Main Protein Research Articles

Extracellular Matrices as Bioactive Materials for In Situ Tissue Regeneration.

Bioactive materials based on a nature-derived extracellular matrix (NECM) represent a category of biomedical devices with versatile therapeutic applications in the realms of tissue repair and engineering. With advancements in decellularization technique, the inherent bioactive molecules and the innate nano-structural and mechanical properties are preserved in three-dimensional scaffolds mainly composed of collagens. Techniques such as electrospinning, three-dimensional printing, and the intricate fabrication of hydrogels are developed to mimic the physical structures, biosignalling and mechanical cues of ECM. Until now, there has been no approach that can fully account for the multifaceted properties and diverse applications of NECM. In this review, we introduce the main proteins composing NECMs and explicate the importance of them when used as therapeutic devices in tissue repair. Nano-structural features of NECM and their applications regarding tissue repair are summarized. The origins, degradability, and mechanical property of and immune responses to NECM are also introduced. Furthermore, we review their applications, and clinical features thereof, in the repair of acute and chronic wounds, abdominal hernia, breast deformity, etc. Some typical marketed devices based on NECM, their indications, and clinical relevance are summarized.

Progress of SARS-CoV-2 Main protease peptide-like inhibitors.

The pneumonia outbreak caused by Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) infection poses a serious threat to people worldwide. Although vaccines have been developed, antiviral drugs are still needed to combat SARS-CoV-2 infection due to the high mutability of the virus. SARS-CoV-2 main protein (Mpro ) is a special cysteine protease that is a key enzyme for SARS-CoV-2 replication. It is encoded by peptides and is responsible for processing peptides into functional proteins, making it an important drug target. The paper reviews the structure and peptide-like inhibitors of SARS-CoV-2 Mpro , also the binding mode and structure-activity relationship between the inhibitors and Mpro are introduced in detail. It is hoped that this review can provide ideas and help for the development of anti-coronavirus drugs such as COVID-19, and help to develop broad-spectrum antiviral drug for the treatment of coronavirus diseases as soon as possible.

Preoperative Level of Insulin-Like Growth Factor Binding Protein 2 Predicts The Suboptimal Outcome After Primary Debulking Surgery in Patients with Advance Ovarian Cancer

BACKGROUND: The need for clinically useful biomarkers which can predict the surgical outcome after primary debulking surgery (PDS) in patients with advance ovarian cancer (AOC) is really important. Insulin-like growth factor-binding protein 2 (IGFBP2) is the main binding protein expressed by ovarian cancer cells, which plays a prominent role in promoting proliferation, driving invasion, and suppressing apoptosis. This study was conducted to assess the performance of IGFBP2 in predicting the surgical outcome after PDS in patients with AOC.METHODS: Twenty-four subjects with AOC (Stage IIIc/IV) who underwent PDS were recruited consecutively. Clinicopathologic data were obtained from subjects' medical records. Blood samples were withdrawn form each subject and preoperative level of IGFBP2 were measured using enzyme-linked immunosorbent assay (ELISA). Multivariate analysis was employed to test the performance of multiple predictors of surgical outcome.RESULTS: Eighteen patients (75%) had suboptimal outcome after PDS. Mean IGFBP2 level was significantly higher in the suboptimal group (1157.5±359.9 ng/mL vs. 679.1±504.5 ng/mL, p=0.018). In bivariate model, higher preoperative level of IGFBP2 predict the suboptimal outcome with good accuracy (AUC: 0.796, sensitivity: 83.3%, specificity: 83.3%, p=0.033, optimal threshold level 870 ng/mL). Higher IGFBP2 level was associated with higher risk of suboptimal outcome, although IGFBP2 was not an independent risk factor (adjusted OR: 5.0, 95% CI: 0.43-57.9, p=0.198).CONCLUSION: IGFBP2 is a novel and promising biomarker for surgical outcome prediction following PDS in AOC patients. Since it is predictive for suboptimal outcome, patients with higher preoperative level of IGFBP2 needs more thorough preoperative evaluation as well as meticulous surgical technique to optimize the surgical outcome.KEYWORDS: IGFBP2, advance ovarian cancer, PDS, surgical outcome, predictor

