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AIM OF THE STUDY: The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. MATERIALS AND METHODS: Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 10(6) CFU/ml of Salmonella Typhimurium using the same route of administration. RESULTS: All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 10(4) cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-alpha in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1beta. CONCLUSIONS: LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-alpha). LP inhibited IL-1beta release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application. [ABSTRACT FROM AUTHOR]

Pre-clinical assays demonstrated that a 1% polyvinyl alcohol biomembrane containing latex proteins (10%) from the medicinal plant Calotropis procera was biocompatible and stimulated healing of incisional and excisional wounds in murine models, and the mechanistic aspects were established. The efficacy of the biomembrane (BioMemCpLP) to promote healing of chronic ulcers in leprosy patients was investigated. The study started with 28 volunteers. Five were excluded later due to different disconformities. Ulcers from 15 patients were continuously treated with BioMemCpLP for 56 days. Five patients were treated only with silver sulfadiazine and three patients received plain hydrocolloid wound dressings with high absorption capacity. In all cases, wound dressings were renewed three times a week for 56 days and ulcers were evaluated weekly for contraction and healing progress. The extent of the healed area in the ulcers treated with BioMemCpLP was greater than in the control groups. Approximately 88% of ulcers treated with BioMemCpLP were fully healed before day 56, against 6% in both control groups. This result was not correlated with age/gender, duration or location of ulcers, deformity or whether or not the patient was cured of leprosy. The results showed that BioMemCpLP was beneficial for treatment of ulcers suffered by leprosy patients without noticeable side effects.

Laticifers have been utilized as paradigms to enhance comprehension of specific facets of plant ecology and evolution. From the beginning of seedling growth, autonomous laticifer networks are formed throughout the plant structure, extending across all tissues and organs. The vast majority of identified products resulting from laticifer chemistry and metabolism are linked to plant defense. The latex, which is the fluid contained within laticifers, is maintained under pressure and has evolved to serve as a defense mechanism against both aggressors and invaders, irrespective of their capabilities or tactics. Remarkably, the latex composition varies among different species. The current goal is to understand the specific functions of various latex components in combating plant enemies. Therefore, the study of latex's chemical composition and proteome plays a critical role in advancing our understanding about plant defense mechanisms. Here, we will discuss some of these aspects., Competing Interests: Declaration of Competing Interest No conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: We compared the effects of low-volume combined aerobic and resistance high-intensity interval training (C-HIIT), combined moderate-intensity continuous training (C-MICT) and waitlist control (CON) on vascular health after 8-weeks of supervised training, and an additional 10-months of self-directed training, in adults with type 2 diabetes (T2D)., Methods: Sixty-nine low active adults with T2D were randomised to 8-weeks of supervised C-HIIT (3 times/week, 78-min/week), C-MICT (current exercise guidelines, 4 times/week, 210-min/week) or CON. CON underwent usual care for 8-weeks before being re-randomised to C-HIIT or C-MICT. This was followed by 10-months of self-directed training for participants in C-HIIT and C-MICT. Vascular outcomes were evaluated at baseline, 8-weeks, and 12-months., Results: After 8-weeks, supervised C-HIIT significantly improved relative flow-mediated dilation (FMD) compared with CON (mean difference [MD] 0.8% [0.1, 1.4], p = 0.025). Although not significantly different from CON, the magnitude of change in relative FMD following 8-weeks of supervised C-MICT was similar (MD 0.8% [-0.1, 1.7], p = 0.080). There were no differences in haemodynamic indices, carotid-femoral pulse wave velocity (cfPWV), or aortic reservoir pressure between groups at 8-weeks. After 12-months, there was a significant reduction in haemodynamic indices (time effect, p < 0.05) for both C-HIIT and C-MICT, with no between-group difference. The reduction in cfPWV over 12-months was significantly greater in C-MICT than C-HIIT (group × time effect, p = 0.018). There was no difference in FMD over time or between groups at 12-months., Conclusions: Short-term supervised C-HIIT and C-MICT both increased brachial artery FMD compared with CON. Long-term C-HIIT and C-MICT were beneficial for improving haemodynamic indices, but not brachial artery FMD. C-MICT was superior to C-HIIT for improving cfPWV at 12-months., Trial Registration: Australian New Zealand Clinical Trials Registry Identifier ACTRN12615000475549., (© 2024. The Author(s).)

