Droplet freezing assays using a nanoliter osmometer.

The ability to accurately record the temperature at which ice nucleation occurs is critical for studying biological ice nucleators. Several instruments have been designed and custom built to make such measurements, but they are not yet on the market. Here we reproducibly measure ice nucleation temperatures down close to the homogeneous nucleation temperature of -38 °C with a commercially available nanoliter osmometer, which we routinely use to assay the thermal hysteresis activity of antifreeze proteins. This instrument has both a wide operating temperature range and fine temperature control, while the oil immersion format on 12-well grids prevents droplet evaporation and surface nucleation events. The results obtained are consistent with those reported on other instruments in common use.

Effects of psychosocial interventions on psychological outcomes among caregivers of advanced cancer patients: a systematic review and meta-analysis.

PURPOSE: This systematic review aimed to synthesize the effectiveness of psychosocial interventions on caregivers of advanced cancer patients, in comparison with usual care, on caregivers' quality of life (QoL), anxiety, and depression symptoms. METHODS: Comprehensive searches for published and unpublished studies were performed using nine electronic databases, two trial registers, and reference lists of included studies. Two reviewers independently screened, appraised, and extracted data. The Cochrane risk of bias tool was used to appraise the methodological quality of included studies, while the Cochrane data extraction tool was used to elicit relevant information. Meta-analysis, narrative analysis, and sensitivity analysis were conducted to synthesize data. Standardized mean differences (SMD) represented effects of psychosocial interventions. RESULTS: Fifteen randomized controlled trials were included in this review. At post-intervention, findings revealed a significant small pooled effect size (SMD = 0.45) on QoL and significant moderate effect on depression (SMD = - 0.65). However, a small non-significant pooled effect size was observed on anxiety (SMD = - 0.24). At follow-up assessments, effect sizes of all outcomes were small and non-significant. Overall quality of evidence was rated very low for all outcomes and most studies had unclear or high risk of bias. Thus, results should be interpreted with caution. CONCLUSION: Psychosocial interventions were effective in improving QoL and depression among caregivers of persons with advanced cancer. However, future randomized control trials with lower risk of bias, larger sample size, detailed participant characteristics, and informative interventions are desirable.

Addressing the inappropriate use of force by police in the United States and beyond: A behavioral and social science perspective.

Recent high-profile incidents involving the deadly application of force in the United States sparked worldwide protests and renewed scrutiny of police practices as well as scrutiny of relations between police officers and minoritized communities. In this report, we consider the inappropriate use of force by police from the perspective of behavioral and social science inquiry related to aggression, violence, and intergroup relations. We examine the inappropriate use of force by police in the context of research on modern policing as well as critical race theory and offer five recommendations suggested by contemporary theory and research. Our recommendations are aimed at policymakers, law enforcement administrators, and scholars and are as follows: (1) Implement public policies that can reduce inappropriate use of force directly and through the reduction of broader burdens on the routine activities of police officers. (2) For officers frequently engaged in use-of-force incidents, ensure that best practice, evidence-based treatments are available and required. (3) Improve and increase the quality and delivery of noncoercive conflict resolution training for all officers, along with police administrative policies and supervision that support alternatives to the use of force, both while scaling back the militarization of police departments. (4) Continue the development and evaluation of multicomponent interventions for police departments, but ensure they incorporate evidence-based, field-tested components. (5) Expand research in the behavioral and social sciences aimed at understanding and managing use-of-force by police and reducing its disproportionate impact on minoritized communities, and expand funding for these lines of inquiry.

Global loss of leucine carboxyl methyltransferase-1 causes severe defects in fetal liver hematopoiesis.

Leucine carboxyl methyltransferase-1 (LCMT-1) methylates the C-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In this study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down ∼30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and usually death by embryonic day 16.5. Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit+Lin-Sca1+ cells. The percent cycling cells and mitotic indices of WT and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, noncompetitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multilineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis.

Design and analysis of group-randomized trials in cancer: A review of current practices.

