Responses to salt stress in Juncus acutus and J. maritimus during seed germination and vegetative plant growth

[EN] Responses to increasing salinity, during seed germination and vegetative plant growth, were studied in two related species of Juncus, J. maritimus and J. acutus. In both species, germination was optimal in the absence of salt, reduced by about 50% in the presence of 200 mM NaCl, and completely inhibited by NaCl concentrations above 300 mM. Previous exposure of the seeds to salt, up to 500 mM NaCl, did not affect the germination capacity in J. acutus, and clearly enhanced it in J. maritimus. A concentration-dependent inhibition of plant growth was observed in the presence of NaCl for both species, together with the parallel accumulation of sodium ions in the leaves, as determined by cation exchange HPLC. Regarding the levels of divalent cations, in J. acutus Ca2+ and Mg2+ increased up to about two-fold in plants treated with 500 mM NaCl, as compared to control plants, whereas in J. maritimus they were three-to four-fold higher than in J. acutus in the absence of salt, and did not change significantly with increasing NaCl concentrations. These results suggest that Ca2+ and Mg2+ participate in defence mechanisms against salt stress, which would be constitutive in J. maritimus and salt-inducible in J. acutus.Boscaiu Neagu, MT.; Ballesteros Amat, G.; Naranjo Olivero, MA.; Vicente Meana, Ó.; Boira Tortajada, H. (2011). Responses to salt stress in Juncus acutus and J. maritimus during seed germination and vegetative plant growth. Plant Biosystems. 145(4):770-777. doi:10.1080/11263504.2011.628446S770777145

Analysis of the Calls Received during the COVID-19 Lockdown by the Mental Health Crisis Helpline Operated by the Professional College of Psychology of Aragon

COVID-19 has had a direct impact on the physical and mental health of millions of people worldwide. Therefore, a Mental Health Crisis Helpline (MHCH) was set up and offered free of charge by the Professional College of Psychology of Aragon (COPPA) during the lockdown period. This research aims to study the reasons for the calls, to describe the population segments that used it, and to analyse the possible relationships between the variables studied. A total of 1411 calls were answered and 598 were registered. The main reasons for the calls were: anxiety symptoms, concern for a relative, previous mental health problems, conflicts, and depressive symptoms. Significantly more men called for anxiety symptoms (60.8% vs. 49.5%) versus more women calling regarding a chronic physical illness (3.5% vs. 0.7%), concern about a relative (22.7% vs. 12.4%), care guidelines (6% vs. 1.3%), and bereavement (6.2% vs. 2%). Calls regarding conflict increased slightly as the lockdown period progressed (p < 0.001; r = 0.15), in contrast with calls regarding previous psychological conditions and anxiety symptoms (p = 0.035; r = -0.09; p = 0.005; r = -0.12). These results highlight the intensive use of the MHCH, confirming the need for the implementation of specific psychological care resources in times of crisis. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Avances en la inmunosupresión para el trasplante renal. Nuevas estrategias para preservar la función renal y reducir el riesgo cardiovascular

The development of new immunosuppressants for renal transplantation is aimed not only at improving short-term outcomes, but also at achieving better safety, cardiovascular, and metabolic profiles and at decreasing nephrotoxicity. Belatacept is a fusion protein that inhibits T cell activation by binding to CD80 and CD86 antigens. Clinical trials, particularly the BENEFIT and BENEFIT-EXT studies, have shown that belatacept preserves function and structure in renal grafts. The effects of belatacept provide long-term, sustained results, and the safety and efficacy of this drug have been demonstrated in cases of renal transplantation from expanded criteria donors. Compared to calcineurin inhibitors, belatacept is associated with a lower incidence of chronic allograft nephropathy and a more favourable cardiovascular and metabolic profile

Bacteriophages in bathing wàters: A feasibility study on the development of a method based on bacteriophages for the determination of microbiological quality of bathing waters

Projecte: Project report. BCR Information. EU project KINA19506ENC_001. EUROPEAN COMMISSION. Community Research. DG XII/C - Competitive and Sustainable Growth Programme. Published by EU Directorate General ΧΠ - Science, Research and Development ISBN 92-828-9145-3Informe final projecte europeu aigües de bany i bacteriòfagsMethods for the detection and enumeration of somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis had been standardised and validated. Conditions for the preparation, transport and distribution of bacteriophage reference materials and preservation of samples had been defined. A method based on flocculation with Mg(OH2) with concentration efficiencies from about 40% was settled to concentrate phages from bathing waters. All methods were successfully implemented in routine laboratories all around the EU. Data on the occurrence of bacteriophages as compared to E. coli and Enterococci are available from diverse situations encountered in the EU. Results allow to conclude that the potential of phages for the determination of the microbiological quality of bathing waters merits to be considered since their determination is feasible and their behaviour in natural water differs from the behaviour of bacterial indicators and consequently they add valuable information

