Sick leaves among healthy pregnant Croatian healthcare workers during the COVID-19 pandemic due to loopholes in the occupational safety system.

The aim of this study was to explore occupational safety in pregnant Croatian healthcare workers (HCWs) during the coronavirus disease 2019 (COVID-19) pandemic. To this end we composed an anonymous questionnaire that included pregnancy data, risk assessment and mitigation, and workplace intervention and distributed it to HCWs through social media of their groups and associations. The study includes a total of 173 respondents (71.1 % physicians, 19.7 % nurses, 9.2 % other HCWs) diagnosed with pregnancy in 2020 and 2021. Employers were notified about HCWs' pregnancy at the eighth (IQR 7.0-11.0) week of pregnancy, which delayed workplace risk assessment and mitigation beyond the first trimester. Only 19.6 % of the participants had the risk assessed and mitigated, mostly on their own initiative (76.5 %). After notifying employers about pregnancy, 37.0 % of participants opted for temporary work incapacity (TWI) due to "pregnancy complications" despite healthy pregnancy, 16.8 % were granted a pregnant worker's paid leave at the expense of the employer, while 5.8 % continued to work at the same workplace. Nurses used the TWI benefit more frequently than physicians (58.8 % vs 30.1 %, P=0.004). Our findings suggest that occupational safety of pregnant HCWs in Croatia lacks clear-cut and transparent strategies to protect pregnant HCWs, forcing them to misuse the healthcare system.

Correlation between neural response telemetry measurements and fitting levels.

INTRODUCTION: The neural response telemetry (NRT) is a standard procedure in cochlear implantation mostly used to determine the functionality of implanted device and to check auditory nerve responds to the stimulus. Correlation between NRT measurements and subjective threshold (T) and maximum comfort (C) levels has been reported but results are inconsistent, and it is still not clear which of the NRT measurements could be the most useful in predicting fitting levels. PURPOSE: In our study we aimed to investigate which NRT measurement corresponds better to fitting levels. Impedance (IMP), Evoked Action Potential (ECAP) threshold and amplitude growth function (AGF) slope values were included in the study. Also, we tried to identify cochlear area at which the connection between NRT measurements and fitting levels would be the most pronounced. MATERIALS AND METHODS: Thirty-one children implanted with Cochlear device were included in this retrospective study. IMP, ECAP thresholds and AGF were obtained intra-operatively and 12 months after surgery at electrodes 5, 11 and 19 as representative for each part of cochlea. Subjective T and C levels were obtained 12 months after the surgery during cochlear fitting. RESULTS: ECAP thresholds obtained 12 months after surgery showed statistically significant correlation to both T and C levels at all 3 selected electrodes. IMP correlated with C levels while AGF showed tendency to correlate with T levels. However, these correlations were not statistically significant for all electrodes. CONCLUSION: ECAP threshold measurements correlated to T and C values better than AGF slope and IMP. Measurements obtained twelve months after surgery seems to be more predictive of T and C values compared to intra-operative measurements. The best correlation between ECAP threshold and T and C values was found at electrode 11 suggesting NRT measurements at mid-portion cochlear region to be the most useful in predicting fitting levels.

Associations of burnout with job demands/resources during the pandemic in health workers from Southeast European countries.