Peroxidases (EC 1.11.1.7) are involved in a wide range of physiological processes, hence their broad distribution across biological systems. These proteins can be classified as haem or non-haem enzymes. According to the RedOxiBase database, haem peroxidases are approximately 84 % of all known peroxidase enzymes. Class III plant peroxidases are haem-enzymes that share similar three-dimensional structures and a common catalytic mechanism for hydrogen peroxide degradation. They exist as large multigene families and are involved in metabolizing Reactive Oxygen Species (ROS), hormone synthesis and decomposition, fruit growth, defense, and cell wall synthesis and maintenance. As a result, plant peroxidases gained attention in research and became one of the most extensively studied groups of enzymes. This review provides an update on the database, classification, phylogeny, mechanism of action, structure, and physiological functions of class III plant peroxidases., Competing Interests: Declaration of competing interest The authors confirm that the contents of this article pose no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Background: Patients aged over 50 years require four times more surgical interventions than younger groups. Many guidelines recommend the performance of preoperative electrocardiogram (ECG) in this population., Objectives: To determine the value of preoperative ECG in patients aged over 50 years and classified as ASA I-II (surgical risk)., Methods: Patients older than 50 years, without comorbidities, who underwent surgical intervention and general anesthesia were included in the study. Patients were randomized to undergo ECG (group A, n=214) or not (group B, n=213) in the preoperative period. The following variables were analyzed: sex, age, ECG, chest x-ray and laboratory tests results, surgical risk, surgery duration, adverse events and in-hospital mortality. The level of significance was set at 5%., Results: Adverse outcomes were reported in 23 (5.4%) patients, with a significant number of adverse events in male patients (OR=7.91 95%CI 3.3-18.90, p<0.001) and in those undergoing major surgeries (OR=30.02 95%CI 4.01-224.92, p<0.001). No differences were observed between patients who underwent ECG and those who did not (OR=1.59, 95%CI, 0.67-3.75, p=0.289). No significant differences were found in the other variables. In multivariate logistic regression, male sex (OR = 6.49; 95%CI 2.42-17.42, p<0.001) and major surgery (OR=22.62; 95%CI 2.95-173.41, p=0.002) were independent predictors of adverse outcomes, whereas undergoing (or not) ECG (OR=1.09; IC95% 0.41-2.90, p=0.867) remained without statistical significance., Conclusion: Our findings suggest that preoperative ECG could not predict an increased risk of adverse outcomes in our study population during the hospital phase.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1β). STEC stimulated neutrophils to release elevated levels of IL-1β through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1β secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1β secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1β secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation., Competing Interests: JC is inventor of a patent related to Ecotin. The patent “Immunomodulating and immunostimulating polypeptides for drug-delivery” was filled on February 8, 2019 by the authors’ National Research Council (CONICET) and the University of San Martin (UNSAM) in the United States patent and trademark Office (US2021009371), the European patent office (PCT/IB2019/051026, EP3749364), the China National intellectual property administration (CN111936166), and the National Institute of Intellectual Property from Argentina (AR114683). The filing of the patent did not have any role in experimental design, data collection and analysis, decision to publish, or preparation of this manuscript. The authors have no financial conflicts of interest., (Copyright: © 2023 Sabbione et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although latex fluids are found in >20,000 plant species, the biochemical composition and biological function of their proteins are still poorly explored. Thus, this work aimed to conduct a proteomic analysis of Cryptostegia grandiflora latex (CgLP) for subsequent purification and characterization of an antifungal protein. After 2D-SDS-PAGE and mass spectrometry, 27 proteins were identified in CgLP, including a polygalacturonase inhibitor, cysteine peptidases, pathogenesis-related proteins (PR-4), and osmotins. Then, two osmotin isoforms (CgOsm) were purified, and a unique N-terminal sequence was determined ( 1 ATFDIRSNCPYTVWAAAVPGGGRRLDRGQTWTINVAPGTA 40 ). The PCR products revealed a cDNA sequence of 609 nucleotides for CgOsm, which encoded a polypeptide with 203 amino acid residues. The structure of CgOsm has features of typical osmotin or thaumatin-like proteins (TLPs), such as 16 conserved Cys residues, REDDD and FF motifs, an acidic cleft, and three main domains. Atomic force microscopy (AFM) and bioinformatics suggested that CgOsm is associated with three chain units. This result was interesting since the literature describes osmotins and TLPs as monomers. AFM also showed that Fusarium falciforme spores treated with CgOsm were drastically damaged. Therefore, it is speculated that CgOsm forms pores in the membrane of these cells, causing the leakage of cytoplasmic content., Competing Interests: Declaration of competing interest The authors confirm that the contents of this article pose no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This work investigated the healing properties of proteins extracted of latex (HdLP) on excisional wounds. Cell toxicity of HdLP was investigated carried out in murine fibroblasts after incubation with HdLP (12.5-100 μg/ml). The dermal irritability test was performed to evaluate dermal reactions. The wounds were performed and treated with vehicle or HdLP (0.5 %, 1.0 %, and 2.0 %). The macroscopic parameters, histological analysis and measurement of inflammatory markers and mediators were evaluated. HdLP did not exhibit cytotoxicity and did not induce skin irritation . HdLP stimulated the release of IL-1β at the beginning of the inflammatory phase. This effect probably favored the earlier release of IL-10 by macrophages, during the proliferative phase. The shortening and completeness of healing were characterized by fibroblast proliferation and the presence of newly synthesized collagen fibers. This was accompanied by well-organized re-epithelialization. The involvement of latex proteins in this activity is reported for the first time., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (© 2023 The Authors.)

