Pllans-II: Unveiling the Action Mechanism of a Promising Chemotherapeutic Agent Targeting Cervical Cancer Cell Adhesion and Survival Pathways.

Despite advances in chemotherapeutic drugs used against cervical cancer, available chemotherapy treatments adversely affect the patient's quality of life. For this reason, new molecules from natural sources with antitumor potential and few side effects are required. In previous research, Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, has shown selective attack against the HeLa and Ca Ski cervical cancer cell lines. This work suggests that the cytotoxic effect generated by Pllans-II on HeLa cells is triggered without affecting the integrity of the cytoplasmic membrane or depolarizing the mitochondrial membranes. The results allow us to establish that cell death in HeLa is related to the junction blockage between α5ß1 integrins and fibronectin of the extracellular matrix. Pllans-II reduces the cells' ability of adhesion and affects survival and proliferation pathways mediated by intracellular communication with the external environment. Our findings confirmed Pllans-II as a potential prototype for developing a selective chemotherapeutic drug against cervical cancer.

Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses.

Focusing electric pulse effects away from electrodes is a challenge because the electric field weakens with distance. Previously we introduced a remote focusing method based on bipolar cancellation, a phenomenon of low efficiency of bipolar nanosecond electric pulses (nsEP). Superpositioning two bipolar nsEP into a unipolar pulse canceled bipolar cancellation ("CANCAN" effect), enhancing bioeffects at a distance despite the electric field weakening. Here, we introduce the next generation (NG) CANCAN focusing with unipolar nsEP packets designed to produce bipolar waveforms near electrodes (suppressing electroporation) but not at the remote target. NG-CANCAN was tested in CHO cell monolayers using a quadrupole electrode array and labeling electroporated cells with YO-PRO-1 dye. We routinely achieved 1.5-2 times stronger electroporation in the center of the quadrupole than near electrodes, despite a 3-4-fold field attenuation. With the array lifted 1-2 mm above the monolayer (imitating a 3D treatment), the remote effect was enhanced up to 6-fold. We analyzed the role of nsEP number, amplitude, rotation, and inter-pulse delay, and showed how remote focusing is enhanced when re-created bipolar waveforms exhibit stronger cancellation. Advantages of NG-CANCAN include the exceptional versatility of designing pulse packets and easy remote focusing using an off-the-shelf 4-channel nsEP generator.

Pllans-II Induces Cell Death in Cervical Cancer Squamous Epithelial Cells via Unfolded Protein Accumulation and Endoplasmic Reticulum Stress.

Due to the lack of chemotherapeutic drugs that selectively affect cervical cancer cells, natural sources such as snake venom are currently being investigated for molecules with antitumor potential. Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, induced cell death in a cervical cancer cell line-Ca Ski-related to dysfunction in the ability to resolve endoplasmic reticulum stress, evidenced by sub-expression of genes such as PERK, ERO1 PDIs, HSP70, and CHOP. Western blot analysis validated the last two genes' sub-expression at the protein level. In addition, Pllans-II presented a dose-dependent cytotoxic effect on cancer cells and an insignificant effect on healthy endothelial cells (HUVEC). Additionally, Pllans-II inhibited cancer cells' adhesion and migration capacity, induced cell cycle arrest in the G2/M phase, and induced apoptosis stimulated possibly by the extrinsic route. These results demonstrate for the first time that Pllans-II has an antitumor effect on a squamous epithelial cervical cancer cell line and represents a possible biotechnological tool for designing a prominent antitumor agent.

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers.

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength-duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Evaluation of the systemic alterations triggers by Porthidium lansbergii lansbergii snake venom.

