24-h Incubation Research Articles

Identification of cytochrome P450 gene family and functional analysis of HgCYP33E1 from Heterodera glycines.

The cytochrome P450 (CYP) genes of nematode play a crucial role in the metabolic detoxification of xenobiotics including pesticides. Heterodera glycines, also known as the soybean cyst nematode, is a sedentary endoparasite that infests plant roots, causing high annual economic losses in soybean production regions globally. In this study, we identified 36 CYP genes at a genome-wide level of the H. glycines isolate TN10 using all CYPs from Caenorhabditis elegans as queries. Subsequently, a full-length cDNA of HgCYP33E1 which was significantly up-regulated by the conventional nematicide abamectin was initially cloned from H. glycines. It presented significantly higher expressions in the second-stage juvenile (J2) compared to other parasitic stages of H. glycines. qRT-PCR analysis suggested that the expression of HgCYP33E1 was also xenobiotically induced by soybean root exudate and the metabolites of biocontrol agents. Using RNA interference (RNAi), we investigated the function of HgCYP33E1 in H. glycines parasitism and nematicide selectivity. Compared to the control and dsGFP-treated group, silencing of HgCYP33E1 did not affect the J2 behaviors and the early invasion ability, while it decreased the number of J4s in soybean roots after 18-d inoculation with the dsHgCYP33E1-treated nematodes. In addition, knockdown of HgCYP33E1 in H. glycines resulted in an increase in J2 mortality after 24-h incubation with abamectin compared to the GFP dsRNA-soaked and the control group. These findings revealed the potential role of HgCYP33E1 in the xenobiotic detoxification pathway of H. glycines. Moreover, our data also provided valuable gene information for studying the functions of the CYP family in H. glycines host adaption.

Cytotoxicity of poly-guanidine in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common pediatric brain tumor. The therapy frequently causes serious side effects, and new selective therapies are needed. MB expresses hyper sialylation, a possible target for selective therapy.The cytotoxic efficacy of a poly guanidine conjugate (GuaDex) incubated with medulloblastoma cell cultures (DAOY and MB-LU-181) was investigated. The cells were incubated with 0.05-8µM GuaDex from 15min to 72h. A fluorometric cytotoxicity assay (FMCA) measured the cytotoxicity. Labeled GuaDex was used to study tumor cell interaction. FITC-label Sambucus nigra confirmed high expression of sialic acid (Sia). Immunofluorescence microscopy was used to visualize the cell F-actin and microtubules. The cell interactions were studied by confocal and fluorescence microscopy. Annexin-V assay was used to detect apoptosis. Cell cycle analysis was done by DNA content determination. A wound-healing migration assay determined the effects on the migratory ability of DAOY cells after GuaDex treatment.IC50 for GuaDex was 223.4 -281.1nM. FMCA showed potent growth inhibition on DAOY and MB-LU-181 cells at 5 uM GuaDex after 4h of incubation. GuaDex treatment induced G2/M phase cell cycle arrest.S. nigraFITC-label lectin confirmed high expression of Sia on DAOY medulloblastoma cells. The GuaDex treatment polymerized the cytoskeleton (actin filaments and microtubules) and bound to DNA, inducing condensation. The Annexin V assay results were negative. Cell migration was inhibited at 0.5µM GuaDex concentration after 24h of incubation.GuaDex showed potent cytotoxicity and invasion-inhibitory effects on medulloblastoma cells at low micromolar concentrations. GuaDex efficacy was significant and warrants further studies.

