Evidence of thermophilization in Afromontane forests.

Thermophilization is the directional change in species community composition towards greater relative abundances of species associated with warmer environments. This process is well-documented in temperate and Neotropical plant communities, but it is uncertain whether this phenomenon occurs elsewhere in the tropics. Here we extend the search for thermophilization to equatorial Africa, where lower tree diversity compared to other tropical forest regions and different biogeographic history could affect community responses to climate change. Using re-census data from 17 forest plots in three mountain regions of Africa, we find a consistent pattern of thermophilization in tree communities. Mean rates of thermophilization were +0.0086 °C·y-1 in the Kigezi Highlands (Uganda), +0.0032 °C·y-1 in the Virunga Mountains (Rwanda-Uganda-Democratic Republic of the Congo) and +0.0023 °C·y-1 in the Udzungwa Mountains (Tanzania). Distinct from other forests, both recruitment and mortality were important drivers of thermophilzation in the African plots. The forests studied currently act as a carbon sink, but the consequences of further thermophilization are unclear.

Liver kinase B1 (LKB1) regulates the epigenetic landscape of mouse pancreatic beta cells.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic ß-cell identity and function. Elimination of Lkb1 from the ß-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of ß cell- selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from ß-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors important for ß-cell identity, such as FOXA, MAFA or RFX6 and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates ß-cell identity and function.

Identifying keystone connectivity spots under climate change: Implications to conservation and management of riparian systems.

Climate change has intensified the effects of habitat fragmentation in many ecosystems, particularly exacerbated in riparian habitats. Therefore, there is an urgent need to identify keystone connectivity spots to ensure long-term conservation and sustainable management of riparian systems as they play a crucial role for landscape connectivity. This paper aims to identify critical areas for connectivity under two contrasting climate change scenarios (RCP 4.5 and RCP 8.5 models) for the years 2030, 2050 and 2100 and to group these critical areas by similar connectivity in keystone spots for sustainable management. A set of analyses comprising climate analysis, drainage network analysis, configuration of potential riparian habitats, riparian habitat connectivity, data clustering, and statistical analysis within a Spanish river basin (NW Spain) were applied. The node and link connectivity would be reduced under the two climate change scenarios (≈2.5 % and 4.4 % reduction, respectively), intensifying riparian habitat fragmentation. Furthermore, 51 different clusters (critical areas) were obtained and classified in five classes (keystone spots) with similar connectivity across the different scenarios of climate change. Each keystone spot obtained by hierarchical classification was associated with one or more climate scenarios. One of these keystone spots was especially susceptible to the worst climate change scenario. Key riparian connectivity spots will be crucial for the management and restoration of highly threatened riparian systems and to ensure long-term biodiversity conservation.

Carbapenemase-Producing Klebsiella pneumoniae in COVID-19 Intensive Care Patients: Identification of IncL-VIM-1 Plasmid in Previously Non-Predominant Sequence Types.

During the COVID-19 pandemic, intensive care units (ICUs) operated at or above capacity, and the number of ICU patients coinfected by nosocomial microorganisms increased. Here, we characterize the population structure and resistance mechanisms of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn) from COVID-19 ICU patients and compare them to pre-pandemic populations of CP-Kpn. We analyzed 84 CP-Kpn isolates obtained during the pandemic and 74 CP-Kpn isolates obtained during the pre-pandemic period (2019) by whole genome sequencing, core genome multilocus sequence typing, plasmid reconstruction, and antibiotic susceptibility tests. More CP-Kpn COVID-19 isolates produced OXA-48 (60/84, 71.4%) and VIM-1 (18/84, 21.4%) than KPC (8/84, 9.5%). Fewer pre-pandemic CP-Kpn isolates produced VIM-1 (7/74, 9.5%). Cefiderocol (97.3-100%) and plazomicin (97.5-100%) had the highest antibiotic activity against pandemic and pre-pandemic isolates. Sequence type 307 (ST307) was the most widely distributed ST in both groups. VIM-1-producing isolates belonging to ST307, ST17, ST321 and ST485, (STs infrequently associated to VIM-1) were detected during the COVID-19 period. Class 1 integron Int1-blaVIM-1-aac(6')-1b-dfrB1-aadAI-catB2-qacEΔ1/sul1, found on an IncL plasmid of approximately 70,000 bp, carried blaVIM-1 in ST307, ST17, ST485, and ST321 isolates. Thus, CP-Kpn populations from pandemic and pre-pandemic periods have similarities. However, VIM-1 isolates associated with atypical STs increased during the pandemic, which warrants additional monitoring and surveillance.

Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq".

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SIGNIFICANCE: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

Urine collection methods in precontinent children treated at the paediatric emergency department.

AIM: To describe the urine collection methods used in precontinent children presenting at the Paediatric Emergency Department (PED) and compare results and contamination rates. METHODS: Retrospective observational cohort study that included 1678 urine cultures collected in infants <24 months of age between January 2016 and December 2019. Urine cultures were compared based on collection technique, sex and patient age. RESULTS: In total, 60.4% of samples were collected by clean-catch urine collection (CCUC), 26.4% by urethral catheterisation (UC) and 13.2% by urine bag (UB). Contamination rates were 2.9% (95% CI 1.3, 4.4) for UC, 11.3% (95% CI 9.3, 13.2) for CCUC and 23.4% (95% CI 17.8, 29.0) for UB. Significant differences in contamination rates were found between UC and CCUC in the 6-12-month age group (1.9% [95% CI 0.0-4.0] versus 12.0% [95% CI 7.2-16.8] [p < 0.0009]), and between UC and UB for all ages. CONCLUSIONS: CCUC is the most common method for urine culture collection in infants <24 months of age at the PED in our centre. UC has the lowest contamination rates, but significant differences were only observed between CCUC and UC in the 6-12-month age group. CCUC is a non-invasive alternative for urine collection in infants.

Two stage hepatectomy (TSH) versus ALPPS for initially unresectable colorectal liver metastases: A systematic review and meta-analysis.

BACKGROUND: Although numerous comparisons between conventional Two Stage Hepatectomy (TSH) and Associating Liver Partition and Portal Vein Ligation for staged hepatectomy (ALPPS) have been reported, the heterogeneity of malignancies previously compared represents an important source of selection bias. This systematic review and meta-analysis aimed to compare perioperative and oncological outcomes between TSH and ALPPS to treat patients with initially unresectable colorectal liver metastases (CRLM). METHODS: Main electronic databases were searched using medical subject headings for CRLM surgically treated with TSH or ALPPS. Patients treated for primary or secondary liver malignancies other than CRLM were excluded. RESULTS: A total of 335 patients from 5 studies were included. Postoperative major complications were higher in the ALPPS group (relative risk [RR] 1.46, 95% confidence interval [CI] 1.04-2.06, I2 = 0%), while no differences were observed in terms of perioperative mortality (RR 1.53, 95% CI 0.64-3.62, I2 = 0%). ALPPS was associated with higher completion of hepatectomy rates (RR 1.32, 95% CI 1.09-1.61, I2 = 85%), as well as R0 resection rates (RR 1.61, 95% CI 1.13-2.30, I2 = 40%). Nevertheless, no significant differences were achieved between groups in terms of overall survival (OS) (RR 0.93, 95% CI 0.68-1.27, I2 = 52%) and disease-free survival (DFS) (RR 1.08, 95% CI 0.47-2.49, I2 = 54%), respectively. CONCLUSION: ALPPS and TSH to treat CRLM seem to have comparable operative risks in terms of mortality rates. No definitive conclusions regarding OS and DFS can be drawn from the results.

Glucose-Dependent miR-125b Is a Negative Regulator of ß-Cell Function.

Impaired pancreatic ß-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of ß cells. We have previously shown that ß cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in ß cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in ß cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human ß-cell line EndoCß-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in ß cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg ß cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.