Effectiveness and cost-effectiveness of a lifestyle modification programme in the prevention and treatment of subclinical, mild and moderate depression in primary care: A randomised clinical trial protocol

Introduction Major depression is a highly prevalent pathology that is currently the second most common cause of disease-induced disability in our society. The onset and continuation of depression may be related to a wide variety of biological and psychosocial factors, many of which are linked to different lifestyle aspects. Therefore, health systems must design and implement health promotion and lifestyle modification programmes (LMPs), taking into account personal factors and facilitators. The main objective of this protocol is to analyse the clinical effectiveness, cost-effectiveness and cost utility of an LMP and an LMP with information and communication technologies (ICTs) as adjunctive treatment for depression in primary care patients. The secondary objectives are to analyse the clinical effectiveness in the subgroup that presents comorbidity and to analyse the correlation between personal factors on health behaviour and lifestyle patterns. Methods and analysis A randomised, multicenter pragmatic clinical trial with three parallel groups consisting of primary healthcare patients suffering from subclinical, mild or moderate depression. The following interventions will be used: (1) Usual antidepressant treatment with psychological advice and/or psychotropic drugs prescribed by the general practitioner (treatment as usual (TAU)). (2) TAU+LMP. A programme to be imparted in six weekly 90-minute group sessions, intended to improve the following aspects: behavioural activation+daily physical activity+adherence to the Mediterranean diet pattern+sleep hygiene+careful exposure to sunlight. (3) TAU+LMP+ICTs: healthy lifestyle recommendations (TAU+LMP)+monitoring using ICTs (a wearable smartwatch). The primary outcome will be the depressive symptomatology and the secondary outcomes will be the quality of life, the use of health and social resources, personal factors on health behaviour, social support, lifestyle patterns and chronic comorbid pathology. Data will be collected before and after the intervention, with 6-month and 12-month follow-ups. Ethics and dissemination This study has been approved by the Clinical Research Ethics Committee of Aragón (approval number: C.P.-C.I. PI18/286) and the Research Ethics Committee of the Balearic Islands (IB3950/19 PI). Data distribution will be anonymous. Results will be disseminated via conferences and papers published in peer-reviewed, open-access journals. Trial registration number ClinicalTrials.gov Registry (NCT03951350)

Belatacept and long-term outcomes in kidney transplantation

Background: in previous analyses of BENEFIT, a phase 3 study, belatacept-based immunosuppression, as compared with cyclosporine-based immunosuppression, was associated with similar patient and graft survival and significantly improved renal function in kidney-transplant recipients. Here we present the final results from this study. Methods: we randomly assigned kidney-transplant recipients to a more-intensive belatacept regimen, a less-intensive belatacept regimen, or a cyclosporine regimen. Efficacy and safety outcomes for all patients who underwent randomization and transplantation were analyzed at year 7 (month 84). Results: a total of 666 participants were randomly assigned to a study group and underwent transplantation. Of the 660 patients who were treated, 153 of the 219 patients treated with the more-intensive belatacept regimen, 163 of the 226 treated with the less-intensive belatacept regimen, and 131 of the 215 treated with the cyclosporine regimen were followed for the full 84-month period; all available data were used in the analysis. A 43% reduction in the risk of death or graft loss was observed for both the more-intensive and the less-intensive belatacept regimens as compared with the cyclosporine regimen (hazard ratio with the more-intensive regimen, 0.57; 95% confidence interval [CI], 0.35 to 0.95; P=0.02; hazard ratio with the less-intensive regimen, 0.57; 95% CI, 0.35 to 0.94; P=0.02), with equal contributions from the lower rates of death and graft loss. The mean estimated glomerular filtration rate (eGFR) increased over the 7-year period with both belatacept regimens but declined with the cyclosporine regimen. The cumulative frequencies of serious adverse events at month 84 were similar across treatment groups. Conclusions: seven years after transplantation, patient and graft survival and the mean eGFR were significantly higher with belatacept (both the more-intensive regimen and the less-intensive regimen) than with cyclosporine. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT00256750)

Morphological and molecular analysis of natural hybrids between the diploid Centaurea aspera L. and the tetraploid C. seridis L. (Compositae)