Introduction: Despite several studies assessing job demands and burnout in countries from the Southeast European (SEE) region, there is still a lack of data about the psychological impact of the pandemic on health workers (HWs). Aims: The present study aimed to demonstrate and compare levels of burnout dimensions in HWs from SEE countries and to reveal the burnout-job demands/resources relationships in these workers during the pandemic. Materials and methods: During the autumn of 2020, this online multicentric cross-sectional survey studied a large group (N = 4.621) of HWs working in SEE countries. The Maslach Burnout Inventory was used for the measurement of burnout dimensions. We analyzed the job demands by using the Hospital Experience Scale. Remuneration and relationships with superiors were measured using the Questionnaire sur les Ressources et Contraintes Professionnelles (English version). Results: A series of ANOVA comparisons of means revealed the countries in which respondents showed higher mean values of emotional exhaustion (Bosnia and Herzegovina, Bulgaria, Croatia, Moldova, Montenegro, and North Macedonia) and the countries in which respondents showed lower mean values of this burnout dimension (Israel and Romania) (Welch F = 17.98, p < 0.001). We also found differences among HWs from different countries in job demands and job resources. The testing of hierarchical regression models, which have been controlled for certain confounding factors, clearly revealed that emotional exhaustion was predicted by job demands (R2 = 0.37) and job resources (R2 = 0.16). Conclusion: Preventive measures for the improvement of mental health in HWs during the pandemic and beyond have to take into account the differences between countries regarding the country context and current scientific knowledge. A modified stress test should be implemented in hospitals regarding future shocks that might include new pandemics, terrorism, catastrophes, or border conflicts.

Comparison of Intratympanic Steroid and Hyperbaric Oxygen Salvage Therapy Hearing Outcomes in Idiopathic Sudden Sensorineural Hearing Loss: A Retrospective Study.

OBJECTIVES: Systemic steroids are the most common first-line therapy in sudden sensorineural hearing loss (SSNHL), with significant improvement in hearing outcomes in over 60% of patients. It is unknown why 40% of patients do not respond to systemic steroid therapy. Salvage treatment includes intratympanic steroids (ITS) and hyperbaric oxygenation (HBO) therapy, with inconsistent results reported. This study aimed to compare the results of ITS and HBO therapy in patients with SSNHL that previously failed systemic steroid therapy. DESIGN: This is a comparative retrospective nonrandomized interventional cohort study, enrolling 126 patients with SSNHL. Out of these, 35 patients received HBO therapy, 43 patients received ITS, and 48 patients did not receive any second-line therapy (control group). Pure-tone audiograms were performed before and after the salvage therapy in the IT and HBO groups and at the same time interval in the control group. Study variables included age, time until therapy initiation, tinnitus status, and hearing outcomes, with a cutoff criteria of cumulative >30 dB improvement on all frequencies indicating recovery. RESULTS: ITS and HBO therapy were associated with statistically significant hearing recovery at all frequencies compared to systemic steroids. The results show an average hearing improvement of 13.6 dB overall frequencies (250 to 8000 Hz) after ITS therapy and 7.4 dB in HBO therapy in comparison to the control group. Presence of significant hearing improvement positively correlated with age, ITS therapy, and HBO therapy. Presence of tinnitus before therapy was negatively correlated with hearing improvement. Patients with tinnitus present at the start of therapy improve 4.67 dB less on average compared to those without tinnitus. ITS therapy significantly reduced tinnitus compared to the other two treatment options. Patients with tinnitus present before therapy significantly improve hearing at low frequencies, compared to the control group. CONCLUSIONS: ITS and HBO therapy show superior hearing results compared to observation alone after failed oral steroid therapy for SSNHL. ITS shows an additional positive impact on tinnitus reduction and shows superior hearing outcomes after salvage therapy.

Protein and RNA ADP-ribosylation detection is influenced by sample preparation and reagents used.

The modification of substrates with ADP-ribose (ADPr) is important in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts containing ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the reactivity of available reagents with diverse ADP-ribosylated protein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross-reactivity with adenylylated RNA, AMPylated proteins, and metabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample preparation methods and have defined the reagents suitable for specific methods and substrates.

Overtraining Syndrome as a Risk Factor for Bone Stress Injuries among Paralympic Athletes.

