Poly(I:C) transfection induces a pro-inflammatory cascade in murine mammary carcinoma and fibrosarcoma cells.

Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.

Gene electrotransfer of plasmid AMEP, an integrin-targeted therapy, has antitumor and antiangiogenic action in murine B16 melanoma.

Gene therapy with Plasmid AMEP (antiangiogenic metargidin peptide) has recently been studied as a potential targeted therapy for melanoma. This plasmid is designed to downregulate α5ß1 and αvß3 integrins. In our study, electroporation was used as a nonviral delivery system. We investigated the antiangiogenic and direct antitumor effectiveness of this gene therapy on low and highly metastatic B16 melanoma variants. In vitro, the antiangiogenic effectiveness as determined by tube formation assay on endothelial cells was predominantly dependent on AMEP expression levels. In vivo, antitumor effectiveness was mediated by the inhibition of proliferation, migration and invasion of melanoma cells and correlated with the expression of integrins on tumor cells after intratumor delivery. In addition, reduced metastatic potential was shown. Intramuscular gene electrotransfer of Plasmid AMEP, for AMEP systemic distribution, had no antitumor effect with this specific preventive treatment protocol, confirming that direct tumor delivery was more effective. This study confirms our previous in vitro data that the expression levels of integrins on melanoma cells could be used as a biomarker for antitumor effectiveness in integrin-targeted therapies, whereas the expression levels of AMEP peptide could be a predictive factor for antiangiogenic effectiveness of Plasmid AMEP in the treatment of melanoma.

Ecthyma gangrenosum and ecthyma-like lesions: review article.

The generally accepted definition of ecthyma gangrenosum (EG) states that this condition is pathognomonic of Pseudomonas septicemia (Pseudomonas aeruginosa) and that it should usually be seen in immunocompromised patients, particularly those with underlying malignant disease. The cases described in the literature present a somewhat different picture. Our objective was to analyze this controversy. The review analyzes 167 cases of EG that were described in the literature from 1975 to 2014. All articles on EG cases with EG-specific tissue defect that had signs of general and/or local infection and skin necrosis were included and analyzed, whatever the etiology detected. Necrotic lesions of the skin diagnosed as EG have various microbiological etiology, can occur in immunocompetent or even healthy persons, and are not necessarily connected with septicemia. In published cases, P. aeruginosa was detected in 123 cases (73.65%); of them, there were only 72 cases (58.5%) with sepsis. Other bacterial etiology was detected in 29 cases (17.35%) and fungi were detected in 15 cases (9%). While the clinical picture of the disease and the treatment strategy remain the same, there is no need to invent two separate definitions for Pseudomonas and non-Pseudomonas cases. We suggest accepting a broader definition of EG.

Responsiveness of water-sector regulation in Brazil to the Covid-19 pandemic: A view through the human rights lens.

This paper provides an overview of the regulatory responses to Covid-19 from the regulatory authorities of Brazilian states. This paper aims to provide new insights into the operationalization of the human rights to water and sanitation in the Brazilian regulatory authorities' actions during a health emergency. We find that communities located in unserved areas were not mentioned in the regulatory responses, nor were people in vulnerable situations. Equity and non-discrimination principles were correlated more with economic measures. This study has also identified the absence of responses related to access to sanitation facilities, with normative contents related to the issue not appearing in the content analysis.

Inactivation of bacterial opportunistic skin pathogens by nonthermal DC-operated afterglow atmospheric plasma.

AIMS: Multidrug-resistant opportunistic pathogens are clinically significant and require the development of new antimicrobial methods. In this study, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus cells were exposed to atmospheric plasma on agar plates and in vitro on porcine skin for the purpose of testing bacterial inactivation. METHODS AND RESULTS: Microbial inactivation at varying exposure durations was tested using a nonthermal plasma jet generated with a DC voltage from ambient air. The observed reduction in colony forming units was quantified as log(10) reductions. CONCLUSIONS: Direct plasma exposure significantly inactivated seeded bacterial cells by approx. 6 log(10) on agar plates and 2-3 log(10) on porcine skin. On agar plates, an indirect 'bystander' inactivation outside the plasma delivery area was also observed. The reduced inactivation observed on the skin surface was most likely due to cell protection by the variable surface architecture. SIGNIFICANCE AND IMPACT OF STUDY: Atmospheric plasma has potential for clinical application as a disinfectant of patient skin and medically relevant surfaces.