[EN] Polyploidy and hybridisation are the basis of the evolution of Centaurea (Compositae). At the El Saler dune field (eastern Spain), the diploid Centaurea aspera ssp. stenophylla and the tetraploid C. seridis ssp. maritima form a polyploid complex in which C. x subdecurrens individuals occur. This polyploid complex was analysed morphologically and genetically, using random amplified polymorphic DNA (RAPD) and tubulin-based polymorphism (TBP) markers. Flow cytometry showed that the hybrids are triploid, which is a rare finding in Centaurea. Morphologically, in contrast to leaf characters, flowering characters clearly discriminated the three taxa. The genetic analyses confirm that C. x subdecurrens is a result of the hybridisation between Centaurea aspera ssp. stenophylla and C. seridis ssp. maritima, and suggest that backcrossing events and gene flow are very rare or absent. Although the hybrids likely represent true F1 offspring, they displayed some genetic diversity that is probably due to the combination of alleles. Genetic diversity was higher in diploid than in tetraploid individuals. This fact, and the high degree of sterility of the triploid hybrids, may reflect a cytotype minority exclusion effect. This may cause spatial segregation, which effectively takes place in the study area. Dune disturbance may lead to an overlapping of the parents' distribution areas, facilitating hybridisation.This work is posthumously dedicated to Antonio Samo Lumbreras, to whom we are very grateful for all his help. This study was sponsored by the Valencian Government (Research Project GVPRE/2008/130) and the Universitat Politecnica de Valencia (Research Project Ref. 3241).Ferriol Molina, M.; Garmendia, A.; Ruiz, J.; Merle Farinós, HB.; Boira Tortajada, H. (2012). Morphological and molecular analysis of natural hybrids between the diploid Centaurea aspera L. and the tetraploid C. seridis L. (Compositae). Plant Biosystems. 146(1):86-100. https://doi.org/10.1080/11263504.2012.727878S86100146

Lipid-lowering drugs in ischaemic heart disease : a quasi-experimental uncontrolled before-and-after study of the effectiveness of clinical practice guidelines

Background: Cardiovascular diseases(CVD), specifically ischaemic heart disease(IHD), are the main causes of death in industrialized countries. Statins are not usually prescribed in the most appropriate way. To ensure the correct prescription of these drugs, it is necessary to develop, disseminate and implement clinical practice guidelines(CPGs), and subsequently evaluate them. The main objective of this study is to evaluate the effectiveness of the implementation of consensual Lipid-lowering drugs (LLD) prescription guidelines in hospital and primary care settings, to improve the control of Low-Density Lipoprotein Cholesterol (LDL-C) levels in patients with IHD in the Terres de l'Ebre region covered by the Catalonian Health Institute. Secondary bjectives are to assess the improvement of the prescription profile of these LLDs, to assess cardiovascular morbimortality and the professional profile and participant centre characteristics that govern the control of LDL-C. Methods/Design Design: Quasi-experimental uncontrolled before and after study. The intervention consists of the delivery of training strategies for guideline implementation (classroom clinical sessions and on-line courses) aimed at primary care and hospital physicians. The improvement in the control of LDL-C levels in the 3,402 patients with IHD in our territory is then assessed. Scope: Primary care physicians from 11 basic health areas(BHAs) and two hospital services (internal medicine and cardiology). Sample: 3,402 patients registered with IHD in the database of the Catalan Institute of Health(E-cap) before December 2008 and patients newly diagnosed during 2009-2010. Variables: Percentage of patients achieving good control of LDL-C, measured in milligrams per decilitre. The aim of the intervention is to achieve levels of LDL-C < 100 mg/dl in patients with IHD. Secondary variables measure type and time of diagnosis of IHD, type and dose of prescribed cholesterol-lowering drugs, level of physician participation in training activities and their professional profile. Discussion: The development of prescription guidelines previously agreed by various medical specialists involved in treating IHD patients have usually improved drug prescription. The guideline presented in this study aims to improve the control of LDL-C by training physicians through presential and on-line courses on the dissemination of this guideline, and by providing feedback on their personal results a year after this training intervention

Influence of phenological barriers and habitat differentiation on the population genetic structure of the balearic endemic Rhamnus ludovici-salvatoris Chodat and R. alaternus L

