Types Of Protease Inhibitors Research Articles

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

Improvement of hepatic innate immunity in chemically-injured livers to develop hepatocarcinoma by a serine type-protease inhibitors enriched extract from Chenopodium quinoa.

Food ingredients have critical effects on the maturation and development of the immune system, which innate - lymphoid (ILCs) and myeloid - cells play key roles as important regulators of energy storage and hepatic fat accumulation. Therefore, the objective of this study is to define potential links between a dietary immunonutritional induction of the selective functional differentiation of monocytes-derived macrophages, ILCs and lipid homeostasis in hepatocarcinoma (HCC)-developing mice. Hepatic chemically injured (diethylnitrosamine/thiacetamide) Rag2-/- and Rag2-/-Il2-/- mice were administered with serine-type protease inhibitors (SETIs) obtained from Chenopodium quinoa. Early HCC-driven immunometabolic imbalances (infiltrated macrophages, glucose homeostasis, hepatic lipid profile, ILCs expansion, inflammatory conditions, microbiota) in animals put under a high-fat diet for 2 weeks were assessed. It was also approached the potential of SETIs to cause functional adaptations of the bioenergetics of human macrophage-like cells (hMLCs) in vitro conditioning their capacity to accumulate fat. It is showed that Rag2-/-Il2-/- mice, lacking ILCs, are resistant to the SETIs-induced hepatic macrophages (CD68+F4/80+) activation. Feeding SETIs to Rag2-/- mice, carrying ILCs, promoted the expansion towards ILC3s (CD117+Nkp46+CD56+) and reduced that of ILC2s (CD117+KLRG1+) into livers. In vitro studies demonstrate that hMLCs, challenged to SETIs, develop a similar phenotype of that found in mice and bioenergetic adaptations leading to increased lipolysis. It is concluded that SETIs promote liver macrophage activation and ILCs adaptations to ameliorate HCC-driven immunometabolic imbalances.

Non-Alcoholic fatty liver disease and metabolic syndrome in patients with HIV/AIDS and its correlation with antiretroviral therapy and severity of disease

Introduction: Metabolic syndrome (MetS) and Non-alcoholic fatty liver disease (NAFLD) are two major causes of morbidity in chronic HIV infected patients on antiretroviral therapy (ART). This study was done on HIV infected individuals by comparing ART naive patients with patients on different ART regimens and evaluating the effect of ART on Metabolic syndrome and NAFLD.Method: It was a cross-sectional observational study done on 120 HIV infected individuals in a tertiary care centre in New Delhi. All cases with hypertension, diabetes, chronic kidney or liver disease, thyroid disorders or on any drugs except ART were excluded. The risk markers for metabolic syndrome were assessed and compared within groups on different ART regimens.Results: Metabolic syndrome and NAFLD were found to be significantly more in cases on ART as compared to ART naïve cases. Metabolic syndrome was found to be associated with type of ART protease inhibitors (ATV/r)> nonucleoside reverse transcriptase inhibitors (NNRTI) > no ART] and low CD4 cell counts (p=0.01). In those patients who were on ART, these parameters were found to be more in those on second line ART [i.e., protease inhibitor (PI)(ATV/r) based regimens] as compared to those on first line ART, [i.e., nonnucleoside reverse transcriptase inhibitors (NNRTI) based regimen]. 15% of cases on 2nd line ART (group C) had MetS as compared to 12.5% in those on 1st line ART (group B) and nil in ART naïve cases (group A). One third (34%) of all 120 cases were found to have NAFLD. A significantly higher number of cases (45%) in group C had NAFLD as compared to 32.5% in group B and 25% in group A respectively. Insulin resistance and metabolic risk markers were also significantly higher in cases on ART as compared to ART naïve.Conclusions: In HIV patients, the use of antiretroviral therapy (ART) is linked to an increase in the prevalence of metabolic risk factors, including insulin resistance, lipoatrophy and dystrophy, dyslipidaemia, and abnormalities of fat distribution. Although care of Opportunistic infections and recently CVD has received a lot of attention, it is equally important to address the metabolic abnormalities such as metabolic syndrome and NAFLD brought on by ART.

Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1–P4′ was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1–P4’ positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.

Characterization of the role of Kunitz-type protease inhibitor domain in dimerization of amyloid precursor protein.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo- and hetero-dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo-dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero-dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP-APP interactions using coarse-grained models with the Martini force field. Though the model initial ternary complexes (KPI-E1, KPI-KPI, KPI-E2, E1-E1, E2-E2, and E1-E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane-bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo-dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.

Combining elicitor treatment of chitosan, methyl jasmonate, and cyclodextrin to induce the generation of immune response bioactive peptides in peanut hairy root culture

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1–4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and β-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1–4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries.

A brief review on oryzacystatin: a potent phytocystatin for crop management.

Phytocystatins are a type of proteinase inhibitor which are extensively studied for their specific inhibitory action against cysteine protease enzymes (CP) of insects and pathogens. Oryzacystatins (OC), a phytocystatin from rice inhibits CP in a reversible manner with its conserved tripartite wedge. OCs have important role in plant innate defense mechanism through phytohormonal signalling pathways. OC are induced in response to both biotic and abiotic stress conditions and are used to develop transgenic plants exhibiting resistance against stress conditions. In this review, we focus on the structure and mechanism of action of oryzacystatins, their possible role in plant physiology, biotic and abiotic stress tolerance mechanism in plants and their potential application strategies for future crop management studies.

The Development of an Ontology Platform to Detect Serine Protease Involvement in COVID-19-Induced Co-Morbid BALT Lymphoma

Mucosal-associated lymphoid tissue (MALT) lymphoma is a rare, low-grade non-Hodgkin lymphoma that develops from B cells and often is diagnosed incidentally. Although it possesses diverse subvariants, such as bronchial-associated lymphoid tissue (BALT), its pathogenesis remains unclear. Currently, little consensus exists on a standard treatment approach partially due to their rare occurrences and heterogeneities, but also due to good prognosis. Studies over the past decades have identified an association between coronaviral infection and BALT formation, whereby mutations in serine proteases increase the risk of BALT lymphoma. The aim of this study was to use a computational approach to validate and further elucidate this relationship specifically between SARS-CoV-2 and BALT lymphoma development. To address this, ontologies for serine proteolytic homeostasis were constructed utilizing keywords and abstracts from PubMed, which were then used with parsing and logical "OR" functions to generate search inputs for the European Bioinformatics Institute (EBI) COVID-19 ontology and ClinicalTrials.gov (as in Table 1), specifically for and between "BALT", "lymphoma", "serine protease", and "heat shock protein" as a representative type of serine protease inhibitor (SerPIn) class. The Heat Shock Protein 70 family and its more specific subtype Binding immunoglobulin protein (BiP) (a.k.a. GRP-78, HSPA5, and Byun1), were also manually identified as an alternative results marker for efficacy comparison. Results showed that this approach successfully demonstrated a literary relationship between serine proteases (SERPINE1) with heat shock proteins (HS90) and COVID-19, with aryl hydrocarbon (Row 4 of Table 1) potentially acting as this bridge between BALT lymphomas, transmembrane bound serine proteases, and SARS-CoV-2. This demonstrates a viable approach to automate the development of future ontologies to detail the pathogenesis between SARS-CoV-2, BALT lymphoma, and serine proteases. However, it was limited in its ability to yield positive hit results for HSP70 or GRP-78, likely due to the narrow focus of the EBI COVID-19 ontology and PubMed for covering full text papers. Nonetheless, this platform still offers a wealth of genetic, proteomic, and clinical trials information that can effectively be deployed toward characterizing disease pathogenesis, even in rare hematological malignancies. Forward looking, future studies will continue to investigate the application of serine proteolytic homeostasis ontologies to BALT lymphoma to better characterize its pathogenesis and interplay with other malignancies, which can then be utilized to develop targeted therapeutic solutions for critical BALT and MALT malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bioinformatics analysis of prognostic value and immune cell infiltration of SERPINA1 gene in cutaneous melanoma.

