Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD) and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

Acute pancreatitis (AP) is one common clinical acute abdominal disease, for which specific pharmacological or nutritional therapies remain elusive. Lactose, a macronutrient and an inducer of host innate immune responses, possesses immune modulatory functions. The current study aimed to investigate potential modulatory effects of lactose and the interplay between the nutrient and pancreatic immunity during experimentally induced AP in mice. We found that either prophylactic or therapeutic treatment of lactose time-dependently reduced the severity of AP, as evidenced by reduced pancreatic edema, serum amylase levels, and pancreatic myeloperoxidase activities, as well as by histological examination of pancreatic damage. Overall, lactose promoted a regulatory cytokine milieu in the pancreas and reduced infiltration of inflammatory neutrophils and macrophages. On acinar cells, lactose was able to suppress caerulein-induced inflammatory signaling pathways and to suppress chemoattractant tumor necrosis factor (TNF)-α and monocyte chemotactic protein-1 production. Additionally, lactose acted on pancreas-infiltrated macrophages, increasing interleukin-10 and decreasing tumor necrosis factor alpha production. Notably, lactose treatment reversed AP-associated infiltration of activated neutrophils. Last, the effect of lactose on neutrophil infiltration was mimicked by a galectin-3 antagonist, suggesting a potential endogenous target of lactose. Together, the current study demonstrates an immune regulatory effect of lactose to alleviate AP and suggests its potential as a convenient, value-added therapeutic macronutrient to control AP, and lower the risk of its systemic complications.

Acute brain injury leads to the recruitment and activation of immune cells including resident microglia and infiltrating peripheral myeloid cells (MC), which contribute to the inflammatory response involved in neuronal damage. We previously reported that TLR2 stimulation by peptidoglycan (PGN) from Staphylococcus aureus, in vitro and in vivo, induced microglial cell activation followed by autophagy induction. In this report, we evaluated if phosphatidyl-inositol-3 kinase (PI3K) pharmacological inhibitors LY294200 and 3-methyladenine (3-MA) can modulate the innate immune response to PGN in the central nervous system. We found that injection of PGN into the mouse brain parenchyma (caudate putamen) triggered an inflammatory reaction, which involved activation of microglial cells, recruitment of infiltrating MC to injection site, production of pro-inflammatory mediators, and neuronal injury. In addition, we observed the accumulation of LC3B+ CD45+ cells and colocalization of LC3B and lysosomal-associated membrane protein 1 in brain cells. Besides, we found that pharmacological inhibitors of PI3K, including the classical autophagy inhibitor 3-MA, reduced the recruitment of MC, microglial cell activation, and neurotoxicity induced by brain PGN injection. Collectively, our results suggest that PI3K pathways and autophagic response may participate in the PGN-induced microglial activation and MC recruitment to the brain. Thus, inhibition of these pathways could be therapeutically targeted to control acute brain inflammatory conditions.

The integrative omics approach is crucial to identify the molecular mechanisms underlying high-temperature response in non-model species. Based on future scenarios of heat increase, Pinus radiata plants were exposed to a temperature of 40°C for a period of 5 days, including recovered plants (30 days after last exposure to 40°C) in the analysis. The analysis of the metabolome using complementary mass spectrometry techniques (GC-MS and LC-Orbitrap-MS) allowed the reliable quantification of 2,287 metabolites. The analysis of identified metabolites and highlighter metabolic pathways across heat time exposure reveal the dynamism of the metabolome in relation to high-temperature response in P. radiata, identifying the existence of a turning point (on day 3) at which P. radiata plants changed from an initial stress response program (shorter-term response) to an acclimation one (longer-term response). Furthermore, the integration of metabolome and physiological measurements, which cover from the photosynthetic state to hormonal profile, suggests a complex metabolic pathway interaction network related to heat-stress response. Cytokinins (CKs), fatty acid metabolism and flavonoid and terpenoid biosynthesis were revealed as the most important pathways involved in heat-stress response in P. radiata, with zeatin riboside (ZR) and isopentenyl adenosine (iPA) as the key hormones coordinating these multiple and complex interactions. On the other hand, the integrative approach allowed elucidation of crucial metabolic mechanisms involved in heat response in P. radiata, as well as the identification of thermotolerance metabolic biomarkers (L-phenylalanine, hexadecanoic acid, and dihydromyricetin), crucial metabolites which can reschedule the metabolic strategy to adapt to high temperature.