[EN] Rhamnus ludovici-salvatoris, endemic to the Gymnesian Islands, coexists with the related and widespread R. alaternus in Mallorca and Menorca. In both species, the population genetic structure using RAPD, and flowering during a 3-year period to check for possible phenological barriers, were analyzed. Rhamnus ludovici-salvatoris showed lower genetic diversity and stronger population structure than R. alaternus, the Cabrera population being less diverse and the most differentiated. Rhamnus ludovici-salvatoris flowered one month later, although flowering of both species coincided sporadically. These congeners seem to have diverged through isolation by time and differentiation in habitat. The population genetic structure of R. ludovici-salvatoris could mainly be due to the existence of small populations on the one hand, and a gene flow caused by rare hybridization events on the other, which may also explain the presence of morphologically intermediate individuals in Menorca. The conservation of R. ludovici-salvatoris populations may include population reinforcements and other in situ interventions.Ferriol Molina, M.; Llorens García, L.; Gil, L.; Boira Tortajada, H. (2009). Influence of phenological barriers and habitat differentiation on the population genetic structure of the balearic endemic Rhamnus ludovici-salvatoris Chodat and R. alaternus L. Plant Systematics and Evolution. 277(1-2):105-116. doi:10.1007/s00606-008-0110-3S1051162771-2Affre L, Thompson JD, Debussche M (1997) Genetic structure of continental and island populations of the Mediterranean endemic Cyclamen balearicum (Primulaceae). Amer J Bot 84(4): 437–451BOIB (2005) Decreto 75/2005. BOIB 106: 29–32Bolmgren K, Oxelman B (2004) Generic limits in Rhamnus L. s.l. (Rhamnaceae) inferred from nuclear and chloroplast DNA sequence phylogenies. Taxon 53(2):383–390Bolòs O, Molinier R (1958) Recherches phytosociologiques dans l’île de Majorque. Collectanea Botanica 34:699–865Cardona MA (1979) Consideracions sobre l’endemisme i l’origen de la flora de las Illes Balears. Butlletí del Institut Catalá de Historia Natural 44 (Sec. Bot. 3):7–15Cardona MA, Contandriopoulos J (1979) Endemism and evolution in the islands of the Western Mediterranean. In: Bramwell D (ed) Plants and islands. Academic Press, London, pp 133–169Chodat L (1924) Contributions à la Géo-Botanique de Majorque. PhD Thesis, Université de Genève—Institut de Botanique, SwitzerlandCollins D, Mill RR, Moller M (2003) Species separation of Taxus baccata, T. canadensis, and T. cuspidata (Taxaceae) and origins of their reputed hybrids inferred from RAPD and cpDNA data. Amer J Bot 90(2):175–182Cronk QCB (1997) Islands: stability, diversity, conservation. Biodivers Conserv 6(3):477–493Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Ducarme V, Wesselingh RA (2005) Detecting hybridization in mixed populations of Rhinanthus minor and Rhinanthus angustifolius. Folia Geobot 40(2/3):151–161Englishloeb GM, Karban R (1992) Consequences of variation in flowering phenology for seed head herbivory and reproductive success in Erigeron glaucus (Compositae). Oecologia 89:588–595Gautier F, Caluzon G, Suk JP, Violanti D (1994) Age et durée de la crise de salinité Messinienne. Comptes Rendus de l’Académie des Sciences de Paris 318:1103–1109Gerard PR, Fernandez-Manjarres JF, Frascaria-Lacoste N (2006) Temporal cline in a hybrid zone population between Fraxinus excelsior L. and Fraxinus angustifolia Vahl. Molec Ecol 15:3655–3667Gil L, Llorens L, Tébar FJ, Costa M (1995) La vegetación de la isla de Cabrera. In: Guía de la excursión geobotánica de las XV Jornadas de Fitosociología. Datos sobre la vegetación de Cabrera. Palma de Mallorca: Universitat de les Illes Balears, pp 51–77Gulías J, Flexas J, Abadía A, Medrano H (2002) Photosynthetic responses to water deficit in six Mediterranean sclerophyll species: possible factors explaining the declining distribution of Rhamnus ludovici-salvatoris, and endemic Balearic species. Tree Physiol 22:687–697Gulías J, Traveset A, Riera N, Mus M (2004) Critical stages in the recruitment process of Rhamnus alaternus L. Ann Bot 93:723–731Gustafsson S, Sjögren-Gulve P (2002) Genetic diversity in the rare orchid, Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea. Conserv Genet 3:225–234Gustafsson S (2003) Population genetic analyses in the orchid genus Gymnadenia—a conservation genetic perspective. PhD Thesis, Uppsala University, SwedenGustafsson S, Lönn M (2003) Genetic differentiation and habitat preference of flowering-time variants within Gymnadenia conopsea. Heredity 91:284–292Harris W (1996) Genecological aspects of flowering patterns of populations of Kunzea ericoides and K. sinclairii (Myrtaceae). New Zealand J Bot 34:333–354Hendry AP, Dray T (2005) Population structure attributable to reproductive time: isolation by time and adaptation by time. Molec Ecol 14:901–916Hosokawa K, Minami M, Kawahara K, Nakamura I, Shibata T (2000) Discrimination among three species of medicinal Scutellaria plants using RAPD markers. Pl Med 66:270–272Huang Z, Liu L, Zhou T, Ju B (2005) Effects of environmental factors on the population genetic structure in chukar partridge (Alectoris chukar). J Arid Environ 62:427–434Juan A, Crespo MB, Cowan RS, Lexer C, Fay F (2004) Patterns of variability and gene flow in Medicago citrina, an endangered endemic of islands in the western Mediterranean, as revealed by amplified fragment length polymorphism (AFLP). Molec Ecol 13:2679–2690Krijgsman W, Hilgen FJ, Raffi I, Sierro FJ, Wilson DS (1999) Chronology, causes and progression of the Messinian salinity crisis. Nature 400:652–655Lamont BB, He T, Enright NJ, Krauss SL, Miller BP (2003) Anthropogenic disturbance promotes hybridization between Banksia species by altering their biology. J Evol Biol 16:551–557Lennartsson T (1997) Seasonal differentiation—a conservative reproductive barrier in two grassland Gentianella (Gentianaceae) species. Pl Syst Evol 208:45–69Martinez-Solis I, Iranzo J, Estrelles E, Ibars AM (1993) Leaf domatia in the section Alaternus (Miller) DC. of the genus Rhamnus (Rhamnaceae). Bot J Linn Soc 112:311–318McIntosh ME (2002) Flowering phenology and reproductive output in two sister species of Ferocactus (Cactaceae). Pl Ecol 159:1–13Nei M (1973) Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA 70:3321–3323Nei M (1978) Estimation of average heterozigosity and genetic distance from a small number of individuals. Genetics 89:583–590Nei M, Li W (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 79:5269–5273Nybom H, Bartish IV (2000) Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspect Pl Ecol Evol Syst 3(2):93–114Oostermeijer JGB, Luijten SH, Ellis-Adam AC, den Nijs JCM (2002) Future prospects for the rare, late-flowering Gentianella germanica and Gentianopsis ciliata in Dutch nutrient-poor calcareous grasslands. Biol Conserv 104:339–350Pease CM, Lande R, Bull JJ (1989) A model of population growth, dispersal and evolution in a changing environment. Ecology 70(6):1657–1664Perron M, Gordon AG, Bousquet J (1995) Species-specific RAPD fingerprints for the closely related Picea mariana and P. rubens. Theor Appl Genet 91:142–149Pierce S, Ceriani RM, Villa M, Cerabolini B (2006) Quantifying relative extinction risks and targeting intervention for the orchid flora of a natural park in the European prealps. Conserv Biol 20(6):1804–1810Richardson JE, Fay MF, Cronk QCB, Bowman D, Chase MW (2000) A phylogenetic analysis of Rhamnaceae using rbcL and trnL-F plastid DNA sequences. Amer J Bot 87(9):1309–1324Roselló JA, Sáez L (2000) Index Balearicum: an annotated check-list of the vascular plants described from the Balearic Islands. Collect Bot 25(1):3–203Roselló JA, Cebrián MC, Mayol M (2002) Testing taxonomic and biogeographical relationships in a narrow mediterranean endemic complex (Hippocrepis balearica) using RAPD markers. Ann Bot 89:321–327Sales E, Nebauer SG, Mus M, Segura J (2001) Population genetic study in the Balearic plant species Digitalis minor (Scrophulariaceae) using RAPD markers. Amer J Bot 88(10):1750–1759Sherwin WB, Moritz C (2000) Managing and monitoring genetic erosion. In: Young AG, Clarke GM (eds) Genetics, demography and viability of fragmented populations. Cambridge University Press, Cambridge, pp 9–34Sneath PHA, Sokal RR (1973) Numerical taxonomy. Freeman and Co., San FranciscoTraveset A, Gulías J, Riera N, Mus M (2003) Transition probabilities from pollination to establishment in a rare dioecious shrub species (Rhamnus ludovici-salvatoris) in two habitats. J Ecol 91:427–437Tutin TG, Heywood VH, Burges NA, Valentine DH, Walters SM, Webb DA (eds) (2001) Flora Europaea, vol 2. Rosaceae to Umbelliferae. Cambridge University Press, CambridgeWright S (1931) Evolution in Mendelian populations. Genetics 16:97–159Zimmerman M (1980a) Reproduction in Polemonium: pre-dispersal seed predation. Ecology 61:502–506Zimmerman M (1980b) Reproduction in Polemonium: competition for pollinators. Ecology 61:497–50

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks