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This paper interrogates the actuality of 'ruang terbuka hijau kota'(urban green open space - UGOS) in Denpasar Bali. This debate is critical because it is directly linked to two conditions. First, is an ever increasing population, and second, is a diminishing public realm. While the social, economic, and environmental significances of the UGOS in the formation of a liveable city is widely acknowledged, both conditions, in reality, do not go in line with the importance of UGOS to urban living. The paper argues that while UGOS are legitimated by the existing urban planning procedures and reflected in spatial planning, there is no policy directive on securing the deliverance of sustainable UGOS that guarantee the public interest. The following study is supported by relevant case studies that illustrate and demonstrate the above claims, predominantly across Denpasar, the capital city of both Bali Province and Denpasar city. The conclusion to the paper becomes increasingly self-evident as it progresses. It is clear that having new categories of land use will accomplish nothing. Similarly the key is not to re-designate land uses or manipulate existing codes, but to reconstitute how the planning system itself functions in relation to a sustainable public realm and environmental justice.

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1 , and the DNA damage repair gene PARP1 , identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts. Isogenic pairs of cell lines are powerful tools but time-consuming to generate. Here the authors conduct genome-wide genetic interactions screens of ‘anchor’ genes with SaCas9 and SpCas9.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2 . Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models. Improved Cas12a variants and sgRNA design rules enhance genome-wide screens.

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2 , identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest. Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here the authors make comparisons of numerous Cas9 variants, nominate options for base editing screens with denser coverage with A>G and C>T base editing screens and identify loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2 .

Background Pregnancy-related anxiety (PrA) has been identified as a construct distinct from general stress and anxiety with a negative impact on birth and child outcomes. Validated instruments with good psychometric properties to assess pregnancy-related anxiety in German-speaking expectant mothers are still lacking. The Pregnancy-Related Anxiety Questionnaire revised for its use independent of parity (PRAQ-R2) assesses fear of giving birth (FoGB), worries of bearing a physically or mentally handicapped child (WaHC) and concerns about own appearance (CoA). The aim of this study was to investigate the psychometric properties of the PRAQ-R2 in a German sample of pregnant women in their third pregnancy trimester. Methods The PRAQ-R2 and several questionnaires measuring different forms of anxiety as well as depressive symptoms and perceived general self-efficacy were administered cross-sectionally in a sample of nulliparous and parous women ( N  = 360) in the third trimester of pregnancy. Results Reliability was satisfactory to excellent for the PRAQ-R2 total scale (Cronbach’s α  = .85) and the subscales ( α  = .77 to .90). Confirmatory and exploratory factor analysis confirmed the three-factorial structure of the instrument. The three factors together explained 68% of variance. Construct validity was confirmed by positive low- to moderate-sized correlations of the PRAQ-R2 total score and the subscales with measurements of anxiety and depression and by negative low correlations with general self-efficacy. Conclusions The German version of the PRAQ-R2 is a valid and feasible measurement for pregnancy-related anxiety for research and clinical practice.

The microbiome, an intriguing component of the human body, composed of trillions of microorganisms, has prompted scientific exploration to identify and understand its function and role in health and disease. As associations between microbiome composition, disease, and symptoms accumulate, the future of medicine hinges upon a comprehensive knowledge of these microorganisms for patient care. The oral microbiome may provide valuable and efficient insight for predicting future changes in disease status, infection, or treatment course. The main aim of this pilot study was to characterize the oral microbiome in patients with severe aplastic anemia (SAA) during their therapeutic course. SAA is a hematologic disease characterized by bone marrow failure which if untreated is fatal. Treatment includes either hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). In this study, we examined the oral microbiome composition of 24 patients admitted to the National Institutes of Health (NIH) Clinical Center for experimental SAA treatment. Tongue brushings were collected to assess the effects of treatment on the oral microbiome. Twenty patients received standard IST (equine antithymocyte globulin and cyclosporine) plus eltrombopag. Four patients underwent HSCT. Oral specimens were obtained at three time points during treatment and clinical follow-up. Using a novel approach to 16S rRNA gene sequence analysis encompassing seven hypervariable regions, results demonstrated a predictable decrease in microbial diversity over time among the transplant patients. Linear discriminant analysis or LefSe reported a total of 14 statistically significant taxa (p < 0.05) across time points in the HSCT patients. One-way plots of relative abundance for two bacterial species (Haemophilus parainfluenzae and Rothia mucilaginosa) in the HSCT group, show the differences in abundance between time points. Only one bacterial species (Prevotella histicola) was noted in the IST group with a p value of 0.065. The patients receiving immunosuppressive therapy did not exhibit a clear change in diversity over time; however, patient-specific changes were noted. In addition, we compared our findings to tongue dorsum samples from healthy participants in the Human Microbiome Project (HMP) database and found among HSCT patients, approximately 35% of bacterial identifiers (N = 229) were unique to this study population and were not present in tongue dorsum specimens obtained from the HMP. Among IST-treated patients, 45% (N = 351) were unique to these patients and not identified by the HMP. Although antibiotic use may have likely influenced bacterial composition and diversity, some literature suggests a decreased impact of antimicrobials on the oral microbiome as compared to their effect on the gut microbiome. Future studies with larger sample sizes that focus on the oral microbiome and the effects of antibiotics in an immunosuppressed patient population may help establish these potential associations.