Dynamic quantification of drought response is a key issue both for variety selection and for functional genetic study of rice drought resistance. Traditional assessment of drought resistance traits, such as stay-green and leaf-rolling, has utilized manual measurements, that are often subjective, error-prone, poorly quantified and time consuming. To relieve this phenotyping bottleneck, we demonstrate a feasible, robust and non-destructive method that dynamically quantifies response to drought, under both controlled and field conditions. Firstly, RGB images of individual rice plants at different growth points were analyzed to derive 4 features that were influenced by imposition of drought. These include a feature related to the ability to stay green, which we termed greenness plant area ratio (GPAR) and 3 shape descriptors [total plant area/bounding rectangle area ratio (TBR), perimeter area ratio (PAR) and total plant area/convex hull area ratio (TCR)]. Experiments showed that these 4 features were capable of discriminating reliably between drought resistant and drought sensitive accessions, and dynamically quantifying the drought response under controlled conditions across time (at either daily or half hourly time intervals). We compared the 3 shape descriptors and concluded that PAR was more robust and sensitive to leaf-rolling than the other shape descriptors. In addition, PAR and GPAR proved to be effective in quantification of drought response in the field. Moreover, the values obtained in field experiments using the collection of rice varieties were correlated with those derived from pot-based experiments. The general applicability of the algorithms is demonstrated by their ability to probe archival Miscanthus data previously collected on an independent platform. In conclusion, this image-based technology is robust providing a platform-independent tool for quantifying drought response that should be of general utility for breeding and functional genomics in future.

The intestine is a high cellular turnover tissue largely dependent on the regenerative function of stem cell throughout life, and a signaling center for the health and viability of organisms. Therefore, better understanding of the mechanisms underlying the regulation of intestinal stem cell (ISC) regenerative potential is essential for the possible intervention of aging process and age-related diseases. Drosophila midgut is a well-established model system for studying the mechanisms underlying ISC regenerative potential during aging. Here, we report the requirement of Drosophila phosphatidylethanolamine binding protein 1 (PEBP1) in ISC regenerative potential. We showed that PEBP1 was strongly expressed in enterocytes (ECs) of guts and its decrease with age and oxidative stress. Furthermore, the downregulation of PEBP1 in ECs accelerates ISC aging, as evidenced by ISC hyper-proliferation, γH2AX accumulation, and centrosome amplification, and intestinal hyperplasia. The decrease in PEBP1 expression was associated with increased extracellular signal-regulated kinase (ERK) activity in ECs. All these phenotypes by EC-specific depletion of PEBP1 were rescued by the concomitant inhibition of ERK signaling. Our findings evidence that the age-related downregulation of PEBP1 in ECs is a novel cause accelerating ISC aging and that PEBP1 is an EC-intrinsic suppressor of epidermal growth factor receptor (EGFR)/ERK signaling. Our study provides molecular insights into the tight regulation of EGFR/ERK signaling in niches for stem cell regenerative potential.

Neuroblastoma is the most common diagnosed tumor in infants and the second most common extracranial tumor of childhood. The survival rate of patients with high-risk neuroblastoma is still very low despite intensive multimodal treatments. Therefore, new treatment strategies are needed. In recent years, miRNA-based anticancer therapy has received growing attention. Advances in this novel treatment strategy strongly depends on the identification of candidate miRNAs with broad-spectrum antitumor activity. Here, we identify miR-193b as a miRNA with tumor suppressive properties. We show that miR-193b is expressed at low levels in neuroblastoma cell lines and primary tumor samples. Introduction of miR-193b mimics into nine neuroblastoma cell lines with distinct genetic characteristics significantly reduces cell growth in vitro independent of risk factors such as p53 functionality or MYCN amplification. Functionally, miR-193b induces a G1 cell cycle arrest and cell death in neuroblastoma cell lines by reducing the expression of MYCN, Cyclin D1 and MCL-1, three important oncogenes in neuroblastoma of which inhibition has shown promising results in preclinical testing. Therefore, we suggest that miR-193b may represent a new candidate for miRNA-based anticancer therapy in neuroblastoma.