BackgroundCutaneous melanoma (CM) has a poor overall prognosis. Immune checkpoint inhibitor (ICI) therapy effectively improves overall survival in individuals with advanced melanoma, but only some patients benefit. Serpin Family A Member 1 (SERPINA1), a type of proteinase inhibitor that is used for many targets, is abnormally expressed and plays a vital role in multiple cancers. However, little is known about the clinical significance of SERPINA1 in CM.MethodsThe Cancer Genome Atlas (TCGA) and the gene expression omnibus (GEO) datasets were used to compare SERPINA1 expression levels. The association between SERPINA1 and other clinical factors were examined with R software, and receiver operating characteristic (ROC) curves for identification was developed. The Tumor IMmune Estimation Resource (TIMER) was used to examine the invasion of immune cells, markers for immune cells, and immunological checkpoints. The predictive value of SERPINA1 DNA methylation levels for every CpG was analyzed with the MethSurv web tool. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to assess the roles of genes that interacted with SERPINA1. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to predict SERPINA1’s response to ICIs.ResultsSERPINA1 was substantially expressed in CM. Overexpression of SERPINA1 was significantly associated with CM severity. The outcome for individuals with elevated SERPINA1 expression was good (HR =0.54, P<0.001). Using SERPINA1 expression levels, tumors and normal tissues could be reliably differentiated [area under the curve (AUC) =0.889]. Positive associations were found between SERPINA1 in CM and the infiltration of immune cells and immunological checkpoints [programmed cell death-1 (PD-1) and CTLA-4]. The efficacy of immune checkpoint blockade (ICB) in patients with a low expression of SERPINA1 was good. The GO pathway enrichment analysis showed that activation of neutrophil granulocytes participated in enrichment in the immune response pathway. Patients with low SERPINA1 expression had low TIDE scores.ConclusionsSERPINA1 is involved not only in the development and progression of CM but also in the immunological control of CM. Thus, SERPINA1 may serve as a possible biomarker for CM diagnosis, as well as its therapeutic target.

A TIL-Type Serine Protease Inhibitor Involved in Humoral Immune Response of Asian Corn Borer Ostrinia furnaculis.

To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response.

Analytical Techniques for the Analysis of Lopinavir and Ritonavir in Pharmaceutical Dosage Form and Biological Matrices: A Review

Background: Lopinavir and Ritonavir are protease inhibitor type of anti-retroviral drugs. Both are used for the treatment of HIV/AIDS. This paper reviews many analytical methods for the analysis of LPV and RTV in pharmaceutical formulations (tablet, capsule, syrup, and bulk) and biological fluids (human plasma, serum, cerebrospinal fluid, rat plasma, and human hair). Objective: The study aims to summarize various analytical techniques, such as chromatography and spectrophotometry, and also hyphenated techniques, such as LC-MS/MS and UPLC-MS, for the analysis of Lopinavir and Ritonavir. Method: The review deals with comprehensive details regarding the type of various analytical techniques, such as spectroscopy (UV), chromatography (RP-HPLC, HPTLC, UPLC), and hyphenated techniques, i.e., LC-MS/MS and UPLC-MS, for the analysis of lopinavir and ritonavir. These techniques are either explored for the quantification and detection of metabolite or for stability studies of the LPV and RTV. Conclusion: The studies presented revealed that the HPLC technique along with spectroscopy have been most widely used for the analysis. Out of the developed methods, hyphenated UPLCMS and LC-MS are very sensitive and help in the easy estimation of drugs compared to other techniques. This review may provide comprehensive details to the researchers working in the area of analytical research of LPV and RTV.

Physicochemical Properties and Elimination of the Activity of Anti-Nutritional Serine Protease Inhibitors from Mulberry Leaves.