Combined antimicrobial effect of phage-derived endolysin and depolymerase against biofilm-forming Salmonella Typhimurium

This study was designed to evaluate the antimicrobial activity of phage-derived endolysin (LysPB32) and depolymerase (DpolP22) against planktonic and biofilm cells of Salmonella Typhimurium (STKCCM). Compared to the control, the numbers of STKCCM were reduced by 4.3 and 5.9 log, respectively, at LysPB32 and LysPB32 + DpolP22 in the presence of polymyxin B (PMB) after 48-h incubation at 37 °C. LysPB32 + DpolP22 decreased the relative fitness (0.8) and the cross-resistance of STKCCM to chloramphenicol (CHL), cephalothin (CEP), ciprofloxacin (CIP), and tetracycline (TET) in the presence of PMB. The MICtrt/MICcon ratios of CHL, CEP, CIP, PMB, and TET were between 0.25 and 0.50 for LysPB32 + DpolP22 in the presence of PMB. These results suggest that the application of phage-encoded enzymes with antibiotics can be a promising approach for controlling biofilm formation on medical and food-processing equipment. This is noteworthy in that the application of LysPB32 + DpolP22 could increase antibiotic susceptibility and decrease cross-resistance to other antibiotics.

Dual functionalized hyaluronic acid micelles loading paclitaxel for the therapy of breast cancer.

Although many carriers for the delivery of chemotherapeutic drugs have been investigated, the disadvantages of passive targeting and uncontrolled drug release limit their utility. Herein, hyaluronic acid (HA) was hydrophobically modified to serve as a carrier for binding to cluster determinant 44 (CD44) overexpressed on tumor cell surfaces. Specifically, after deacetylation, HA was grafted to dodecylamine or tetradecylamine to afford amphiphilic zwitterionic polymer micelles, designated dHAD and dHAT, respectively, for the delivery of paclitaxel (PTX). The micelles were negatively charged at pH 7.4 and positively charged at pH 5.6, and this pH sensitivity facilitated PTX release under acidic conditions. The cell uptake efficiencies of the dHAD-PTX and dHAT-PTX micelles by MCF-7 cells after 4h of incubation were 96.9% and 95.4%, respectively, and their affinities for CD44 were twice that of HA. Furthermore, the micelles markedly inhibited tumor growth both in vitro and in vivo, with IC50 values of 1.943μg/mL for dHAD-PTX and 1.874μg/mL for dHAT-PTX for MCF-7 cells; the tumor inhibition rate of dHAD-PTX (92.96%) was higher than that of dHAT-PTX (78.65%). Importantly, dHAD and dHAT micelles showed negligible systemic toxicity. Our findings suggest that these micelles are promising delivery vehicles for antitumor drugs.

PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10

Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

Due to antibiotic tolerance of microbes within a biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for short-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid (T2DA), which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. Titanium coupons were drop coated with 0.75mg of DSF in ethanol and dried. Surface characteristics and presence of DSF were confirmed with Fourier Transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), and water contact angle. Antimicrobial assays on 12-well plates analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-hour incubation, but only sustained through 72 hours for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-hour time point, but not through 72 hours for E. coli. Although approximately 60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. In this study we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Toward Understanding the Environmental Risks of Combined Microplastics/Nanomaterials Exposures: Unveiling ZnO Transformations after Adsorption onto Polystyrene Microplastics in Environmental Solutions.

Over recent decades, there has been a dramatic increase in the manufacture of engineered nanomaterials, which has inevitably led to their environmental release. Zinc oxide (ZnO) is among the more abundant nanomaterial manufactured due to its advantageous properties, used for piezoelectric, semiconducting, and antibacterial purposes. Plastic waste is ubiquitous and may break down or delaminate into smaller microplastics, leaving open the question of whether these small polymers may alter the fate of ZnO through adsorption within aquatic media (tap-water and seawater). Here, scanning electron microscopy analysis confirms the effective Zn nano/microstructures adsorption onto polystyrene surfaces after only 24-h incubation in the aquatic media. After pre-aging the nanomaterials for 7-days in different environmental media, nanoprobe X-ray absorption near-edge spectroscopy analysis reveals significant ZnO transformation toward Zn-sulfide and Zn-phosphate. The interaction between a commercial ZnO-based sunscreen with polystyrene and a cleanser consumer containing microbeads with ZnO nanomaterials is also studied, revealing the adsorption of transformed Zn-species in the microplastics surfaces, highlighting the environmental relevancy of this work. Understanding the structural and functional impacts of the microplastics/ZnO complexes, and how they evolve, will provide insights into their chemical nature, stability, transformations, and fate, which is key to predicting their bioreactivity in the environment.

