S-phase checkpoint prevents leading strand degradation from strand-associated nicks at stalled replication forks.

The S-phase checkpoint is involved in coupling DNA unwinding with nascent strand synthesis and is critical to maintain replication fork stability in conditions of replicative stress. However, its role in the specific regulation of leading and lagging strands at stalled forks is unclear. By conditionally depleting RNaseH2 and analyzing polymerase usage genome-wide, we examine the enzymology of DNA replication during a single S-phase in the presence of replicative stress and show that there is a differential regulation of lagging and leading strands. In checkpoint proficient cells, lagging strand replication is down-regulated through an Elg1-dependent mechanism. Nevertheless, when checkpoint function is impaired we observe a defect specifically at the leading strand, which was partially dependent on Exo1 activity. Further, our genome-wide mapping of DNA single-strand breaks reveals that strand discontinuities highly accumulate at the leading strand in HU-treated cells, whose dynamics are affected by checkpoint function and Exo1 activity. Our data reveal an unexpected role of Exo1 at the leading strand and support a model of fork stabilization through prevention of unrestrained Exo1-dependent resection of leading strand-associated nicks after fork stalling.

Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the emergence of novel B3-derived lineages, ENPEN multicentre study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019.

BackgroundVarious pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels.AimIn this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail.MethodsCerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed.ResultsAmong 23 idiopathic cases, TOSV was identified in eight (all male; median age: 39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event.ConclusionsOur study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.

Study of the Effect of Wild-Type and Transiently Expressing CXCR4 and IL-10 Mesenchymal Stromal Cells in a Mouse Model of Peritonitis.

Sepsis due to peritonitis is a process associated with an inflammatory state. Mesenchymal stromal cells (MSCs) modulate the immune system due to the paracrine factors released and may be a therapeutic alternative. Three treatment groups were developed in a murine model of peritonitis to verify the effect of human adipose mesenchymal stem cell (hASCs). Additionally, a temporary modification was carried out on them to improve their arrival in inflamed tissues (CXCR4), as well as their anti-inflammatory activity (IL-10). The capacity to reduce systemic inflammation was studied using a local application (peritoneal injection) as a treatment route. Comparisons involving the therapeutic effect of wild-type ASCs and ASCs transiently expressing CXCR4 and IL-10 were carried out with the aim of generating an improved anti-inflammatory response for sepsis in addition to standard antibiotic treatment. However, under the experimental conditions used in these studies, no differences were found between both groups with ASCs. The peritoneal administration of hASCs or genetically modified hASCs constitutes an efficient and safe therapy in our model of mouse peritonitis.

A Mixed-Methods Systematic Review on Syndemics in Rheumatology.

OBJECTIVE: We aimed to assess the use of framework and corresponding methodology to document syndemics and its impact in rheumatic and musculoskeletal diseases (RMDs). METHODS: Using a mixed-methods systematic review, studies using the syndemic framework approach for RMDs were identified and published from January 2003 to January 2021. The Joanna Briggs Institute, Cochrane Collaboration, and PRISMA guidelines were followed to search, retrieve, revise, and analyze. RESULTS: A total of 658 potential articles were identified, but only 10 were initially eligible. After a full-text review, 4 were included. Following a full-text review, 2 quantitative, 1 qualitative, and 1 mixed-methods study were included. In the first, network analysis found that RMDs were associated with comorbidities, unhealthy habits, low educational level, living in rural areas, socioeconomic conditions, and health inequality in indigenous communities. In the second, SSEM and cluster analysis demonstrated an association between low back pain and factors, such as comorbidities and indigenous status, among others, in urban/rural communities. The qualitative study examined 3 fishing family generations and reported less syndemic vulnerability. The mixed-methods study focused on osteoarthritis with multimorbidities in African American population, where lack of education added to worsening outcomes. CONCLUSIONS: Even though the insights syndemic studies have given to other areas, its use in rheumatology is scarce. The complexity of the clinical and social determinants related to RMDs makes it necessary to conduct further studies from a syndemic perspective.

Prevalence and genetic characterization of noroviruses in children with acute gastroenteritis in Senegal, 2007-2010.

