. Screening of the Rv1984c, Tn1Rv1984c and Tn2Rv1984c against 231 sera samples from the culture positive TB patients showed sensitivities of 34.2%, 49.4% and 26.8%, respectively. Another antigen Rv1352 was analyzed for the location of epitopes, which had not been reported before. A fusion molecule consisting of Tn1Rv1984c and Rv1352, expressed in E. coli, showed enhanced sensitivity of 62.8%. Joining another antigen Rv2031c to the N-terminus of Tn1Rv1984c-Rv1352, improved sensitivity to 71.4%. The fusion construct Rv2031c-Tn1Rv1984c-Rv1352 showed comparatively higher sensitivity of 73.4% in the male group as compared to 67% in the female group. Data derived for the secondary structure analysis through Circular Dichroism (CD) spectroscopy and prediction on the basis of molecular modelling was also in agreement. This construct can be a potential base for producing constructs with greater sensitivity through fusion of epitopes from additional Mtb antigens., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (Cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as Cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant Cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of Cel6A.CBC which revealed thermostability of Cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of Cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Purpose: An early and accurate diagnosis of ovarian carcinoma (OC) may reduce morbidity and mortality of the patients. To improve the clinical outcome in OC patients, the present study is aimed at identifying robust biomarkers for early OC diagnosis. EXPERIMENTAL DESIGN: In order to look for early-stage protein markers, a systematic protein profiling approach involving 2-dimensional electrophoresis coupled with mass spectrometric analyses of human malignant and non-malignant ovarian biopsy samples, is performed., Results: Six 2D gel spots, corresponding to five proteins, display statistically significant differential expression in the tumor tissues compared to benign controls (FDR ≤ 0.05; PMF score ≥ 79). Ingenuity pathway analysis predicts two proteins, that is, Ca 2+ -dependent membrane-binding protein annexin A6 (AnxA6) and the metabolic enzyme l-lactate dehydrogenase A chain, as potential predictive biomarkers. Increased expression of AnxA6 is further ascertained by Western blot and enzyme linked immunosorbent assay in the resected tissues and the plasma samples. The expression is found markedly increasing particularly in the advanced stage tumors., Conclusions and Clinical Relevance: The significant upregulation of AnxA6 in OC, reported for the first time, is likely to provide insight into the mechanism of OC progression, which may lead to the design of potential diagnostic and therapeutic strategies., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7-, 5- and 4.8-fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hair, being one of the most important components of the beauty care processes, attracts the use of a variety of hair treating cosmetics. Conventional hair treating cosmetics are not satisfactory for one reason or the other. Commercially used keratins are isolated from wool or chicken feathers. As these lack complete sequence identity with human hair keratin, are likely to be less efficient than the human hair keratin. K31, a type I acidic keratin, is a major protein of human hair keratin complex and it is essential for maintaining the hair tensile strength. In this study keratin K31 (46 kDa) gene was expressed in Escherichia coli at a level of approximately 35% of the total cell proteins. The protein, however, was expressed as insoluble inclusion bodies. The expressed protein was refolded and purified by FPLC using an anion-exchange column. The CD analysis results showed that the K31 was properly refolded. MALDI-TOF mass spectroscopy showed the characteristics expected for human K31 keratin. The refolded and partially purified K31 protein, when applied on chemically damaged hairs, increased the diameter of the hair up to 49%. The mechanical strength of the bleached hair increased by almost 2 fold after a single treatment of K31. The protein also straightened curly hair efficiently on a single treatment for one hour. Application of K31 resulted in marked improvements in smoothness, diameter and mechanical strength of the damaged hair.

The most frequently reported genetic aberration among polycythemia vera (PV) patients is a gain of function mutation V617F in exon 14 of Janus kinase 2 (JAK2) gene. However in many investigations, V617F negative PV patients have been reported to harbor mutations in JAK 2 exon 12. We investigated 24 patients with PV (diagnosed following 2016 WHO guidelines) to detect V617F mutation through allele specific PCR. The frequency of which was found to be 19/24 (79.2 %). Later on JAK2 exon 12 and 14 was amplified by conventional PCR in V617F negative patients and subjected to sequence analysis. A total of 03 mutated sites in exon 12 were detected in only two V617F-negative patients 2/5 (40%). All three substitutions were heterozygous i.e. F537F/I found in both patients and R528R/T, which is a novel mutation. In addition, one patient 1/5 (10%) manifested amino acid substitution V617A in JAK2 exon 14. Hematological parameters of individuals harboring mutations do not vary significantly than rest of the PV patients. Previous history and 2.3 years of follow-up studies reveal 15-year survival of V617F positive patients (n=19) to be 76%, while it is 94% for wild type V617 patients (n=05). Mean TLC of the patient cohort was 17.6± 9.1 x 109/L, mean platelet count was 552± 253 x 109/L, mean hemoglobin was 16.9± 3.2 g/dl, mean corpuscular volume (MCV) was 77.2± 13.0 fl and mean corpuscular hemoglobin (MCH) was 25.6± 3.9 pg. This is the very first attempt from Pakistan to screen JAK2-exon 12 mutations in PV patients. We further aim to investigate Jak2 exon 12 mutations in larger number of PV patients to assess their clinical relevance and role in disease onset, progression and transformation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Cel5A of Thermotoga maritima, a 37 kDa cellulase of the family GH5, was expressed in partially soluble state in E. coli. However, the truncated version tCel5A1, produced by removing ten residues from the C-terminal of Cel5A, was expressed in a completely soluble form. tCel5A1 showed 7.3- fold increased specific activity against carboxy methyl cellulose while the increase in activities against regenerated amorphous cellulose and Avicel were 21 and 16 fold, respectively. tCel5A1 is stable at 60 °C for more than 2 hr and it showed temperature and pH optima 70 °C and 6.0, respectively, under the assay conditions used. These characteristics are similar to those of the native enzyme. As expected, CD spectral analysis showed that C-terminal truncation has little effect on the secondary structure of the molecule. tCel5A1 showed higher binding to pretreated rice straw (84%) as compared to the native form (46%). Molecular modelling analysis of tCel5A1 showed that the removal of C-terminal residues exposed the active site residues Glu253, Trp286, and Phe292, which are located in the catalytic cavity close to the C-terminus. Making these residues more accessible to the substrate would result in increased activity. The ratio of 10.01 between the soluble to the insoluble reducing groups produced from RAC on treatment with tCel5A1, and the presence of cellobiose as the major end product in the hydrolysate showed that tCel5A1 is a processive cellulase. Although other processive cellulases belonging to the family GH5, mainly of the fungal origin, have been reported, but tCel5A1, to our knowledge, is the first processive cellulase from an extreme thermophile reported so far., (© 2018 Wiley Periodicals, Inc.)