Mulberry leaf is an excellent protein resource that can be used as feed additive for livestock and poultry. Nevertheless, the use of mulberry leaves in animal diets is limited by its protease inhibitors, tannic acid and other anti-nutritional factors. This study systematically analyzed the type and activity of serine protease inhibitors (SPIs) from the leaves of 34 mulberry varieties, aiming to reveal the physicochemical properties and inactivation mechanism of SPIs. The types and activities of trypsin inhibitors (TIs) and chymotrypsin inhibitors (CIs) exhibited polymorphisms among different mulberry varieties. The highest number of types of inhibitors was detected in Jinshi, with six TIs (TI-1~TI-6) and six CIs (CI-1~CI-6). TIs and CIs exhibited strong thermal and acid–base stability. High-temperature and high-pressure treatment could reduce the activities of TIs and CIs to a certain extent. β-mercaptoethanol treatment could completely abolish TIs and CIs, suggesting that the disulfide bridges were critical for their inhibitory activities. The Maillard reaction could effectively eliminate the inhibitory activities of TI-1~TI-4 and CI-1~CI-4. This study reveals the physicochemical properties and inactivation mechanisms of the anti-nutritional SPIs from mulberry leaves, which is helpful to exploit mulberry-leaf food with low-activity SPIs, promote the development and utilization of mulberry-leaf resources in animal feed and provide reference for mulberry breeding with different functions.

Cysteine and Aspartic Proteases Underlie the Digestion of Egg Yolk Proteins during the Development of Columba livia domestica Embryo

Yolk proteins undergo digestion either inside the egg yolk or in the surrounding yolk sac membrane (YSM) before being consumed by the developing avian embryo. However, the mechanisms underlying the digestion of yolk proteins during embryogenesis are largely unexplored in the pigeon Columba livia domestica. To better understand these mechanisms, the present study examined the classes of activated proteases in the egg yolk and the gene expression patterns of cathepsin B (CTSB) and cathepsin D (CTSD), which encode for lysosomal cysteine and aspartic proteases, respectively, in the YSM. We investigated the activated proteases by applying different types of protease inhibitors to yolk samples taken from incubation day 16. Then, we detected the mRNA levels of CTSB and CTSD in the YSM at incubation days 6, 8, 10, and 12-17. Both cysteine and aspartic proteases appeared to be activated in the egg yolk. Moreover, CTSB expression increased progressively and reached the maximum value on day 13; however, it decreased significantly on days 14 and 15 and further reduced toward hatching (day 17). In contrast, CTSD expression was weak and fluctuated insignificantly during development. Our results suggest that the degradation of yolk proteins at late developmental stages largely occurs in the egg yolk itself, probably by the activated cysteine and aspartic proteases. Furthermore, cathepsin B in the YSM seems to have a primary role in protein digestion, but this role decreases toward hatching.

The Angiostrongylus vasorum Excretory/Secretory and Surface Proteome Contains Putative Modulators of the Host Coagulation.

Angiostrongylus vasorum is a cardiopulmonary nematode of canids and is, among others, associated with bleeding disorders in dogs. The pathogenesis of such coagulopathies remains unclear. A deep proteomic characterization of sex specific A. vasorum excretory/secretory proteins (ESP) and of cuticular surface proteins was performed, and the effect of ESP on host coagulation and fibrinolysis was evaluated in vitro. Proteins were quantified by liquid chromatography coupled to mass spectrometry and functionally characterized through gene ontology and pathway enrichment analysis. In total, 1069 ESP (944 from female and 959 from male specimens) and 1195 surface proteins (705 and 1135, respectively) were identified. Among these were putative modulators of host coagulation, e.g., von Willebrand factor type D domain protein orthologues as well as several proteases, including serine type proteases, protease inhibitors and proteasome subunits. The effect of ESP on dog coagulation and fibrinolysis was evaluated on canine endothelial cells and by rotational thromboelastometry (ROTEM). After stimulation with ESP, tissue factor and serpin E1 transcript expression increased. ROTEM revealed minimal interaction of ESP with dog blood and ESP did not influence the onset of fibrinolysis, leading to the conclusion that Angiostrongylus vasorum ESP and surface proteins are not solely responsible for bleeding in dogs and that the interaction with the host’s vascular hemostasis is limited. It is likely that coagulopathies in A. vasorum infected dogs are the result of a multifactorial response of the host to this parasitic infection.

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5mM IPTG after 4h incubation at 37°C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35kDam.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of β-pleated sheets and β-turns in RbL protein structure. β-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

PINIR: a comprehensive information resource for Pin-II type protease inhibitors

BackgroundSerine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases.ResultsWe have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family.ConclusionPINIR (https://pinir.ncl.res.in) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.

Characterization of a Bowman\u2013Birk type trypsin inhibitor purified from seeds of Solanum surattense

A Bowman–Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman–Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1–10) and temperatures (10–100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10−8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Treatment Failure and Associated Factors among Individuals on Second Line Antiretroviral Therapy in Eastern Uganda: A Retrospective Cohort Study.

Background:ART failure is a growing public health problem and a major threat to the progress of HIV/AIDS control. In Uganda however, little is documented on treatment outcomes and their associated factors among individuals on second line ART regimen. The rapid scale-up of ART over the past has resulted in substantial reductions in morbidity and mortality. However, as millions of people must be maintained on ART for life, individuals with ART treatment failure are increasingly encountered and the numbers are expected to rise. This could be attributed to factors such as sub-standard regimens, limited access to routine viral load monitoring, treatment interruptions, suboptimal adherence, among others. The purpose of this study was to estimate 5-year cumulative treatment failure and the associated factors among individuals on second line ART regimen Eastern Uganda.Materials and methods:A retrospective analysis of 541 records of HIV positive individuals, switched to second line ART regimen from January 2012 to December 2017. Inferential statistics including the Chi square test and multivariable logistic regression analysis was applied to determine associations of treatment failure against of the selected demographic, laboratory and clinical factors was performed. Associations between treatment failure and the predictors was based on a P-value of less than 5% and confidence intervals level of 95%.Results:We reviewed 541 records of individuals on second line ART regimen, of which 350 (64.7%) were female, 226 (41.8%) were married, and 197 (36.4%) were older than 35 years. The mean age at ART initiation was 30 years (SD = 14.8), while the mean weight at ART initiation was 47 kg (SD = 18.6), (range 4-97 kg). The overall proportion of treatment failure was 23%. The cumulative mortality risk for 5 years was 12.4% and the mortality rate was 2.5 deaths per 100 individuals per year. The odds of developing treatment failure among individuals switched to ATV/r-based regimen were 44% lower as compared to individuals who were switched to LPV/r (ORadj0.56, 95% CI 0.35-0.90, P = .016). while the odds of experiencing treatment failure among individuals that used AZT at ART initiation were 43% lower as compared to individuals that used a TDF based regimen at ART initiation (ORadj0.57, 95% CI 0.33-0.98, P = .041).Conclusion:The 5 year cumulative incidence of treatment failure in a cohort of 541 individuals was 23%. The type of protease inhibitor (PI) used in second line regimen and use of AZT at ART initiation were significantly associated with treatment failure. Our study also shows that the cumulative mortality risk while on second line ART regimen was 12.4% while the mortality rate was 2.5 deaths per 100 individuals per year. Given the high level of treatment failure among individuals on second line ART regimen, yet the current ART protocols limits the use of third line ART regimens to only regional referral hospitals, the Ministry of Health should strengthen the surveillance systems for identifying individuals failing on second line ART regimen even at district hospitals and lower health facilities to facilitate timely switch to optimal regimen. The Ministry of health through the Quality Improvement Division should conduct routine onsite support supervision to sites offering ART to ensure that treatment guides and other standard of care like timely switch to appropriate regimens among others are being adhered to. Knowledge gaps identified can also be addressed through onsite Continuous Medical Educations.

Comparative expression profiling reveals the role of the hot pepper apoplast under drought conditions

Drought stress is the most important limiting factor of crop cultivation around the world and stress conditions will modulate the expression of many genes that play a significant role in the plant growth and development and to stress adaptation. The apoplast is a dynamic environment subject and considered as a ‘key information bridge’ between the environment and cells. In the present study we aimed to measure the changes in the expression levels of genes by RT-qPCR studies.Analysis shown the down-regulation of Peptidylprolyl isomerase and Pin II type protease inhibitor and there observed the huge increase in the expression of Pectinesterase and Alpha L fucosidase under treated conditions. These changes in the expression level of proteins under stress conditions implicate their possible role in stress tolerance and would prove to be useful for protein phenotyping.