Effects of okra (Abelmoschus esculentus L.) leaves, fruits and seeds extracts on European sea bass (Dicentrarchus labrax) leukocytes, and their cytotoxic, bactericidal and antioxidant properties.

Okra, Abelmoschus esculentus L., is a popular vegetable crop with many bioactive compounds. The in vitro immunostimulant, cytotoxic, bactericidal and antioxidant activities of ethanolic extracts obtained from different parts of okra (leaves, fruits, and seeds) were studied. Phytochemical screening of hydroalcoholic extracts of okra leaves, fruits and seeds revealed a significant content of total phenols and flavonoids. Notable effects on the activities of leukocytes in the head kidney of European sea bass (Dicentrarchus labrax) (viability, phagocytic ability and capacity, and respiratory burst), as well as on peroxidase leukocyte contents were detected after incubation for 24h with different concentrations (0.01-1mgmL-1) of the extracts. The mean concentrations (0.1 and 0.5mgmL-1) of the different extracts increased the phagocytic ability and respiratory activity of head kidney leukocytes. However, the mean concentrations (0.1mgmL-1) of leaf and fruit extracts significantly decreased the peroxidase activity of leukocytes. In addition, all ethanolic okra extracts at higher concentrations (1mgmL-1) produced a marked reduction in the viability of the DLB-1cell line compared to the viability recorded in the control samples. In addition, ethanolic extracts used at 0.5 and 1mgmL-1had a significant cytotoxic effect on the viability of PLHC-1cells. Finally, all doses of seed and leaf extracts at higher concentrations (0.5 and 1mgmL-1) showed significant bactericidal activity on two fish pathogenic bacteria, Vibrio anguillarum and V. harveyi strains. Finally, a remarkable antioxidant activity was detected on the ethanolic extracts. All these results point to their possible use as an alternative to chemical compounds in farmed fish.

Parafilm as an efficient transport matrix for corneal scrapings.

Acanthamoeba spp. are free-living parasites increasingly implicated in causing Acanthamoeba keratitis (AK). AK is diagnosed by demonstration of parasites in corneal samples by direct microscopy, culture, and nucleic acid amplification. Most commonly, corneal scrapings are sent to the laboratory smeared between two glass slides. These scrapings are suitable for direct microscopy but less suitable for culture and polymerase chain reaction (PCR) which, in turn, are more sensitive for the diagnosis of AK. The aim of the study was to explore better alternatives for transporting corneal scrapings from the point-of-care eye center to the concerned laboratories. The study used small Parafilm (Bemis Company Inc., USA) squares (PSs) of 1 cm each prepared by cutting Parafilm using a surgical blade under sterile conditions. Each of the four different dilutions of Acanthamoeba suspension (15, 30, 60, and 120 cells) was used in this study. Each dilution was added onto the surface of 36 PSs and kept at room temperature for 24-h, 48-h, and 72-h incubation. The PSs for one particular time point and dilution were used for calcofluor white staining, its inoculation onto the surface of nonnutrient agar having a lawn of Escherichia coli, and Acanthamoeba-specific PCR amplification. In addition, two PSs inoculated with 30 cells and incubated for 24 h and 72 h were used for scanning electron microscopy (SEM). All three diagnostic techniques, i.e. microscopy, culture, and PCR, detected the presence of Acanthamoeba at all the tested concentrations and time points. However, the growth pattern on culture changed directly in proportion to increased incubation periods and increased concentration of inoculum. In addition, the adherence of Acanthamoeba to the Parafilm was confirmed by SEM; these results suggest the use of these PSs as a suitable matrix for the transport of corneal scrapings.

Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2)

Article Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2) Haishan Xu 1,#, Jinhong Duan 1,#, Jun Tao 2, Wen Wang 3, Yunqing Wu 1,^, Shunling Dai 1,*, and Jun Ren 4,5, 1 Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005 China 2 Department of Cardiovascular Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510000 China 3 Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 4 Department of Cardiology, Zhongshan Hospital Fudan University, Shanghai 200032, China 5 National Clinical Research Center for Interventional Medicine, Shanghai 200032, China # These two authors contributed equally to this work ^ Deceased * Correspondence: daishunling@aliyun.com (Shunling Dai); corresponding author:jren_aldh2@outlook.com (Jun Ren) Abstract: It is perceived that oxidized low density lipoprotein (oxLDL) perturbs endothelial function and fosters endothelin-1 (ET-1) secretion although the underlying mechanism remains elusive. This study was designed to decipher potential mechanisms underscoring oxLDL-evoked regulation of ET-1 and signaling pathways involved in endothelial cells. ET-1 mRNA expression, secretion and promoter function were determined using RT-PCR, enzyme immunometric and luciferase assays, respectively. GO and GSEA bioinformatics analyses depicted differentially expressed genes (DEGs) mainly associated with cell proliferation, cell division, cellular structure, energy supply, and apoptosis in oxLDL-challenged endothelial cells. Incubation of oxLDL overtly increased ROS production, apoptosis, mRNA level, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), the effects were mitigated by N-Acetyl Cysteine (NAC). Moreover, oxLDL challenge evoked phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) in HUVECs, the effect was reversed by NAC and MEK inhibitor PD98059. NAC and PD98059 nullified oxLDL- induced rises in mRNA expression, secretion and promoter activity of ET-1. Truncation of 5’-flanking sequence of ET-1 (–566 bpLuc to –250 bpLuc) displayed elevated luciferase activity with 24-h oxLDL incubation. Fusion plasmid from –233 and –185 bp Luc drastically dampened luciferase activity in basal and oxLDL-challenged HUVECs. Transfection of reporter construct –250 bp Luc with a 2 bp mutation at AP-1 locus, removed basal and oxLDL- evoked rises in ET-1 promoter activity. Collectively, our findings support that oxLDL evoked activation of ERK1/2 signaling likely through ROS production, en route to upregulation of endothelial transcriptional factor AP-1, resulting in expression and secretion of ET-1.

P-078 In vitro effects of polycarbophil vaginal moisturizing gel on sperm motility and vitality

Abstract Study question The aim of this study was to evaluate the possible effects of intravaginal polycarbophil gel in sperm quality in an in vitro model. Summary answer Incubation of human sperm samples with a vaginal moisturizer containing polycarbophil gel 10% resulted in important reduction of sperm motility and vitality. What is known already The possible effect of lubricants on sperm quality has been a matter of debate for over 50 years. Despite the publication of a few studies, the real impact of lubricants on male gametes is remains unknown. Some commercial lubricants have been tested but so far there is a lack of studies evaluating the impact of vaginal moisturizers on seminal quality. Vaginal dryness is a recurring and significant complaint affecting couples trying to conceive who may use lubricants, vaginal moisturizers or even natural oils that could advsersely interfere with seminal quality. Study design, size, duration This paired prospective study involved seminal samples obtained fom 14 men undergoing fertility treatment at a private IVF clinic in Brazil from August/2020 to July/2022. The study was approved by the local review board and each participant signed an informed consent. Each sample was divided into two aliquots: 20% polycarbophil gel (final dilution 10%) or medium only. Statistical analysis was performed using D’Agostino/ Pearson, General Linear Model, Wilcoxon, Friedman’s. P values <0.05 were considered significant. Participants/materials, setting, methods Normozoospermic samples of 14 patients (alpha 0.05, power 0.8)were collected by masturbation, 2 to 5 days after the last ejaculation. After liquefaction ,10 μmL were analyzed using a Makler chamber according to World Helath Organization (WHO) guidelines. Samples were mixed 1:1 v/v with 20% policarbophil gel or with culture medium only, then incubated for 24h at 34 °c. Analysis were performed at 0, 15, 30 min and 24h to determine progressive motility and vitality (eosin-nigrosin test) Main results and the role of chance There was a significant decrease of sperm motility according to the WHO classification in both experimental conditions over time, but the reduction was more evident in the samples treated with polycarbophil gel (p < 0.0001 group x time, General LinearModel for Repeated Measure). The proportion of progressively motile sperm reduced immediately upon contact of the seminal sample with polycarbophil gel (time point 0 min) and decreased throughout the experiment, contrasting to incubation with culture medium (control), which only affected substantially sperm motility after 24h. In the validation cohort, the proportion of progressively motile sperm decreased from a median of 52% in fresh samples to 11% after 24h incubation with culture medium and 0% after 24h incubation with polycarbophil gel (p < 0.0001). Sperm vitality at the end of the experiment also differed significantly between treatments, with medians of 62.5% after exposure to culture medium and 47% after exposure to polycarbophil gel (p = 0.020, Wilcoxon’s test) Limitations, reasons for caution Results represent samples from a selected group of men undergoing fertility treatment at a private center in Brazil. The effect of polycarbophil gel in fertile males may be different. Moreover, the in vitro setting used here does not accurately mimic the vaginal environment which could interfere with the results. Wider implications of the findings Polycarbophil-based gels and similar products may harm sperm vitality and motility, therefore woment trying to conceive should avoid the use of such products until more studies prove otherwise. Trial registration number Not applicable