Introduction: Hemolytic uremic syndrome (HUS) is a condition that results in acute kidney failure mainly in children, which is caused by Shiga toxin-producing Escherichia coli and inflammatory response. Although anti-inflammatory mechanisms are triggered, studies on the implication in HUS are scarce. Interleukin-10 (IL-10) regulates inflammation in vivo , and the interindividual differences in its expression are related to genetic variants. Notably, the single nucleotide polymorphism (SNP) rs1800896 -1082 (A/G), located in the IL-10 promoter, regulates cytokine expression., Methods: Plasma and peripheral blood mononuclear cells (PBMC) were collected from healthy children and HUS patients exhibiting hemolytic anemia, thrombocytopenia, and kidney damage. Monocytes identified as CD14 + cells were analyzed within PBMC by flow cytometry. IL-10 levels were quantified by ELISA, and SNP -1082 (A/G) was analyzed by allele-specific PCR., Results: Circulating IL-10 levels were increased in HUS patients, but PBMC from these patients exhibited a lower capacity to secrete this cytokine compared with those from healthy children. Interestingly, there was a negative association between the circulating levels of IL-10 and inflammatory cytokine IL-8. We observed that circulating IL-10 levels were threefold higher in HUS patients with -1082G allele in comparison to AA genotype. Moreover, there was relative enrichment of GG/AG genotypes in HUS patients with severe kidney failure., Discussion: Our results suggest a possible contribution of SNP -1082 (A/G) to the severity of kidney failure in HUS patients that should be further evaluated in a larger cohort., 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., (© 2023 Mongelos, Sosa, Pineda, Fiorentino, Santiago, Abelleyro, Rossetti, Exeni, De Brasi, Palermo and Ramos.)

Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections., Materials and Methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo., Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo , and surviving mice presented high titres of anti-H7 and Stx antibodies., Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors., 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 © 2023 Bernal, Sosa, Todero, Montagna, Vermeulen, Fernández-Brando, Ramos, Errea, Rumbo and Palermo.)

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin., 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 © 2022 Elsevier Ltd. All rights reserved.)

