Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR^® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr?≤?1.65%, R² values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

     

Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties

Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments where they are concomitantly subjected to several types of stress, such as toxicity, scarce nutrient availability, and high or low temperature extremes. Their ability to assimilate alkylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions, including on railway ties. Samples were collected using cotton swabs, premoistened with physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt extract agar supplemented with chloramphenicol, and incubated at 26 °C for 4 weeks. Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P = 0.02), tested positive for fungi. Exophiala crusticola was found to be the most common species (n = 13), followed by Exophiala phaeomuriformis (n = 7) and Exophiala heteromorpha (n = 4). These results suggest that hydrocarbons, particularly creosote-treated oak woods, support the growth of black yeasts, some of which are opportunists in humans.     

The Use of Albino Adult Hair and Blond Prepubertal Hair Yields Equivalent Results in an In Vitro Hair Perforation Test to Differentiate Between Different Dermatophytic Fungi

An in vitro hair perforation test is used to differentiate isolates of Trichophyton mentagrophytes and Trichophyton rubrum complexes because morphological criteria are insufficient. Here, we performed in vitro hair perforation tests using blond prepubertal hair and albino adult hair to determine whether they differentiate between fungal species. We tested 43 well-characterized dermatophyte strains, Arthroderma spp. [n = 4], Epidermophyton floccosum [n = 1], Microsporum spp. [n = 8], and Trichophyton spp. [n = 30], and examined hair perforation at 3–30 days postinoculation (p.i.). The perforation times were not significantly different between the two hair types (P > 0.05). The T. mentagrophytes complex strains perforated hair 4–5 days p.i., whereas T. rubrum complex strains perforated hair 13–30 days p.i., except for Trichophyton violaceum, which perforated hair after 6–7 days. Thus, the hair perforation test is highly sensitive (100 %) and specific (100 %) for differentiating T. mentagrophytes from T. rubrum complexes 5 days p.i. At 14 and 30 days, the sensitivity and negative predictive value of the test remained unchanged (100 %), but the specificity was reduced (64.3 and 14.3 %, respectively). Consistent with previous reports, we observed “perforating organs” of zoophilic Microsporum canis and geophilic Microsporum gypseum at 4 and 3 days, respectively. This paper offers a “low-cost” and “low-tech” alternative to differentiating dermatophyte species where standard morphological techniques fail and/or where molecular techniques are not a viable option.     

In vitro antimicrobial activity of propolis samples from different geographical origins against certain oral pathogens

Propolis is an agent having antimicrobial properties, however, its composition can vary depending on the area where it is collected. In the present study, the antimicrobial activity of five propolis samples, collected from four different regions in Turkey and from Brazil, against nine anaerobic strains was evaluated. Ethanol extracts of propolis (EEP) were prepared from propolis samples and we determined minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEP on the growth of test microorganisms by using agar dilution method. All strains were susceptible and MIC values ranged from 4 to 512 microg/ml for propolis activity. Propolis from Kazan-Ankara showed most effective MIC values to the studied microorganisms. MBC values of Kazan-Ankara EEP samples were ranged from 8 to 512 microg/ml. Death was observed within 4 h of incubation for Peptostreptococcus anaerobius and micros and Lactobacillus acidophilus and Actinomyces naeslundii, while 8 h for Prevotella oralis and Prevotella melaninogenica and Porphyromonas gingivalis, 12 h for Fusobacterium nucleatum, 16 h for Veillonella parvula. It was shown that propolis samples were more effective against Gram positive anaerobic bacteria than Gram negative ones. The organic chemical compositions of EEPs were determined by high-resolution gas chromatography coupled to mass spectrometry (GC-MS). The main compounds of EEPs were flavonoids such as pinobanksin, quercetin, naringenin, galangine, chrysin and aromatic acids such as cafeic acid. Because of increased antimicrobial resistance, propolis may be kept in mind in the treatment of oral cavity diseases.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.     

High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3M^TM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.     

Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule

The ability to osmoregulate is fundamental to life. Adult Drosophila melanogaster maintain hemolymph osmolarity within a narrow range. Osmolarity modulates transepithelial ion and water flux in the Malpighian (renal) tubules of the fly, which are in direct contact with hemolymph in vivo, but the mechanisms causing increased transepithelial flux in response to hypotonicity are unknown. Fly renal tubules secrete a KCl-rich fluid. We have previously demonstrated a requirement for Ncc69, the fly sodium-potassium-2-chloride cotransporter (NKCC), in tubule K⁺ secretion. Mammalian NKCCs are regulated by a kinase cascade consisting of the with-no-lysine (WNK) and Ste20-related proline/alanine-rich (SPAK)/oxidative stress response (OSR1) kinases. Here, we show that decreasing Drosophila WNK activity causes a reduction in K⁺ flux. Similarly, knocking down the SPAK/OSR1 homolog fray also decreases K⁺ flux. We demonstrate that a hierarchical WNK-Fray signaling cascade regulates K⁺ flux through Ncc69, because (i) a constitutively active Fray mutant rescues the wnk knockdown phenotype, (ii) Fray directly phosphorylates Ncc69 in vitro, and (iii) the effect of wnk and fray knockdown is abolished in Ncc69 mutants. The stimulatory effect of hypotonicity on K⁺ flux is absent in wnk, fray, or Ncc69 mutant tubules, suggesting that the Drosophila WNK-SPAK/OSR1-NKCC cascade is an essential molecular pathway for osmoregulation, through its effect on transepithelial ion flux and fluid generation by the renal tubule.     

The regulatory effects of clomiphene and tamoxifen on mTOR and LC3-II expressions in relation to autophagy in experimental polycystic ovary syndrome (PCOS)

Molecular Biology Reports - Polycystic ovary syndrome (PCOS) is a metabolic disease that causes infertility due to anovulation in women in reproductive age. It is known that clomiphene citrate (CC)...