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Abstract The KM3NeT research infrastructure is unconstruction in the Mediterranean Sea. KM3NeT will study atmospheric and astrophysical neutrinos with two multi-purpose neutrino detectors, ARCA and ORCA, primarily aimed at GeV–PeV neutrinos. Thanks to the multi-photomultiplier tube design of the digital optical modules, KM3NeT is capable of detecting the neutrino burst from a Galactic or near-Galactic core-collapse supernova. This potential is already exploitable with the first detection units deployed in the sea. This paper describes the real-time implementation of the supernova neutrino search, operating on the two KM3NeT detectors since the first months of 2019. A quasi-online astronomy analysis is introduced to study the time profile of the detected neutrinos for especially significant events. The mechanism of generation and distribution of alerts, as well as the integration into the SNEWS and SNEWS 2.0 global alert systems, are described. The approach for the follow-up of external alerts with a search for a neutrino excess in the archival data is defined. Finally, an overview of the current detector capabilities and a report after the first two years of operation are given.

Abstract This article presents the potential of a combined analysis of the JUNO and KM3NeT/ORCA experiments to determine the neutrino mass ordering. This combination is particularly interesting as it significantly boosts the potential of either detector, beyond simply adding their neutrino mass ordering sensitivities, by removing a degeneracy in the determination of ∆ m 31 2 $$ {m}_{31}^2 $$ between the two experiments when assuming the wrong ordering. The study is based on the latest projected performances for JUNO, and on simulation tools using a full Monte Carlo approach to the KM3NeT/ORCA response with a careful assessment of its energy systematics. From this analysis, a 5σ determination of the neutrino mass ordering is expected after 6 years of joint data taking for any value of the oscillation parameters. This sensitivity would be achieved after only 2 years of joint data taking assuming the current global best-fit values for those parameters for normal ordering.

Abstract KM3NeT/ORCA is a next-generation neutrino telescope optimised for atmospheric neutrino oscillations studies. In this paper, the sensitivity of ORCA to the presence of a light sterile neutrino in a 3+1 model is presented. After three years of data taking, ORCA will be able to probe the active-sterile mixing angles θ 14, θ 24, θ 34 and the effective angle θ μe , over a broad range of mass squared difference ∆ m 41 2 $$ \Delta {m}_{41}^2 $$ ∼ [10 −5 , 10] eV2, allowing to test the eV-mass sterile neutrino hypothesis as the origin of short baseline anomalies, as well as probing the hypothesis of a very light sterile neutrino, not yet constrained by cosmology. ORCA will be able to explore a relevant fraction of the parameter space not yet reached by present measurements.

According to the Swiss legislation on maternity protection in the workplace (OProMa), if pregnant workers are exposed to occupational hazards and no protective measures are taken, a gynecologist will prescribe a certificate of preventive leave and the women must stop working. Returning to work is only possible if job adjustments are made. This study aims to evaluate the burden of absences on companies and to examine the predictors of the return to work for pregnant workers on preventive leave, by examining both the probability of return to work and the time required to return to work. The study sample includes data on 258 workplaces of pregnant workers on preventive leave, collected during an occupational medicine consultation aimed at supporting the implementation of the OProMa. Information is available on the worker (age, date of consultation), the hazards to which she is exposed, the company's knowledge of the OProMa and whether a risk analysis exists. Descriptive statistics and multivariate regression analysis are carried out. In 58% of the workplaces, it was not possible to return to work before the end of the pregnancy. This corresponds to an average absence of 4.5 months. In 42% of the workplaces, a return to work was possible thanks to workplace adaptations. A conforming risk analysis and a full knowledge of the OProMa for companies, and an early visit to the occupational medicine consultation for workers are good predictors of the likelihood of returning to work. Younger age and exposure to certain types of risks are factors that influence the duration of preventive leave. The implementation of OProMa in Switzerland poses serious challenges, but early identification of occupational hazards and practices that anticipate compliance with the law in the company increase the return to work in safety for pregnant workers., Competing Interests: The authors have read the journal’s policy: KM, LB, AAZ, IP, SCR, and JV have declared that they have no competing interests. PK one of the author s of the manuscript, has the following competing interests: financial (paid employment). PK is one of the occupational health physicians who is working at the specialized occupational medicine consultation in the canton of Vaud, Switzerland. PK is employed by Unisanté, a university centre for general medicine and public health (public institution) based in Lausanne, Switzerland. Unisanté provides several services, of which this consultation is one., (Copyright: © 2024 Moschetti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Most women continue to work during pregnancy. However, some of them have to stop working before giving birth. Absence from work poses several challenges for employers and employees, as well as for society. The literature on absence from work during pregnancy and its determinants remains inconsistent and rather scarce. To conduct a narrative literature review on the factors associated with work interruption and on existing interventions aimed at reducing the absence prevalence during pregnancy. The review refers to published peer-reviewed articles dealing with all types of work interruption among pregnant women. Keyword searches were performed in the electronic databases PubMed, EMBASE, and Google Scholar, covering the period 2000-2022. The review, which includes 42 papers, presents a broad and comprehensive picture of factors and interventions associated with absence from work among pregnant workers. The factors appear at different levels and include factors related to the pregnant women, such as individual health and socio-demographic factors; employer and workplace-related factors, such as risk exposures and working conditions; factors related to the role of the healthcare provider; and factors related to the national context (social benefits/insurance). The determinants of absence from work during pregnancy are complex and multifactorial and involve multiple stakeholders. The discussion addresses gaps and needs in the literature on pregnancy at work and in the field of occupational health. Int J Occup Med Environ Health. 2023;36(3):303-23., (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)