Background: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolysis, thrombocytopenia, and thrombus formation leading to tissue injury. HUS is classified according to its etiology as post-diarrheal or atypical HUS. Differential diagnosis of both entities continues to be a challenge for pediatric physicians., Methods: The aim was to improve the rapid etiological diagnosis of post-diarrheal HUS cases based on the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection by screening of stx 1/ stx 2 and rfb O157 in cultured stools by multiplex PCR, and the additional detection of anti-lipopolysaccharide (anti-LPS) O157, O145, and O121 antibodies by Glyco-iELISA test. In addition, we studied patients' relatives to detect circulating pathogenic strains that could contribute to HUS diagnosis and/or lead to the implementation of measures to prevent dissemination of familial outbreaks. This study describes the diagnosis of 31 HUS patients admitted to Hospital Municipal de Niños Prof Dr Ramón Exeni during the 2017-2020 period., Results: Stool PCR confirmed the diagnosis of STEC associated with HUS in 38.7% of patients (12/31), while anti-LPS serology did in 88.9% (24/27). In those patients in which both methods were carried out (n = 27), a strong association between the results obtained was found. We found that 30.4% of HUS patients had at least one relative positive for STEC., Conclusions: We could identify 96.3% (26/27) of HUS cases as secondary to STEC infections when both methods (genotyping and serology) were used. The results demonstrated a high circulation of STEC in HUS families and the prevalence of the STEC O157 serotype (83%) in our pediatric cohort. A higher-resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Objective: Calotropis procera latex protein (CpLP) is a popular anti-inflammatory and therefore we aimed to study its effects on inflammatory bone loss., Design: Male Wistar rats were subjected to a ligature of molars. Groups of rats received intraperitoneally CpLP (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) or saline (0.9% NaCl) one hour before ligature and then daily up to 11 days, compared to naïve. Gingiva was evaluated by myeloperoxidase activity and interleukin-1 beta (IL-1β) expression by ELISA. Bone resorption was evaluated in the region between the cement-enamel junction and the alveolar bone crest. The histology considered alveolar bone resorption and cementum integrity, leukocyte infiltration, and attachment level, followed by immunohistochemistry bone markers between 1 st and 2 nd molars. Systemically, the weight of the body and organs, and a leukogram were performed., Results: The periodontitis significantly increased myeloperoxidase activity and the IL-1β level. The increased bone resorption was histologically corroborated by periodontal destruction, leukocyte influx, and attachment loss, as well as the increasing receptor activator of the nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio, and Tartrate-resistant acid phosphatase (TRAP)+ cells when compared to naïve. CpLP significantly reduced myeloperoxidase activity, level of IL-1β, alveolar bone resorption, periodontal destruction, leukocyte influx, and attachment loss. The CpLp also reduced the RANKL/OPG ratio and TRAP+ cells, when compared with the saline group, and did not affect the systemic parameters., Conclusions: CpLP exhibited a periodontal protective effect by reducing inflammation and restricting osteoclastic alveolar bone resorption in this rat model., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The extraction and commercialization of palm hearts is the most profitable activity involving the peach palm (Bactris gasipaes ), while consumption of its fruits is limited to Amazonian communities. The excessive attention paid to the implementation of germplasm banks contributed to the lack of development of high-performance varieties, limiting the production and consumption of peach palm fruits and by-products. In addition, with the fragmentation of the Amazonian rainforest, wild populations are in danger of extinction. The species domestication, initiated by Native Amazonians, generated a large variety of peach palm populations, as evidenced by the diversity in fruit sizes and quality. Some advances in agronomic traits also took place. However, more research needs to be conducted to understand the implications of climatic changes on plant physiological performance. Indeed, the key point is that the exploitation of the full potential of B. gasipaes has not been completely exploited. Therefore, understanding the state-of-the-art research on the peach palm with a focus on its underutilized resources is essential for expanding plantations and, consequently, promoting the market expansion of the peach palm as a fruit crop.

Background: The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions., Purpose: This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model., Methods: Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes., Results: Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10., Conclusion: This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model., (Copyright © 2022. Published by Elsevier GmbH.)

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites., (© 2022 John Wiley & Sons Ltd.)