A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme was optimally active at 70 °C and pH 6.0. It was stable up to 70 °C while the CD spectroscopy analysis showed thermal unfolding at 80 °C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate were found to be 126,480, 10,350 and 17,250 U μmol -1 , respectively. Xyn10B was highly active producing xylobiose and xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry cereals, bio-bleaching of wood pulp and degradation of plant biomass.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c +/- (Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention.

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Objective: Low back pain is a frequent problem faced by the majority of people at some point in their lifetime. Exercise therapy has been advocated an effective treatment for chronic low back pain. However, there is lack of consensus on the best exercise treatment and numerous studies are underway. Conclusive studies are lacking especially in this part of the world. Thisstudy was designed to compare the effectiveness of specific stabilization exercises with routine physical therapy exerciseprovided in patients with nonspecific chronic mechanical low back pain., Methods: This is single blinded randomized control trial that was conducted at the department of physical therapy Orthopedic and Spine Institute, Johar Town, Lahore in which 120 subjects with nonspecific chronic low back pain participated. Subjects with the age between 20 to 60 years and primary complaint of chronic low back pain were recruited after giving an informed consent. Participants were randomly assigned to two treatment groups A & B which were treated with core stabilization exercise and routine physical therapy exercise respectively. TENS and ultrasound were given as therapeutic modalities to both treatment groups. Outcomes of the treatment were recorded using Visual Analogue Scale (VAS) pretreatment, at 2 nd , 4 th and 6 th week post treatment., Results: The results of this study illustrate that clinical and therapeutic effects of core stabilization exercise program over the period of six weeks are more effective in terms of reduction in pain, compared to routine physical therapy exercise for similar duration. This study found significant reduction in pain across the two groups at 2 nd , 4 th and 6 th week of treatment with p value less than 0.05. There was a mean reduction of 3.08 and 1.71 on VAS across the core stabilization group and routine physical therapy exercise group respectively., Conclusion: Core stabilization exercise is more effective than routine physical therapy exercise in terms of greater reduction in pain in patients with non-specific low back pain.

Tuberculosis is one of the top ten causes of deaths worldwide. The cause of tuberculosis is a bacterium, Mycobacterium tuberculosis, which has been surviving for centuries. Immunological tests based on detecting the presence of antibodies in the sera of active TB patients against various antigens of M. tuberculosis are useful for diagnosis of TB and offer simple, rapid and cost effective methods most suitable for poor and developing countries. Several recombinant antigens have been reported so far with varying sensitivity individually, yet none had shown sensitivity higher enough to be used in a commercial test. There is a trend of utilizing recombinant DNA technology to make polypeptide chain with two or more different antigenic regions, in order to increase the diagnostic efficiency. In this review, we have made an attempt to combine current studies on the usefulness of the multi-component Mycobacterium tuberculosis antigens in serological tests for the diagnosis of tuberculosis., (Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30-70%, and 93-99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer., Biological Significance: Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Better and sensitive biomarkers are needed to help understand the mechanism of disease onset, progression, prognosis and monitoring of the therapeutic response. Aim of this study was to identify the candidate circulating markers of chronic-phase chronic myeloid leukemia (CP-CML) manifestations, having potential to develop into predictive- or monitoring-biomarkers. A proteomic approach, two-dimensional gel electrophoresis in conjunction with mass spectrometry (2DE-MS), was employed for this purpose. Based on the spot intensity measurements, six proteins were found to be consistently dysregulated in CP-CML subjects compared to the healthy controls [false discovery rate (FDR) threshold ≤0.05]. These were identified as α-1-antichymotrypsin, α-1-antitrypsin, CD5 molecule-like, stress-induced phosphoprotein 1, vitamin D binding protein isoform 1 and transthyretin by MS analysis [PMF score ≥79; data accessible via ProteomeXchange with identifier PXD002757]. Quantitative ELISA, used for validation of candidate proteins both in the pre-treated and nilotinib-treated CP-CML cases, demonstrate that CD5 molecule-like, transthyretin and alpha-1-antitrypsin may serve as useful predictive markers and aid in monitoring the response of TKI-based therapy (ANOVA p < 0.0001). Two of the circulating marker proteins, identified in this study, had not previously been associated with chronic- or acute-phase myeloid leukemia. Exploration of their probable association with CP-CML, in a larger study cohort, may add to our understanding of the disease mechanism besides developing clinically useful biomarkers in future.