The purpose of this paper is to summarize current practices for the design and analysis of group-randomized trials involving cancer-related risk factors or outcomes and to offer recommendations to improve future trials. We searched for group-randomized trials involving cancer-related risk factors or outcomes that were published or online in peer-reviewed journals in 2011-15. During 2016-17, in Bethesda MD, we reviewed 123 articles from 76 journals to characterize their design and their methods for sample size estimation and data analysis. Only 66 (53.7%) of the articles reported appropriate methods for sample size estimation. Only 63 (51.2%) reported exclusively appropriate methods for analysis. These findings suggest that many investigators do not adequately attend to the methodological challenges inherent in group-randomized trials. These practices can lead to underpowered studies, to an inflated type 1 error rate, and to inferences that mislead readers. Investigators should work with biostatisticians or other methodologists familiar with these issues. Funders and editors should ensure careful methodological review of applications and manuscripts. Reviewers should ensure that studies are properly planned and analyzed. These steps are needed to improve the rigor and reproducibility of group-randomized trials. The Office of Disease Prevention (ODP) at the National Institutes of Health (NIH) has taken several steps to address these issues. ODP offers an online course on the design and analysis of group-randomized trials. ODP is working to increase the number of methodologists who serve on grant review panels. ODP has developed standard language for the Application Guide and the Review Criteria to draw investigators' attention to these issues. Finally, ODP has created a new Research Methods Resources website to help investigators, reviewers, and NIH staff better understand these issues.

Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes.

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

UNLABELLED: Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. IMPORTANCE: dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.

Treatment Patterns and Clinical Outcomes Among Patients With Metastatic Prostate Cancer Harboring Homologous Recombination Repair Mutations.

BACKGROUND: There is currently limited literature assessing the real-world treatment patterns and clinical outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) and homologous recombination repair (HRR) mutations. METHODS: Medical charts were abstracted for mCRPC patients with ≥ 1 of 12 HRR somatic gene alterations treated at US oncology centers participating in the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange. Treatment patterns and clinical outcomes were assessed from the initiation of first-line or later (1L+) mCRPC therapy received on or after July 1, 2014. RESULTS: Among 138 patients included in the study, the most common somatic HRR mutations were CDK12 (47.8%), BRCA2 (22.5%), and ATM (21.0%). Novel hormonal therapy and taxane chemotherapy were most commonly used in 1L; taxane use increased in later lines. Median overall survival (95% confidence interval [CI]) was 36.3 (30.7-47.8) months from initiation of 1L therapy and decreased for subsequent lines. Similarly, there was a trend of decreasing progression-free survival and prostate-specific antigen response from 1L to 4L+ therapy. CONCLUSIONS: Treatment patterns identified in this study were similar to those among patients with mCRPC regardless of tumor HRR mutation status in the literature.

Case Studies of Small-Medium Food Enterprises around the World: Major Constraints and Benefits from the Implementation of Food Safety Management Systems.

Global food safety and security are key principles to be followed in the context of the implementation of food safety management systems. The objective of this paper is to assess the contemporary developments of Food Safety Management System standards (FSMS) worldwide and to identify the primary constraints and advantages associated with their implementation by small and medium-sized enterprises across different regions. The effectiveness of these systems has also been evaluated. 116 case studies have been employed across developing and developed regions worldwide across 27 primary food sectors. After the implementation of FSMS, there was a significant increase in the percentage of companies that have implemented the international FSMS, both in developed (16.7% to 63.9%) and developing countries (26.6% to 48.1%). Certification has also increased from 34.2% to 59.6% in the total sample, namely from 33.3% to 61.1% in developed countries and from 34.6% to 59.0% in developing countries. There was a significant increase in medium vs. small company size (57.1% to 62.3%, p = 0.046), only in developing countries. Food safety culture and manager leadership implementation were increased to over 80% after FSMS implementation in both developed and developing countries (p < 0.001). Training, resources, and technology adequacy were also increased in all companies (p < 0.001).

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins of common environmental bacteria like Pseudomonas syringae and P. borealis. However, individually, these 100-kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an ice nucleation protein gene into Escherichia coli led to efficient ice nucleation. Here we demonstrate that a positively-charged sub-domain at the C-terminal end of the central beta-solenoid of the ice nucleation protein is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the ice nucleation protein multimers form fibres that are ~ 5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins (INPs) of common environmental bacteria like Pseudomonas syringae and Pseudomonas borealis. However, individually, these 100 kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an INP gene into Escherichia coli led to efficient ice nucleation. Here, we demonstrate that a positively charged subdomain at the C-terminal end of the central ß-solenoid of the INP is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the INP multimers form fibres that are ~5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Genomic Landscapes and Hallmarks of Mutant RAS in Human Cancers.

The RAS family of small GTPases represents the most commonly activated oncogenes in human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that are coaltered in RAS-mutant tumors, we analyzed targeted next-generation sequencing data of 607,863 mutations from 66,372 tumors in 51 cancer types in the AACR Project GENIE Registry. Bayesian hierarchical models were implemented to estimate the cancer-specific prevalence of RAS and non-RAS somatic mutations, to evaluate co-occurrence and mutual exclusivity, and to model the effects of tumor mutation burden and mutational signatures on comutation patterns. These analyses revealed differential RAS prevalence and comutations with non-RAS genes in a cancer lineage-dependent and context-dependent manner, with differences across age, sex, and ethnic groups. Allele-specific RAS co-mutational patterns included an enrichment in NTRK3 and chromatin-regulating gene mutations in KRAS G12C-mutant non-small cell lung cancer. Integrated multiomic analyses of 10,217 tumors from The Cancer Genome Atlas (TCGA) revealed distinct genotype-driven gene expression programs pointing to differential recruitment of cancer hallmarks as well as phenotypic differences and immune surveillance states in the tumor microenvironment of RAS-mutant tumors. The distinct genomic tracks discovered in RAS-mutant tumors reflected differential clinical outcomes in TCGA cohort and in an independent cohort of patients with KRAS G12C-mutant non-small cell lung cancer that received immunotherapy-containing regimens. The RAS genetic architecture points to cancer lineage-specific therapeutic vulnerabilities that can be leveraged for rationally combining RAS-mutant allele-directed therapies with targeted therapies and immunotherapy. SIGNIFICANCE: The complex genomic landscape of RAS-mutant tumors is reflective of selection processes in a cancer lineage-specific and context-dependent manner, highlighting differential therapeutic vulnerabilities that can be clinically translated.

Comparison of lasting life changes after cancer and BMT: perspectives of long-term survivors and spouses.

OBJECTIVE: This qualitative follow up of long-term (>5 years) cancer survivor and spouse participants from a large, previous study of quality of life after blood and marrow transplantation (BMT) was designed to gain a deeper understanding of lasting life changes they experienced. METHODS: Thirty spouse-survivor pairs, an average of 13 years post-BMT, were individually interviewed to identify lasting life changes. Participants were asked about their most significant long-lasting change since cancer/BMT, most significant positive change and negative change, and whether the experience had affected them and their spouse differently. RESULTS: Spouses and survivors spontaneously identified both positive and negative changes. Spouses reported a higher proportion of negative changes (24%) than did survivors (15%), and survivors a higher proportion of positive changes (85%) than spouses (76%). For both groups, the most frequent positive change was in 'perspective/outlook on life' and negative change was 'lingering health effects,' although survivors mentioned the latter twice as often as did spouses. Spouses were more likely to talk about changes in the first-person plural (we, us) that were largely emotional or in relation to the survivor, whereas survivors spoke of changes in the first-person singular (I, me) that occurred to them directly and were largely physical. CONCLUSIONS: Although both spouses and survivors described similar negative and positive long-lasting changes that continued an average of 13 years post-BMT, they reported differences in the ways they were impacted by the experience, which was reflected in the language they used. Implications for future studies, family education, and couples-based interventions are discussed.

Implementation of Food Safety Management Systems along with Other Management Tools (HAZOP, FMEA, Ishikawa, Pareto). The Case Study of Listeria monocytogenes and Correlation with Microbiological Criteria.

The food industry's failure in planning and designing of and in implementing a Food Safety Management System and its foundation elements leads, in most instances, to compromised food safety and subsequent foodborne illness outbreaks. This phenomenon was noticed, worldwide, for all food processors, but with a much higher incidence in the medium- and small-sized food processing plants. Our study focuses on the importance of Food Safety Management System (FSMS), Critical Control Points Hazard Analysis (HACCP) and the Prerequisite Programs (PRPs) as the foundation of HACCP, in preventing foodborne outbreaks. For emphasis, we make use of the example of organizational food safety culture failures and the lack of managerial engagement which resulted in a multi-state listeriosis outbreak in USA. Moreover, we correlate this with microbiological criteria. Implementation of food safety management systems (ISO 22000:2018) along with incorporation of management tools such as HAZOP, FMEA, Ishikawa and Pareto have proved to be proactive in the maintenance of a positive food safety culture and prevention of cross-contamination and fraud.

Confirmatory factor analysis of the Posttraumatic Growth Inventory with a sample of soldiers previously deployed in support of the Iraq and Afghanistan wars.

