A High-Power Dielectric Biconical Antenna for Treatment of Subcutaneous Targets

A dielectric biconical antenna (DiBiCA) for radiating subnanosecond pulses to treat subcutaneous tissue was designed, constructed, and tested. It is composed of a conical wave launcher and truncated conical emitter. In between, there is a short cylinder that provides a space for a ring terminating resistor. The material of the antenna has a dielectric constant of 28, so its size is small (length: 7 cm and aperture diameter: 2.2 cm). It was housed in an oil container to withstand high voltages and avoid surface flashover. The radiated electric field, measured in water, increased as the input voltage increased up to 30 kV but leveled off for higher voltages up to 50 kV, presumably because of losses in the antenna dielectric. The maximum field was 1.5 kV/cm for a depth of 5 mm and 1.0 kV/cm for a depth of 20 mm. Although the dielectric loss mechanism remains to be investigated, the antenna can be useful for noninvasive delivery of subnanosecond pulses to induce biological responses on subcutaneous targets. The DiBiCA radiated pulses were shown to change the viabilities of dendritic cells and macrophages for 10-min exposure. Bioelectromagnetics. 2020;41:413–424. © 2020 Bioelectromagnetics Society.     

Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system.

The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.     

Crystal structure of the pyrazinamidase of Mycobacterium tuberculosis: insights into natural and acquired resistance to pyrazinamide

Pyrazinamidase (PncA) activates the first-line antituberculous drug pyrazinamide into pyrazinoic acid. The crystal structure of the Mycobacterium tuberculosis PncA protein has been determined, showing significant differences in the substrate binding cavity when compared to the pyrazinamidases from Pyrococcus horikoshii and Acinetobacter baumanii. In M. tuberculosis, this region was found to hold a Fe(2+) ion coordinated by one aspartate and three histidines, one of them corresponding to His57 which is replaced by Asp in Mycobacterium bovis, a species naturally resistant to pyrazinamide. The binding cavity also contains a Cys138-Asp8-Lys96 motif evocating a cysteine-based catalytic mechanism. Mutants have been constructed and investigated by kinetic and thermal shift assays, highlighting the importance of protein folding and thermal stability in the pyrazinamidase activity.     

Natural products reveal cancer cell dependence on oxysterol-binding proteins

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.     

Sometimes sperm whales (Physeter macrocephalus) cannot find their way back to the high seas: a multidisciplinary study on a mass stranding

Background

Mass strandings of sperm whales (Physeter macrocephalus) remain peculiar and rather unexplained events, which rarely occur in the Mediterranean Sea. Solar cycles and related changes in the geomagnetic field, variations in water temperature and weather conditions, coast geographical features and human activities have been proposed as possible causes. In December 2009, a pod of seven male sperm whales stranded along the Adriatic coast of Southern Italy. This is the sixth instance from 1555 in this basin.

Methodology/Principal Findings

Complete necropsies were performed on three whales whose bodies were in good condition, carrying out on sampled tissues histopathology, virology, bacteriology, parasitology, and screening of veins looking for gas emboli. Furthermore, samples for age determination, genetic studies, gastric content evaluation, stable isotopes and toxicology were taken from all the seven specimens.The animals were part of the same group and determined by genetic and photo-identification to be part of the Mediterranean population. Causes of death did not include biological agents, or the “gas and fat embolic syndrome”, associated with direct sonar exposure. Environmental pollutant tissue concentrations were relatively high, in particular organochlorinated xenobiotics. Gastric content and morphologic tissue examinations showed a prolonged starvation, which likely caused, at its turn, the mobilization of lipophilic contaminants from the adipose tissue. Chemical compounds subsequently entered the blood circulation and may have impaired immune and nervous functions.

Conclusions/Significance

A multi-factorial cause underlying this sperm whales'' mass stranding is proposed herein based upon the results of postmortem investigations as well as of the detailed analyses of the geographical and historical background. The seven sperm whales took the same “wrong way” into the Adriatic Sea, a potentially dangerous trap for Mediterranean sperm whales. Seismic surveys should be also regarded as potential co-factors, even if no evidence of direct impact has been detected.     

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer

The Class III β-tubulin isotype (βIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while βIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.     

Annexin A1 N-Terminal Derived Peptide Ac2-26 Stimulates Fibroblast Migration in High Glucose Conditions

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG) or high glucose (HG) we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i) Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii) N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.     

TLQP-21, a VGF-derived peptide, stimulates exocrine pancreatic secretion in the rat

The aims of this paper were to study: (1) the effects of TLQP-21 (non-acronic name), the C-terminal region of the VGF (non-acronic name), polypeptide (from residue 557 to 576 of VGF), on in vitro amylase release from rat isolated pancreatic lobules and acinar cells; (2) the mechanism through which TLQP-21 regulates exocrine pancreatic secretion, by using the muscarinic receptor antagonist atropine (10(-6)M) and the cyclo-oxygenase inhibitor, indomethacin (10(-6)M). On pancreatic lobules of rats, concentrations of TLQP-21 from 10(-7) to 10(-5)M significantly (p<0.05) induced a 2-3-fold increase of baseline pancreatic amylase release, measured at the end of 60 min incubation period. Co-incubation with atropine 10(-6)M did not antagonise the enzyme outflow induced by the peptide. On the contrary, co-incubation of TLQP-21 (10(-7) and 10(-6)M) with indomethacin, at concentration of 10(-6)M, which alone did not modify enzyme secretion, completely suppressed the increase of amylase evoked by TLQP-21 on pancreatic lobules. On rat pancreatic acinar cells, TLQP-21, at all the concentrations tested, was unable to affect exocrine pancreatic secretion, indicating an indirect mechanism of action on acinar cells. These results put in evidence, for the first time, that TLQP-21, a VGF-derived peptide, modulates exocrine pancreatic secretion in rats through a stimulatory mechanism involving prostaglandin release. In conclusion, TLQP-21 could be included among the neurohumoral signals regulating pancreatic exocrine secretion, and increases the knowledge concerning the systems controlling this function.     

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

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