P-018 Structural and biocompatibility analysis of gold nanoparticle integrated polymeric scaffolds for infertility diagnosis

Abstract Study question Can infertility diagnosis and sperm selection be improved by using bio- nanotechnologies? Summary answer Gold nanoparticle integrated biopolymeric scaffolds show good biocompatibility when interacting with human sperm cells, being suitable to be used as cell sorting and biosensing platforms. What is known already Microfluidic platforms are used in sperm selection for increasing the in-vitro fertilization success but they are limited by the exclusive analysis of sperm cell motility. The integration of microfluidic platforms with nanomaterials such as nanopatterned substrates can greatly enhance the selectivity and the sensitivity of the detection. The accuracy can be further improved by employing the intrinsic properties of gold nanoparticles (optical properties, high biocompatibility, surface chemistry favoring the conjugation of specific biomolecules), which makes them suitable for the successful development of precise and reliable detection assays, so far used to a limited extent in infertility research and clinical approaches. Study design, size, duration Concentration, motility and morphology of spermatozoa were analyzed in semen samples collected for spermogram. The normozoospermic and teratozoospermic samples were selected and prepared by density gradient centrifugation. The sediment obtained contained 100% motile spermatozoa, which were incubated on the thin films for 24h. After 24h, the probe from each incubation dish was collected and the survival rate was analyzed using the Makler chamber. Participants/materials, setting, methods Thin films of atactic polystyrene, chitosan and mixtures of each of the two polymers with gold nanoparticles were used to produce scaffolds of microchannels. The substrates were morphologically characterized by optical microscopy, scanning transmission electron microscopy and atomic force microscopy. Normozoospermic and teratozoospermic sperm samples were collected and incubated on the scaffolds. After 24h incubation, the viability was assessed by counting the motile spermatozoa under microscopic observation. Statistical analysis was performed using two-way ANOVA test. Main results and the role of chance Analysis of the sperm cell viability incubated on the polymeric scaffolds revealed good biocompatibility with higher survival rate in the case of chitosan-gold nanoparticle integrated samples. In multiple comparisons of normozoospermic samples, the survival rates of the sample incubated on the chitosan thin films to the control sample were 18% to 80% (-62%) (p = 0.0143), 16.66% to 45% (-28.34%) (p = 0.0143), 0% to 15.38% (-15.38%) (p = 0.0239), 4.76% to 55.35% (-50.59%) (p = 0.0239). In multiple comparisons of teratozoospermic samples, the survival rates of the sample incubated on the chitosan thin films to the control sample were 14.67% to 41% (-26.33%) (p = 0.0143), 0% to 44.82% (-33.82%) (p = 0.0239). For polystyrene, the viability was 0% which excludes its further use in infertility-related assays due to its high cytotoxicity. Overall, the colloidal gold nanoparticles integrated into the polymeric matrix improved the biocompatibility of the films. For example, citrate gold nanoparticles of spherical shape and sizes of about 20 nm increased the survival rate of normozoospermic samples, while PEGylation of nanoparticles showed further improvement of the biocompatibility. The influence of the particle type, composition and coating material onto the spermatozoa viability is currently under investigation for optimizing the effectiveness of the polymeric scaffolds. Limitations, reasons for caution NA Wider implications of the findings Due to the unique properties of gold nanoparticles, the nanoparticle-polymer scaffold systems have the potential to become the foundation of a precise biosensor that can enhance the accuracy of the current approaches in infertility assessment. Trial registration number Not applicable