5-FU chemotherapy is a current strategy to treat pancreatic cancer (PC), but unfortunately chemoresistance is eventually developed in most patients. Obesity is a risk factor for PC that could affect 5-FU effectiveness through the adipokine leptin, which is a known proliferation, survival factor and Notch inducer. We investigated whether leptin signaling affects 5-FU cytotoxicity on PC. To this end, tumorspheres developed from BxPC-3 and MiaPaCa-2 PC cells were treated with 5-FU, leptin, inhibitors for Notch (DAPT) and leptin signaling (IONP-LPrA2) and ATP-binding cassette of proteins (Probenecid). Leptin treatment decreased 5-FU cytotoxicity, and increased cell proliferation, colony forming ability, stem cell, pluripotency, EMT markers, drug efflux proteins (ABCC5, ABCC11) and Notch. In addition, leptin reduced the 5-FU effects on apoptosis by decreasing pro-apoptotic (Bax, Caspase-3 activation and PARP degradation) and increasing anti-apoptotic factors (RIP and Bcl-XL). Leptin's effects on PC tumorspheres treated with 5-FU were reduced by IONP-LPrA2 and were mainly Notch signaling- dependent and more evident in MiaPaCa-2-derived tumorspheres. Present results suggest that leptin could impair 5-FU cytotoxicity and promote chemoresistance. Therefore, targeting the leptin-Notch axis could be a novel way to improve 5-FU therapy for PC patients, especially in obesity context.

Melanoma is an aggressive cancer type with a high mortality rate and an elevated resistance to conventional treatment. Recently, promising new tools for anti-melanoma targeted therapy have emerged including inhibitors directed against frequently overexpressed receptors of growth factors implicated in the progression of this cancer. The ineffectiveness of single-targeted therapy prompted us to study the efficacy of treatment with a combination of foretinib, a MET (hepatocyte growth factor receptor) inhibitor, and gefitinib or lapatinib, EGFR (epidermal growth factor receptor) inhibitors. We observed a synergistic cytotoxic effect for the combination of foretinib and lapatinib on the viability and proliferation of the examined melanoma cell lines. This combination of inhibitors significantly decreased Akt and Erk phosphorylation, while the drugs used independently were insufficient. Additionally, after treatment with pairs of inhibitors, cells became larger, with more pronounced stress fibers and abnormally shaped nuclei. We also noticed the appearance of polyploid cells and massive enrichment in the G2/M phase. Therefore, combination treatment was much more effective against melanoma cells than a single-targeted approach. Based on our results, we conclude that both EGFR and MET receptors might be effective targets in melanoma therapy. However, variation in their levels in patients should be taken into consideration.

Since desmoid tumors (DT) exhibit an unpredictable clinical course, with stabilization and/or spontaneous regression, an initial "wait-and-see" policy is the new standard of care-thus, the actual challenge is to identify early factors of progression. We present a method of detection of CTNNB1 mutations using a targeted digital droplet PCR (ddPCR) on cell-free DNA (cfDNA) extracted from blood samples of 31 DT patients. Furthermore, we analyzed the correlation between DT evolution and plasmatic concentration of total and mutated cfDNA at the time of diagnosis. Circulating copies of CTNNB1 mutants (ctDNA) were detected in the plasma of 6 patients (33%) but their concentration was not correlated with evolution of the tumor. Concentration of total cfDNA was higher in the plasma of patients with progressive desmoids (p = 0,0009). Using a threshold <900 copies/mL of plasma to detect indolent desmoid and a threshold >1375, it was possible to predict desmoid evolution for 65% of patients by measuring the quantity of circulating DNA in their plasma as early as the time of diagnosis. Albeit showing that the detection of CTNNB1 mutants is possible in the plasma of patients harboring a desmoid tumor, the results of this preliminary study raise the hypothesis that most of the circulating DNA detected in their plasma is derived from non-neoplastic cells, most likely normal neighboring tissues being actively invaded. Our results open the perspective of using cfDNA as a biomarker to predict prognosis at the time of diagnosis and assess tumor dynamics to optimize the treatment strategy.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

The genetic architecture of bone and soft tissue sarcomas susceptibility is yet to be elucidated. We aimed to comprehensively collect and meta-analyze the current knowledge on genetic susceptibility in these rare tumors.