Research has documented the impact of combat trauma on psychological functioning but less is known about the measurement of positive changes after military deployments. This study examined the factor structure of the Posttraumatic Growth Inventory (PTGI; Tedeschi & Calhoun, 1996) on a sample of active duty soldiers (n = 3537) exposed to combat in Iraq or Afghanistan. Confirmatory factor analyses (CFA) were conducted to test a 5-factor model and a single higher-order factor model. CFA results indicated that both models fit the data equally well and provide support for using both the whole scale and a multidimensional scale. The use of the PTGI in military research and the limitations of the current study are discussed.

Characteristics and Outcome of AKT1 E17K-Mutant Breast Cancer Defined through AACR Project GENIE, a Clinicogenomic Registry.

AKT inhibitors have promising activity in AKT1 E17K-mutant estrogen receptor (ER)-positive metastatic breast cancer, but the natural history of this rare genomic subtype remains unknown. Utilizing AACR Project GENIE, an international clinicogenomic data-sharing consortium, we conducted a comparative analysis of clinical outcomes of patients with matched AKT1 E17K-mutant (n = 153) and AKT1-wild-type (n = 302) metastatic breast cancer. AKT1-mutant cases had similar adjusted overall survival (OS) compared with AKT1-wild-type controls (median OS, 24.1 vs. 29.9, respectively; P = 0.98). AKT1-mutant cases enjoyed longer durations on mTOR inhibitor therapy, an observation previously unrecognized in pivotal clinical trials due to the rarity of this alteration. Other baseline clinicopathologic features, as well as durations on other classes of therapy, were broadly similar. In summary, we demonstrate the feasibility of using a novel and publicly accessible clincogenomic registry to define outcomes in a rare genomically defined cancer subtype, an approach with broad applicability to precision oncology. SIGNIFICANCE: We delineate the natural history of a rare genomically distinct cancer, AKT1 E17K-mutant ER-positive breast cancer, using a publicly accessible registry of real-world patient data, thereby illustrating the potential to inform drug registration through synthetic control data.See related commentary by Castellanos and Baxi, p. 490.

A Machine Learning Approach to Identify NIH-Funded Applied Prevention Research.

INTRODUCTION: To fulfill its mission, the NIH Office of Disease Prevention systematically monitors NIH investments in applied prevention research. Specifically, the Office focuses on research in humans involving primary and secondary prevention, and prevention-related methods. Currently, the NIH uses the Research, Condition, and Disease Categorization system to report agency funding in prevention research. However, this system defines prevention research broadly to include primary and secondary prevention, studies on prevention methods, and basic and preclinical studies for prevention. A new methodology was needed to quantify NIH funding in applied prevention research. METHODS: A novel machine learning approach was developed and evaluated for its ability to characterize NIH-funded applied prevention research during fiscal years 2012-2015. The sensitivity, specificity, positive predictive value, accuracy, and F1 score of the machine learning method; the Research, Condition, and Disease Categorization system; and a combined approach were estimated. Analyses were completed during June-August 2017. RESULTS: Because the machine learning method was trained to recognize applied prevention research, it more accurately identified applied prevention grants (F1 = 72.7%) than the Research, Condition, and Disease Categorization system (F1 = 54.4%) and a combined approach (F1 = 63.5%) with p<0.001. CONCLUSIONS: This analysis demonstrated the use of machine learning as an efficient method to classify NIH-funded research grants in disease prevention.

NIH Primary and Secondary Prevention Research in Humans During 2012-2017.

INTRODUCTION: This paper provides the first detailed analysis of the NIH prevention research portfolio for primary and secondary prevention research in humans and related methods research. METHODS: The Office of Disease Prevention developed a taxonomy of 128 topics and applied it to 11,082 projects representing 91.7% of all new projects and 84.1% of all dollars used for new projects awarded using grant and cooperative agreement activity codes that supported research in fiscal years 2012-2017. Projects were coded in 2016-2018 and analyzed in 2018. RESULTS: Only 16.7% of projects and 22.6% of dollars were used for primary and secondary prevention research in humans or related methods research. Most of the leading risk factors for death and disability in the U.S. were selected as an outcome in <5% of the projects. Many more projects included an observational study, or an analysis of existing data, than a randomized intervention. These patterns were consistent over time. CONCLUSIONS: The appropriate level of support for primary and secondary prevention research in humans from NIH will differ by field and stage of research. The estimates reported here may be overestimates, as credit was given for a project even if only a portion of that project addressed prevention research. Given that 74% of the variability in county-level life expectancy across the U.S. is explained by established risk factors, it seems appropriate to devote additional resources to developing and testing interventions to address those risk factors.