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0–11.0) and thermal tolerance (4–60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30–4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Preparation of injectable hydrophilic dextran/AgNPs nanocomposite product: White light active biomolecules as an antitumor agent

Incidence of various cancers including melanoma continues to rise worldwide. While treatment options have expanded in the recent years, the benefit of these treatments suffer from short period of duration for many patients. Hence, new treatment options are highly desired. Here, we propose a method combining a Dextran/reactive-copolymer/AgNPs nanocomposite and a harmless visible light approach to obtain a plasma substitute carbohydrate-based nanoproduct (D@AgNP) that shows strong antitumor activity. Light-driven polysaccharide-based nanocomposite provided essential conditions for extra small (8-12nm) AgNPs capping with subsequent specific self-assembly into spherical-like cloud nanostructures. Obtained biocompatible D@AgNP are stable over six months at room temperature and demonstrated absorbance peak at 406 nm. New formulated nanoproduct revealed efficient anticancer properties against A375 with IC50 0.0035 mg/mL following 24-h incubation; complete cell death is achieved at 0.001 mg/mL and 0.0005 mg/mL by 24- and 48-h time points, respectively. SEM examination shows that D@AgNP altered the shape of the cell structure and damaged the cell membrane. TEM finding shows that D@AgNP are mostly localized at vesicles such as the endosomes, lysosomes and mitochondria. It is anticipated that the introduced new method serves as the cornerstone for improving the generation of biocompatible hydrophilic carbohydrate-based anticancer drugs.

Determination of the Cytotoxicity and Antibiofilm Potential Effect of Equisetum arvense Silver Nanoparticles.

This study aimed to synthesize and characterize silver nanoparticles (AgNPs) by green synthesis from Equisetum arvense (Ea) extracts and to investigate their cytotoxicity, antibiofilm activity, and α-glucosidase enzyme inhibition. Diverse characterization techniques were applied to verify the production of nanoparticles. SEM examination confirmed that the size of nanoparticles is in the range of 40-60nm. Also, interactions between silver and natural compounds of plant extract were confirmed through FT-IR and EDX analyses. It was determined that Equisetum arvense silver nanoparticles had antibiofilm activity against three different clinical strains with high biofilm-forming ability. AgNPs reduced the biofilm-forming capacity of clinical A. baumannii isolate with strong biofilm-forming capacity by approximately twofold, while the capacity of clinical K.pneumonaie and E.coli isolates decreased by 1.5 and 1.2 fold, respectively. The α-glucosidase enzyme inhibition potential of the AgNPs, which is determined as 93.50%, was higher than the plant extract with, and the α- 30.37%. MTT was performed to assess whether incubation of nanoparticles with A549 and ARPE-19 cell lines affected their viability, and a dramatic reduction in cell growth inhibition of both A549 and ARPE-19 cells was observed. It has been shown that A549 cells treated with 200 and 150µg/mL nanoparticles had less cell proliferation compared to control cells at 24-h and 48-h incubation time. According to these results, Ea-derived AgNPs appear to have potential anticancer activity against A549 cancer cells. Investigating the effects of green synthesis nanoparticles on microbial biofilm and various tumors may be important for developing new therapies. The outcomes of this study have showed that Ea-AgNPsmay have a high potential both in the treatment of pathogenic strains that form biofilms, as well as in anticancer therapy use.