The systemic effects generated by Porthidium lansbergii lansbergii envenoming, a species found in the northern region of Colombia, is poorly known. The present study aimed to analyze for the first time the mice's behavior, the histological alterations, and changes in biochemical markers levels resulting from the intraperitoneal injection of an LD50 of P. lansbergii lansbergii snake venom on mice. The envenoming mice displayed hypodynamic condition, clonic head movements, accompanied by bradypnea and thoracoabdominal imbalance. After 7 h of envenoming, the mice showed an ecchymotic region at the injection site, including bleeding in the pleural, liver, and kidney capsules. The effect on the brain revealed a micro-hemorrhage in the sensorimotor cortex with substantial loss of neurons. The venom caused dilated blood vessels in lung tissue, with endothelial necrosis associated with alveolar rupture. The liver showed parenchyma alteration with many extravasated erythrocytes. The kidneys exhibited renal tubules necrosis and a statistically significant increase in creatinine concentration. ALP and ALT's enzymatic activities remained constant at 7 h after envenoming but increased at 12 h. AST and LDH were significantly increased at 7 h but decreased to the near baseline 12 h after venom administration. Massive hemorrhages could trigger a hypovolemic shock, which could lead to death after several h without treatment. Knowledge of P. lansbergii lansbergii snake bites' injuries is essential to make the appropriate diagnostic in human envenoming cases by this snake.

Comparative analysis of plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane in critical mandibular bone defects: An experimental study on rats.

Repair of bone deficiencies in the craniofacial skeleton remains a challenging clinical problem. The aim of this study was to evaluate and compare the effects of a plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane implanted into experimental mandibular defects. Bilateral mandibular defects were created in twelve immunodeficient rats. The bone defect was filled with serum scaffold alone in left sides and scaffold combined with human alveolar osteoblast in right side defects. Implanted areas were closed directly in Group 1 (n = 6) and covered by a resorbable polyglycolic-polylactic acid membrane in Group 2 (n = 6). Bone regeneration was determined at 12 weeks as measured by and exhaustive multiplanar computed tomography analysis and histological examination. No significant differences in bone density were observed between defects transplanted with scaffold alone or scaffold seeded with osteoblasts. The use of membrane did not result in a determining factor in the grade of bone regeneration between Groups 1 and 2. Based on these results, it could be concluded that the albumin scaffold alone has osteoinductive capacity but presence of seeded ostogenic cells accelerates defect repair without being significantly influenced by covering the defect with a resorbable membrane.

Bone regeneration with autologous adipose-derived mesenchymal stem cells: A reliable experimental model in rats.

The adult mesenchymal stem cell (MSC) has been proposed to be the definitive tool in regenerative medicine due to its multi-differentiation potential and expansion capacity ex vivo. The use of MSCs on bone regeneration has been assessed in several studies, obtaining promising results. However, the endless combinations that can be tested and the heterogeneity in the experimental conditions become a drawback when comparing results between authors. Moreover, it is very hard to find autologous studies using adipose-derived MSCs (AD-MSC) in rodents, which is the most used preclinical animal model. In this article an experimental model for basic bone tissue engineering research is described and justified, on which adult AD-MSCs are safely isolated from the rat dorsal interscapular fat pad, allowing ex vivo expansion and autogenous orthotopic reimplantation in a bilateral mandibular bone defect made in the same animal. This reliable and reproducible model provides a simple way to perform basic experimentation studies in a small animal model using autologous MSC for bone regeneration or cell therapy techniques prior to improve the research on large animal models.•Predictable and safe harvest of adipose-derived MSC. No need of animal sacrifice.•Allows for autologous studies with the most frequently used animal model: the rat. No need of allogeneic or human MSC use and, therefore, immunological concerns are avoided.•Bilateral mandibular critical size defect to allow direct control/experimental comparison.

Antitumoral Potential of Lansbermin-I, a Novel Disintegrin from Porthidium lansbergii lansbergii Venom on Breast Cancer Cells.