In vitro and in vivo correlation of skin and cellular responses to nucleic acid delivery.

Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer.

Evaluation of delivery conditions for cutaneous plasmid electrotransfer using a multielectrode array.

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field strength and pulse width on transgene expression and duration using a plasmid encoding the luciferase reporter gene delivered by intradermal injection in a guinea pig model followed by EP with the MEA. As expected, the level of luciferase expression increased with the magnitude and duration of the voltage applied. In addition to adjusting transgene expression levels by altering fielding strength, levels could also be controlled by adjusting the plasmid dose. Our results indicate that the design of the MEA is a viable option for cutaneous plasmid DNA delivery by in vivo EP to a large surface area.

Growth environment influences B16.F10 mouse melanoma cell response to gene electrotransfer.

We developed and characterized a 3D collagen hydrogel model for B16.F10 melanoma tumors. Cells in this 3D environment exhibited lower proliferation than cells in the conventional 2D culture environment. Interestingly, the basal expression levels of several genes varied when compared to conventionally grown cells. In each growth environment, a significant number of melanoma cells were transfected by plasmid electroporation (electrotransfer), although expression could only be ascertained on the surface of the 3D constructs. Cellular responses to plasmid entry as demonstrated by pro-inflammatory cytokine and chemokine upregulation varied based on the growth environment, as did the mRNA levels of several putative DNA-specific pattern recognition receptors (DNA sensors). Unexpectedly, when plasmid DNA was delivered while cells where attached in the 2D or 3D environments, the mRNAs of the DNA sensor p204 and the inflammatory mediator TNFα were regulated in cells receiving pulses only. However, we were unable to confirm coordinate upregulation of TNFα and p204 proteins. This study confirms that cell responses differ significantly based on their environment, and demonstrates the difficulty of extending experimental observations between cell environments.

Electroporation-mediated delivery of a naked DNA plasmid expressing VEGF to the porcine heart enhances protein expression.

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly increased cardiac expression of VEGF compared with injection of plasmid alone. This is the first report showing successful intracardiac delivery, through in vivo EP, of a protein expressing plasmid in a large animal.

Mechanism for the Autocatalytic Formation of Optically Active Compounds under Abiotic Conditions.

The bromination of chiral crystalline sam ples of 4,4'-dimethylchalcone was reinvestigated. In the presence of the optically active reaction product, (+)-or (-)-chalcone dibromide, crystallization from solutions of the achiral chalcone is specifically directed toward one-handedness. A feedback mechanism can thus be envisaged where optically active compounds are formed, generate additional material of the same chirality, and communicate this chirality to other regions, simply by cycles of solidification, reaction, and liquefaction.

A method for prioritization of areas for pesticides surveillance on surface waters: a study in Minas Gerais, Brazil.

The occurrence of pesticides in drinking water is a matter of growing concern in several parts of the world, mainly in developing countries, due the possible adverse effects on human health. Pesticides applied in the agriculture are an important source of contamination and are rarely monitored in surface water in developing countries, either by water supply operators or health authorities, often not accomplishing the legal issues regarding the quality control of raw waters. The paper discusses a method for prioritization of surveillance actions of pesticides in surface waters, through multicriteria analysis. Five criteria were defined and a range of weight was established for each criterion. For validation of the method, it was applied in five sub-basins of Grande River Basin - MG. This application allowed ordering priority sub-basins for pesticides surveillance, suggesting two sub-basins as priorities. The validation performed enabled the evaluation and adjustment of the method, mainly regarding the availability of information. The method showed a practical alternative for the environmental surveillance, targeting priority areas. Moreover, its structure allows the application in other different areas and for other pollutants.

Endemic Cryptosporidium infection and drinking water source: a systematic review and meta-analyses.

Cryptosporidium is a well-known cause of diarrhoea in humans. Little is known about risk factors associated with endemic cryptosporidiosis, which constitutes the majority of cases. We carried out meta-analyses to verify if drinking water is also associated with endemic infection and to assess the magnitude of the associations. The global meta-analysis suggests that there is an increased risk of Cryptosporidium infection among unsafe water users (OR 1.40 [1.15, 1.72]). Studies were stratified, according to the exposure to different sources of safe drinking water, due to the heterogeneity presented. The consumption of non-well and unboiled water was associated with an increased chance of endemic cryptosporidiosis, though only the latter was significant (OR 1.45 [0.95, 2.20]; OR 1.61 [1.09, 2.38]). Drinking non-bottled water did not present a risk factor associated with endemic cryptosporidiosis (OR 0.87 [0.72, 1.05]). These meta-analyses present results that could be useful to clarify the epidemiology of Cryptosporidium. We recommend that other risk factors could also be studied by this approach.