Biosynthesis of diisooctyl 2,5-furandicarboxylate by Candida antarctica lipase B (CALB) immobilized on a macroporous epoxy resin.

Diisooctyl 2,5-furandicarboxylate (DEF), an ester derivative of 2,5-furandicarboxylic acid (FDCA, a bio-based platform chemical), resembles the physical and chemical properties of phthalates. Due to its excellent biodegradability, DEF is considered a safer alternative to the hazardous phthalate plasticizers. Although FDCA esters are currently mainly produced by chemical synthesis, the enzymatic synthesis of DEF is a green, promising alternative. The current study investigated the biosynthesis of DEF by Candida antarctica lipase B (CALB) immobilized on macroporous resins. Out of five macroporous resins (NKA-9, LX-1000EP, LX-1000HA, XAD-7HP, and XAD-8) evaluated, the LX-1000EP epoxy resin was identified as the best carrier for CALB, and the XAD-7HP weakly polar resin was identified as the second best. The optimal immobilization conditions were as follows: CALB (500μL) and LX-1000EP (0.1g) were incubated in phosphate butter (20mM, pH 6.0) for 10h at 35°C. The resulting immobilized CALB (EP-CALB) showed an activity of 639U/g in the hydrolysis of p-nitrophenyl acetate, with an immobilization efficiency of 87.8% and an activity recovery rate of 56.4%. Using 0.02g EP-CALB as the catalyst in 10mL toluene, and the molar ratio of 2,5-dimethyl furanediformate (1mmol/mL) and isooctyl alcohol (4mmol/mL) that was 1:4, a DEF conversion rate of 91.3% was achieved after a 24-h incubation at 50°C. EP-CALB had similar thermal stability and organic solvent tolerance compared to Novozym 435, and both were superior to CALB immobilized on the XAD-7HP resin. EP-CALB also exhibited excellent operational stability, with a conversion rate of 52.6% after 10 repeated uses. EP-CALB could be a promising alternative to Novozym 435 in the biomanufacturing of green and safe plasticizers such as DEF.

Peripheral (lung-to-brain) exposure to diesel particulate matter induces oxidative stress and increased markers for systemic inflammation

Combustion-derived air pollution is a complex environmental toxicant that has become a global health concern due to urbanization. Air pollution contains pro-inflammatory stimulants such as fine and ultrafine particulate matter, gases, volatile organic compounds, and metals. This study is focused on the particulate phase, which has been shown to induce systemic inflammation after chronic exposure due to its ability to travel to the lower airway, resulting in the activation of local immune cell populations, releasing acute phase reactants to mitigate ongoing inflammation. The systemic response is a potential mechanism for the co-morbidity associated with regions with high pollution and neuropathology. We exposed diesel particulate matter (DPM) to a pulmonary cell-derived in vitro model where macrophages mimic the diffusion of cytokines into the peripheral circulation to microglia. Alveolar macrophages (transformed U937) were inoculated with resuspended DPM in an acute exposure (24-h incubation) and analyzed for MCP-1 expression and acute phase reactants (IL-1β, IL-6, IL-8, and TNF-α). Post-exposure serum was collected and filtered from cultured alveolar macrophages, introduced to a healthy culture of microglial cells (HMC3), and measured for neurotoxic cytokines, oxidative stress, and pattern recognition receptors. After DPM exposure, the macrophages significantly upregulated all measured acute phase reactants, increased H2O2 production, and increased MCP-1 expression. After collection and filtration to remove excess particulates, microglia cells were incubated with the collected serum for 48 h to allow for cytokine diffusion between the periphery of microglia. Microglia significantly upregulated IL-6, IL-8, and oxidative stress with a moderate increase in IL-1β and TNF-α. As a marker required for signaling tissue damage, CD14 indicated that compared to direct inoculation of DPM, peripheral exposure resulted in the potent activation of microglia cells. The specificity and potency of the response have implications for neuropathology through lung-to-brain mechanisms after inhalation of environmental pollutants.