Background: Previous studies have shown that latex proteins from Plumeria pudica (LPPp) have anti-inflammatory and antioxidant activity. Therefore, the aim of this study was to evaluate the effects in rats of LPPp on ligature-induced periodontitis, an inflammatory disease., Methods: The animals were divided into groups: saline (animals without induction of periodontitis), periodontitis (induced periodontitis and untreated) and LPPp (induced periodontitis and treated with 40 mg/kg). The following parameters were evaluated after 20 consecutive days of treatment: gingival bleeding index (GBI), probing pocket depth (PPD), alveolar bone height (ABH) and gingival myeloperoxidase (MPO) activity. In the hepatic tissue, malondialdehyde (MDA), glutathione (GSH) and histopathological alterations were evaluated. Blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured., Results: Significant reduction in GBI, PPD and gingival MPO activity and ABH was seen in animals treated with LPPp compared with periodontitis. Values of GSH, MDA, ALT and histopathological evaluation were preserved in animals treated with LPPp., Conclusions: Treatment with LPPp improved clinical aspects of periodontitis, reduced the blood and hepatic alterations and prevented alveolar bone loss. Data suggest that LPPp have potential for treatment of periodontitis., (© 2021 Wiley Periodicals LLC.)

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme:  substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

SARS-CoV-2-specific humoral response was analyzed over time in a group of healthcare workers with or without exposure to SARS-CoV-2, who underwent vaccination with BBIBP-CorV (Sinopharm) vaccine in Argentina. Seroconversion rates in unexposed subjects after the first and second doses were 40 % and 100 %, respectively, showing a significant increase in antibody concentrations from dose 1 to dose 2 (p < 0.0001). The highest antibody concentrations were found in younger subjects and women, remaining significantly associated in a multivariable linear regression model (p = 0.005). A single dose of the BBIBP-CorV vaccine induced a strong antibody response in individuals with prior SARS-CoV-2infection, while a second dose did not increase this response. A sharp increase in antibody concentrations was observed following SARS-CoV-2 infection in those participants who became infected after the first and second doses (p = 0.008). Individuals with SARS-CoV-2 exposure prior to vaccination showed significantly higher anti-spike IgG antibody levels, at all-time points, than those not exposed (p < 0.001). Higher antibody titers were induced by a single dose in previously SARS-CoV-2 infected individuals than those induced in naïve subjects by two doses of the vaccine (p < 0.0001). Three months after the second dose both groups showed a decline in antibody levels, being more abrupt in unexposed subjects. Overall, our results showed a trend towards lower antibody concentrations over time following BBIBP-CorV vaccination. Sex and age seem to influence the magnitude of the humoral response in unexposed subjects while the combination of exposure to SARS-CoV-2 plus vaccination, whatever the sequence of the events was, produced a sharp increase in antibody levels. Evaluation of the humoral responses over time and the analysis of the induction and persistence of memory B and T cell responses, are needed to assess long-term immune protection induced by BBIBP-CorV vaccine., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis., Objective: The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer., Methods: LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions., Results: Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1β, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA., Conclusion: We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera., Objective: We aimed to evaluate the interaction between cardenolides and Na + /K + ATPases from herbivores., Methods: The Na + /K + ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera., Results: The replacement of serine in sensitive Na + /K + ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides., Conclusion: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance., Hypothesis/purpose: In this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis., Study Design: Swiss mice were infected with L. monocytogenes and then treated with CFL., Methods: Adult Swiss mice weighing with 30-40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 10 7 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection., Results: We observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection., Conclusion: The lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The chronification of ulcers or sores may result in a dramatic outcome such as amputation. Currently, the search for plant based treatments of various diseases/disorders, including complicated ones, is getting the attention of researchers worldwide. The soluble latex protein fraction (CpLP) obtained from Calotropis procera (Apocynaceae) was previously demonstrated to accelerate wound healing by topical application or when incorporated in a polyvinyl alcohol biomembrane (BioMemCpLP). Here, in vitro assays were performed to investigate and characterize the biocompatibility and bioactivity of latex proteins dressing. Macrophages (RAW 264.7), fibroblasts (L929) and keratinocytes (HaCaT) cell lines were used to evaluate the effect of CpLP. These cell lines were exposed to concentrations of CpLP comparable to those found in BioMemCpLP during 24-72 h. The cytotoxicity, proliferation, release of wound healing mediators (TGF-β, VEGF, IL-10, IL-6, IL-1β, TNF-α and NO) and migration of cells (E-cadherin and β-catenin) incubated with CpLP was assessed and the cell adhesion to BioMemCpLP as well. The results showed that CpLP has no cytotoxic effects. It induced a suitable balance between pro- and anti-inflammatory mediators, enhanced proliferation and re-epithelialization in all cell lines, but the intensity of each effect was different at various doses in all cell strains. The BioMemCpLP stimulated cell adhesion to PVA substrate. The CpLP-PVA based biomembrane can be a good option for healing of different wounds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The medicinal uses of Calotropis procera are diverse, yet some of them are based on effects that still lack scientific support. Control of diabetes is one of them. Recently, latex proteins from C. procera latex (LP) have been shown to promote in vivo glycemic control by the inhibition of hepatic glucose production via AMP-activated protein kinase (AMPK). Glycemic control has been attributed to an isolated fraction of LP (CpPII), which is composed of cysteine peptidases (95%) and osmotin (5%) isoforms. Those proteins are extensively characterized in terms of chemistry, biochemistry and structural aspects. Furthermore, we evaluated some aspects of the mitochondrial function and cellular mechanisms involved in CpPII activity. The effect of CpPII on glycemic control was evaluated in fasting mice by glycemic curve and glucose and pyruvate tolerance tests. HepG2 cells was treated with CpPII, and cell viability, oxygen consumption, PPAR activity, production of lactate and reactive oxygen species, mitochondrial density and protein and gene expression were analyzed. CpPII reduced fasting glycemia, improved glucose tolerance and inhibited hepatic glucose production in control animals. Additionally, CpPII increased the consumption of ATP-linked oxygen and mitochondrial uncoupling, reduced lactate concentration, increased protein expression of mitochondrial complexes I, III and V, and activity of peroxisome-proliferator-responsive elements (PPRE), reduced the presence of reactive oxygen species (ROS) and increased mitochondrial density in HepG2 cells by activation of AMPK/PPAR. Our findings strongly support the medicinal use of the plant and suggest that CpPII is a potential therapy for prevention and/or treatment of type-2 diabetes. A common epitope sequence shared among the proteases and osmotin is possibly the responsible for the beneficial effects of CpPII., Competing Interests: 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., (© 2021 The Author(s).)

