Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKC alpha, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKC alpha directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residueopen

Safety, immunogenicity, and protective efficacy of the intranasal live pertussis vaccine GamLPV in an infant monkey model (<i>Papio hamadryas</i>)

INTRODUCTION. The current progressive increase in pertussis incidence and infant mortality rates is due to the insufficient effectiveness of existing vaccines, both in Russia and worldwide. Previous clinical trials showed that healthy adult volunteers developed long-term antibacterial immunity after vaccination with GamLPV, an intranasal recombinant live pertussis vaccine developed by the National Research Center for Epidemiology and Microbiology named after the honorary academician N. F. Gamaleya. Further clinical development of GamLPV in paediatric volunteers, including infants, requires preclinical studies in a newborn monkey model.AIM. This study aimed to evaluate the safety, immunogenicity, and protective efficacy of the GamLPV vaccine in infant hamadryas baboons (Papio hamadryas) challenged with pertussis after intranasal vaccination.MATERIALS AND METHODS. The study used 20 hamadryas baboons, including 7 infants aged 1–1.5 months, 7 mothers of these infants, and 6 control animals. The study examined the time course of changes in serum levels of specific IgG antibodies to pertussis toxin (PT) and filamentous haemagglutinin (FHA) by enzyme-linked immunosorbent assay (ELISA), monitored changes in serum levels of Bordetella pertussis antibodies by agglutination immunoassay, and detected B. pertussis DNA in oropharyngeal aspirates by real-time polymerase chain reaction.RESULTS. Intranasal GamLPV administration to infant baboons induced the production of specific IgG antibodies to PT and FHA (ELISA) and an increase in the total pertussis antibody titre (agglutination immunoassay). GamLPV did not cause any injection site or systemic reactions. There were no changes in complete blood counts and serum biochemistry profiles after vaccination. The protective efficacy of GamLPV against B. pertussis was demonstrated in challenge tests, where immunised animals had no clinical signs or laboratory findings indicative of pertussis in contrast to controls.CONCLUSIONS. The study demonstrated the safety and immunogenicity of the intranasal live pertussis vaccine GamLPV in newborn hamadryas baboons. GamLPV shows promise in the primary vaccination of infants, the revaccination of children and adults, and the development of herd immunity against pertussis in families

Neurodevelopment of children exposed in utero to treatment of maternal malignancy

Cancer is the second most common cause of death during the reproductive years, complicating approximately 1/1000 pregnancies. The occurrence of cancer during gestation is likely to increase as a result of a woman's tendency to delay childbearing. Improved diagnostic techniques for malignancies increases detection of cancer during pregnancy. Malignant conditions during gestation are believed to be associated with an increase in poor perinatal and fetal outcomes that are often due to maternal treatment. Physicians should weigh the benefits of treatment against the risks of fetal exposure. To date, most reports have focused on morphologic observations made very close to the time of delivery with little data collected on children's long-term neurodevelopment following in utero exposure to malignancy and treatment. Because the brain differentiates throughout pregnancy and in early postnatal life, damage may occur even after first trimester exposure. The possible delayed effects of treatment on a child's neurological, intellectual and behavioural functioning have never been systematically evaluated. The goal of this report was to summarize all related issues into one review to facilitate both practitioners' and patients' access to known data on fetal risks and safety. © 2001 Cancer Research Campaign http://www.bjcancer.co

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Antineoplastic Drugs as a Potential Risk Factor in Occupational Settings: Mechanisms of Action at the Cell Level, Genotoxic Effects, and Their Detection Using Different Biomarkers