Background and Objectives: In this review, we have explored the relationship between overtraining syndrome (OTS) and bone stress injuries among paralympic athletes. OTS is a complex condition that arises from an imbalance between training volume, nutrition, and recovery time, leading to significant negative effects on paralympic athlete's performance and overall well-being. On the other hand, bone stress injuries occur when abnormal and repetitive loading is applied to normal bone, resulting in microdamage accumulation and potential. The prevalence of overtraining syndrome and bone stress injuries among athletes highlights the need for a better understanding of their relationship and implications for prevention and management strategies. Methods: A literature review from the PubMed, Web of Science, and Google Scholar databases including the MeSH keywords "overtraining syndrome", "bone", and "paralympic athletes". Results: Studies have consistently shown that athletes engaged in endurance sports are particularly susceptible to overtraining syndrome. The multifactorial nature of this condition involves not only physical factors, but also psychological and environmental determinants. In addition, the diagnosis and management of OTS and bone stress injuries present challenges in clinical practice. Conclusions: Currently, there are no definitive biochemical markers for overtraining syndrome. The diagnosis is based on a combination of subjective measures such as questionnaires, symptoms checklists, and objective biomarkers, including hormone levels, inflammatory markers, and imaging studies. However, these diagnostic approaches have limitations regarding their specificity and sensitivity.

ADP-ribosylation of RNA in mammalian cells is mediated by TRPT1 and multiple PARPs.

RNA function relies heavily on posttranscriptional modifications. Recently, it was shown that certain PARPs and TRPT1 can ADP-ribosylate RNA in vitro. Traditionally, intracellular ADP-ribosylation has been considered mainly as a protein posttranslational modification. To date, it is not clear whether RNA ADP-ribosylation occurs in cells. Here we present evidence that different RNA species are ADP-ribosylated in human cells. The modification of cellular RNA is mediated by several transferases such as TRPT1, PARP10, PARP11, PARP12 and PARP15 and is counteracted by different hydrolases including TARG1, PARG and ARH3. In addition, diverse cellular stressors can modulate the content of ADP-ribosylated RNA in cells. We next investigated potential consequences of ADP-ribosylation for RNA and found that ADPr-capped mRNA is protected against XRN1 mediated degradation but is not translated. T4 RNA ligase 1 can ligate ADPr-RNA in absence of ATP, resulting in the incorporation of an abasic site. We thus provide the first evidence of RNA ADP-ribosylation in mammalian cells and postulate potential functions of this novel RNA modification.

Are PARPs promiscuous?

Post-translational modifications exist in different varieties to regulate diverse characteristics of their substrates, ultimately leading to maintenance of cell health. The enzymes of the intracellular poly(ADP-ribose) polymerase (PARP) family can transfer either a single ADP-ribose to targets, in a reaction called mono(ADP-ribosyl)ation or MARylation, or multiple to form chains of poly(ADP-ribose) or PAR. Traditionally thought to be attached to arginine or glutamate, recent data have added serine, tyrosine, histidine and others to the list of potential ADP-ribose acceptor amino acids. PARylation by PARP1 has been relatively well studied, whereas less is known about the other family members such as PARP7 and PARP10. ADP-ribosylation on arginine and serine is reversed by ARH1 and ARH3 respectively, whereas macrodomain-containing MACROD1, MACROD2 and TARG1 reverse modification of acidic residues. For the other amino acids, no hydrolases have been identified to date. For many PARPs, it is not clear yet what their endogenous targets are. Better understanding of their biochemical reactions is required to be able to determine their biological functions in future studies. In this review, we discuss the current knowledge of PARP specificity in vitro and in cells, as well as provide an outlook for future research.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Salivary cortisone as potential predictor of occupational exposure to noise and related stress.