Norovirus is the leading cause of sporadic and epidemic acute gastroenteritis (AGE) in children and adults around the world. We investigated the molecular diversity of noroviruses in a pediatric population in Senegal between 2007 and 2010 before the rotavirus vaccine implementation. Stool samples were collected from 599 children under 5 years of age consulting for AGE in a hospital in Dakar. Specimens were screened for noroviruses using the Allplex™ GI-Virus Assay. Positive samples were genotyped after sequencing of conventional reverse transcription-polymerase chain reaction products. Noroviruses were detected in 79 (13.2%) of the children, with GII.4 (64%) and GII.6 (10%) as the most frequently identified genotypes. Our study describes the distribution of genotypes between 2007 and 2010 and should be a baseline for comparison with more contemporary studies. This could help decision-makers on possible choices of norovirus vaccines in the event of future introduction.

Mapping sites of gibberellin biosynthesis in the Arabidopsis root tip.

Root elongation depends on the action of the gibberellin (GA) growth hormones, which promote cell production in the root meristem and cell expansion in the elongation zone. Sites of GA biosynthesis in the roots of 7-d-old Arabidopsis thaliana seedlings were investigated using tissue-specific GA inactivation in wild-type (Col-0) or rescue of GA-deficient dwarf mutants. Tissue-specific GA depletion was achieved by ectopic expression of the GA-inactivating enzyme AtGA2ox2, which is specific for C19 -GAs, and AtGA2ox7, which acts on C20 -GA precursors. In addition, tissue-specific rescue of ga20ox triple and ga3ox double mutants was shown. Furthermore, GUS reporter lines for major GA20ox, GA3ox and GA2ox genes were used to observe their expression domains in the root. The effects of expressing these constructs on the lengths of the root apical meristem and cortical cells in the elongation zone confirmed that roots are autonomous for GA biosynthesis, which occurs in multiple tissues, with the endodermis a major site of synthesis. The results are consistent with the early stages of GA biosynthesis within the root occurring in the meristematic region and indicate that the penultimate step of GA biosynthesis, GA 20-oxidation, is required in both the meristem and elongation zone.

Prevalence and Genetic Diversity of Aichi Virus 1 from Urban Wastewater in Senegal.

Aichi virus 1 (AiV-1) has been proposed as a causative agent of human gastroenteritis. In this study, raw, decanted, and treated wastewater samples from a wastewater treatment plant in an urban area of Dakar, Senegal, were collected. AiV-1 was detected in raw (70%, 14/20), decanted (68.4%, 13/19), and treated (59.3%, 16/27) samples, revealing a noticeable resistance of AiV-1 to chlorine-based treatment. Phylogenetic analysis revealed that all sequences clustered within genotype B. Our study presents the first report on the detection of AiV-1 in the environment of Dakar and constitutes indirect evidence of virus circulation in the population.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

An imported case of vaccine-derived poliovirus type 2, Spain in the context of the ongoing polio Public Health Emergency of International Concern, September 2021.

The monthly retrospective search for unreported acute flaccid paralysis (AFP) cases conducted as a complementary component of the Spanish AFP surveillance system identified a case of AFP in a child admitted in Spain from Senegal during August 2021. Vaccine-derived poliovirus 2 was identified in the stool in September 2021. We present public health implications and response undertaken within the framework of the National Action Plan for Polio Eradication and the Public Health Emergency of International Concern.

[Human resource management and vaccination strategies in primary care in Europe during COVID-19 pandemic]. / Gestión de los recursos humanos y estrategias de vacunación en atención primaria en Europa en la pandemia COVID-19.

This article describes the management of human resource and the vaccination strategies in primary care in twelve European countries in relation to the COVID-19 pandemic. All the countries have found solutions to increase their workforce in primary care. Other healthcare professionals were incorporated to support family doctors assuming their tasks, under their supervision and coordination. The European Commission had a crucial role in the production, purchase and distribution of the vaccines. The engagement of primary care in the vaccination campaign has had an unequal participation in the different countries, although the greatest burden has been managed from the government's public health departments.

[The role of primary care during COVID-19 pandemic: A European overview]. / El papel de la atención primaria en la pandemia COVID-19: Una mirada hacia Europa.