BACKGROUND: Disintegrins from snake venoms bind with high specificity cell surface integrins, which are important pharmacological targets associated with cancer development and progression. OBJECTIVE: In this study, we isolated a disintegrin from the Porthidium lansbergii lansbergii venom and evaluated its antitumoral effects on breast cancer cells. METHODS: The isolation of the disintegrin was performed on RP-HPLC and the inhibition of platelet aggregation was evaluated on human platelet-rich plasma. The inhibition of cell adhesion was also evaluated in vitro on cultures of cell lines by the MTT method as well as the inhibition of breast cancer cell migration by the wound healing assay. The binding of the disintegrin to integrin subunits was verified by flow cytometry and confocal microscopy. Finally, inhibition of angiogenesis was assessed in vitro on HUVEC cells and the concentration of VEGF was measured in the cellular supernatants. RESULTS: The disintegrin, named Lansbermin-I, is a low molecular weight protein (< 10 kDa) that includes an RGD on its sequence identified previously. Lansbermin-I showed potent inhibition of ADP and collagen-induced platelet aggregation on human plasma and also displayed inhibitory effects on the adhesion and migration of breast cancer MCF7 and MDA-MB 231cell lines, without affecting nontumorigenic breast MCF-10A and lung BEAS cells. Additionally, Lansbermin-I prevented MCF7 cells to adhere to fibronectin and collagen, and also inhibited in vitro angiogenesis on human endothelial HUVEC cells. CONCLUSION: Our results display the first report on the antitumor and anti-metastatic effects of an RGDdisintegrin isolated from a Porthidium snake venom by possibly interfering with α2 and/or ß1-containing integrins. Thus, Lansbermin-I could be an attractive model to elucidate the role of disintegrins against breast cancer development.

Phylogeny and toxicological assessments of two Porthidium lansbergii lansbergii morphotypes from the caribbean region of Colombia.

After a snakebite accident, species identification is of vital importance. However, the existence of intraspecific differences in the body coloration patterns of venomous snakes can generate confusion and delay a convenient and effective treatment. This is the situation for Porthidium lansbergii lansbergii from Colombia, for which two distinctive color morphs occur, and the relationship of these morphs with venom toxicity is unknown. Therefore, venom samples from specimens of these two morphs were collected from the Colombian Caribbean region, and their protein profiles compared. Likewise, their venom functional activities were evaluated in vitro and in vivo in BALB/C mice. Additionally, using sequences of the mitochondrial cytochrome b (Cyt-b) gene, the relationship between these Colombian P. lansbergii lansbergii morphotypes was investigated, and their phylogenetic positions were determined for the first time using Bayesian inference. Despite the noticeable coloration divergence between the individuals analyzed, similar protein profiles of their venoms were observed. Additionally, neither their lethality nor biochemical activities were notably different. In general, both venoms were highly proteolytic, lacked a coagulant effect in vitro, and extended the clotting time due to the action of venom components, such as disintegrins and proteases, that induce defibrination. These results agreed with the result of our phylogenetic analysis, suggesting that the two chromatic morphs do not represent isolated populations. The phylogenetic analyses also supported the currently recognized P. lansbergii lansbergii subspecies as a monophyletic complex. In conclusion, the results of this investigation suggest similar clinical manifestations regardless of body coloration after a P. lansbergii lansbergii envenomation, and pools can therefore be used for antivenom development, medical treatments, and further research efforts.

Repair of maxillary cystic bone defects with mesenchymal stem cells seeded on a cross-linked serum scaffold.

Tissue engineering combining cross-linked serum scaffolds with bone-derived mesenchymal stem cells has displayed excellent results for repair of maxillofacial bone defects in animal models, but it had not been tested in humans yet. We present here a pilot clinical trial using autologous bone-derived mesenchymal stem cells (H-MSV) grown in a serum cross-linked scaffold (BioMax) for treatment of maxillary cysts in 9 patients. Cells obtained from alveolar bone were seeded in the BioMax scaffold prepared from autologous serum, expanded under GMP conditions, and subjected to osteogenic differentiation for 3-4 weeks before application. Evolution of the cystic cavity was followed by computerized tomography (CT) for 7 months. There was no inflammation or other adverse effects, and the CT density of the cyst interior increased significantly after the treatment. The ratio of the CT values after/before treatment was (mean ± SE) 2.52 ± 0.45; in contrast, the density of the contralateral control area of spongy alveolar bone without treatment did not change (ratio after/before, 0.99 ± 0.14). In conclusion, cell therapy with BioMax could be considered as an alternative therapy for maxillary bone defects and other losses of bone substance. Further research with allogeneic cells would be useful for reducing costs and improving logistics. CLINICAL TRIAL REGISTRATION NUMBERS: EudraCT 2010-024246-30 and NCT01389661.