Fertility reduction due to sub-fertile males is a major concern in breeder flocks. Phenotypic traits of broiler breeder males and their relationships with fertility can be used as reliable indicators for identification and removal of sub-fertile males from the breeder flocks. This study was conducted to investigate semen traits (semen volume, sperm motility, sperm viability and sperm count) and testes histomorphometric features including tubule differentiation index (TDI), spermiation index (SPI), Sertoli cell index (SCI) and mitotic index (MI) of broiler breeder males with the same age but different phenotypic traits. According to phenotypic traits, 12 broiler breeder males (Ross-308 strain) were classified into three equal groups. Group 1: roosters with fertile phenotypic traits (fertile), group 2: roosters with the lowest fertile phenotypic traits (sub-fertile) and group 3: roosters with moderate fertile phenotypic traits (moderate). The results confirmed potential relationship between phenotypic traits and fertility in broiler breeder males. Semen traits and histomorphometric features of broiler breeder males' testis of the group 3 were more similar to those of the fertile roosters. Therefore, it can be concluded that exclusion of these roosters from the breeder flock may have undesirable effects on flock fertility.

Lysophosphatidic acid (LPA) known as a serum-derived growth factor, is involved in several cell physiological functions in the female reproductive system including: oocyte maturation, in vitro fertilization and embryo implantation by its transmembrane G protein-coupled receptors. The aim of the present study was to examine the effect of LPA on in vitro follicular development of mouse ovarian tissue. Neonatal mouse ovarian tissues were cultured in five different concentrations of LPA (0, 5, 10, 20 and 40 µM). The developmental competence and the function of cultured ovarian tissue were assessed by morphological study using hematoxylin and eosin staining and hormonal analysis. The expression of LPA receptor (LPAR 1-4) genes were analyzed by real-time RT-PCR. The proportion of preantral follicles and the level of E2 hormone were significantly higher in the 20 µM LPA-treated group than those in the other treatment groups. There was a significant difference in the expression of LPAR 1-4 genes in 20 µM LPA treated group in comparison with 0 µM LPA (control group) treated and non-cultured groups. In addition, the expression of LPAR1 gene was higher than other receptor genes in all studied groups. In conclusion supplementation of the media with 20 µM LPA, could improve the survival and developmental potential of follicles and it had positive effects on cell function and stimulation of E2 synthesis in mouse whole ovarian tissues.

In recent years, use of edible coatings as carriers of food additives and antimicrobial compounds has been considered in fishery products. This study was carried out to evaluate the effects of 2.00% chitosan coating singly and combined with 0.10% grape seed extract (GSE) on microbial (mesophils and psychrophils counts), chemical (thiobarbituric acid; TBA), pH and peroxide value (PV) and sensorial properties of rainbow trout fillet stored at 4 °C over a period of 15 days. The coating had a significant effect in reducing aerobic mesophilic and psychrophilic bacteria counts. The TBA, PV and pH of samples of chitosan coating alone and with GSE were lower than control ones indicating a significant influence of coating on fillet shelf-life. Moreover, chitosan coating represented an equal sensorial quality with controls. It can be concluded that chitosan coating containing GSE can help to maintain the sensorial quality and increase the shelf-life of rainbow trout fillets at refrigerated conditions.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and swollen head syndrome in chickens. Four aMPV subgroups (A-D) have been reported previously based on their genetic and antigenic differences. Evidence suggests that the live bird markets (LBMs) play an important role in the epidemiology of the avian viral diseases. A total number of 450 oropharyngeal samples from eight different species of birds (migratory and local) were collected from LBMs of Gilan province, Iran, from October to December 2016. The presence of aMPV was determined by reverse transcription polymerase chain reaction (RT-PCR) based on nucleoprotein gene. The aMPV was detected in 30.60% of the examined birds including chickens (37.00%), turkey (33.00%), Eurasian teal (25.00%), common blackbird (33.00%), and Eurasian woodcock (25.00%). Bioinformatics analysis and a phylogenetic tree based on partial nucleotide sequences of the N gene showed that the detected aMPVs were belonged to subtype B. This is the first report of aMPV in non-commercial birds in Iran. Knowledge of the frequency and types of infected birds with pneumoviruses allow a better understanding of the epidemiology of aMPV in Iran.