Metals bioaccumulation in fish captured from Araguari River upper section (Amazon biome), and risk assessment to human health resulting from their consumption

BackgroundThe Araguari River is one of the most important rivers from Amapá State and its waters are essential as source of drinking water and food, especially for the traditional Amazonian populations. These populations have fish as their main protein source; however, studies have shown the metals bioaccumulation in fish caught in the Araguari River and the risks to human health resulting from fish consumption. Our objective was to investigate the bioaccumulation of Cd, Pb, Cr, Ni, Fe, Mn, Cu, Zn, and Hg in muscle tissue from the fish species Ageneiosus inermis and Hoplias aimara, and to evaluate the risks to human health resulting from their consumption. MethodsFish sampling was carried out between September and October 2018 at two sampling sites located in the Araguari River upper section. The metal concentrations quantification was performed by Atomic Absorption Spectrometry, except for Hg whose concentrations were determined using a hydride generator coupled to Inductively Coupled Plasma Optical Emission Spectrometry. For risk assessment of each metal individually we use the risk quotient (RQ) approach. To assess the risk of metal mixtures, present in fish muscle samples, we used the risk index (RI) approach. We also calculate the estimated daily intake (EDI). ResultsThere was no statistical difference in the bioaccumulation of metals between both fish species. Only Cd and Pb presented average concentrations higher than the maximum limits established by Brazilian legislation aimed at protecting human health. The RI values obtained for both fish species were > 25, which indicates high risks to human health. The EDI values obtained for Hg in both fish species exceeded their reference dose (RfD). Taken together, our results show that these fish species are unfit for consumption. ConclusionsBased on our results we recommend that the population does not consume the species A. inermis and H. aimara. When suspending consumption of both species is not possible, reducing their consumption is mandatory to reduce the exposure of human populations to toxic metals bioaccumulated in fish muscle tissue and thus reduce health risks.

Entopallium Lost GFAP Immunoreactivity during Avian Evolution: Is GFAP a “Condition Sine Qua Non”?

Introduction: The present study demonstrates that in the same brain area the astroglia can express GFAP (the main cytoskeletal protein of astroglia) in some species but not in the others of the same vertebrate class. It contrasts the former opinions that the distribution of GFAP found in a species is characteristic of the entire class. The present study investigated birds in different phylogenetic positions: duck (Cairina moschata domestica), chicken (Gallus gallus domesticus), and quails (Coturnix japonica and Excalfactoria chinensis) of Galloanserae; pigeon (Columba livia domestica) of a group of Neoaves, in comparison with representatives of other Neoaves lineages, which emerged more recently in evolution: finches (Taeniopygia guttata and Erythrura gouldiae), magpie (Pica pica), and parrots (Melopsittacus undulatus and Nymphicus hollandicus). Methods: Following a perfusion with 4% buffered paraformaldehyde, immunoperoxidase reactions were performed with two types of anti-GFAP: monoclonal and polyclonal, on floating sections. Results: The entopallium (formerly “ectostriatum,” a telencephalic area in birds) was GFAP-immunopositive in pigeon and in the representatives of Galloanserae but not in songbirds and parrots, which emerged more recently in evolution. The lack of GFAP expression of a brain area, however, does not mean the lack of astroglia. Lesions induced GFAP expression in the territory of GFAP-immunonegative entopallia. It proved that the GFAP immunonegativity is not due to the lack of capability, but rather the suppression of GFAP production of the astrocytes in this territory. In the other areas investigated besides the entopallium (optic tectum and cerebellum), no considerable interspecific differences of GFAP immunopositivity were found. It proved that the immunonegativity of entopallium is due to neither the general lack of GFAP expression nor the incapability of our reagents to detect GFAP in these species. Conclusion: The data are congruent with our proposal that a lack of GFAP expression has evolved in different brain areas in vertebrate evolution, typically in lineages that emerged more recently. Comparative studies on GFAP-immunopositive and GFAP-immunonegative entopallia may promote understanding the role of GFAP in neural networks.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus

BackgroundEctromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method.ResultsIn this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement.ConclusionsIn conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Simvastatin induces degradation of the extracellular matrix in human leiomyomata: novel in vitro, in vivo, and patient level evidence of matrix metalloproteinase involvement

ObjectivesTo assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. DesignLaboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. SettingAcademic research center. Patient(s)Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. Intervention(s)Simvastatin treatment. Main Outcome Measure(s)Serum concentrations, xenograft volumes, and protein expression. ResultsMice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls, 12 treated with activated simvastatin at 10 mg/kg body weight, and 15 at 20 mg/kg body weight. Simvastatin was detected in serum from mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration, p < 0.05). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase (MT1-MMP) was increased in vitro and in vivo. Matrix metalloproteinase 2 (MMP2) and low density lipoprotein receptor related protein 1 (LRP1) were increased in vitro. ConclusionsSimvastatin exhibited antitumoral activity, with extracellular matrix degradation and decreased leiomyoma tumor volume in vivo. Activation of the MMP2/MT1-MMP/LRP1 pathway may explain these findings.

Mechanism of Yixishu lotion in the treatment of vaginitis based on network pharmacology combined with experimental validation: an experimental research study

Objective: Yixishu lotion (YXSL) originates from the summary of traditional Chinese medicine clinical experience and constantly improves in practice in clinical validation of the exact efficacy of traditional Chinese medicine prescription. To explore the mechanism of YXSL in treating vaginitis and the potential mechanisms based on network pharmacology and experimental verification. Methods: The active components and drug-related targets of YXSL were retrieved from the TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform) database, and the target was predicted by the UniProt database. Searching for genes related to ‘vaginitis’ disease in the GeneCards database, a total of 2581 drug targets were obtained. The interaction between proteins (PPI – protein–protein interaction) relationship was obtained by STRING database and visualized by Cytoscape software. Finally, the ‘Bioconductor’ installation package in R software was used to analyze the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of the target. Results: In this study, by the method of network pharmacology, the key active components of YXSL were flavonoids such as quercetin, apigenin, kaempferol, luteolin, β-sitosterol; the main core proteins included MAPK14, TP53, FGF2, ESR1, MAPK3, MAPK1, VEGFA, JUN, IL-6, and the KEGG pathway was mainly involved in MAPK pathway, Th17 pathway, Malaria, TNF pathway, and other signaling pathways. Animal experiments showed that the clinical symptoms and vaginal tissue lesions of the YXSL group and the fluconazole group were improved, and the levels of TNF-α (tumor necrosis factor alpha), IL-6 (interleukin-6), MDA (malondialdehyde), SOD (superoxide dismutase), IL-4, and IFN-γ (interferon-γ) in vaginal tissue and serum were better than the model group. Conclusion: YXSL may achieve its therapeutic effect on vaginitis by reducing the inflammatory response, improving oxidative stress response, and improving body immunity, and it provides a theoretical basis for further research on its pharmacodynamic material basis and mechanism of action.

Comparison of the Serum Protein Electrophoretic Pattern and Concentrations of Acute Phase Proteins in Bitches with and Without Mammary Gland Tumors

Abstract Alterations in the serum protein pattern may be associated with many diseases, including neoplastic processes. In veterinary medicine, these changes are poorly understood. Therefore, this study was aimed at the analysis of the distribution of blood serum protein fractions separated by agarose gel electrophoresis, and at the determination of the concentrations of main acute phase proteins in bitches with mammary gland neoplasia. The evaluation was conducted on twelve female dogs with palpable single or multiple nodules in the parenchyma of the mammary gland and on ten tumor-free clinically healthy bitches to compare the possible differences in the obtained results. Blood serum was used to perform agarose gel electrophoresis of the main blood serum protein fractions and to analyze the concentrations of total serum proteins and the following canine acute phase proteins: serum amyloid A, haptoglobin, C-reactive protein and α1-acid glycoprotein. The concentrations of total serum proteins were slightly higher in bitches with mammary gland tumors. Serum protein electrophoresis showed lower mean concentrations of albumin and α1-globulins in the affected dogs, while the concentrations of α2-and β1-globulins were significantly higher (P=0.0032 and P=0.0021, respectively) compared to dogs without mammary gland tumors. In the concentrations of acute phase proteins, significantly higher mean concentrations of C-reactive protein and haptoglobin were obtained in dogs with mammary tumors (P=0.0025 and P=0.0002, respectively). The values of α1-acid glycoprotein did not vary markedly between the bitches with and without mammary tumors. Presented data suggest that neoplastic processes in the mammary gland may also alter the electrophoretic pattern of blood serum proteins and induce changes in the production of some inflammatory proteins.

Impact of Foliar Spray with Gibberellic Acid GA3 and Nauta Fertilizer on the Some Vital and Mineral Components of Three Cultivars of Grape (Vitis vinifera L.)

The University of Mosul’s Department of Horticulture and Landscape Design / College of Agriculture and Forestry vineyard hosted this study. During the growing season 2022, to study the effects of foliar fertilization with Nauta fertilizer containing amino acids and seaweed extracts in three concentrations (0, 250, and 500 mg l-1) and foliar spraying with gibberellic acid in three concentrations (0, 50, and 100 mg l-1) on three table grape varieties, Halwany Lebanese, Kamali, and Black Khalili, and their interactions and effects on some It is possible to summarize the most important results obtained: According to this study, Halwany surpassed Kamali and Black Khalili in petiole iron concentration and grape variety superiority. Halwany and Kamali were over the Black Khalili variety in the average concentration of potassium in the petioles, just as the grape varieties were over the Halwany variety in the average protein and phosphorus concentrations. The Black Khalili variety also outperformed the Halwany and Kamali varieties in zinc content and carbohydrate percentage. In leaf petioles, nitrogen content increased significantly after foliar application of Nauta organic fertilizer at 250 mg l-1. In addition to the superiority of foliar spraying at 500 mg l-1 of the same fertilizer in main stem carbohydrates, protein, phosphorus, and iron in leaf petioles. Foliar spraying with gibberellic acid, especially at a concentration of 100 mg l-1, led to a significant increase in the average of protein and the concentration of phosphorus, potassium, zinc and iron in the leaf petioles.

Cognitive impairment acceleration after late‐life depression in a model of Alzheimer´s disease

AbstractBackgroundClinical studies suggest that depressive symptoms could be considered an important risk factor for the future development of cognitive impairment and even Alzheimer’s disease (AD). In fact, there is a strong association between depression in later life and AD. The age of onset of AD has been shown to be accelerated in patients with mild cognitive impairment (MCI) with a history of depression, and women appear to be particularly more vulnerable to this condition. In addition, individuals with MCI who present with depressive symptoms have an elevated burden of amyloid‐beta (Aβ), the main toxic protein associated with Alzheimer’s pathology, and a higher risk of developing AD compared to non‐depressed MCI patients. However, it is unknown whether depression can be considered a risk factor for the development of AD. Although it has been described that some transgenic models of AD can develop signs similar to depression in advanced stages, the induction of Alzheimer’s pathology due to a depressive process has not been studied under experimental conditions to emulate late‐life depression as a risk factor for AD.MethodIn this study, we induced chronic unpredictable mild stress (CUMS) in P301S tau transgenic mice to determine whether depression is a cause, rather than a consequence, of the development of AD.ResultThe results of our study indicate that the induction of CUMS in transgenic animals of the disease give rise to changes in depressive state of the animals.ConclusionThe findings generated in this project could provide evidence of depression as a risk factor for AD, its mechanisms of action, use as early biomarkers, as well as the discovery of new therapies for AD.The objectives proposed in this research have specific funding from the Brain and Behavior Research Foundation (27565 2018 NARSAD), the B1‐2019_06 of the Own Plan of the University of Malaga, and the UMA20‐FEDERJA‐ 104FEDER Operational Program Andalusia 2014‐2020, as well as the Ramón y Cajal Program (RYC‐2017‐21879), all awarded to Dr. Inés Moreno.