Salivary cortisone strongly correlates with serum cortisol, and since it is less invasive to measure salivary cortisone than serum cortisol and easier than to measure cortisol in saliva, as its concentrations are much lower, we wanted to compare salivary cortisone and cortisol levels as markers of noise-induced stress reaction. The study included 104 participants aged 19-30 years, 50 of whom were exposed to occupational noise ≥85 dB(A) and 54 non-exposed, control students. All participants took samples of their saliva with Salivette® Cortisol synthetic swabs on three consecutive working days first thing in the morning. Salivary cortisone and cortisol levels were determined with high-performance liquid chromatography. In addition, they completed a 10-item Perceived Stress Scale (PSS-10) questionnaire, and occupationally noise-exposed participants also completed the Health and Safety Executive (HSE) questionnaire on occupational psychosocial risks. The exposed participants had significantly higher cortisone (P<0.001) and cortisol (P<0.001) levels than controls, and the correlation between cortisone and cortisol levels in the exposed participants was strong (ϱ =0.692, P<0.001), which suggests that salivary cortisone can replace cortisol measurements in saliva as a more reliable method than salivary cortisol and less invasive than serum cortisol. However, the level of perceived stress scored on PSS-10 in the exposed participants did not differ significantly from stress reported by controls, but correlated negatively with cortisone levels, which is contrary to our expectations and raises questions as to why.

Work from home and musculoskeletal pain in telecommunications workers during COVID-19 pandemic: a pilot study.

One of the side-effects of the COVID-19 pandemic is a global change in work ergonomic patterns as millions of people replaced their usual work environment with home to limit the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection. The aim of our cross-sectional pilot study was to identify musculoskeletal pain that may have resulted from this change and included 232 telecommunications company workers of both genders [121 (52.2 %) men aged 23-62 (median 41; interquartile range 33-46 yrs.) and 111 (47.8 %) women aged 23-53 (median 40; interquartile range 33-44)] who had been working from home for eight months (from 16 March to 4 December 2020) before they joined the study. The participants were asked to fill in our web-based questionnaire by self-assessing their experience of hand, lower back, and upper back/neck pain while working at home and by describing their work setting and physical activity. Compared to previous work at the office, 90 (39.1 %) participants reported stronger pain in the lower back, 105 (45.7 %) in the upper back/neck, and 63 (27.2 %) in their hands. Only one third did not report any musculoskeletal problems related to work from home. Significantly fewer men than women reported hand, lower back, and upper back/ neck pain (p=0.033, p=0.001 and p=0.013, respectively). Sixty-nine workers (29.9 %) reported to work in a separate room, 75 (32.4 %) worked in a separate section of a room with other household members, whereas 87 (37.7 %) had no separate work space, 30 of whom most often worked in the dining room. Ninety-five participants (40.9 %) had no office desk to work at, and only 75 (32.3 %) used an ergonomic chair. Of those who shared their household with others (N=164), 116 (70.7 %) complained about constant or occasional disturbances. Over a half of all participants (52 %) said that they worked longer hours from home than at work, predominantly women (p=0.05). Only 69 participants (29.9 %) were taking frequent breaks, predominantly older ones (p=0.006). Our findings clearly point to a need to inform home workers how to make more ergonomic use of non-ergonomic equipment, use breaks, and exercise and to inform employers how to better organise working hours to meet the needs of work from home.

Knowledge of and attitudes to occupational and sports medicine among medical students in Zagreb, Croatia.

Occupational and sports medicine (OSM) education is poorly represented in Croatian university undergraduate medical curricula. Zagreb University medical students are required to take OSM classes for a week on their final year of studies. The classes are organised around team-based learning (TBL). Given that students who attend TBL classes have significantly higher exam scores than students who take lectures ex cathedra, the aim of this study was to assess students' knowledge and attitudes immediately after TBL OSM classes. This cross-sectional study included 162 final-year Zagreb University School of Medicine students taking TBL classes in OSM in the academic year of 2019/2020. They were recruited from 30 September 2019 to 4 March 2020. Participants filled in a 20-item questionnaire compiled by the authors and adapted to the Croatian legislation. Their answers demonstrated positive attitude toward OSM classes and negative attitude toward occupational medicine practice and OSM specialty. They showed moderate interest only for the job of sports physician. Even though they showed sufficient knowledge of OSM immediately after the course was completed, they were moderately satisfied with their knowledge. Our findings call for rethinking the practical aspects of teaching OSM classes in order to promote OSM practice among medical students or at least raise awareness about the importance of prevention of numerous work or sport-related disorders.