U članku je prikazana osnovna podjela antineoplastičnih lijekova prema mehanizmima djelovanja na razini stanice. Objašnjeni su mehanizmi genotoksičnosti najvažnijih vrsta lijekova koji se primjenjuju u okviru uobičajenih protokola za liječenje zloćudnih novotvorina. Navedena je važeća klasifi kacija antineoplastika prema kancerogenom potencijalu, podaci o mutagenom potencijalu te je prikazana njihova podjela u skladu s anatomsko-terapijsko-kemijskim sustavom klasifi kacije. Sustavno su prikazani najvažniji rezultati svjetskih i hrvatskih istraživanja na populacijama radnika izloženih antineoplasticima, provedenih u razdoblju 1980.-2009. s pomoću četiri najčešće primjenjivane metode: analize izmjena sestrinskih kromatida, analize kromosomskih aberacija, mikronukleus-testa i komet-testa. Objašnjena su osnovna načela navedenih metoda te raspravljene njihove prednosti i nedostaci. Biološki pokazatelji daju važne podatke o individualnoj osjetljivosti profesionalno izloženih ispitanika koji mogu poslužiti unaprjeđenju postojećih uvjeta rada i upravljanju rizicima pri izloženosti genotoksičnim agensima. Na osnovi prednosti i nedostataka citogenetičkih metoda zaključeno je da je mikronukleus-test, koji podjednako uspješno dokazuje klastogene i aneugene učinke, jedna od najboljih metoda dostupnih za otkrivanje štetnih djelovanja antineoplastičnih lijekova koji su u aktivnoj primjeni.This article brings an overview of the mechanisms of action of antineoplastic drugs used in the clinical setting. It also describes the genotoxic potentials of the most important classes of antineoplastic drugs involved in standard chemotherapy protocols. Classifi cation of antineoplastic drugs according to the IARC monographs on the evaluation of carcinogenic risks to humans is accompanied by data on their mutagenicity and the most recent updates in the Anatomical Therapeutic Chemical (ATC) Classifi cation System. We report the main fi ndings of biomonitoring studies that were conducted in exposed healthcare workers all over the world between 1980 and 2009 using four biomarkers: sister chromatid exchanges, chromosome aberrations, micronuclei. and the comet assay. The methods are briefl y explained and their advantages and disadvantages discussed. Biomarkers provide important information on individual genome sensitivity, which eventually might help to improve current working practices and to manage the risks related with exposure to genotoxic agents. Taking into consideration all known advantages and drawbacks of the existing cytogenetic methods, the micronucleus assay, which is able to detect both clastogenic and aneugenic action, is the most suitable biomarker for assessing harmful effects of antineoplastic drugs currently used in health care

The correlation between intestinal microbiota and colorectal cancer

The study of intestinal and oral microflora and their metabolites playing an important role in intestinal homeostasis, has led to the identification of species closely related to the development of colorectal cancer, intracellular correlations of fungi and bacteria compared to control. The correlation between oral microbiota and intestinal microflora, as well as associated with the mucous membrane of the large intestine, was revealed. It was noted that the use of eu- and probiotics improved the immunological indices and the structure of the intestinal microbiota. Thus, studying the oral and intestinal microbiota and its metabolites may prove to be a simple, accessible and informative method for the early diagnosis of colon cancer. However, most studies indicate only changes in the quantitative and qualitative composition of the microbiota, hardly revealing its cause-effect relations with the processes of tumor formation in the colon. Therefore, it is necessary to continue studies of this problem.</jats:p

High Resolution Mass Spectrometry Analysis of the Sertraline Residues Contained in the Tissues of Rainbow Trout Reared in Model Experimental Conditions

The growing consumption of pharmaceuticals in the human population and the insufficient efficiency of their elimination in waste water has a long-term negative impact on the environment of aquatic ecosystems, including the organisms that inhabit them. A significant contributor is the consumption of anti-depressants from the SSRI group, which corresponds to their increasing concentration in the environment. The aim of this work was to determine if antidepressant sertraline is able to be stored in fish organisms and to evaluate the content of residues in various body tissues. Rainbow trout (Oncorhynchuss mykkis) was selected as the test organism and was artificially exposed to the antidepressant for 1 month (concentrations 0; 4.2; 44 and 400 ng.g-1 sertraline in the feed). Liver, kidney, brain and muscle tissue biopsies samples were taken for analysis. Detection was performed using an Accela 1250 LC pump and an Accela autosampler coupled with a high-performance mass analyzer with a heated electrospray ionization source Q-Exactive Orbitrap, operating in positive ionization mode and in PRM mode (m/z 306.08108→275.03888 and 309.009991→275.03888 for sertraline and internal standard, respectively). The limit of quantification of the method was 0.1 ng.g-1 of sertraline and the calibration curve showed a good linearity up to 20 ng.g-1. From the collected data, amount of residues was found in the liver, kidney and brain. In contrast, the incidence of residues in muscle tissue was not detected in all groups, which is favorable from the point of view of fish meat consumption, by humans.</jats:p

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta.

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG