A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells.

The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.

Voice hearing as a social barometer: Benevolent persuasion, ancestral spirits, and politics in the voices of psychosis in Shanghai, China.

The comparative study of voice hearing is in its early stages. This approach is important due to the observation that the content of voices differs across different settings, which suggests that voice hearing may respond to cultural invitation and, ultimately, to learning. Our interview-based study found that persons diagnosed with schizophrenia in China (Shanghai), compared to those diagnosed with schizophrenia in the United States, Ghana, and India, reported voices that were strikingly concerned with politics. Compared to participants in the United States in particular, voices seemed to be experienced more relationally: Shanghai participants reported voices notable for a sense of benevolent persuasion rather than harsh command, and knew the identities of their voices more so than in the United States. The voices were striking as well for their religious content, despite the previous prohibition of religion in China. Our findings further support the hypothesis that voice hearing seems to be shaped by context, and we observe that this shaping may affect not only conceptual content but the emotional valence of the experience.

Coordinated peptidoglycan synthases and hydrolases stabilize the bacterial cell wall.

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.

Student engagement in flipped classroom in nursing education: An integrative review.

AIM: To summarize the effect of the flipped classroom method (FCM) on student engagement in nursing education and provide the implications for future practice. BACKGROUND: Technological advancement of learning approaches including flipped classroom has become increasingly popular in nursing education. However, no integrative review has been published that specially explore the behaviour engagement, cognitive engagement and emotional engagement of flipped classroom use in nursing education. METHODS: Population, Intervention, Comparison, Outcomes and Study (PICOS) strategies were implemented to investigate the literature from published peer-reviewed papers from 2013 to 2021 in CINAHL, MEDLINE and Web of Science. RESULTS: The initial search identified 280 potentially relevant articles. After comprehensively reviewing the initial catchment using several analytical phases, 16 articles were considered for the final review. Most of articles targeted on undergraduate nursing students and were conducted in the USA and Australia. The review primarily identified positive learning outcomes among nursing students in term of student engagement. However, a few studies reported contrasting findings, possibly due to students are still dependent on traditional lecture-based classroom. CONCLUSION: This review suggests that use of the FCM in nursing education could promote students' behavioral engagement and cognitive engagement but yield a mixed outcome in emotional engagement. This review contributed to our understanding of the effect of the flipped classroom method on student engagement in nursing education, the strategies for promoting student engagement in future flipped classroom practice and recommendation for future research on flipped classroom.

Flipped versus traditional classroom and student achievement and cognitive engagement in an associate degree nursing fundamental course.

BACKGROUND: There is a call for instructional transformation in nursing education to include an innovative approach. This study aimed to investigate the effects of the flipped classroom method on student achievement and engagement in an associate degree nursing course. This study investigated associate degree nursing studies students' achievement and cognitive engagement under traditional learning (n = 141) and flipped learning (n = 130). This study was conducted in two first-year cohorts enrolled in their nursing fundamental course, before and during the COVID-19 pandemic respectively at a Hong Kong community college. METHOD: A mixed methods approach was used with quantitative (examination scores) and qualitative (semi-structured focus group interview) measures applied. RESULTS: There was a significant increase in final exam scores for the flipped classroom, all p < 0.01, with a medium-to-large (d = 0.60) effect size. Students commented flipped classroom method promoted their cognitive engagement and the benefits that contributed to such a positive student achievement included more peer interaction and more opportunity to apply their knowledge. CONCLUSION: This study contributed to our understanding of the effect of the flipped classroom method on student engagement in nursing education.

The Use of Extracorporeal Membrane Oxygenation for Burns: A Systematic Review and Meta-Analysis.

Burns are among the leading causes of trauma worldwide, and acute respiratory distress syndrome (ARDS) is a common cause of death in burn patients. Some patients develop hypoxemia refractory to conventional therapies and may be initiated on extracorporeal membrane oxygenation (ECMO) as rescue therapy. We conducted a meta-analysis for studies reporting on survival rates of patients receiving ECMO for burns or inhalation injuries, which was the primary outcome. Secondary outcomes included the hospital and intensive care unit length of stay and duration of ECMO. Random-effects (DerSimonian and Laird) were conducted. The pooled survival from 10 studies was 53.6% (95% confidence interval [CI]: 37.6-69.2%, high certainty). Survival was significantly associated with age (regression coefficient [B]: -0.0088, 95% CI: -0.0155 to -0.0021, p = 0.011) and the proportion of male patients (B: -1.0137 95% CI: -1.9695 to -0.0580, p = 0.038). Patients were cannulated on ECMO for a mean of 8.4 days (95% CI: 6.1-10.7) and remained in the ICU for a mean of 40.4 days (95% CI: 11.4-69.3). Mean hospital length of stay was 45.4 days (95% CI: 31.7-59.0). In conclusion, patients with burn and inhalation injuries who develop ARDS refractory to conservative management have a survival rate of 54% when placed on ECMO.

Bilateral Choroidal Osteoma in a Teenage Girl With Chronic Posterior Scleritis.

A 14-year-old girl with bilateral chronic posterior scleritis was referred to us for poor control of ocular inflammation. There was an incidental finding of choroidal osteoma bilaterally whereby the choroidal mass in her right eye demonstrated a significant tumor growth in a short duration of two months. The right eye choroidal tumor was orangy in color with a well-defined margin, two-disc diameter in size, and located at the macula encroaching the fovea. Multimodal imaging assessments, including serial color fundus photo, enhanced depth imaging optical coherence tomography (EDI-OCT), and B scan ultrasonography monitoring, confirmed a continuous rapid growth of choroidal osteoma with episodes of scleritis flare-ups. Furthermore, intolerance toward second-line immunosuppressants and loss of follow-ups during the coronavirus disease 2019 (COVID-19) pandemic lockdown led to frequent relapses of her posterior scleritis. Therefore, a new treatment plan was designed, and close monitoring of choroidal osteoma growth and control of posterior scleritis were initiated. Subsequently, bilateral posterior scleritis remained quiescent, and her vision remained stable with stagnant growth of choroidal osteoma.

Effect of Socio-Economic Status on Perioperative Outcomes After Robotic-Assisted Pulmonary Lobectomy.

BACKGROUND:  Lower socioeconomic status (SES) has been correlated with poor survival rates and surgical outcomes following lung cancer resection. This study sought to determine whether this disparity exists perioperatively in lung cancer patients following robotic-assisted video-thoracoscopic pulmonary lobectomy.  Methods: We retrospectively reviewed 447 consecutive patients who underwent robotic-assisted pulmonary lobectomy by one surgeon for known or suspected lung cancer. Ten patients were excluded due to incomplete data. We used median income by residential ZIP code as a surrogate for SES status and grouped patients based on whether ZIP-based median income was less than (Group 1) or greater than (Group 2) 300% of the federal poverty income level. The effects of SES status groups on incidence of postoperative complications, chest tube duration, hospital length of stay (LOS), and in-hospital mortality were evaluated by the logistic regression model and Inverse Gaussian regression model, respectively. RESULTS:  Without adjustment, Group 1 tended to have a higher rate of postoperative complications, with 54% of patients experiencing complications compared to 34% of patients in Group 2 (p=0.007). Median chest tube duration and hospital LOS were also significantly longer in Group 1 than in Group 2 (p=0.034). In multivariable logistical regression analysis, while controlling for covariates and considering effect modifications, lower SES was significantly and positively associated with postoperative complications (odds ratio (OR)=1.98, p=0.039). Preoperative chronic obstructive pulmonary disease (COPD) was also a positive and significant predictor of postoperative complications (OR=1.89, p=0.017), chest tube duration (p=0.020), and LOS (p=0.010). CONCLUSIONS:  Lower median income is associated with a greater number of postoperative complications following pulmonary resection for lung cancer when controlling for covariates.

A Comprehensive Analysis of the Role of Oxidative Stress in the Pathogenesis and Chemoprevention of Oral Submucous Fibrosis.

Oral submucous fibrosis (OSMF) is a chronic oral potentially malignant disorder (OPMD). It is described as a scarring disease of the oral mucosa associated with excess oxidants and insufficient antioxidants. While it is becoming increasingly accepted that oxidative stress results in excessive accumulation of collagen and progressive fibrosis of the submucosal tissues, there is limited data regarding the moderation of oxidative stress to initiate or prevent OSMF. To assess the scope for mechanism-based approaches to prevent or reverse OSMF, we systematically evaluated the existing literature and investigated the role of oxidative stress in the pathogenesis and chemoprevention of OSMF. A search for relevant articles on PubMed and Scopus was undertaken using pre-defined inclusion and exclusion criteria. A total of 78 articles were selected in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. The articles eligible for assessment investigated both OSMF and/or oxidative stress biomarkers or specific antioxidants. Both in vitro and human studies consistently demonstrated variations in oxidative stress biomarker levels in OSMF and revealed an increase in oxidative stress, paralleling the development of the disease. Furthermore, the use of antioxidant supplements was overall associated with an improvement in clinical outcomes. Having identified the significance of oxidative stress in OSMF and the therapeutic potential of antioxidant supplements, this scoping review highlights the need for further well-designed studies in the development of mechanism-based interventions for managing OSMF.

Cavernous Sinus Thrombosis and Blindness Complicating Dental Infection.

A 32-year-old gentleman with underlying hypertension presented with left eye ptosis and diplopia for two weeks. He also complained of the left eye progressive blurring of vision. One week of left-sided toothache, headache, and fever preceded these symptoms. He visited a dental clinic for the toothache and was prescribed oral metronidazole before scheduling tooth extraction. However, the disease progressed with ocular symptoms. On examination, his visual acuity was 20/20 on the right and perception to light on the left. The left eye pupil was sluggish, and relative afferent pupillary reflex was positive. There was partial ptosis, mild proptosis, and ophthalmoplegia involving cranial nerve III, IV, and VI over the left. Hypoesthesia over the left V1 region was also present. Bilateral anterior and posterior segments were unremarkable. Blood investigations revealed an elevated total white cell count and C- reactive protein. Hence, an urgent computed tomography of the brain was requested and demonstrated left cavernous sinus thrombosis with diffuse thickening and enhancement extended anteriorly to the left orbital apex. He was admitted for intravenous ceftriaxone and subcutaneous enoxaparin. He was hemodynamically stable and allowed home with new direct anti-coagulants. He sustained the permanent sequelae of a left blind eye and residual cranial nerve palsies despite the treatment.

Similarities and differences in concepts of mental life among adults and children in five cultures.

How do concepts of mental life vary across cultures? By asking simple questions about humans, animals and other entities - for example, 'Do beetles get hungry? Remember things? Feel love?' - we reconstructed concepts of mental life from the bottom up among adults (N = 711) and children (ages 6-12 years, N = 693) in the USA, Ghana, Thailand, China and Vanuatu. This revealed a cross-cultural and developmental continuity: in all sites, among both adults and children, cognitive abilities travelled separately from bodily sensations, suggesting that a mind-body distinction is common across diverse cultures and present by middle childhood. Yet there were substantial cultural and developmental differences in the status of social-emotional abilities - as part of the body, part of the mind or a third category unto themselves. Such differences may have far-reaching social consequences, whereas the similarities identify aspects of human understanding that may be universal.

Ritual futures: Spirit mediumship as chronotopic labor.

This essay reflects on the still-present difficulty in approaching contemporary rural mediumship as coeval with their urban psychotherapeutic counterparts. Drawing on ethnographic work in rural Henan province in central China, I describe how both rurality and spirit mediumship have been rendered anachronistic through national imaginaries, anti-superstition campaigns, and psychiatric discourses. The essay centers on the case of a spirit medium located in the psychiatric unit, and the social evolutionary and developmentalist temporalities condensed in her cultural psychiatric diagnosis. I then turn to the medium's ritual work and cosmological account, which invert mediumship's position in space and time. The essay approaches mediumship's rituals as a form of chronotopic labor, which reworks the spatio-temporal coordinates they inherit from within. It closes by bringing together the conundrums of rural mediumship and those of urban psychotherapeutic and diasporic worlds, to consider psychic landscapes of dislocation, and other formulations of futures to come.

Equivalence Curves for Healthy Lifestyle Choices.

BACKGROUND: Understanding equivalence of time-use trade-offs may inform tailored lifestyle choices. We explored which time reallocations were associated with equivalent changes in children's health outcomes. METHODS: Participants were from the cross-sectional Child Health CheckPoint Study (N = 1179; 11-12 years; 50% boys) nested within the population-based Longitudinal Study of Australian Children. Outcomes were adiposity (bioelectrical impedance analysis, BMI and waist girth), self-reported health-related quality of life (HRQoL; Pediatric Quality of Life Inventory), and academic achievement (standardized national tests). Participants' 24-hour time use (sleep, sedentary behavior, light physical activity, and moderate-to-vigorous physical activity [MVPA]) from 8-day 24-hour accelerometry was regressed against outcomes by using compositional log-ratio linear regression models. RESULTS: Children with lower adiposity and higher HRQoL had more MVPA (both P ≤ .001) and sleep (P = .001; P < .02), and less sedentary time (both P < .001) and light physical activity (adiposity only; P = .03), each relative to remaining activities. Children with better academic achievement had less light physical activity, relative to remaining activities (P = .003). A 0.1 standardized decrease in adiposity was associated with either 52 minutes more sleep, 56 minutes less sedentary time, 65 minutes less light physical activity, or 17 minutes more MVPA. A 0.1 standardized increase in HRQoL was associated with either 68 minutes more sleep, 54 minutes less sedentary time, or 35 minutes more MVPA. CONCLUSIONS: Equivalent differences in outcomes were associated with several time reallocations. On a minute-for-minute basis, MVPA was 2 to 6 times as potent as sleep or sedentary time.

Sensing the presence of gods and spirits across cultures and faiths.

Hearing the voice of God, feeling the presence of the dead, being possessed by a demonic spirit-such events are among the most remarkable human sensory experiences. They change lives and in turn shape history. Why do some people report experiencing such events while others do not? We argue that experiences of spiritual presence are facilitated by cultural models that represent the mind as "porous," or permeable to the world, and by an immersive orientation toward inner life that allows a person to become "absorbed" in experiences. In four studies with over 2,000 participants from many religious traditions in the United States, Ghana, Thailand, China, and Vanuatu, porosity and absorption played distinct roles in determining which people, in which cultural settings, were most likely to report vivid sensory experiences of what they took to be gods and spirits.

The mitochondria-targeted peptide SS-31 binds lipid bilayers and modulates surface electrostatics as a key component of its mechanism of action.

Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 µm, versus 9.5 µm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 µm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 µm, observed with P106L alone, to 10.3 µm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.

Novel form of the Michaelis-Menten equation that enables accurate estimation of (kcat/KM)*KI with just two rate measurements; utility in directed evolution.

One of applications of directed evolution is to desensitize an enzyme to an inhibitor. kcat,1/KM and KI are three dimensions that when multiplied measure an enzyme's intrinsic capacity for catalysis in the presence of an inhibitor. The ideal values for the individual dimensions depend on substrate and inhibitor concentrations under the conditions of the application. When attempting to optimize those values by directed evolution, (kcat/KM)*KI can be an informative parameter for evaluating libraries of variants, but throughput is limited. We describe a manipulation of the Michaelis-Menten equation for competitive inhibition that isolates (kcat/KM)*KI on one side of the equation. If velocity is measured at constant enzyme and substrate concentrations with two different inhibitor concentrations (one of which can be 0), the data are sufficient to calculate (kcat/KM)*KI with just two rate measurements. The procedure is validated by correlating values obtained by the rapid method with those obtained by substrate saturation kinetics.

Comparison of pulmonary function tests and perioperative outcomes after robotic-assisted pulmonary lobectomy vs segmentectomy.

BACKGROUND: Lobectomy is standard treatment for early-stage lung cancer, but sublobar resection remains debated. We compared outcomes after robotic-assisted video-assisted thoracoscopic (R-VATS) segmentectomy vs lobectomy. METHODS: We retrospectively analyzed data from 251 consecutive patients who underwent R-VATS lobectomy (n = 208) or segmentectomy (n = 43) by a single surgeon over 36 months. Pulmonary function tests and perioperative outcomes were compared using Chi-squared test, unpaired Student t test, or Kruskal-Wallis test, with significance at P ≤ .05. RESULTS: Intraoperative complications were not significantly different, but median operative times were longer for R-VATS segmentectomies (P < .01). Postoperative complications were not significantly different, except for increased rates of pneumothorax after chest tube removal (P = .032) and of effusions or empyema (P = .011) after R-VATS segmentectomies. Predicted changes for forced expiratory volume in 1 second and diffusion constant of the lung for carbon monoxide are significantly less after R-VATS segmentectomy (P < .001). CONCLUSIONS: R-VATS segmentectomy should be considered as an alternative to lobectomy for conserving lung function in respiratory-compromised lung cancer patients, although oncologic efficacy remains undetermined.