Specific working conditions may endanger pregnant women's and their children's health. Switzerland has specific legislation for the protection of pregnant and breastfeeding employees. However, the implementation of these provisions presents important shortcomings. Attending physicians or gynaecologists-obstetricians who care for pregnant women have a central role in controlling the efficacy of protective measures put in place for their patients at work. What are the occupational exposures at risk and their health impact ? What are the collaboration with the different actors involved in the protection of maternity at work ? What steps should be taken if occupational dangers are identified? What tools can be used ? This article answers and explore these questions., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci . In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆ traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Berger, Michaelis, Probst, Sagmeister, Petrowitsch, Puchner, Pavkov-Keller, Gesslbauer, Grohmann and Keller.)

Background: Switzerland's maternity protection legislation aims to protect the health of pregnant employees and their unborn children by regulating their potential occupational exposure to hazards and strenuous activities. This legislation provides a role for obstetricians, but not for midwives., Aims: Identify the practices of Switzerland's French-speaking midwives that favour the implementation of maternity protection legislation and reflect on the profession's role in supporting pregnant employees., Methods: 356 midwives answered an online questionnaire. The analysis focuses on the 205 midwives who perform pregnancy consultations in their practice. Data were analysed in two stages using STATA software: 1) simple descriptive and correlational statistics and 2) hierarchical cluster analysis to identify typologies of practices by grouping similar responses., Findings: Despite having no officially defined role in Switzerland's maternity protection legislation, its midwives actively participate in protecting pregnant employees , especially those with more knowledge of the legislation, those with more years of experience and those practicing independently. The barriers that midwives face when trying to provide greater support for pregnant employees are linked significantly to their lack of knowledge about the legislation, a lack of recognition for their role in the current legislation and a lack of continuing education about the occupational health risks associated with pregnancy at work., Conclusions and Implications for Practice: Their profession and specific practices give midwives privileged access to pregnant employees. Midwives' knowledge of the legislation, their awareness of the occupational risks and hazards facing pregnant employees and the conviction that their profession has the potential to make a difference could all be improved. The role of midwives should be-and deserves to be-formally and legally recognised and integrated into Switzerland's maternity protection legislation., Competing Interests: Declaration of Competing Interest None Declared, (Copyright © 2021. Published by Elsevier Ltd.)

Background: In accordance with the International Labour Organization’s Maternity Protection Convention (No. 183) and European Union Directive 92/857CEE (1992), Switzerland’s Labour Law and its Maternity Protection Ordinance (OProMa) aim to protect the health of pregnant employees and their future children while enabling them to pursue their working activities. Gynaecologists-obstetricians have a key role in this legislation, particularly through the prescription of preventive leave for patients who would otherwise face dangerous or arduous tasks in the absence of an adequate risk analysis or suitable protective measures. However, international and national literature suggests that gynaecologists-obstetricians may encounter difficulties in fulfilling their role., Aims: This study aimed to: (1) describe the practices and difficulties encountered by gynaecologists-obstetricians in the practical implementation of the OProMa; and (2) compare the evolution of these practices and difficulties between 2008 and 2017., Methods: A survey by questionnaire was conducted in 2008 and repeated in 2017. Both surveys focused on gynaecologists-obstetricians working in the French-speaking part of Switzerland (in private practices, hospitals or both). Descriptive and comparative analyses were carried out., Results: 83 gynaecologists-obstetricians responded in 2008 and 93 in 2017: response rates of 47% and 32%, respectively. In 2017, gynaecologists-obstetricians were more likely to ask questions about occupational risks faced by their patients when consulted by working mothers about their pregnancies. The estimated percentage of patients exposed to an occupational risk remained constant (20% in 2008 and 22% in 2017). Communication and collaboration with employers were reported to be difficult in both surveys, even though these are key elements in the implementation of the OProMa. Collaboration with occupational physicians, however, was more frequent in 2017., Conclusion: In 2017, gynaecologists-obstetricians showed a greater awareness of occupational risks and collaborated more frequently with occupational health specialists. However, the application of the OProMa remained limited over the studied time period. Improving training of gynaecologists-obstetricians in this field could be a significant factor in encouraging better implementation of the current legislation. Moreover, gynaecologists-obstetricians need to be given the necessary support to enable their clinical practice to evolve towards a more preventive type of medicine. Collaboration with relevant stakeholders, including occupational physicians, midwives and workers, should be encouraged.

Background: Switzerland's Ordinance on Maternity Protection at Work (OProMa) requires that companies take the necessary measures to ensure that pregnant employees can continue working without danger., Objective: To investigate the extent of compliance with OProMa within companies in French-speaking Switzerland as well as factors which facilitate and obstruct the ordinance's implementation., Methods: A stratified random telephone survey of 202 companies from the healthcare and food industry was conducted. Descriptive and correlational statistics were calculated. Responses to open questions were analysed thematically., Results: Only a minority of companies performed risk analyses or adapted employees' workstations, as per the legislation. OProMa was implemented more effectively in larger companies than smaller ones, in public rather than private ones, in the healthcare sector rather than the food industry, and when the person responsible for the wellbeing of pregnant employees within the company had undergone specific training on the subject. Data extrapolation suggested that only 2% of pregnant employees in French-speaking Switzerland's food industry and 12% in its healthcare sector are properly protected according to OProMa's provisions., Conclusions: Maternity protection in French-speaking Switzerland's companies urgently requires improvement. In addition to the apparent need for stronger incentives and for monitoring of companies, our findings indicate a need to provide them with resources to meet OProMa's provisions.

Background: In several countries, maternity protection legislations (MPL) confer an essential role to gynecologist-obstetricians (OBGYNs) for the protection of pregnant workers and their future children from occupational exposures. This study explores OBGYNs' practices and difficulties in implementing MPL in the French-speaking part of Switzerland., Methods: An online survey was sent to 333 OBGYNs. Data analysis included: 1) descriptive and correlational statistics and 2) hierarchical cluster analysis to identify patterns of practices., Results: OBGYNs evoked several problems in MPL implementation: absence of risk analysis in the companies, difficult collaboration with employers, lack of competencies in the field of occupational health. Preventive leave was underused, with sick leave being prescribed instead. Training had a positive effect on OBGYNs' knowledge and implementation of MPL. Hierarchical cluster analysis highlighted three main types of practices: 1) practice in line with legislation; 2) practice on a case-by-case basis; 3) limited practice. OBGYNs with good knowledge of MPL more consistently applied its provisions., Conclusion: The implementation of MPL appears challenging for OBGYNs. Collaboration with occupational physicians and training might help OBGYNs to better take on their role in maternity protection. MPL in itself could be improved., Competing Interests: The authors have declared that no competing interests exist.