Opposing effects on regulated insulin secretion of acute vs chronic stimulation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. METHODS: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. RESULTS: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). CONCLUSIONS/INTERPRETATION: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators.

Pharmaceutical pollution of the world's rivers.

Environmental exposure to active pharmaceutical ingredients (APIs) can have negative effects on the health of ecosystems and humans. While numerous studies have monitored APIs in rivers, these employ different analytical methods, measure different APIs, and have ignored many of the countries of the world. This makes it difficult to quantify the scale of the problem from a global perspective. Furthermore, comparison of the existing data, generated for different studies/regions/continents, is challenging due to the vast differences between the analytical methodologies employed. Here, we present a global-scale study of API pollution in 258 of the world's rivers, representing the environmental influence of 471.4 million people across 137 geographic regions. Samples were obtained from 1,052 locations in 104 countries (representing all continents and 36 countries not previously studied for API contamination) and analyzed for 61 APIs. Highest cumulative API concentrations were observed in sub-Saharan Africa, south Asia, and South America. The most contaminated sites were in low- to middle-income countries and were associated with areas with poor wastewater and waste management infrastructure and pharmaceutical manufacturing. The most frequently detected APIs were carbamazepine, metformin, and caffeine (a compound also arising from lifestyle use), which were detected at over half of the sites monitored. Concentrations of at least one API at 25.7% of the sampling sites were greater than concentrations considered safe for aquatic organisms, or which are of concern in terms of selection for antimicrobial resistance. Therefore, pharmaceutical pollution poses a global threat to environmental and human health, as well as to delivery of the United Nations Sustainable Development Goals.

Molecular Mechanisms of Nutrient-Mediated Regulation of MicroRNAs in Pancreatic ß-cells.

Pancreatic ß-cells within the islets of Langerhans respond to rising blood glucose levels by secreting insulin that stimulates glucose uptake by peripheral tissues to maintain whole body energy homeostasis. To different extents, failure of ß-cell function and/or ß-cell loss contribute to the development of Type 1 and Type 2 diabetes. Chronically elevated glycaemia and high circulating free fatty acids, as often seen in obese diabetics, accelerate ß-cell failure and the development of the disease. MiRNAs are essential for endocrine development and for mature pancreatic ß-cell function and are dysregulated in diabetes. In this review, we summarize the different molecular mechanisms that control miRNA expression and function, including transcription, stability, posttranscriptional modifications, and interaction with RNA binding proteins and other non-coding RNAs. We also discuss which of these mechanisms are responsible for the nutrient-mediated regulation of the activity of ß-cell miRNAs and identify some of the more important knowledge gaps in the field.

High aboveground carbon stock of African tropical montane forests.

Tropical forests store 40-50 per cent of terrestrial vegetation carbon1. However, spatial variations in aboveground live tree biomass carbon (AGC) stocks remain poorly understood, in particular in tropical montane forests2. Owing to climatic and soil changes with increasing elevation3, AGC stocks are lower in tropical montane forests compared with lowland forests2. Here we assemble and analyse a dataset of structurally intact old-growth forests (AfriMont) spanning 44 montane sites in 12 African countries. We find that montane sites in the AfriMont plot network have a mean AGC stock of 149.4 megagrams of carbon per hectare (95% confidence interval 137.1-164.2), which is comparable to lowland forests in the African Tropical Rainforest Observation Network4 and about 70 per cent and 32 per cent higher than averages from plot networks in montane2,5,6 and lowland7 forests in the Neotropics, respectively. Notably, our results are two-thirds higher than the Intergovernmental Panel on Climate Change default values for these forests in Africa8. We find that the low stem density and high abundance of large trees of African lowland forests4 is mirrored in the montane forests sampled. This carbon store is endangered: we estimate that 0.8 million hectares of old-growth African montane forest have been lost since 2000. We provide country-specific montane forest AGC stock estimates modelled from our plot network to help to guide forest conservation and reforestation interventions. Our findings highlight the need for conserving these biodiverse9,10 and carbon-rich ecosystems.

