Cold Atmospheric Plasma, Platelet-Rich Plasma, and Nitric Oxide Synthesis Inhibitor: Effects Investigation on an Experimental Model on Rats

The evolution of reconstructive methods for defects of the human body cannot yet replace the use of flap surgery. Research is still preoccupied with the ideal techniques for offering the best chances of survival of the flaps. In our study, we investigated the effects of cold atmospheric plasma (CAP), N-nitro-L-arginine methyl ester (L-NAME), and platelet-rich plasma (PRP) injectable solutions on flap survival using an in vivo model. Twenty-four Wistar rats (four groups) had the McFarlane flap raised and CAP, L-NAME, and PRP substances tested through a single dose subcutaneous injection. The control group had only a saline solution injected. To the best of our knowledge, this is the first study that evaluated a CAP activated solution through injection on flaps. The flap survival rate was determined by clinical examination (photography documented), hematology, thermography, and anatomopathological tests. The image digital analysis performed on the flaps showed that the necrosis area (control—49.64%) was significantly lower for the groups with the three investigated solutions: CAP (14.47%), L-NAME (18.2%), and PRP (23.85%). Thermography exploration revealed less ischemia than the control group on the CAP, L-NAME, and PRP groups as well. Anatomopathological data noted the best degree of angiogenesis on the CAP group, with similar findings on the L-NAME and PRP treated flaps. The blood work did not indicate infection or a strong inflammatory process in any of the subjects. Overall, the study shows that the CAP activated solution has a similar (better) impact on the necrosis rate (compared with other solutions with known effects) when injected on the modified dorsal rat skin flap, and on top of that it can be obtained fast, in unlimited quantities, non-invasively, and through a standardized process.

Necrotic debris and STING exert therapeutically relevant effects on tumor cholesterol homeostasis

Malignant tumors commonly display necrosis, which invariably triggers an inflammatory response that supports tumor growth. However, the effect on tumor cells of necrotic debris, or damage-associated molecular patterns (DAMPs) released by dying cells is unknown. Here, we addressed the effect of DAMPs on primary Ewing sarcoma (EwS) cells and cell lines grown in 3D (spheroids) and 2D culture. We show that DAMPs promote the growth of EwS spheroids but not 2D cultures and that the underlying mechanism implicates an increase in cholesterol load in spheroids. In contrast, stimulation of the nucleic acid sensor signaling platform STING by its ligand cyclic GMP-AMP decreases the tumor cell cholesterol load and reduces their tumor initiating ability. Overexpression of STING or stimulation with cyclic GMP-AMP opposes the growth stimulatory effect of DAMPs and synergizes with the cholesterol synthesis inhibitor simvastatin to inhibit tumor growth. Our observations show that modulation of cholesterol homeostasis is a major effect of necrotic cell debris and STING and suggest that combining STING agonists with statins may help control tumor growth.

Melatonin Participates in Selenium-Enhanced Cold Tolerance of Cucumber Seedlings

Melatonin is an important and widespread plant hormone. However, the underlying physiological and molecular mechanisms of melatonin as a secondary messenger in improving cold tolerance by selenium are limited. This study investigated the effects of selenite on the cold stress of cucumber seedlings. The results showed that exogenous application of selenite improved the cold tolerance of cucumber seedlings, which was dependent on the concentration effect. In the present experiment, 1 μM of selenite showed the best effect on alleviating cold stress. Interestingly, we found that in the process of alleviating cold stress, selenite increased the content of endogenous melatonin by regulating the expression of melatonin biosynthesis genes (TDC, T5H, SNAT, and COMT). To determine the interrelation between selenite and melatonin in alleviating cold stress, melatonin synthesis inhibitor p-chlorophenylalanine and melatonin were used for in-depth study. This study provides a theoretical basis for cucumber cultivation and breeding.

Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 μM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 μM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A

Fanconi Anemia (FA) is a rare recessive genetic disorder characterized by aplastic anemia due to a defective DNA repair system. In addition, dysfunctional energy metabolism, lipid droplets accumulation, and unbalanced oxidative stress are involved in FA pathogenesis. Thus, to modulate the altered metabolism, Fanc-A lymphoblast cell lines were treated with quercetin, a flavonoid compound, C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid), a fatty acid synthesis inhibitor, and rapamycin, an mTOR inhibitor, alone or in combination. As a control, isogenic FA cell lines corrected with the functional Fanc-A gene were used. Results showed that: (i) quercetin recovered the energy metabolism efficiency, reducing oxidative stress; (ii) C75 caused the lipid accumulation decrement and a slight oxidative stress reduction, without improving the energy metabolism; (iii) rapamycin reduced the aerobic metabolism and the oxidative stress, without increasing the energy status. In addition, all molecules reduce the accumulation of DNA double-strand breaks. Two-by-two combinations of the three drugs showed an additive effect compared with the action of the single molecule. Specifically, the quercetin/C75 combination appeared the most efficient in the mitochondrial and lipid metabolism improvement and in oxidative stress production reduction, while the quercetin/rapamycin combination seemed the most efficient in the DNA breaks decrement. Thus, data reported herein suggest that FA is a complex and multifactorial disease, and a multidrug strategy is necessary to correct the metabolic alterations.

Dose Finding and Food Effect Studies of a Novel Abiraterone Acetate Formulation for Oral Suspension in Comparison to a Reference Formulation in Healthy Male Subjects

Currently approved formulations of the androgen synthesis inhibitor abiraterone acetate (AA) consist of multiple tablets administered daily in a fasted state. Removing the food effect and switching to a suspension formulation is expected to improve the pharmacokinetic profile and facilitate drug administration for patients with late-stage prostate cancer. Two four-sequence, four-period randomized crossover investigations were undertaken to establish the pharmacokinetic profiles of single doses of commercially available Zytiga®, as the reference AA (R-AA), and a novel tablet for oral suspension (TOS). Four single doses of TOS (from 62.5 to 250 mg) were compared in study C01, and two single doses each of TOS (250 mg) and R-AA (1000 mg) were compared under fasted and fed (modified fasted for R-AA) conditions in C02. Plasma concentrations of abiraterone over time were measured, and pharmacokinetic parameters were calculated. Each doubling of the dose of TOS was associated with a greater than 3-fold increase in exposure. A single dose of TOS (250 mg) exhibited similar exposure over 24 h, whether given fasted (625 ng × h/mL) or fed (485 ng × h/mL). A single dose of TOS (250 mg) was associated with higher (fasted, p = 0.028) or equivalent exposure (fed) compared to 1000 mg R-AA fasted (532 ng × h/mL). Substantially higher exposures were seen with 1000 mg R-AA under modified fasted conditions compared to TOS, irrespective of prandial status (p < 0.001). TOS was generally safe and well tolerated in the study. A 250 mg dose of a novel AA formulation for oral suspension demonstrated bioequivalence to 1000 mg R-AA under fasted conditions. This novel TOS formulation also addresses some of the limitations of current AA treatment, including low bioavailability, high variability in systemic exposure and a large food effect. It may offer an alternative for patients with dysphagia or discomfort with swallowing large pills.

Unraveling Autocrine Signaling Pathways through Metabolic Fingerprinting in Serous Ovarian Cancer Cells

Focusing on defining metabolite-based inter-tumoral heterogeneity in ovarian cancer, we investigated the metabolic diversity of a panel of high-grade serous ovarian carcinoma (HGSOC) cell-lines using a metabolomics platform that interrogate 731 compounds. Metabolic fingerprinting followed by 2-dimensional and 3-dimensional principal component analysis established the heterogeneity of the HGSOC cells by clustering them into five distinct metabolic groups compared to the fallopian tube epithelial cell line control. An overall increase in the metabolites associated with aerobic glycolysis and phospholipid metabolism were observed in the majority of the cancer cells. A preponderant increase in the levels of metabolites involved in trans-sulphuration and glutathione synthesis was also observed. More significantly, subsets of HGSOC cells showed an increase in the levels of 5-Hydroxytryptamine, γ-aminobutyrate, or glutamate. Additionally, 5-hydroxytryptamin synthesis inhibitor as well as antagonists of γ-aminobutyrate and glutamate receptors prohibited the proliferation of HGSOC cells, pointing to their potential roles as oncometabolites and ligands for receptor-mediated autocrine signaling in cancer cells. Consistent with this role, 5-Hydroxytryptamine synthesis inhibitor as well as receptor antagonists of γ-aminobutyrate and Glutamate-receptors inhibited the proliferation of HGSOC cells. These antagonists also inhibited the three-dimensional spheroid growth of TYKNU cells, a representative HGSOC cell-line. These results identify 5-HT, GABA, and Glutamate as putative oncometabolites in ovarian cancer metabolic sub-type and point to them as therapeutic targets in a metabolomic fingerprinting-based therapeutic strategy.