For parents working in the transportation industry, atypical schedules are often a daily puzzle. Schedule planning systems allowing workers to choose shifts may affect job strenuousness and work-family balance (WFB) for both female and male workers. How could ergonomic interventions related to the implementation of those systems better consider gender dynamics regarding WFB strategies, and minimize inequities among workers? This article presents a joint analysis of two independent case studies related to ergonomic interventions in transport companies in Canada and Switzerland. Direct observation and semi-structured interviews shed light on the characteristics of schedule planning systems and their interaction with men's and women's WFB strategies. Issues related to each step of the planning process (shift construction, schedule choice, day-to-day schedule management) are discussed to inform interventions aimed at facilitating WFB, and ultimately gender equity, in atypical schedule contexts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Most industrialized countries have introduced maternity protection legislation (MPL) to protect the health of pregnant workers and their unborn children from workplace exposure. This review aimed to assess this legislation's level of implementation, barriers and facilitators to it, and its expected or unexpected effects., Methods: A realist narrative review was conducted. Keyword searches of the PubMed, CINAHL, PsycINFO, MIDIRS, Sociological abstracts and Google Scholar electronic databases were performed in March 2018., Results: The 42 publications included show that the implementation of MPL is deficient in most countries. Allowing pregnant women to withdraw from work on preventive leave or sick leave is favored over workplace adaptations or worker reassignments. The publications highlight mechanisms which encourage or obstruct the enforcement of legislation at the levels of the individual, the physical and social environment, and the macrosocial context. The delay between the conception and implementation of maternity protection measures appears to be a major barrier to the efficacy of MPL. The literature also suggests that unexpected adverse effects, such as degradation in working relationships or discrimination can obstruct the implementation of protective measures., Conclusions: This study showed the need for a better implementation of MPL during pregnancy. Further research and recommendations for improvements in MPL should consider the diverse mechanisms and effects of its implementation. Barriers and adverse effects of this implementation do not only ensure a lack of information or awareness about MPL, but are also linked to contradictions between requirements to protect employment and protect pregnancy.

The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein-DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.

Introduction: Like most industrialised countries, Switzerland has introduced legislation to protect the health of pregnant workers and their unborn children from workplace exposure. This legislation provides for a risk assessment, adaptations to workplaces and, if the danger is not eliminated, preventive leave (prescribed by a gynaecologist). This study's first objective is to analyse the degree to which companies, gynaecologists and midwives implement the law. Its second objective is to understand the obstacles and resources of this implementation, with a focus on how relevant stakeholders perceive protective measures and their involvement with them., Methods and Analysis: Data will be collected using mixed methods: (1) online questionnaires for gynaecologists and midwives; telephone questionnaires with company human resources (HR) managers in the healthcare and food production sectors; (2a) case studies of 6-8 companies in each sector, including interviews with stakeholders such as women workers, HR managers and occupational health physicians; (2b) two focus groups, one involving occupational physicians and hygienists, one involving labour inspectors.Quantitative data will be analysed statistically using STATA software V.15. Qualitative data will be transcribed and thematically analysed using MaxQDA software., Ethics and Dissemination: The Human Research Ethics Committee of the Canton Vaud (CER-VD) has certified that this research study protocol falls outside of the field of application of the Swiss Federal Act on Research Involving Humans.The publications and recommendations resulting from this study will form the starting point for future improvements to the protection of pregnant women at work and their unborn children.This study started in February 2017 and will continue until January 2020., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

For many gram-positive pathogens, conjugative plasmid transfer is an important means of spreading antibiotic resistance . Therefore, the search for alternative treatments to fight and prevent infections caused by these bacteria has become of major interest. In the present study, we evaluated the protein TraM, from the conjugative plasmid pIP501, as a potential vaccine candidate. Anti-TraM antiserum mediated in vitro opsonophagocytic killing of the strain harboring the pIP501 plasmid and also proved to be cross-reactive against other clinically relevant enterococcal and staphylococcal strains. Specificity of antibodies toward TraM was confirmed by results of an opsonophagocytic inhibition assay and Western blot. In addition, conjugative transfer experiments proved that TraM is essential for the transfer of pIP501. Finally, immunization with either TraM or anti-TraM antiserum reduced significantly the colony counts in mice livers, demonstrating that TraM is a promising vaccine candidate against enterococci and other gram-positive pathogens., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21(st) century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. We report the NMR structure and functional characterization of the transfer protein TraH encoded by the conjugative Gram-positive broad-host range plasmid pIP501. The structure exhibits a striking similarity to VirB8 proteins of Gram-negative secretion systems where they play an essential role in the scaffold of the secretion machinery. Considering TraM as the first VirB8-like protein discovered in pIP501, TraH represents the second protein affiliated with this family in the respective transfer operon. A markerless traH deletion in pIP501 resulted in a total loss of transfer in Enterococcus faecalis as compared with the pIP501 wild type (wt) plasmid, demonstrating that TraH is essential for pIP501 mediated conjugation. Moreover, oligomerization state and topology of TraH in the native membrane were determined providing insights in molecular organization of a Gram-positive T4SS.

Actinobacillus (A.) pleuropneumoniae is among the most important pathogens in pig. The agent causes severe economic losses due to decreased performance, the occurrence of acute or chronic pleuropneumonia, and an increase in death incidence. Since therapeutics cannot be used in a sustainable manner, and vaccination is not always available, new prophylactic measures are urgently needed. Recent research has provided evidence for a genetic predisposition in susceptibility to A. pleuropneumoniae in a Hampshire × German Landrace F2 family with 170 animals. The aim of the present study is to characterize the expression response in this family in order to unravel resistance and susceptibility mechanisms and to prioritize candidate genes for future fine mapping approaches. F2 pigs differed distinctly in clinical, pathological, and microbiological parameters after challenge with A. pleuropneumoniae. We monitored genome-wide gene expression from the 50 most and 50 least susceptible F2 pigs and identified 171 genes differentially expressed between these extreme phenotypes. We combined expression QTL analyses with network analyses and functional characterization using gene set enrichment analysis and identified a functional hotspot on SSC13, including 55 eQTL. The integration of the different results provides a resource for candidate prioritization for fine mapping strategies, such as TF, TFRC, RUNX1, TCN1, HP, CD14, among others.

Actinobacillus pleuropneumoniae is among the most important pathogens worldwide in pig production. The agent can cause severe economic losses due to decreased performance, acute or chronic pleuropneumonia and an increased incidence of death. Therapeutics cannot be used in a sustainable manner, and vaccination is not always available, but discovering more about host defence and disease mechanisms might lead to new methods of prophylaxis. The aim of the present study was to detect quantitative trait loci (QTL) associated with resistance/susceptibility to A. pleuropneumoniae. Under controlled conditions, 170 F2 animals of a Hampshire/Landrace family, with known differences in founder populations regarding A. pleuropneumoniae resistance, were challenged with an A. pleuropneumoniae serotype 7 aerosol followed by a detailed clinical, radiographic, ultrasonographic, pathological and bacteriological examination. F2 pigs were genotyped with 159 microsatellite markers. Significant QTL were identified on Sus scrofa chromosomes (SSC) 2, 6, 12, 13, 16, 17 and 18. They explained 6-22% of phenotypic variance. One QTL on SSC2 reached significance on a genome-wide level for five associated phenotypic traits. A multiple regression analysis revealed a combinatory effect of markers SWR345 (SSC2) and S0143 (SSC12) on Respiratory Health Score, Clinical Score and the occurrence of death. The results indicate the genetic background of A. pleuropneumoniae resistance in swine and provide new insights into the genetic architecture of resistance/susceptibility to porcine pleuropneumonia. The results will be helpful in identifying the underlying genes and mechanisms.

Clinical studies have proved the therapeutic potential of hepatocyte transplantation as a promising alternative to whole organ liver transplantation in the treatment of hereditary or end-stage liver disease. However, donor shortage seriously restricts cell availability, and the lack of appropriate cell culture protocols for the storage and maintenance of donor cells constitutes a significant obstacle. The aim of this study was to stimulate mature hepatocytes in culture to multiply in vitro and track their fate on transplantation. Rat hepatocytes isolated nonenzymatically were cultured serum free for up to 10 days. They were stimulated into proliferation in the presence of growth factors and conditioned media from nonparenchymal and hepatocyte culture supernatants, as well as 10 mM lithium chloride (LiCl). Cell proliferation was assessed by determining DNA content. Additionally, the extent of cell differentiation was estimated using immunofluorescence staining of hepatic, biliary, progenitor, and mesenchymal markers and gene expression analyses. Transplantation studies were performed on the Fischer CD26-mutant rat following pretreatment with retrorsine and partial hepatectomy. Proliferating hepatocytes increasingly adopted precursor characteristics, expressing progenitor (OV6, CD133), hepatic lineage (CK18), biliary (CD49f, CK7, CK19), and mesenchymal (vimentin) markers. The supplement of LiCl further enhanced the proliferative capacity by 30%. Transplantation studies revealed extensive repopulation by large donor hepatocyte clusters. Furthermore, bile duct-like structures deriving from donor cells proved to be immunoreactive to ductular markers and formed in close proximity to endogenous bile ducts. Mature hepatocytes reveal their potential to "switch" between phenotypes, adopting progenitor characteristics during proliferation in vitro. Following transplantation, these "retrodifferentiated" cells further expanded in vivo, thereby generating bipotentially differentiated progenies (hepatocytes and bile duct-like structures). This apparent plasticity of mature hepatocytes may open new approaches for cell-based strategies to treat liver disease.

Paracetamol (acetaminophen) is a widely used analgesic drug. It interacts with various enzyme families including cytochrome P450 (CYP), cyclooxygenase (COX), and nitric oxide synthase (NOS), and this interplay may produce reactive oxygen species (ROS). We investigated the effects of paracetamol on prostacyclin, thromboxane, nitric oxide (NO), and oxidative stress in four male subjects who received a single 3 g oral dose of paracetamol. Thromboxane and prostacyclin synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F(1α), respectively. Endothelial NO synthesis was assessed by measuring nitrite in plasma. Urinary 15(S)-8-iso-prostaglanding F(2α) was measured to assess oxidative stress. Plasma oleic acid oxide (cis-EpOA) was measured as a marker of cytochrome P450 activity. Upon paracetamol administration, prostacyclin synthesis was strongly inhibited, while NO synthesis increased and thromboxane synthesis remained almost unchanged. Paracetamol may shift the COX-dependent vasodilatation/vasoconstriction balance at the cost of vasodilatation. This effect may be antagonized by increasing endothelial NO synthesis. High-dosed paracetamol did not increase oxidative stress. At pharmacologically relevant concentrations, paracetamol did not affect NO synthesis/bioavailability by recombinant human endothelial NOS or inducible NOS in rat hepatocytes. We conclude that paracetamol does not increase oxidative stress in humans.

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.

During liver regeneration in vivo carbon monoxide (CO) and nitric oxide (NO) are supposed to play a significant role. We raise the question whether CO and NO are involved in the growth process of cultured hepatocytes. Rat hepatocytes were stimulated into proliferation, growth being estimated by DNA content, mRNA by quantitative RT-PCR, and inducible NO synthase (iNOS) activity by GC-MS. Dexamethasone proved obligatory for fast proliferation. It suppressed the spontaneous rise of iNOS-mRNA in cultures devoid of glucocorticoids, but did not counteract the rise in mRNA in actively dividing cultures. Expression of iNOS-mRNA and cell growth were further enhanced by LiCl (10 mM). NOS activity was completely suppressed by the iNOS-specific inhibitors N-(3-(aminomethyl)benzyl) acetamidine (1400 W,100 microM) and L-N(6)-(1-iminoethyl)lysine (L-NIL, 500 microM), however, without a decrease in hepatocyte growth. Proliferation was attenuated only by very high concentrations (>0.5 mM) of N-nitro-L-arginine methyl ester (L-NAME) and asymmetric dimethylarginine (ADMA). Various NO donors (at 100 microM) did not stimulate cell growth. The furoxan CAS 1609 stimulated growth, decreased iNOS-mRNA expression and transiently increased haem oxygenase-1 (HO-1)-mRNA without releasing considerable amounts of NO. 1H-[1,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ) attenuated the action of CAS 1609. Proliferation was stimulated by Co-protoporphyrin and tricarbonyldichlororuthenium(II) dimer (CORM-2). We conclude that CAS 1609 triggers hepatocyte mitosis most likely via direct, NO-independent induction of HO-1 expression, pointing to CO as a growth-promoting signal in the proliferation cascade in cultured hepatocytes., (Copyright 2010 Elsevier Inc. All rights reserved.)