Rapid high performance liquid chromatography method for erlotinib quantification in vitro: Application to study the effect of resveratrol on metabolism and cellular uptake of erlotinib.

Erlotinib is a selective epidermal growth factor receptor inhibitor that is used for the treatment of non-small cell lung cancer and pancreatic cancer. Its metabolism is mainly mediated by cytochrome P450 3A (CYP 3A). Resveratrol, a natural compound found in many plants and supplements, is known to inhibit CYP3A enzyme, therefore, it may act as an inhibitor for the metabolism of erlotinib. Development of a rapid high performance liquid chromatography with photodiode array detection (HPLC-PDA) method for the quantification of erlotinib in liver microsomes and cancer cells and its application to study resveratrol effect on metabolism and cellular uptake of erlotinib. HPLC-PDA was used to develop an efficient bioanalytical method with a 2.5-min runtime preceded by a simple protein precipitation step. The method was validated according to the European Medicines Agency guidelines. Erlotinib metabolic stability and resveratrol effect on erlotinib metabolite formation were evaluated in rat liver microsomes. Furthermore, the method was used to measure the intracellular concentrations of erlotinib in cancer colorectal cells and investigating resveratrol effect on the cellular uptake of erlotinib. A rapid HPLC-PDA method was developed and validated for the first time to address potential drug interaction of erlotinib with resveratrol. Resveratrol was a strong inhibitor of erlotinib metabolism in vitro with IC50 = 4.03 μM. Resveratrol, however, had no effect on erlotinib cellular uptake after 1h incubation in human colorectal cancer cells. The study suggests that resveratrol may produce a potential herb-drug interaction with erlotinib at the metabolism level and should be investigated in patients in the clinic.

A step forward on the in vitro and in vivo assessment of a novel nanomedicine against melanoma

Melanoma is the most aggressive form of skin cancer, with increasing incidence and mortality rates. To overcome current treatment limitations, a hybrid molecule (HM) combining a triazene and a sulfur L-tyrosine analogue, was recently synthesized, incorporated in long blood circulating liposomes (LIP HM) and validated in an immunocompetent melanoma model. The present work constitutes a step forward in the therapeutic assessment of HM formulations. Here, human melanoma cells, A375 and MNT-1, were used and dacarbazine (DTIC), a triazene drug clinically available as first-line treatment for melanoma, constituted the positive control. In cell cycle analysis, A375 cells, after 24-h incubation with HM (60 μM) and DTIC (70 μM), resulted in a 1.2 fold increase (related to control) in the percentage of cells in G0/G1 phase. The therapeutic activity was evaluated in a human murine melanoma model (subcutaneously injected with A375 cells) to most closely resemble the human pathology. Animals treated with LIP HM exhibited the highest antimelanoma effect resulting in a 6-, 5- and 4-fold reduction on tumor volume compared to negative control, Free HM and DTIC groups, respectively. No toxic side effects were detected. Overall, these results constitute another step forward in the validation of the antimelanoma activity of LIP HM, using a murine model that more accurately simulates the pathology that occurs in human patients.

Interaction of Graphene Oxide Nanoparticles with Human Mononuclear Cells in the Cell-IQ System.

The interaction of graphene oxide nanoparticles with human peripheral blood mononuclear cells was studied using the Cell-IQ continuous monitoring system for living cells. We used graphene oxide nanoparticles of various sizes coated with linear or branched polyethylene glycol (PEG) in concentrations of 5 and 25 μg/ml. After 24-h incubation with graphene oxide nanoparticles, the increase in the number of peripheral blood mononuclear cells at visualization points decreased; nanoparticles coated with branched PEG more markedly suppressed cell growth in culture. In the presence of graphene oxide nanoparticles, peripheral blood mononuclear cells retained high viability in culture after daily monitoring in the Cell-IQ system. The studied nanoparticles were engulfed by monocytes and the type of PEGylation had no effect on this process. Thus, graphene oxide nanoparticles reduced the increase in peripheral blood mononuclear cell mass during dynamic observation in the Cell-IQ system without reducing their viability.