Uso do curativo hemostático de gelatina absorvível no cuidado pós-operatório do pedículo vascular dos retalhos interpolados / Use of absorbable hemostatic gelatin sponge in postoperative care of the vascular pedicle of interpolated flaps

Os retalhos interpolados têm sido um dos mais importantes e funcionais retalhos no arsenal da cirurgia plástica reconstrutiva, tornando-se uma opção segura mesmo nos casos mais difíceis. O pedículo do retalho interpolado necessita de curativo para evitar sangramento e a contaminação local. Este curativo frequentemente falha na prevenção de pequenos sangramentos que ocorrem durante as primeiras 24-48 horas, forçando a troca recorrente do mesmo, em média de três a cinco trocas. A técnica proposta neste trabalho consiste na aplicação direta de uma camada de GELFOAM®, envolto por gaze petrolizada, para prevenção do sangramento da área cruenta do pedículo do retalho, acarretando melhor hemostasia e menos manipulação do pedículo vascular.

Interpolated flaps are among the most important and functional flaps in reconstructive plastic surgery, representing a safe option even in the most difficult cases. The pedicle of the interpolated flap requires a dressing to avoid bleeding and local contamination. This dressing often fails to prevent minor bleedings, which occurs within the first 24-48 hours. As a result, it needs to be continuously changed, from three to five times on average. The technique proposed in this study consists in a direct application of a GELFOAM® layer. This is subsequently wrapped with petroleum gauze to prevent bleeding of the open area in the pedicle flap, improving hemostasis and reducing the manipulation of the vascular pedicle.

Divergent functional profiles of acidic and basic phospholipases A2 in the venom of the snake Porthidium lansbergii lansbergii.

The Lansberg's hognose pitviper, Porthidium lansbergii lansbergii, inhabits northern Colombia. A recent proteomic characterization of its venom (J. Proteomics [2015] 114, 287-299) revealed the presence of phospholipases A2 (PLA2) accounting for 16.2% of its proteins. The two most abundant PLA2s were biochemically and functionally characterized. Pllans-I is a basic, dimeric enzyme with a monomer mass of 14,136 Da, while Pllans-II is an acidic, monomeric enzyme of 13,901 Da. Both have Asp49 in their partial amino acid sequences and, accordingly, are catalytically active upon natural or synthetic substrates. Nevertheless, these two enzymes differ markedly in their bioactivities. Pllans-I induces myonecrosis, edema, and is lethal by intracerebro-ventricular injection in mice, as well as cytolytic and anticoagulant in vitro. In contrast, Pllans-II is devoid of these effects, except for the induction of a moderate edema. In spite of lacking myotoxicity, Pllans-II enhances the muscle damaging action of Pllans-I in vivo. Altogether, results further illustrate the divergent functional profiles of basic and acidic PLA2s in viperid venoms, and suggest that Pllans-I plays a myotoxic role in envenomings by P. l. lansbergii, whereas Pllans-II, apparently devoid of toxicity, enhances muscle damage caused by Pllans-I.

Integrated Toolset for WSN Application Planning, Development, Commissioning and Maintenance: The WSN-DPCM ARTEMIS-JU Project.

In this article we present the main results obtained in the ARTEMIS-JU WSN-DPCM project between October 2011 and September 2015. The first objective of the project was the development of an integrated toolset for Wireless sensor networks (WSN) application planning, development, commissioning and maintenance, which aims to support application domain experts, with limited WSN expertise, to efficiently develop WSN applications from planning to lifetime maintenance. The toolset is made of three main tools: one for planning, one for application development and simulation (which can include hardware nodes), and one for network commissioning and lifetime maintenance. The tools are integrated in a single platform which promotes software reuse by automatically selecting suitable library components for application synthesis and the abstraction of the underlying architecture through the use of a middleware layer. The second objective of the project was to test the effectiveness of the toolset for the development of two case studies in different domains, one for detecting the occupancy state of parking lots and one for monitoring air concentration of harmful gasses near an industrial site.

Proteomic and functional analyses of the venom of Porthidium lansbergii lansbergii (Lansberg's hognose viper) from the Atlantic Department of Colombia.

The venom of the Lansberg's hognose pitviper, Porthidium lansbergii lansbergii, a species found in the northern region of Colombia, is poorly known. Aiming to increase knowledge on Porthidium species venoms, its proteomic analysis and functional evaluation of in vitro and in vivo activities relevant to its toxicity were undertaken. Out of 51 protein components resolved by a combination of RP-HPLC and SDS-PAGE, 47 were assigned to 12 known protein families. In similarity with two previously characterized venoms from species within this genus, Porthidium nasutum and Porthidium ophryomegas, that of P. lansbergii lansbergii was dominated by metalloproteinases, although in lower proportion. A common feature of the three Porthidium venoms appears to be a high content of disintegrins. Proteins not previously observed in Porthidium venoms belong to the vascular endothelium growth factor, phosphodiesterase, and phospholipase B families. P. lansbergii lansbergii venom showed relatively weak lethal activity to mice, and induced a moderate local myotoxicity, but considerable hemorrhage. Its isolated VEGF component showed potent edema-inducing activity in the mouse footpad assay. Significant thrombocytopenia, but no other major hematological changes, were observed in envenomed mice. In vitro, this venom lacked coagulant effect on human plasma, and induced a potent inhibition of platelet aggregation which was reproduced by its purified disintegrin components. Phospholipase A2 and proteolytic activities were also demonstrated. Overall, the compositional and functional data herein described for the venom of P. lansbergii lansbergii may contribute to a better understanding of envenomings by this pitviper species, for which specific clinical information is lacking. BIOLOGICAL SIGNIFICANCE: Porthidium lansbergii lansbergii is estimated to be responsible for nearly 20% of snakebite envenoming cases at the Atlantic Department of Colombia, but the identity and functional properties of its venom components are largely unknown. This study provides the first combined proteomic and functional analyses of the venom of this pitviper, which may contribute to a better understanding of the features of envenomings by this species.

[GUIDE DEVELOPMENT OF A PROCESS MODEL OF NURSE APPLYING VIRGINIA HENDERSON AND TAXONOMY NANDA]. / Guía de elaboración de un proceso enfermero aplicando el modelo de Virginia Henderson y la taxonomía NANDA.

This study aims to develop a practical guide to applying the nur- sing model recognized Virginia Henderson NANDA taxonomy and nursing process.

Dietary fiber down-regulates colonic tumor necrosis factor alpha and nitric oxide production in trinitrobenzenesulfonic acid-induced colitic rats.

Previous studies have revealed the beneficial effects exerted by dietary fiber in human inflammatory bowel disease, which were associated with an increased production of SCFA in distal colon. The aim of the present study was to elucidate the probable mechanisms involved in the beneficial effects of a fiber-supplemented diet (5% Plantago ovata seeds) in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis, with special attention to its effects on the production of some of the mediators involved in the inflammatory response, such as tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO). Rats were fed the fiber-supplemented diet for 2 wk before TNBS colitis induction and thereafter until colonic evaluation 1 wk later. The results obtained showed that dietary fiber supplementation facilitated recovery from intestinal insult as evidenced both histologically, by a preservation of intestinal cytoarchitecture, and biochemically, by a significant reduction in colonic myeloperoxidase activity and by restoration of colonic glutathione levels. This intestinal anti-inflammatory effect was associated with lower TNFalpha levels and lower NO synthase activity in the inflamed colon, showing significant differences when compared with nontreated colitic rats. Moreover, the intestinal contents from fiber-treated colitic rats showed a significantly higher production of SCFA, mainly butyrate and propionate. We conclude that the increased production of these SCFA may contribute to recovery of damaged colonic mucosa because they constitute substrates for the colonocyte and, additionally, that they can inhibit the production of proinflammatory mediators, such as TNFalpha and NO.

Meningitis por Neisseria meningitidis después de traumatismo craneoencefálico / Neisseria meningitidis meningitis

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