Understanding the complexity of Tityus serrulatus venom: A focus on high molecular weight components

Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and ß), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil.

Actividad de fosfolipasas y proteasas en aislamientos de especies de Candida colonizadoras y causantes de vulvovaginitis en mujeres gestantes / Phospholipase and proteinase activities of isolatesof colonizing Candida spp. causing vulvovaginitis in pregnant women

Introducción. Las proteasas y las fosfolipasas son factores de virulencia de Candida spp. que cumplen un papel importante en la invasión de los tejidos. Entre los factores relacionados con el huésped, se encuentran algunos asociados con las características ambientales y otros con la colonización. Objetivo. Determinar la actividad de fosfolipasas y proteasas en aislamientos de especies colonizadoras y patógenas de Candida spp., aisladas de mujeres gestantes de Cartagena de Indias. Materiales y métodos. Se determinó la actividad de fosfolipasas y proteasas en 56 aislamientos mediante degradación del sustrato y cálculo del coeficiente de actividad enzimática. Se compararon las actividades de fosfolipasas y proteasas, entre los aislamientos colonizadores y los patógenos. Resultados. La actividad de la fosfolipasa fue "muy alta" (< 0,69) en 34 aislamientos e, igualmente, la de la proteasa en 14. No hubo diferencias significativas al comparar las actividades de las fosfolipasas y de las de las proteasas, entre los aislamientos colonizadores y los patógenos. Conclusiones. La actividad de las fosfolipasas predominó como factor de virulencia en los aislamientos estudiados. No obstante, no se encontró una diferencia significativa entre los grupos de aislamientos colonizadores y los patógenos, en cuanto a las actividades de fosfolipasas y proteasas.

Introduction. Proteases and phospholipases are virulence factors of Candida spp. that play an important role in tissue invasion. Among the factors related to the host some are associated with environmental characteristics and others with Candida colonization. Objectives. To determine phospholipase and protease activities in colonizing and pathogenic strains, isolated from pregnant women in Cartagena de Indias. Materials and methods. Phospholipase and protease activity was determined in 56 isolates, evaluating substrate degradation and calculating the enzyme activity coefficient. Phospholipase and protease activities were compared between colonizing and pathogenic strains. Results. "Very high" (<0.69) phospholipase and protease activity was found in 34 and 14 isolates, respectively. There was no significant difference when comparing phospholipase and protease activities between colonizing and pathogenic isolates. Conclusions. Phospholipase activity predominated as a virulence factor in the studied strains, but no significant difference was found between colonizing and pathogenic strains for phospholipase and protease activities.

Novel Pathogenic Mutation of PNPLA1 Identified in Autosomal Recessive Congenital Ichthyosis: A Case Report / 中国医学科学杂志(英文版)

Autosomal recessive congenital ichthyosis (ARCI) is characterized by being born as collodion babies, hyperkeratosis, and skin scaling. We described a collodion baby at birth with mild ectropion, eclabium, and syndactyly. Whole exome sequencing showed a compound heterozygous variant c.[56C>A], p.(Ser19X) and c.[100G>A], p.(Ala34Thr) in the PNPLA1 gene [NM_001145717; exon 1]. The protein encoded by PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. The variant was located in the patatin core domain of PNPLA1 and resulted in a truncated protein which could disrupt the function of the protein. This case report highlights a novel compound heterozygous mutation in PNPLA1 identified in a Chinese child.

Synergistic interaction of fluconazole/sodium bicarbonate on the inhibition of Candida glabrata phospholipase gene

Abstract Candida glabrata infections are responsible for deaths of people globally. Fluconazole is known to be less effective against C. glabrata, which developed many strategies to evade being destroyed by fluconazole. To achieve enhanced efficacy of fluconazole against C. glabrata, the interaction of fluconazole with sodium bicarbonate was investigated using the CLSI guidelines. The efficacy of fluconazole alone and in combination with sodium bicarbonate was evaluated using the time-kill and phospholipase production assays. Eventually, the expression of PLB was assessed using semi-quantitative RT-PCR to investigate the inhibitory properties of fluconazole alone and in combination with sodium bicarbonate against C. glabrata. The fluconazole/sodium bicarbonate combination displayed synergistic and antagonistic effects (FICI= 0.375-4.25). In C. glabrata ATCC, SN 152, and SN 164, the fluconazole/sodium bicarbonate combination exhibited a significant fungicidal activity (p< 0.05) but antagonistic effect in the case of SN 283. With exception of SN 283, a significant reduction was noted in phospholipase production in clinical isolates of C. glabrata treated with fluconazole/sodium bicarbonate combination. The PLB was down-regulated significantly by 0.168-0.515 fold in C. glabrata treated with fluconazole/sodium bicarbonate. The results suggested fluconazole/sodium bicarbonate to have a potential synergistic interaction in C. glabrata, and the underlying mechanism may be associated with phospholipase gene

High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Phlogiellus bundokalbo spider venom: Its neuroactive, phospholipase a2 and cytotoxic components against human breast adenocarcinoma (MCF-7)

@#Spider venoms and toxins are valuable sources of lead compounds for drug development due to their essential role in cellular and physiological processes targeting various receptors. Here, we present the protein profile of the venom of Phlogiellus bundokalbo, an endemic Philippine tarantula, to screen and characterize its cytotoxicity against MCF-7 cells, secretory phospholipase a2 (sPLA2), and neurotoxicity to evaluate its potential anticancer properties. Spider venom was extracted via electrical stimulation. Venom components were fractionated by reversed-phase high-performance liquid chromatography and characterized through liquid chromatography-mass spectrometry (LC-MS) and SDS-PAGE analysis before assay. The resulting five venom fractions were amphiphilic peptides showing cytotoxicity against MCF-7 cells in a concentrationdependent manner (IC50 ranging from 52.25μg/ml to 110.20μg/ml) after 24-hour incubation. Cells appeared detached, rounded, and shrunk with cytoplasmic condensation upon overnight incubation with venom fractions. The sPLA2 was observed in all the venom fractions tested for cytotoxicity. Venom fractions revealed a predominant mass of ~3-5 kDa with LC-MS analysis. Results showed distinct similar mass as μ- theraphotoxin-Phlo1a, an Australian tarantula, Phlogiellus sp. toxin with inhibitor cystine knot motif. The venom fractions exhibit excitatory neurotoxins that might activate presynaptic voltage-gated ion channels, such as an agonist or gating modifier toxins that slow down the channel inactivation similar to spider toxins. In conclusion, the spider venom of P. bundokalbo exhibits cytotoxic, phospholipase A2, and neuroactive properties suggesting that its venom components, upon further purification and structure-function analysis, can be potential tools in the development of targeted breast chemotherapeutics.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2⁺-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression

BACKGROUND: Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated.METHODS: The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2⁺-oscillation was also investigated.RESULTS: RMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2⁺-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2.CONCLUSIONS: RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.(AU)

Severity of Nonalcoholic Fatty Liver Disease in Type 2 Diabetes Mellitus: Relationship between Nongenetic Factors and PNPLA3/HSD17B13 Polymorphisms

BACKGROUND: The prevalence of nonalcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM) is high, though its severity is often underestimated. Our aim is to provide an estimate of the prevalence of severe NAFLD in T2DM and identify its major predictors. METHODS: T2DM patients (n=328) not previously known to have NAFLD underwent clinical assessment, transient elastography with measure of liver stiffness (LS) and controlled attenuation parameter (CAP), and genotyping for patatin like phospholipase domain containing 3 (PNPLA3) and 17β-hydroxysteroid-dehydrogenase type 13 (HSD17B13). RESULTS: Median LS was 6.1 kPa (4.9 to 8.6). More than one-fourth patients had advanced liver disease, defined as LS ≥7.9 kPa (n=94/238, 29%), and had a higher body mass index (BMI) than those with a LS <7.9 kPa. Carriage of the G allele in the PNPLA3 gene was associated with higher LS, being 5.9 kPa (4.7 to 7.7) in C/C homozygotes, 6.1 kPa (5.2 to 8.7) in C/G heterozygotes, and 6.8 kPa (5.8 to 9.2) in G/G homozygotes (P=0.01). This trend was absent in patients with ≥1 mutated HSD17B13 allele. In a multiple linear regression model, BMI and PNPLA3 genotype predicted LS, while age, gender, disease duration, and glycosylated hemoglobin did not fit into the model. None of these variables was confirmed to be predictive among carriers of at least one HSD17B13 mutated allele. There was no association between CAP and polymorphisms of PNPLA3 or HSD17B13. CONCLUSION: Advanced NAFLD is common among T2DM patients. LS is predicted by both BMI and PNPLA3 polymorphism, the effect of the latter being modulated by mutated HSD17B13.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

New Perspectives in Pediatric Nonalcoholic Fatty Liver Disease: Epidemiology, Genetics, Diagnosis, and Natural History / 대한소아소화기영양학회지

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in children. The global prevalence of pediatric NAFLD from general populations is 7.6%. In obese children, the prevalence is higher in Asia. NAFLD has a strong heritable component based on ethnic difference in the prevalence and clustering within families. Genetic polymorphisms of patatin-like phospholipase domain–containing protein 3 (PNPLA3), transmembrane 6 superfamily member 2, and glucokinase regulatory protein (GCKR) are associated with the risk of NAFLD in children. Variants of PNPLA3 and GCKR are more common in Asians. Alterations of the gut microbiome might contribute to the pathogenesis of NAFLD. High fructose intake increases the risk of NAFLD. Liver fibrosis is a poor prognostic factor for disease progression to cirrhosis. Magnetic resonance spectroscopy and magnetic resonance proton density fat fraction are more accurate for steatosis quantification than ultrasound. Noninvasive imaging methods to assess liver fibrosis, such as transient elastography, shear-wave elastography, and magnetic resonance elastography are useful in predicting advanced fibrosis, but they need further validation. Longitudinal follow-up studies into adulthood are needed to better understand the natural history of pediatric NAFLD.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca2+ in Cat Esophageal Smooth Muscle Cells

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.

Inhibitory effects of 2,6-di-tert-butyl-4-hydroxymethylphenol on asthmatic responses to ovalbumin challenge in conscious guinea pigs

This study evaluated the anti-asthmatic activities of 2,6-di-tert-butyl-4-hydroxymethylphenol (DBHP) that is a potent phenolic antioxidant in edible vegetable oil. The effects of DBHP on bronchial asthma were evaluated by determining the specific airway resistance (sRaw) and tidal volume (TV) during the immediate asthmatic response (IAR) and the late-phase asthmatic response (LAR) in guinea pigs with aerosolized ovalbumin-induced asthma. Recruitment of leukocytes and the levels of biochemical inflammatory mediators were determined in the bronchoalveolar lavage fluids (BALFs), and histopathological surveys performed in lung tissues. DBHP significantly inhibited the increased sRaw and improved the decreased TV on IAR and LAR, and also inhibited recruitment of eosinophils and neutrophils into the lung, and release of biochemical inflammatory mediators such as histamine and phospholipase A₂ from these infiltrated leukocytes, and improved pathological changes. However, anti-asthmatic activities of DBHP at oral doses of 12.5 to 50 mg/kg was less than those of dexamethasone (5 mg/kg, p.o.) and cromoglycate (10 mg/kg, p.o.), but more potent or similar to that of salbutamol (5 mg/kg, p.o.). These results in the present study suggest that anti-asthmatic effects of DBHP in the guinea pigs model of OVA-induced asthmatic responses principally are mediated by inhibiting the recruitments of the leukocytes and the release of biochemical inflammatory mediators from these infiltrated leukocytes.

In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

Abstract INTRODUCTION: Candida parapsilosis complex species, frequently found in hospital environments, have gained importance as etiological agents of candidemia. METHODS: Candida parapsilosis complex isolates from a nosocomial environment were identified and their hydrolitic enzyme activity and ability to form biofilm were characterized. RESULTS: Twenty-two C. parapsilosis sensu stricto isolates produced proteinase and three produced phospholipase. Most Candida metapsilosis isolates produced proteinase and one also produced phospholipase. All 29 isolates formed biofilms. CONCLUSIONS: The nosocomial environment may act as a reservoir for C. parapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients

Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Epidemiology of Nonalcoholic Fatty Liver Disease: Focusing on the Korean Genome Epidemiology Study (KoGES) / 임상당뇨병

The Korean Genome Epidemiology Study (KoGES) is a principal cohort study providing valuable evidence for the prevention of major chronic diseases such as hypertension, obesity, and diabetes in Korea. Since 2001, the Ansan-Anseong cohort is one of the representative cohorts in the KoGES and recruited about 10,000 participants from Ansan and Anseong city to undergo a comprehensive health examination biennially. About 3,000 participants in the Ansan cohort underwent abdominal computed tomography scan to detect the presence of nonalcoholic fatty liver disease (NAFLD). The prevalence of NAFLD was about 23% in this study, and it was twice as high in subjects with diabetes compared to those without. Subjects with NAFLD had early diastolic dysfunction in tissue Doppler study and showed lower vitamin D concentrations than those without. We also found that the palatin-like phospholipase domain containing 3 single nucleotide polymorphisms were significantly associated with NAFLD. Interim prospective analysis after six years showed that NAFLD was associated with worsening of metabolic risk factors and an about 2.6 higher likelihood of developing diabetes than in those without. These results present the clinical importance of the diagnosis and treatment of NAFLD to reduce the future development of diabetes and cardiovascular diseases.

[Detection of phospholipase and type IV pili genes in clinical isolates of multidrug-resistant Pseudomonas aeruginosa]

Background and Objective: Pseudomonas aeruginosa is an opportunistic pathogen with numerous virulence factors such as phospholipase and type IV pili. The emergence of multidrug resistant Pseudomonas aeruginosa has become a serious public health threat worldwide. This study was done to determine the frequency of plcH, plcN, pilA and pilB genes in multi-drug resistant Pseudomonas aeruginosa isolated from clinical samples

Methods: In this cross-sectional study, 93 isolates of Pseudomonas aeruginosa collected from different clinical samples from hospitals of Zanjan, Iran during 2013-14. After identification of isolates by biochemical tests, antibiotic susceptibility testing [Kirby-Bauer] was performed according to CLSI guidelines. Total DNA extracted and PCR was done to detect of plcH, plcN, pilA and pilB genes

Results: Among 93 of Pseudomonas aeruginosa isolates, the highest antibiotic resistance related to Erythromycin and Cefoxitin [95.6%] and the lowest resistance related to Amikacin [26.8%]. 80.6% of isolates were multidrug resistant [MDR]. Out of 75 MDR isolates, the frequency of plcH, plcN, pilA and pilB genes was 97.4%, 49.3%, 26.6% and 17.3%, respectively

Conclusion: According to high frequency of phospholipase C gene [plcH] in MDR Pseudomonas aeruginosa isolates which isolated from different clinical samples, presumably this virulence factor plays an important role in pathogenesis of this bacterium

Dust particles-induced intracellular Ca²⁺ signaling and reactive oxygen species in lung fibroblast cell line MRC5

Epidemiologic interest in particulate matter (PM) is growing particularly because of its impact of respiratory health. It has been elucidated that PM evoked inflammatory signal in pulmonary epithelia. However, it has not been established Ca²⁺ signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular Ca²⁺ signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular Ca²⁺ signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than 10 µm, induced intracellular Ca²⁺ signaling, which was mediated by extracellular Ca²⁺. The PM10-mediated intracellular Ca²⁺ signaling was attenuated by antioxidants, phospholipase blockers, polyADPR polymerase 1 inhibitor, and transient receptor potential melastatin 2 (TRPM2) inhibitors. In addition, PM-mediated increases in reactive oxygen species were attenuated by TRPM2 blockers, clotrimazole (CLZ) and N-(p-amylcinnamoyl) anthranilic acid (ACA). Our results showed that PM10 enhanced reactive oxygen species signal by measuring DCF fluorescence and the DCF signal attenuated by both TRPM2 blockers CLZ and ACA. Here, we suggest functional inhibition of TRPM2 channels as a potential therapeutic strategy for modulation of dust particle-mediated signaling and oxidative stress accompanying lung diseases.