Development of a neuroimaging biomarker of the pro‐coagulant state in Alzheimer’s disease: The BioClotAD Project

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of dementia1. Its multifactorial profile includes a strong vascular factor, partially driven by an early haemostatic dysregulation. Accordingly, fibrin, the main protein component of blood clots, is significantly increased in AD‐patients’ brains2. Furthermore, amyloid‐β contributes to this process by interacting with fibrin and producing degradation‐resistant clots3. Consequently, this pro‐thrombotic milieu intensifies hypoperfusion, neurodegeneration and blood‐brain barrier (BBB) disruption4, but it does not affect all patients. Therefore, early detection of these phenomena would be invaluable to identify patients likely to benefit from anticoagulant therapies5. Therefore, the BioClotAD Project aims to develop an imaging biomarker based on a fibrin binding probe (FBP)6 to non‐invasively identify AD’s pro‐coagulant state.MethodBioClotAD involves four partners in three European countries with complementary expertise. To optimize and test FBP we use extensive in vitro, ex vivo and in vivo assays, including AD animal models and human AD samples. Our project is comprised of three blocks: 1) Testing the FBP to in vivo detect the cerebral occlusions by nuclear imaging. 2) Detecting fibrin deposits inside the brain parenchyma with FBP coupled to a transferrin receptor antibody (FBP‐TfR) to facilitate BBB crossing7. FBP‐TfR will be labelled for optical and nuclear imaging. 3) Validating the most promising FBP probes in frozen brain samples of AD patients by autoradiography or fluorescence microscopy.ResultBioClotAD provides feasible neuroimaging strategies to in vivo detect the fibrin cerebral accumulation of AD models and identify the specific regional distribution in the brain. Our project will set the basis for future clinical trials on neuroimaging of the cerebral fibrin accumulation in AD.ConclusionBioClotAD neuroimaging biomarkers will allow the early detection of the pro‐thrombotic state in AD, opening a window of opportunity to delay the disease progression by personalized anticoagulant therapies. Funding: EU Joint Programme – Neurodegenerative Disease Research (JPND)

Characterization of protein aggregates in cream and skimmed human milk after heat and high-pressure pasteurization treatments.

Preservation processes applied to ensure microbial safety of human milk (HM) can modify the native structure of proteins and their bioactivities. Consequently, this study evaluated the effect of pasteurization methods (Holder pasteurization, high-temperature short-time (HTST), and high hydrostatic pressure (HHP)) of whole human milk (HM) on protein aggregates in skim milk and cream fractions. For heat-treated whole milk, insoluble protein aggregates at milk fat globule membrane (MFGM) were formed by disulfide and non-covalent bonds, but insoluble skim milk protein aggregates were only stabilized by non-covalent interactions. Contrary to heat treatment, the insolubilization of main proteins at the MFGM of HHP-treated HM was only through non-covalent interactions rather than disulfide bonds. Moreover, only heat treatment induced the insoluble aggregation of ⍺-lactalbumin. Overall, compared to heat treatment, HHP produced a milder effect on protein aggregation, validating the use of this process to better preserve the native state of HM bioactive proteins.

CLONING AND EXPRESSION OF GENE ENCODING P46 ANTIGEN OF MYCOPLASMA HYOPNEUMONIAE IN ESCHERICHIA COLI

Enzootic pneumonia is a highly contagious chronic respiratory disease in pigs caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) worldwide. P46 of M. hyopneumoniae is considered one of the main surface antigen proteins. This study aims to successfully clone and express P46 of M. hyopneumoniae in Escherichia coli M15 cells. The gene encoding P46 of M. hyopneumoniae was isolated from lung samples of infected pigs raised in Thua Thien Hue province, Vietnam, and cloned into vector pGEM®-T Easy. The PCR product digested by KpnI and BamHI enzymes was cloned into the pQE30 and expressed in E. coli M15 cells. The results of sequencing show that the nucleotide sequence of the cloned gene encoding P46 antigen protein has a length of 456 bp, corresponding to 152 amino acid residues and 99.34% similarity to the polypeptide chain of recorded M. hyopneumoniae P46 protein in GenBank (accession number: AAZ53879.1). The results of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) analysis show that the molecular mass of the expressed protein is approximately 20 kDa, and anti-histag ELISA confirms that the expressed protein is the 6xHis-P46 recombinant protein.