People with severe mental illness have high rates of obesity, type 2 diabetes, and cardiovascular disease. Emerging evidence suggests that metabolic dysfunction may be causally linked to the risk of severe mental illness. However, more research is needed to identify reliable metabolic markers which may have an impact on mental health outcomes, and to determine the mechanisms behind their impact. In the METPSY research study, we will investigate the relationship between metabolic markers and clinical outcomes of severe mental illness in young adults. We will recruit 120 young adults aged 16–25 years living in Scotland with major depressive disorder, bipolar disorder, schizophrenia, or no severe mental illness (controls) for a prospective observational study. We will assess clinical symptoms at three in-person visits (baseline, 6 months, and 12 months) using the Structured Clinical Interview for DSM-5, and collect blood samples at each of these visits for agnostic profiling of metabolic biomarkers through an untargeted metabolomic screen, using the rapid hydrophilic interaction liquid chromatography ion mobility mass spectrometry method (RHIMMS). Participants will also complete remote assessments at 3 and 9 months after the baseline visit: Ecological Momentary Assessments to measure mental health, wrist actigraphy to measure rhythms of rest and activity, and continuous glucose monitoring to measure metabolic changes. Throughout the 12-month enrolment period, we will also measure objective markers of sleep using a radar sleep monitor (Somnofy). Using advanced statistical techniques and machine learning analysis, we will seek to better understand the mechanisms linking metabolic health with mental health in young adults with schizophrenia, bipolar disorder, and severe depression. Clinical trial number : Not applicable

•Observational studies are required to determine the current state of early mobility in the cardiac surgery intensive care unit.•Sitting in a chair was observed 10-fold more often than any other mode of mobility.•Family was observed to support mobility through active engagement and providing support more than healthcare professionals.•Research is required to support early mobility beyond sitting in a chair supported by family and the healthcare team. Mobilization within 24 h post-cardiac surgery (CS) supports improved patient health outcomes. Despite being safe and recommended, it is unknown how much mobility takes place post-CS in the intensive care unit (ICU). Behaviour mapping was used to describe patterns of patients’ mobility in one CS ICU. Behaviour mapping gathers information on behaviour regularly over a time period. Two authors observed one CS ICU over a sixteen-hour period (0630–2230 h) on four days. Observers collected data on patients’ mobility mode, location, and support at 15-minute intervals. Data aggregated into four-hour time blocks is described. A total of 1342 observations were collected over four days: 487 of mode, 485 of location, and 370 of support. Sitting in a chair was observed 430 of 487 observations, 10-fold more than any other mode of mobility. Mobility within the ICU room was observed in 448 of 485 observations. Family support for mobility was observed in 178 of 370 observations. The most common time block for mobilization was from 0630 to 1030, with 488 of 1342 observations. Research is required to support the integration of early mobility beyond sitting in a chair supported by more team members into local CS ICU clinical care. The existence of early mobility protocols does not mean that they are operational in the CS ICU. Integration of these protocols into CS ICU clinical care requires collaboration among researchers and clinicians.