Cysteine cathepsins normally function in the lysosomal degradation system where they are critical for the maintenance of cellular homeostasis and the MHC II immune response, and have been found to have major roles in several diseases and in tumor progression. Selective visualization of individual protease activity within a complex proteome is of major importance to establish their roles in both normal and tumor cells, thereby facilitating our understanding of the regulation of proteolytic networks. A generally accepted means to monitor protease activity is the use of small molecule substrates and activity-based probes. However, there are eleven human cysteine cathepsins, with a few of them displaying overlapping substrate specificity, making the development of small molecules that selectively target a single cathepsin very challenging. Here, we utilized HyCoSuL, a positional scanning substrate approach, to develop a highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231. Use of this probe enabled us to distinguish the activity of cathepsin L from that of other cathepsins, particularly cathepsin B, which is abundant and ubiquitously expressed in normal and transformed cell types. We found that cathepsin L localization in MDA-MB-231 cells greatly overlaps with that of cathepsin B, however, several cathepsin L-rich lysosomes lacked cathepsin B activity. Overall, these studies demonstrate that HyCoSuL-derived small molecule probes are valuable tools to image cathepsin L activity in living cells. This approach thus enables evaluation of cathepsin L function in tumorigenesis and is applicable to other cysteine cathepsins.

Poly(l-lactide) (PLA) is a bioderived and biodegradable polymer that has limited applications due to its hard and brittle nature. Incorporation of 1,3-trimethylene carbonate into PLA, in a block copolymer fashion, improves the mechanical properties, while retaining the biodegradability of the polymer, and broadens its range of applications. However, the preparation of 1,3-trimethylene carbonate (TMC)/l-lactide (LA) copolymers beyond diblock and triblock structures has not been reported, with explanations focusing mostly on thermodynamic reasons that impede the copolymerization of TMC after lactide. We discuss the preparation of multiblock copolymers via the ring opening polymerization (ROP) of LA and TMC, in a step-wise addition, by a ferrocene-chelating heteroscorpionate zinc complex, {[fc(PPh2)(BH[(3,5-Me)2pz]2)]Zn(μ-OCH2Ph)}2 ([(fcP,B)Zn(μ-OCH2Ph)]2, fc = 1,1'-ferrocenediyl, pz = pyrazole). The synthesis of up to pentablock copolymers, from various combinations of LA and TMC, was accomplished and the physical, thermal, and mechanical properties of the resulting copolymers evaluated.

Amino acid esters are a group of structurally diverse natural products with distinct activities. Some are synthesized through an inter-molecular esterification step catalysed by nonribosomal peptide synthetase (NRPS). In bacteria, the formation of the intra-molecular ester bond is usually catalysed by a thioesterase domain of NRPS. However, the mechanism by which fungal NRPSs perform this process remains unclear. Herein, by targeted gene disruption in Penicillium brevicompactum and heterologous expression in Aspergillus nidulans, we show that two NRPSs, ApmA and ApmB, are sufficient for the synthesis of an amino acid ester, asperphenamate. Using the heterologous expression system, we identified that ApmA, with a reductase domain, rarely generates dipeptidyl alcohol. In contrast, ApmB was determined to not only catalyse inter-molecular ester bond formation but also accept the linear dipeptidyl precursor into the NRPS chain. The mechanism described here provides an approach for the synthesis of new small molecules with NRPS as the catalyst. Our study reveals for the first time a two-module NRPS system for the formation of amino acid esters in nature.