We describe the role of primary care (PC) in 12 European countries in relation to the COVID-19 pandemic. There is no official information at European level on the activity of PC. The findings were: All countries provided COVID-19 information through telephone lines and websites to their citizens. Contact tracing was mainly carried out by Public Health except for Ireland, Portugal and Spain. The epidemiological surveillance task has overlapped with the PC assistance. Active Infection Diagnostic Tests (AIDT) were performed in PC exclusively in Spain. The other countries performed them in external laboratories. Patients were followed-up in PC mostly by remote assessment. Health coverage for vulnerable populations and nursing homes has been regulated in all countries. There is a need for a strategic plan for PC in Europe that responds to the challenges posed.

[The Global Patient Safety Action Plan 2021-2030: Identifying actions for safer primary health care]. / El Plan de acción mundial para la seguridad del paciente 2021-2030: identificando acciones para una atención primaria más segura.

The 74th World Health Assembly adopted in May 2021 the "Global Patient Safety Action Plan: 2021-2030" to enhance patient safety as an essential component in the design, procedures and performance evaluation of health systems worldwide. It is a strategic plan that guides country governments, health sector entities, health organisations and the World Health Organisation secretariat on how to implement the assembly's patient safety resolution. Deployment of the plan will strengthen the quality and safety of health systems worldwide by spanning the entire continuum of people's health care from diagnosis to treatment and care, reducing the likelihood of harm in the course of care. The Declaration on Primary Health Care during the Global Conference on Primary Health Care in Astana, 2018, urged countries to strengthen their primary health care systems as an essential step towards achieving universal health coverage and providing access to safe, quality care without financial loss. The deployment of the Global Patient Safety Action Plan in primary care is therefore a high-priority health policy action. The Action Plan is structured into 6 strategic objectives with 35 strategic actions. We present an analysis of the strategic actions regarding healthcare organizations and the challenges ahead for their particular deployment in primary health care settings.

Effectiveness of a 4-week sophrology program for primary care patients with moderate to high anxiety levels: a randomised controlled trial.

he aim of this study is to determine the effectiveness of an intensive four-week structured group re- laxation-training program (sophrology’s dynamic relaxation) on anxiety and depression symptoms in primary care patients with moderate and high anxiety levels.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid.

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14(-/-) mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16(INK4a) and p21(CIP1/WAF) (1), increased activity of senescence-associated ß-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14(-/-) mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Emergence of Vaccine-Derived Polioviruses during Ebola Virus Disease Outbreak, Guinea, 2014-2015.

During the 2014-2015 outbreak of Ebola virus disease in Guinea, 13 type 2 circulating vaccine-derived polioviruses (cVDPVs) were isolated from 6 polio patients and 7 healthy contacts. To clarify the genetic properties of cVDPVs and their emergence, we combined epidemiologic and virologic data for polio cases in Guinea. Deviation of public health resources to the Ebola outbreak disrupted polio vaccination programs and surveillance activities, which fueled the spread of neurovirulent VDPVs in an area of low vaccination coverage and immunity. Genetic properties of cVDPVs were consistent with their capacity to cause paralytic disease in humans and capacity for sustained person-to-person transmission. Circulation ceased when coverage of oral polio vaccine increased. A polio outbreak in the context of the Ebola virus disease outbreak highlights the need to consider risks for polio emergence and spread during complex emergencies and urges awareness of the challenges in polio surveillance, vaccination, and diagnosis.

Genetic Characterization of Enterovirus A71 Circulating in Africa.

We analyzed whole-genome sequences of 8 enterovirus A71 isolates (EV-A71). We confirm the circulation of genogroup C and the new genogroup E in West Africa. Our analysis demonstrates wide geographic circulation and describes genetic exchanges between EV-A71 and autochthonous EV-A that might contribute to the emergence of pathogenic lineages.

Hyperalgesic and hypoalgesic mechanisms evoked by the acute administration of CCL5 in mice.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1µg) and DAPTA (0.3-3µg), respectively. The prevention of this hyperalgesia by diclofenac (1-10µg), the inhibitors of COX-1 SC-560 (0.1-1µg) or COX-2 celecoxib (1-5µg), the TRPV1 antagonist capsazepine (0.03-0.3µg) or the TRPA1 antagonist HC030031 (10-50µg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17µg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5µg), nor-binaltorphimine (10mg/kg) or an anti-dynorphin A antibody (0.62-2.5ng). The administration of 30ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.