Physical Properties of the Second Type of Aciniform Spidroin (AcSp2) from Neoscona theisi Reveal a pH-Dependent Self-Assembly Repetitive Domain.

Orb-weaving spiders can use an array of specialized silks with diverse mechanical properties and functions for daily survival. Of all spider silk types, aciniform silk is the toughest silk fiber that combines high strength and elasticity. Although aciniform spidroins (AcSp) are the main protein in aciniform silks, their complete genes have rarely been characterized until now. Moreover, the structural and physical properties of AcSp variant proteins within the species are also unclear. Here, we present three full-length AcSp genes (named AcSp1A, AcSp1B, and AcSp2) from the orb-weaving spider Neoscona theisi and investigate the structural and mechanical features of these three AcSp repetitive domains. We demonstrate that all three AcSp proteins have mainly α-helical structural features in neutral solution and high thermal stability. Significantly, the AcSp2 repetitive domain shows a pH-dependent structural transition from α to β conformations and can self-assemble into amyloid fibrils under acidic conditions, which is the first reported AcSp repetitive domain with pH-dependent self-assembly capacity. Compared with the other two AcSp spidroins, AcSp2 demonstrated the lowest expression level in the aciniform gland but had the highest strength for its silk fiber. Collectively, our findings provide new insight into the physical properties of each component of aciniform silk and expand the repertoire of known spidroin sequences for the synthesis of artificial silk materials.

Spidroins under the Influence of Alcohol: Effect of Ethanol on Secondary Structure and Molecular Level Solvation of Silk-Like Proteins.

Future sustainable materials based on designer biomolecules require control of the solution assembly, but also interfacial interactions. Alcohol treatments of protein materials are an accessible means to this, making understanding of the process at the molecular level of seminal importance. We focus here on the influence of ethanol on spidroins, the main proteins of silk. By large-scale atomistically detailed molecular dynamics (MD) simulations and interconnected experiments, we characterize the protein aggregation, secondary structure changes, molecular level origins of them, and solvation environment changes for the proteins, as induced by ethanol as a solvation additive. The MD and circular dichoroism (CD) findings jointly show that ethanol promotes ordered structure in the protein molecules, leading to an increase of helix content and turns but also increased aggregation, as revealed by dynamic light scattering (DLS) and light microscopy. The structural changes correlate at the molecular level with increased intramolecular hydrogen bonding. The simulations reveal that polar amino acids, such as glutamine and serine, are most influenced by ethanol, whereas glycine residues are most prone to be involved in the ethanol-induced secondary structure changes. Furthermore, ethanol engages in interactions with the hydrophobic alanine-rich regions of the spidroin, significantly decreasing the hydrophobic interactions of the protein with itself and its surroundings. The protein solutes also change the microstructure of water/ethanol mixtures, essentially decreasing the level of larger local clustering. Overall, the work presents a systematic characterization of ethanol effects on a widely used, common protein type, spidroins, and generalizes the findings to other intrinsically disordered proteins by pinpointing the general features of the response. The results can aid in designing effective alcohol treatments for proteins, but also enable design and tuning of protein material properties by a relatively controllable solvation handle, the addition of ethanol.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200–350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251–305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262–279. Amino acid 262–279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262–273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

IN SILICO AND MOLECULAR DOCKING STUDIES: AIMING AMYLOID PRECURSOR-LIKE PROTEIN 2 USING ACTIVE PHYTOCHEMICALS FROM WITHANIA SOMNIFERA

Objectives: Due to effective healing properties found in natural chemical compounds obtained from medicinal plants that are employed in curing several diseases, this study aims to exhibit the role of Indian ayurvedic plant Withania somnifera in the management of the Alzheimer’s disease (AD) utilizing the molecular docking, drug-likeness and absorption, distribution, metabolism, and excretion (ADME) analysis. Methods: Alzheimer’s main protein was collected from the PDB database. Molecular docking is achieved using PyRx tool with the removal of the ligands possessing improper binding showing a significant effect on docking. Drug likeness and ADME analysis were evaluated using Swiss-ADME web server and ADMETlab 2.0 web tool. Ramachandran plot analysis for the target protein was achieved using SWISS-MODEL web server. Results: In the protein structure, the distribution of torsion angles ϕ and ψ in a protein is visible. On the basis binding affinity ADME analysis, 27-Deoxywithaferin A is a safe medication and one of the most effective inhibitors of the amyloid precursor protein. It also has drug-like qualities. Conclusion: According to the current research, 27-Deoxywithaferin A has a high affinity for binding, which makes it possible to suppress the major amyloid precursor protein while also managing therapeutic approaches for treating AD.

The nitrate transporter-sensor MtNPF6.8 regulates the branched chain amino acid/pantothenate metabolic pathway in barrel medic (Medicago truncatula Gaertn.) root tip

Nitrogen is the most limiting nutrient for plants, and it is preferentially absorbed in the form of nitrate by roots, which adapt to nitrate fluctuations by remodelling their architecture. Although core mechanisms of the response to nitrate availability are relatively well-known, signalling events controlling root growth and architecture have not all been identified, in particular in Legumes. However, the developmental effect of nitrate in Legumes is critical since external nitrate not only regulates root architecture but also N2-fixing nodule development. We have previously shown that in barrel medic (Medicago truncatula), the nitrate transporter MtNPF6.8 is required for nitrate sensitivity in root tip. However, uncertainty remains as to whether nitrogen metabolism itself is involved in the MtNPF6.8-mediated response. Here, we examine the metabolic effects of MtNPF6.8-dependent nitrate signalling using metabolomics and proteomics in WT and mtnpf6.8 root tips in presence or absence of nitrate. We found a reorchestration of metabolism due to the mutation, in favour of the branched chain amino acids/pantothenate metabolic pathway, and lipid catabolism via glyoxylate. That is, the mtnpf6.8 mutation was likely associated with a specific rerouting of acetyl-CoA production (glyoxylic cycle) and utilisation (pantothenate and branched chain amino acid synthesis). In agreement with our previous findings, class III peroxidases were confirmed as the main protein class responsive to nitrate, although in an MtNPF6.8-independent fashion. Our data rather suggest the involvement of other pathways within mtnpf6.8 root tips, such as Ca2+ signalling or cell wall methylation.

Effect of substituting fish meal with various by-product meals of swine-origin in diet on olive flounder (Paralichthys olivaceus)

The increased global market value of fish meal (FM) and the overfishing of wild fishery resources have boosted efforts to identify protein sources that can be cheaply and reliably supplied as an alternative to FM, which is commonly used as a main protein source in fish diets. Numerous studies over the years have demonstrated that animal by-product meals are the effective alternatives for FM in fish diets. This study aimed to elucidate the effect of substituting FM with various by-product meals of swine-origin in diet on growth, feed utilization, chemical composition, plasma biochemical parameters, and superoxide dismutase (SOD) and lysozyme activity of juvenile olive flounder (P. olivaceus). All experimental diets were formulated with five isonitrogenous (56.5%) and isolipidic (10.0%) diets. The control (Con) diet included 65% FM and 12% dehulled soybean meal as the protein sources and 4.2% fish oil as a lipid source. Thirty percent of FM was replaced with plasma powder, heme powder, globin powder, and pork greaves meal, named as the PP, HP, GP, and PM diets, respectively. Three hundred and seventy-five juvenile (9.2 ± 0.01 g; mean ± SD) fish were placed into 15 flow-through tanks with three replicates. Fish were carefully hand-fed to apparent satiation twice a day for 56 days. At the end of the feeding period, weight gain and specific growth rate of fish fed the GP and PM diets were comparable to fish fed the Con diet. Feed consumption of fish fed the PP, GP, and PM diets was comparable to fish fed the Con diet. Feed utilization and condition factor of fish fed the HP diet were the lowest. Plasma biochemical parameters, chemical composition, amino acid profiles, and SOD and lysozyme activity of fish were unaffected by dietary treatments. Thus, GP and PM are the appropriate replacers for FM in the olive flounder diets, wherein 30% FM is substituted by diverse by-product meals of swine-origin.