Characteristics of work-related COVID-19 in Croatian healthcare workers: a preliminary report.

Healthcare workers (HCWs) are considered to run a higher occupational risk of becoming infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and develop coronavirus disease (COVID-19) than the rest of the population. The aim of this study was to describe and analyse the characteristics of work-related COVID-19 in Croatian HCWs. Study participants were HCWs who contacted their occupational physician between 1 May 2020 and 12 November 2020 with a request for the registration of COVID-19 as an occupational disease. All participants filled out our online Occupational COVID-19 in Healthcare Workers Questionnaire. The study included 59 HCWs (median age 45.0, interquartile range 36.0-56.0 years). Most (78 %) were nurses or laboratory technicians, and almost all (94.9 %) worked in hospitals. Hierarchical cluster analysis revealed three clusters of COVID-19-related symptoms: 1) elevated body temperature with general weakness and fatigue, 2) diarrhoea, and 3) headache, muscle and joint pain, anosmia, ageusia, and respiratory symptoms (nasal symptoms, burning throat, cough, dyspnoea, tachypnoea). Almost half (44.6 %) reported comorbidities. Only those with chronic pulmonary conditions were more often hospitalised than those without respiratory disorders (57.1 % vs. 2.5 %, respectively; P=0.001). Our findings suggest that work-related COVID-19 among Croatian HCWs is most common in hospital nurses/laboratory technicians and takes a mild form, with symptoms clustering around three clinical phenotypes: general symptoms of acute infection, specific symptoms including neurological (anosmia, ageusia) and respiratory symptoms, and diarrhoea as a separate symptom. They also support evidence from other studies that persons with chronic pulmonary conditions are at higher risk for developing severe forms of COVID-19.

ADP-ribosylation of RNA and DNA: from in vitro characterization to in vivo function.

The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1-3, PARP10 and tRNA 2'-phosphotransferase (TRPT1). Evidence suggests that these enzymes display different preferences towards different oligonucleotides. These reactions are reversed by ADP-ribosylhydrolases of the macrodomain and ARH families, such as MACROD1, TARG1, PARG, ARH1 and ARH3. Most findings derive from in vitro experiments using recombinant components, leaving the relevance of this modification in cells unclear. In this Survey and Summary, we provide an overview of the enzymes that ADP-ribosylate nucleic acids, the reversing hydrolases, and the substrates' requirements. Drawing on data available for other organisms, such as pierisin1 from cabbage butterflies and the bacterial toxin-antitoxin system DarT-DarG, we discuss possible functions for nucleic acid ADP-ribosylation in mammals. Hypothesized roles for nucleic acid ADP-ribosylation include functions in DNA damage repair, in antiviral immunity or as non-conventional RNA cap. Lastly, we assess various methods potentially suitable for future studies of nucleic acid ADP-ribosylation.

Diagnosing occupational COVID-19 in Croatian healthcare workers.

Coronavirus disease 2019 (COVID-19) can be diagnosed as occupational disease by an occupational health physician (OHP), if supported by relevant work-related and medical documentation. The aim of this study was to analyse such documentation submitted by Croatian healthcare workers (HCWs) and discuss its relevance in view of European and Croatian guidelines. The study included 100 Croatian HCWs who were SARS-CoV-2-positive and requested that their infection be diagnosed as occupational disease by their OHPs from 1 May 2020 to 10 March 2021. As participants they were asked to fill out our online Occupational COVID-19 in Healthcare Workers Questionnaire. For the purpose of this study we analysed answers about the type of close contact at the workplace, COVID-19 symptoms, and enclosed work-related (job description, employer statement about exposure to SARS-CoV-2) and medical documentation (positive SARS-CoV-2 polymerase chain reaction test and patient history confirming the diagnosis of COVID-19). Most participants were working in hospitals (N=95), mostly nurses (N=75), who became infected by a patient (N=68) or colleague (N=31), and had at least one COVID-19 symptom (N=87). Eighty participants did not enclose obligatory documents, 41 of whom failed to submit job description and 31 both job description and employer statement. These findings confirm that the major risk of occupational COVID-19 in HCWs is close contact with patients and colleagues, and points out the need for better cooperation between OHPs, occupational safety experts, employers, and diseased workers.