Introduction Despite the growing use of immune checkpoint inhibitors (ICI) in cancer treatment, data regarding ICI-associated pericardial disease are primarily derived from case reports and case series. ICI related pericardial disease can be difficult to diagnose and is associated with significant morbidity. We conducted a systematic review to further characterize the epidemiology, clinical presentation, and outcomes of this patient population. Methods A search of four databases resulted in 31 studies meeting inclusion criteria. Patients > 18 years old who presented with ICI mediated pericardial disease were included. Intervention was medical + surgical therapy and outcomes were development of cardiac tamponade, morbidity, and mortality. Results Thirty- eight patients across 31 cases were included. Patients were majority male (72%) with a median age of 63. Common symptoms included dyspnea (59%) and chest pain (32%), with 41% presenting with cardiac tamponade. Lung cancer (81%) was the most prevalent, and nivolumab (61%) and pembrolizumab (34%) were the most used ICIs. Pericardiocentesis was performed in 68% of patients, and 92% experienced symptom improvement upon ICI cessation. Overall mortality was 16%. Discussion This study provides the most comprehensive analysis of ICI-mediated pericardial disease to date. Patients affected were most commonly male with lung cancer treated with either Nivolumab or Pembrolizumab. Diagnosis may be challenging in the setting of occult presentation with normal EKG and physical exam as well as delayed onset from therapy initiation. ICI-associated pericardial disease demonstrates high morbidity and mortality, as evidenced by a majority of patients requiring pericardiocentesis.

BackgroundAsymptomatic carotid artery stenosis (ACS) can be treated with carotid endarterectomy (CEA), carotid artery stenting (CAS), or best medical treatment (BMT) only. For all treatment options, optimization of vascular risk factors such as arterial hypertension, hyperlipidemia, smoking, obesity, and insufficient physical activity is essential. Data on adherence to BMT and lifestyle modification in patients with ACS are sparse. The subject of this investigation is the implementation and quality of risk factor adjustment in the context of a randomized controlled trial.MethodsA total of 513 patients in the prematurely terminated, randomized, controlled, multicenter SPACE-2 trial (ISRCTN 78592017) were analyzed within one year after randomization into 3 groups (CEA, CAS, and BMT only) for implementation of prespecified BMT recommendations and lifestyle modifications. Measurement time points were the screening visit and visits after one month (D30), 6 months (M6), and one year (A1). Differences between groups and follow-up visits (FUVs) relative to the screening visit were investigated.FindingsFor all FUVs, a significant increase in statin medication (91% at A1; p < 0.0001) was demonstrated to be associated with a significant decrease (p < 0.01) in cholesterol levels (median 167 mg/dl at A1) and LDL cholesterol levels (median 93 mg/dl at A1). The lowest cholesterol levels were achieved by patients in the BMT group. Seventy-eight percent of all patients reached predefined target cholesterol levels (< 200 mg/dl), with significantly better rates in the BMT group (p = 0.036 at D30). Furthermore, a significant decrease in arterial blood pressure at all FUVs (p < 0.05) was associated with a significant increase in antihypertensive medication (96% at A1, p < 0.0001). However, only 28% of patients achieved the predefined treatment goal of a systolic blood pressure of ≤ 130 mmHg. Forty-two of a total of 100 smokers at the screening visit quit smoking within one year, resulting in a significant increase in nonsmokers at all FUVs (p < 0.0001). Recommended HbA1c levels (< 7%) were achieved in 82% without significant changes after one year. Only 7% of obese (BMI > 25) patients achieved sufficient weight reduction after one year without significant changes at all FUVs (median BMI 27 at A1; p = 0.1201). The BMT group showed significantly (p = 0.024) higher rates of adequate physical activity than the intervention groups. Furthermore, after one year, the BMT group showed a comparatively significantly better implementation of risk factor modification (77%; p = 0.027) according to the treating physician.InterpretationSPACE-2 demonstrated sustained improvement in the noninterventional management of vascular risk factors in patients treated in a clinical trial by general practitioners, internists and neurologists. The best implemented treatment targets were a reduction in cholesterol and HbA1c levels. In this context, a significant increase in statin use was demonstrated. Blood pressure control missed its target but was significantly reduced by intensification of antihypertensive medication. Patients on BMT only had better adjusted lipid parameters and were more physically active. However, all groups failed to achieve sufficient weight reduction. Due to insufficient patient recruitment, the results must be interpreted cautiously.Trial registration: ISRCTN Registry, ISRCTN78592017, Registered 16 June 2007, https://www.isrctn.com/search?q=78592017.