Antibacterial efficacy of a novel plasma reactor without an applied gas flow against methicillin resistant Staphylococcus aureus on diverse surfaces.

The use of nonthermal plasma in the clinic has gained recent interest, as the need for alternative or supplementary strategies are necessary for preventing multi-drug resistant infections. The purpose of this study was to evaluate the antibacterial efficacy of a novel plasma reactor based on a high current version of sliding discharge and operated by nanosecond voltage pulses without an applied gas flow. This modification is advantageous for both portability and convenience. Bacterial inactivation was determined within a chamber by direct quantification of colony Jing units. Plasma exposure significantly inhibited the growth of Escherichia coli and Staphylococcus epidermidis following a 1-min application (p<0.001). S. epidermidis was more susceptible to the plasma after a 5-min exposure compared to E. coli. Temperature and pH measurements taken immediately before and after plasma exposure determined neither heat nor pH changes play a role in bacterial inactivation. Because of the notable effect on S. epidermidis, the effect of plasma exposure on several isolates and strains of the related opportunistic pathogen Staphylococcus aureus was quantified. While S. aureus isolates and strains were efficiently inactivated on an agar surface, subsequent testing on other clinically relevant surfaces demonstrated that the inactivation level, although significant, was reduced. This reduction appeared to depend on both the surface texture and the surface moisture content. These findings suggest this novel plasma source lacking an applied gas flow has potential application for surface bacterial decontamination.

Grain-resolved analysis of localized deformation in nickel-titanium wire under tensile load.

The stress-induced martensitic transformation in tensioned nickel-titanium shape-memory alloys proceeds by propagation of macroscopic fronts of localized deformation. We used three-dimensional synchrotron x-ray diffraction to image at micrometer-scale resolution the grain-resolved elastic strains and stresses in austenite around one such front in a prestrained nickel-titanium wire. We found that the local stresses in austenite grains are modified ahead of the nose cone-shaped buried interface where the martensitic transformation begins. Elevated shear stresses at the cone interface explain why the martensitic transformation proceeds in a localized manner. We established the crossover from stresses in individual grains to a continuum macroscopic internal stress field in the wire and rationalized the experimentally observed internal stress field and the topology of the macroscopic front by means of finite element simulations of the localized deformation.

Blood flow velocity in the right coronary artery: assessment before and after angioplasty.

OBJECTIVES: This study attempted 1) to assess the utility of rest measurements of intracoronary blood flow velocity for the physiologic assessment of coronary stenoses before and after right coronary artery angioplasty, and 2) to compare the phasic flow pattern in the right coronary artery proper with the phasic flow pattern in its major branches to the left ventricle. BACKGROUND: Previous investigations have demonstrated that a reduction in distal blood flow velocity and a loss of distal diastolic predominant flow are characteristic of physiologically significant stenoses and that these indexes normalize after successful coronary artery dilation. However, these studies were predominantly performed in the left coronary artery. The utility of monitoring rest velocity variables during angioplasty of the right coronary artery has not been studied. METHODS: We studied 20 patients undergoing angioplasty of the right coronary artery with use of a Doppler angioplasty guide wire. RESULTS: Values were expressed as the mean value +/- 1 SD. The rest average peak velocity did not decrease distal to angiographically significant right coronary artery stenoses (23.3 +/- 9.4 cm/s proximal vs. 20.2 +/- 11.1 cm/s distal, p = 0.20). The proximal/distal velocity ratio was 1.4 +/- 0.9 before angioplasty and did not significantly decrease after angioplasty (p = 0.58). This study had a 99.4% power to detect a difference between proximal and distal average peak velocity. There was no relation between percent diameter stenosis and proximal/distal velocity ratios (r = 0.15, p = 0.55). Diastolic predominant flow was not observed in the proximal or distal right coronary artery. However, after angioplasty, diastolic predominant flow was observed in the posterolateral and posterior descending coronary arteries. CONCLUSIONS: Rest phasic Doppler flow velocity indexes are not useful for evaluating stenoses in the right coronary artery proper before or after angioplasty. In contrast to the right coronary artery proper, diastolic predominant flow is observed in the posterior descending and posterolateral coronary arteries. The utility of measuring hyperemic Doppler flow velocity indexes, such as distal coronary flow reserve, for assessing right coronary artery stenoses merits further investigation.