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis.

Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell's response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a 'macrodomain' harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

In vitro characterization of zebrafish (Danio rerio) organic anion transporters Oat2a-e.

OATS/Oats are transmembrane proteins that transport a variety of drugs, environmental toxins and endogenous metabolites into the cell. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. In this study we specifically address Oat2 (Slc22a7) family. Conserved synteny analysis showed localization of zebrafish oat2 genes on two chromosomes, 11 and 17. All five zebrafish Oats were localized by live cell imaging in membranes of transiently transfected HEK293-T cells, and Oat2a, b, d, and e were confirmed using western blot analysis. Functional studies using the HEK293T cells overexpressing zebrafish Oats revealed two model fluorescent substrates of three Oats: Lucifer yellow for Oat2a and Oat2d (Km 122, and 49.7µM), and 6-carboxyfluorescein for Oat2b and Oat2d (Km 199.7, and 266.9µM). The initial screening of a series of diverse endo- and xenobiotics showed interaction with a number of compounds, including cGMP and diclofenac (IC50 27.74, and 19.14µM) with Oat2a; estrone-3-sulfate and diclofenac (IC50 30.96, and 12.6µM) with Oat2b; and fumarate and indomethacin (IC50 68.24, and 20.41µM) with Oat2d. This study provides the first comprehensive data set on Oat2 in zebrafish and offers an important basis for more detailed molecular and (eco)toxicological characterizations of these transporters.

Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics.

Organic cation transporters (OCTs) serve as uptake transporters of numerous endo- and xenobiotics. They have been in the focus of medical toxicological research for more than a decade due to their key role in absorption, distribution, metabolism and excretion due to their expression on basolateral membranes of various barrier tissues. OCTs belong to the SLC22A family within the SLC (Solute carrier) protein superfamily, with three co-orthologs identified in humans (OCT1, 2 and 3), and two Oct orthologs in zebrafish (Oct1 and Oct2). The structural and functional properties of zebrafish Octs, along with their toxicological relevance, have still not been explored. In this study, we performed a functional characterization of zebrafish Oct1 using transient and stable heterologous expression systems and model fluorescent substrates as the basis for interaction studies with a wide range of endo- and xenobiotics. We also conducted a basic topology analysis and homology modeling to determine the structure and membrane localization of Oct1. Finally, we performed an MTT assay to evaluate the toxic effects of the seven interactors identified - oxaliplatin, cisplatin, berberine, MPP+, prazosin, paraquat and mitoxantrone - in human embryonic kidney cells (HEK293T) stably expressing zebrafish Oct1 (HEK293T-drOct1 cells). Our results show that the zebrafish Oct1 structure consists of 12 transmembrane alpha helices, which form the active region with more than one active site. Five new fluorescent substrates of Oct1 were identified: ASP+ (Km=26µM), rhodamine 123 (Km=103.7nM), berberine (Km=3.96µM), DAPI (Km=780nM), and ethidium bromide (Km=97nM). Interaction studies revealed numerous interactors that inhibited the Oct1-dependent uptake of fluorescent substrates. The identified interactors ranged from physiological compounds (mainly steroid hormones) to different classes of xenobiotics, with IC50 values in nanomolar (e.g., pyrimethamine and prazosin) to millimolar range (e.g., cimetidine). Cytotoxicity experiments with HEK293T-drOct1 cells enabled us to identify berberine, oxaliplatin and MPP+ as substrates of Oct1. The data presented in this study provide the first insights into the functional properties of zebrafish Oct1 and offer an important basis for more detailed molecular and ecotoxicological characterizations of this transporter.

Serine ADP-Ribosylation Depends on HPF1.

ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

Serine is a new target residue for endogenous ADP-ribosylation on histones.

ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.