[Study of personality and anxiety in children and adolescents with inflammatory bowel disease]. / Estudio de personalidad y ansiedad en niños y adolescentes con enfermedad inflamatoria intestinal.

OBJECTIVE: to analyze the presence of common personality traits and anxiety states in children and adolescents with inflammatory bowel disease (IBD). PATIENTS AND METHOD: Longitudinal, prospecti ve, and analytical study by applying the questionnaires Children's Personality Questionnaire, High School Personality Questionnaire, State-Trait Anxiety Inventory for Children, and State-Trait Anxie ty Inventory for patients with IBD aged between 9 and 18 years seen at reference IBD units in Ara gon, Spain. The participants excluded were those with active disease, defined as a score > 10 on the Pediatric Crohn's Disease Activity Index (PCDAI Score) or > 10 on the Pediatric Ulcerative Colitis Activity Index (PUCAI Score). RESULTS: Twenty-six patients participated (73% male). 61.5% pre sented Crohn's disease (CD) and 38.5% ulcerative colitis (UC). No patient presented active disease. The personality profile as a group was characterized by being open, emotionally stable, calm, sober, sensible, enterprising, impressionable, dependent, serene, perfectionist, and relaxed. 50% of the CD patients were enterprising versus no UC patients (p < 0.05). There were no statistically significant di fferences when comparing the remaining personality factors based on IBD type, age, or sex. Patients with CD tended to be calmer (p = 0.0511) and patients with UC more introverted (p = 0.0549). The sample presented a state anxiety level (A/E) -1.1 ± 0.8 SD compared with the population average. The level of anxiety as a feature (A/R) was -0.6 ± 1 SD. Males had significantly lower levels than females in the case of A/E (p < 0.05). CONCLUSIONS: The presence of common personality traits in the pediatric population with IBD stands out but there was no greater anxiety than in the reference population.

Urine collection methods for infants under 3 months of age in clinical practice.

BACKGROUND: Methods of urine collection used in precontinent children are a controversial issue. Definitive diagnosis of urinary tract infection (UTI) requires an uncontaminated urine culture. We aimed to describe methods used to collect urine for culture in infants under 3 months of age and compare results and contamination rates. METHODS: This retrospective observational cohort study included 721 urine cultures collected from infants <3 months of age at the Hospital Universitario Infanta Sofía, Madrid, between January 2016 and December 2019. Urine cultures were compared based on collection technique, sex, and patient age. RESULTS: Median patient age was 36 days and 54.6% were male. In total, 592 (82.1%) samples were collected using clean-catch urine stimulation technique (CCUST), 77 (10.7%) by urethral catheterization (UC) and 52 (7.2%) by urine bag (UB). Positive cultures were obtained in 11.7% (95% confidence interval [CI] 9.1, 14.3) of CCUST samples and in 28.6% (95% CI 18.5, 38.7) of UC samples (p<0.001). The contamination rate was 13.7% (95% CI 10.9, 16.4] for CCUST, 23.1% (95% CI 11.6, 34.6) for UB and 5.2% (95% CI 0.2, 10.2) for UC, with statistically significant differences (p=0.007) between UB and UC collection. CONCLUSIONS: CCUST is the most commonly used method in our hospital for collecting urine in infants younger than 3 months. The contamination rate of UC is lower but not significantly different to that of CCUST. Urine collection by CCUST serves as a non-invasive alternative to UC for diagnosis of UTI in infants under 3 months of age in routine clinical practice. Graphical abstract.

Complete Mesocolic Excision and D3 Lymphadenectomy versus Conventional Colectomy for Colon Cancer: A Systematic Review and Meta-Analysis.