Sequential teboroxime imaging during and after balloon occlusion of a coronary artery.

OBJECTIVES: We sought to assess whether sequential teboroxime imaging can rapidly evaluate vessel patency and identify the coronary artery occluded in patients undergoing balloon occlusion of a coronary artery. BACKGROUND: Intravenous thrombolytic therapy results in successful reperfusion of the infarct-related artery in only 50% to 80% of cases. A noninvasive technique to serially evaluate coronary perfusion would identify patients who might benefit from other interventions such as emergency percutaneous transluminal coronary angioplasty, coronary artery bypass grafting or increased intensity of thrombolytic therapy. METHODS: Teboroxime scans were performed during balloon occlusion in 15 nonconsecutive patients undergoing angioplasty of a major coronary artery. Equivalent views were repeated after successful angioplasty. RESULTS: The mean time between balloon occlusion and reperfusion imaging was 1.6 +/- 0.6 h. The mean number of defects decreased significantly from 4.13 +/- 1.01 during balloon occlusion to 0.27 +/- 0.44 after reperfusion (p = 0.0006). There was a 30% decrease in the defect/normal zone count/pixel ratios during balloon occlusion and normalization of these ratios after reperfusion (p = 0.0006). The scans correctly identified all nine left anterior descending coronary artery occlusions and both right coronary artery occlusions. One of the four left circumflex coronary artery occlusions was incorrectly identified as a right coronary artery occlusion by scan criteria. Overall, the scans correctly identified the occluded artery 93% of the time (kappa = 0.88). The scan was 100% accurate for distinguishing occlusion of the left anterior descending coronary artery (n = 9) from occlusions of the left circumflex or right coronary artery (n = 6). CONCLUSIONS: We believe that this is the first clinical study to demonstrate that sequential planar imaging with teboroxime can 1) rapidly detect acute coronary artery occlusion and reperfusion, and 2) identify the occluded coronary artery. A trial comparing rapid sequential teboroxime imaging with coronary angiography in patients receiving thrombolytic therapy for acute myocardial infarction is warranted.

Transient increase in release of adenosine during rapid cardiac pacing; transient effects on overdrive suppression of ventricular automaticity.

A study was designed to test the hypothesis that endogenous adenosine concentration may increase during episodes of rapid ventricular pacing and, by virtue of its negative chronotropic effects, contribute to the transient suppression of automaticity that follows the period of overdrive. Isolated, perfused, rat ventricular preparations were subjected to periods of 6.0 Hz overdrive stimulation while adenosine release, oxygen consumption, and subsequent suppression of automaticity were measured. At the end of a 1 min episode of overdrive oxygen consumption and adenosine release were significantly increased, and the initial beating rate after 1 min overdrive was suppressed. At the end of 10 min overdrive oxygen consumption was still increased but adenosine release had returned to control values. Suppression of automaticity after 10 min overdrive was similar to that after 1 min overdrive. The relative magnitude of suppression after 1 min overdrive was decreased by theophylline (10(-4) mol.litre-1), and increased by the adenosine deaminase inhibitor, EHNA (10(-5) mol.litre-1). Neither theophylline nor EHNA had any discernible influence on suppression after 10 min overdrive. It its therefore concluded that endogenous adenosine may contribute to the suppression of ventricular automaticity that follows a 1 min episode of overdrive, but because of the transient nature of the increase in adenosine during overdrive endogenous adenosine does not contribute to the suppression that follows prolonged overdrive.

Neurologic manifestations of progressive systemic sclerosis.

Neurologic involvement in progressive systemic sclerosis is considered uncommon. We retrospectively examined the prevalence and nature of neurologic complications in 50 patients with progressive systemic sclerosis. In 20 (40%), neurologic abnormalities were detected, with a total of 28 neurologic manifestations. All levels of the central and peripheral nervous system were affected: muscle (22%), peripheral nerve (18%), spinal cord (8%), and brain (6%). Of note were the presence of myelopathy in four patients and inclusion-body myositis in two. In 10 patients (20%), no definable cause of the neurologic dysfunction could be identified, apart from progressive systemic sclerosis. Thus, neurologic presentations of progressive systemic sclerosis are much more common than previously reported and may be due to direct involvement of the nervous system by a primary pathologic process in a significant number of patients.