PncA from bacteria improves diet-induced NAFLD by enabling the transition from NAM to NA in mice

Nicotinamide adenine dinucleotide (NAD+) is crucial for energy metabolism, oxidative stress, DNA damage repair, longevity regulation, and several signaling processes. To date, three NAD+ synthesis pathways have been found in microbiota and hosts, but the potential relationship between gut microbiota and their hosts in regulating NAD+ homeostasis remains unknown. Here, we show that an analog of the first-line tuberculosis drug pyrazinamide (a bacterial NAD+ synthesis inhibitor) affected NAD+ levels in the intestines and liver of mice and disrupted the intestinal microecological balance. Furthermore, using microbiota expressing the pyrazinamidase/nicotinamidase (PncA) gene, which is a target of pyrazinamide, hepatic NAD+ levels were greatly increased and significantly increased compared with other NAD+ precursors, and diet-induced non-alcoholic fatty liver disease (NAFLD) in mice was improved. Overall, the PncA gene in microbiota plays an important role in regulating NAD+ synthesis in the host, thereby providing a potential target for modulating the host’s NAD+ level.HighlightsPncA inhibitors disrupt gut microbiome homeostasis and reduce host NAD+ levels but do not affect NAD+ levels in cultured cellsPncA gene in microbiota affects host liver NAD metabolismPncA affects lipid metabolism-related genes and metabolites in mice with NAFLDDiet-induced NAFLD is improved by PncA overexpression in the liver of miceGraphical abstract

Is the suckling period and application pattern relevant for fluazuron against tick infestation in cows and their suckling calves?

Background Fluazuron is a chitin synthesis inhibitor administered as a pour-on formulation in cattle for tick control. This study analyzes under endemic tick infestation, the incidence of the pour-on application pattern on the plasma levels of fluazuron in calves and cows in the lactation period of the beef cow. Two hundred and ninety-two beef cows around parturition were treated with a commercial pour-on formulation of fluazuron at a rate of 2.5 mg/kg of body weight. A total of 4 treatments were carried out on days 0, 32, 77, and 117. At each administration time, the cows were grouped according to the pour-on administration pattern: long (~ 60 cm pour-on application surface) and short (~ 30 cm pour-on application surface). Fluazuron levels in cows and calves plasma were determined before the third and fourth application for each subgroup (n = 10) by HPLC-MS/MS. During the entire study, cow-calf pairs were maintained under field conditions and qualitatively examined for tick infestation on the day of each treatment. Both treatments (long and short) schemes were designed to prevent the annual persistence of ticks. Results No animals with presence of ticks were identified during the first 117 days of the study, except for three cows and one calf at the time of the third application (day 77). There were no differences after 40 days (day 77) post-treatment of the second application (30 ± 5 ppb vs. 28.5 ± 12 ppb, p > 0.05) and 45 days (day 117) after the third application (147 ± 55 ppb vs 140 ± 46 ppb, p > 0.05) between groups of cows treated with the long or short pour-on application, respectively. Plasma concentration of fluazuron at second and third application was increased (3.3 and 2.9 times, respectively) in calves under free suckling compared to cows. Nevertheless, both groups of cows and calves showed a significant increase in plasma concentration of fluazuron between times (4.9 times, p < 0.0001 and 2.8 times, p < 0.0001, respectively). In both groups, tick prevalence was 0% throughout the trial, except for day 77, which reached 1%. Conclusions The main conclusions of this study were the following: 1) Different administration patterns (long vs. short) did not differ in plasma levels of fluazuron.; 2) Given that only the cows were treated and lactating calves presented higher plasma levels of fluazuron than cows, passage through milk appears to be relevant and possibly due to a cumulative effect and continuous drug intake.