BACKGROUNDS: Previous systematic reviews suggest that the implementation of 'complete mesocolon excision' (CME) for colon tumors entails better specimen quality but with limited long-term outcomes. We performed a meta-analysis to compare the pathological, perioperative, and oncological results of CME with conventional surgery (CS) in primary colon cancer. METHODS: Embase, MEDLINE and CENTRAL databases were searched using Medical Subject Headings for CME and D3 lymphadenectomy. The systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: A total of 18,989 patients from 27 studies were included. Postoperative complications were higher in the CME group (relative risk [RR] 1.13, 95% confidence interval [CI] 1.04-1.22, I2 = 0%), while no differences were observed in terms of anastomotic leak (I2 = 0%) or perioperative mortality (I2 = 49%). CME was associated with a higher number of lymph nodes harvested (I2 = 95%), distance to high tie (I2 = 65%), bowel length (I2 = 0%), and mesentery area (I2 = 95%). CME also had positive effects on 3- and 5-year overall survival (RR 1.09, 95% CI 1.04-1.15, I2 = 88%; and RR 1.05, 95% CI 1.02-1.08, I2 = 62%, respectively) and 3-year disease-free survival (RR 1.10, 95% CI 1.04-1.17, I2 = 22%), as well as decreased local (RR 0.35, 95% CI 0.24-0.51, I2 = 51%) and distant recurrences (RR 0.71, 95% CI 0.60-0.85, I2 = 34%). CONCLUSIONS: Limited evidence suggests that CME improves oncological outcomes with a higher postoperative adverse events rate but no increase in anastomotic leak rate or perioperative mortality, compared with CS.

Resistance of African tropical forests to an extreme climate anomaly.

The responses of tropical forests to environmental change are critical uncertainties in predicting the future impacts of climate change. The positive phase of the 2015-2016 El Niño Southern Oscillation resulted in unprecedented heat and low precipitation in the tropics with substantial impacts on the global carbon cycle. The role of African tropical forests is uncertain as their responses to short-term drought and temperature anomalies have yet to be determined using on-the-ground measurements. African tropical forests may be particularly sensitive because they exist in relatively dry conditions compared with Amazonian or Asian forests, or they may be more resistant because of an abundance of drought-adapted species. Here, we report responses of structurally intact old-growth lowland tropical forests inventoried within the African Tropical Rainforest Observatory Network (AfriTRON). We use 100 long-term inventory plots from six countries each measured at least twice prior to and once following the 2015-2016 El Niño event. These plots experienced the highest temperatures and driest conditions on record. The record temperature did not significantly reduce carbon gains from tree growth or significantly increase carbon losses from tree mortality, but the record drought did significantly decrease net carbon uptake. Overall, the long-term biomass increase of these forests was reduced due to the El Niño event, but these plots remained a live biomass carbon sink (0.51 ± 0.40 Mg C ha-1 y-1) despite extreme environmental conditions. Our analyses, while limited to African tropical forests, suggest they may be more resistant to climatic extremes than Amazonian and Asian forests.

Hiperplasia endotelial papilar intravascular en la región supraclavicular / Intravascular papillary endothelial hyperplasia in the supraclavicular region

La hiperplasia endotelial papilar intravascular (IPEH) es una lesión vascular benigna poco frecuente que se presenta habitualmente como una neoformación subcutánea eritemato-violácea inespecífica. El estudio histopatológico, necesario para el diagnóstico de confirmación, muestra proliferación papilar de células endoteliales asociada con material trombótico. La IPEH puede simular otras lesiones como el angiosarcoma, por lo que el diagnóstico correcto de esta entidad es esencial para evitar tratamientos agresivos. La resección con márgenes amplios suele ser suficiente

Intravascular papillary endothelial hyperplasia (IPEH) is a rare benign vascular lesion that usually presents as a nonspecific erythematous-violaceous subcutaneous neoformation. The histopathological study, necessary for the confirmatory diagnosis, shows papillary proliferation of endothelial cells associated with thrombotic material. IPEH can simulate other lesions such as angiosarcoma, so the correct diagnosis of this entity is essential to avoid aggressive treatments. The resection with wide margins is usually enough