Several national regulations and good international practices promote the existence of a central register with the results of individual radiological monitoring. In the majority of countries (in the Latin American region), dosimetry data were neither harmonised nor managed by a unique database, and therefore the possibility of using such data for proper characterisation of the radiological conditions, integrally at national level, in a territory or sector, and by practice or occupational category, was very limited. All these considerations justified the need for developing a prototype of a National Dose Register (NDR) for Latin America. The main objectives were the strengthening of safety supervision in nuclear applications and the provision of a surveillance system for occupational exposure as well as the centralisation of personal dosimetry data, compiling all doses evaluated by the different dosimetric service providers. The experience of Cuba and Brazil in the implementation of their national registers was well known, and was accordingly used as the basis for designing this software. The first version of the NDR prototype, developed in the Spanish language, has been designed, developed and validated. So far, 16 countries have started to implement the NDR. The present work describes aspects such as the technical bases of the NDR design, its characteristics and its functionalities. Experience gained during implementation in Latin America is also addressed. Having a common system of dosimetric information management in the region has opened an important space for scientific exchanges between the countries and their competent authorities. The NDR implementation has provided regulatory authorities with a tool that permits the verification of the level of compliance with dose limits and restrictions, as well as allowing them to carry out statistical assessments of the results of individual radiological surveillance that may permit evaluation of the appropriateness and effectiveness of workplace radiation protection programmes, contributing to their optimisation., (© 2021 Society for Radiological Protection. Published on behalf of SRP by IOP Publishing Limited. All rights reserved.)

Vigna unguiculata is an important source of proteins and energy for humans and animals. However, postharvest losses caused by Callosobruchus maculatus can reach from 20 to 100% of stored seeds. In this study, the insecticide potential of compounds extracted from Himatanthus drasticus latex was assessed. The latex was extracted with ethanol (70%) and then partitioned through sequential use of hexane and chloroform. These fractions were investigated by chromatography to determine their chemical composition. Plumieride, identified in a hydroalcoholic subfraction, was tested for insecticidal activity against C. maculatus . The ethanolic fraction (LC 50 = 0.109; LC 90 = 0.106%) and the plumieride (LC 50 = 0.166; LC 90 = 0.167%) were lethal to larvae. Plumieride (0.25%) delayed larval development, and mortality reached 100%. Its inhibitory action on intestinal α-amylase from larvae was higher (89.12%) than that of acarbose (63.82%). Plumieride (0.1%) inhibited the enzyme α-amylase in vivo in the larval intestine. This result was confirmed by a zymogram test performed by SDS-PAGE when the enzyme electrophoresed on gel copolymerized with starch. When spread on seeds, the hydroalcoholic fraction (1.0%) reduced infestation. The loss of seed mass was 5.26% compared to the control (44.97%). The results confirm the effect of latex compounds in protecting stored seeds against weevil infestation.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism., (Copyright © 2021 American Society for Microbiology.)

Background: Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera., Purpose: A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection., Methods: LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium., Results: LPp2 was not toxic at dosages lower than 62.2 μg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2., Conclusion: We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella., 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 © 2020. Published by Elsevier B.V.)

Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10-/-) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10-/- mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10-/- than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-β levels in IL-10-/- mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the enzymes is reported. The latex was mixed with distilled water and the mixture was cleaned through centrifugation. It was dialyzed with distilled water and centrifuged again to recover the soluble fraction [EP]. The dialyze is a key feature of the process. EP was characterized in terms of protein profile, chemical stability, among other criteria. Wild plants belonging to ten geographic regions and grown in different ecological conditions were used as latex source. Collections were carried out, spaced at three-month, according to the seasons at the site of the study. Proteolytic activity was measured as an internal marker and for determining stability of the samples. EP was also analyzed for metal content and microbiology. EP showed similar magnitude of proteolysis, chromatographic and electrophoretic profiles of proteins. Samples stored at 25 °C exhibited reduced solubility (11%) and proteolytic capacity (11%) after six months. Enzyme autolysis was negligible. Microbiological and metal analyses revealed standard quality of all the samples tested. EP induced milk clotting and hide dehairing after storage for up to six months.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels., Objectives: To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII)., Methods: Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels., Findings: LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood., Main Conclusions: LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical β-barrel fold and two glycosylation sites (Asn 55 and Asn 144 ). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory., Competing Interests: Declaration of Competing Interest The authors confirm that the contents of this article pose no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective and Design: Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM., Materials and Subjects: Male Golden Sirius hamsters were used in all treatments., Treatment: The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later., Methods: Histology, cytokine measurements [ELISA], and macroscopic evaluation [scores] were performed., Results: PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed., Conclusions: Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.

The emergence of antibiotic-resistant microbes has stimulated research worldwide seeking new biologically active molecules. In this respect, synthetic antimicrobial peptides (SAMPs) have been suggested to overcome this problem. Although there are some online servers that provide putative SAMPs from protein sequences, the choice of the best peptide sequences for further analysis is still difficult. Therefore, the goal of this paper is not to launch a new tool but to provide a friendly workflow to characterize and predict potential SAMPs by employing existing tools. Using this proposed workflow, two peptides (PepGAT and PepKAA) were obtained and extensively characterized. These peptides damaged microbial membranes and cell walls, and induced overproduction of reactive oxygen species (ROS). Both peptides were found to assume random coil secondary structure in aqueous solution, organic solvent, and upon binding to negatively charged lipid systems. Peptides were also able to degrade formed biofilms but not to prevent biofilm formation. PepGAT was not resistant to proteolysis, whereas PepKAA was resistant to pepsin but not to pancreatin. Furthermore, both presented no hemolytic activity against red blood cells, even at a 10-fold higher concentration than the antimicrobial concentration. The pipeline proposed here is an easy way to design new SAMPs for application as alternatives to develop new drugs against human pathogenic microorganisms., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Ethnopharmacological Relevance: Himatanthus drasticus is an important medicinal plant whose latex is traditionally used in Northeast Brazil to treat various diseases, including diabetes. The use of α-amylase and α-glucosidase inhibitors can be an effective strategy to modulate levels of postprandial hyperglycemia via control of starch metabolism., Aims of the Study: This study aimed to verify if H. drasticus latex has inhibitory activity against enzymes linked to type 2 diabetes, besides chemically characterizing the metabolites responsible for such activities. In addition, in silico analysis was performed to support the traditional claim of possible antidiabetic activity of this latex., Materials and Methods: Latex from H. drasticus stems was sequentially partitioned with n-hexane (FHDH), CHCl 3 (FHDC) and EtOH (FHDHA). Wash extraction of the FHDHA fraction was performed to obtain the other extract fractions. The FHDHA was submitted to chromatography in a SPE C 18 cartridge using gradient elution with MeOH/H 2 O to produce five fractions: FHDHA1, FHDHA2, FHDHA3, FHDHA4 and FHDHA5. The FHDHA1 was subjected to semi-preparative reverse phase HPLC. Lineweaver-Burk plots were used to investigate the kinetic parameters of α-amylase and α-glucosidase inhibitory activity. The interactions between plumieride and porcine pancreatic α-amylase and α-glucosidase were analyzed through an in silico molecular docking study., Results: Phytochemical identification of compounds present in the FHDHA fraction of H. drasticus latex was possible by 1H, 13C NMR analysis and mass spectrometry, and the results were compared with the literature. The identified compounds were α-ethyl glucoside, protocatechuic acid, 3-O-caffeoylquinic acid, 15-demethylplumieride acid, 5-O-caffeoylquinic acid, caffeic acid, vanillic acid, plumieride, and catechin. The inhibition results of the fractions tested against α-amylase and α-glucosidase showed inhibitory activities dependent on the increase of fractions and compound concentrations. The IC 50 results obtained from FHDHA, FHDHA1 and plumieride fractions against α-amylase were 36.46, 72.61, 33.87 μg/mL respectively. The IC 50 of plumieride was the closest to that of acarbose (22.52 μg/mL), a result similar to that obtained for α-glucosidase. The type of inhibition was competitive for both enzymes., Conclusions: There was strong inhibition of α-amylase and α-glucosidase by FHDHA, FHDHA1 and plumieride, suggesting that these enzymes slow glucose absorption., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Living cells are constantly exchanging momentum with their surroundings. So far, there is no consensus regarding how cells respond to such external stimuli, although it reveals much about their internal structures, motility as well as the emergence of disorders. Here, we report that twelve cell lines, ranging from healthy fibroblasts to cancer cells, hold a ubiquitous double power-law viscoelastic relaxation compatible with the fractional Kelvin-Voigt viscoelastic model. Atomic Force Microscopy measurements in time domain were employed to determine the mechanical parameters, namely, the fast and slow relaxation exponents, the crossover timescale between power law regimes, and the cell stiffness. These cell-dependent quantities show strong correlation with their collective migration and invasiveness properties. Beyond that, the crossover timescale sets the fastest timescale for cells to perform their biological functions.

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx 2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1β and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx 2 -specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections., Competing Interests: The authors declare no conflict of interest.

Background: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet., Objective and Methods: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2)., Results: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide., Conclusion: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest V max , K m , K cat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC 50 of around 50 μg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Severe bacterial infections initiate inadequate inflammation that leads to disseminated intravascular coagulation and death., Objectives: To evaluate the influence of bacterial infection on blood viscosity and red blood cells (RBCs) morphology, and the ability of Calotropis procera proteins (CpLP) to prevent the patho-hemorheology in infected animals., Methods: Rheology of blood, atomic force microscopy measurements on specific blood elements and blood count were performed to examine changes in blood viscosity, RBCs morphology, platelets activation, and RBCs indices., Findings: Infected mice hold their blood rheological behaviour as compared to that of the control group. However, they presented hyperactivated platelets, RBCs at different stages of eryptosis, and variation on RBCs indices. CpLP administration in healthy animals altered blood behaviour from pseudoplastic to Bingham-like fluid. Such effect disappeared over time and by inhibiting its proteases. No alterations were observed in RBCs morphology or platelets. Treatment of infected animals with CpLP prevented the changes in RBCs indices and morphology., Main Conclusions: The inflammatory process triggered by bacterial infection induced pathological changes in RBCs and platelets activation. Treatment of infected animals with CpLP prevented the emergence of RBCs abnormal morphology and this may have implications in the protective effect of CpLP, avoiding animal death.