Rapid, biochemical tagging of cellular activity history in vivo.

Intracellular calcium (Ca 2+ ) is ubiquitous to cell signaling across all biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca 2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely-behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca 2+ in vivo. Ca 2+ -activated Split-TurboID (CaST) labels activated cells within 10 minutes with an exogenously-delivered biotin molecule. The enzymatic signal increases with Ca 2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca 2+ activity. Furthermore, the CaST read-out can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

Social Determinants of Premature Birth.

Social determinants of health have received increasing attention in public health, leading to increased understanding of how social factors-individual and contextual-shape the health of the mother and infant. However, racial differences in birth outcomes persist, with incomplete explanation for the widening disparity. Here, we highlight the social determinants of preterm birth, with special attention to the social experiences among African American women, which are likely attributed to structural racism and discrimination throughout life.

Machine learning-guided engineering of genetically encoded fluorescent calcium indicators.

Here we used machine learning to engineer genetically encoded fluorescent indicators, protein-based sensors critical for real-time monitoring of biological activity. We used machine learning to predict the outcomes of sensor mutagenesis by analyzing established libraries that link sensor sequences to functions. Using the GCaMP calcium indicator as a scaffold, we developed an ensemble of three regression models trained on experimentally derived GCaMP mutation libraries. The trained ensemble performed an in silico functional screen on 1,423 novel, uncharacterized GCaMP variants. As a result, we identified the ensemble-derived GCaMP (eGCaMP) variants, eGCaMP and eGCaMP+, which achieve both faster kinetics and larger ∆F/F0 responses upon stimulation than previously published fast variants. Furthermore, we identified a combinatorial mutation with extraordinary dynamic range, eGCaMP2+, which outperforms the tested sixth-, seventh- and eighth-generation GCaMPs. These findings demonstrate the value of machine learning as a tool to facilitate the efficient engineering of proteins for desired biophysical characteristics.

Prenatal and Pediatric Primary Care-Based Child Obesity Prevention: Effects of Adverse Social Determinants of Health on Intervention Attendance and Impact.

Background: Adverse social determinants of health (SDoHs), specifically psychosocial stressors and material hardships, are associated with early childhood obesity. Less is known about whether adverse SDoHs modify the efficacy of early childhood obesity prevention programs. Methods: We conducted a secondary analysis of publicly insured birthing parent-child dyads with Latino backgrounds participating in a randomized controlled trial of the Starting Early Program (StEP), a child obesity prevention program beginning in pregnancy. We measured baseline adverse SDoHs categorized as psychosocial stressors (low social support, single marital status, and maternal depressive symptoms) and material hardships (food insecurity, housing disrepair, and financial difficulties) individually and cumulatively in the third trimester. Logistic regression models tested effects of adverse SDoHs on StEP attendance. We then tested whether adverse SDoHs moderated intervention impacts on weight at age 2 years. Results: We observed heterogeneous effects of adverse SDoHs on outcomes in 358 parent-child dyads. While housing disrepair decreased odds of higher attendance [adjusted odds ratio (aOR) 0.52, 95% confidence interval (CI): 0.29-0.94], high levels of psychosocial stressors doubled odds of higher attendance (aOR 2.36, 95% CI: 1.04-5.34). Similarly, while certain adverse SDoHs diminished StEP impact on weight (e.g., housing disrepair), others (e.g., high psychosocial stress) enhanced StEP impact on weight. Conclusions: Effects of adverse SDoHs on intervention outcomes depend on the specific adverse SDoH. Highest engagement and benefit occurred in those with high psychosocial stress at baseline, suggesting that StEP components may mitigate aspects of psychosocial stressors. Findings also support integration of adverse SDoH assessment into strategies to enhance obesity prevention impacts on families with material hardships. Trial Registration: This study is registered on clinicaltrials.gov: Starting Early Obesity Prevention Program (NCT01541761); https://clinicaltrials.gov/ct2/show/NCT01541761.

Optimizing pharmacogenomic decision-making by data science.

Healthcare systems have made rapid progress towards combining data science with precision medicine, particularly in pharmacogenomics. With the lack of predictability in medication effectiveness from patient to patient, acquiring the specifics of their genotype would be highly advantageous for patient treatment. Genotype-guided dosing adjustment improves clinical decision-making and helps optimize doses to deliver medications with greater efficacy and within safe margins. Current databases demand extensive effort to locate relevant genetic dosing information. To address this problem, Patient Optimization Pharmacogenomics (POPGx) was constructed. The objective of this paper is to describe the development of POPGx, a tool to simplify the approach for healthcare providers to determine pharmacogenomic dosing recommendations for patients taking multiple medications. Additionally, this tool educates patients on how their allele variations may impact gene function in case they need further healthcare consultations. POPGx was created on Konstanz Information Miner (KNIME). KNIME is a modular environment that allows users to conduct code-free data analysis. The POPGx workflow can access Clinical Pharmacogenomics Implementation Consortium (CPIC) guidelines and subsequently be able to present relevant dosing and counseling information. A KNIME representational state transfer (REST) application program interface (API) node was established to retrieve information from CPIC and drugs that are exclusively metabolized through CYP450, and these drugs were processed simultaneously to demonstrate competency of the workflow. The POPGx program provides a time-efficient method for users to retrieve relevant, patient-specific medication selection and dosing recommendations. Users input metabolizer gene, genetic allele data, and medication list to retrieve clear dosing information. The program is automated to display current guideline recommendations from CPIC. The integration of this program into healthcare systems has the potential to revolutionize patient care by giving healthcare practitioners an easy way to prescribe medications with greater efficacy and safety by utilizing the latest advancements in the field of pharmacogenomics.

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Promoting toddlers' self-regulation and healthy eating habits among families living in poverty: A randomized controlled trial of Recipe 4 Success.

The Recipe 4 Success preventive intervention targeted multiple factors critical to the health and well-being of toddlers living in poverty. This randomized controlled trial, which was embedded within Early Head Start home visits for 12 weeks, included 242 racially and ethnically diverse families (51% girls; toddler mean age = 2.58 years; data collected 2016-2019). Compared to parents in usual practice home visits, parents in Recipe 4 Success displayed greater sensitive scaffolding of toddlers' learning and more responsive food parenting practices (Cohen's d = .21-.30). Toddlers in Recipe 4 Success exhibited greater self-regulation and had healthier eating habits (Cohen's d = |.16-.35|). Results highlight the value of Recipe 4 Success in promoting parent and toddler behavior change that could have life-long benefits.

Long-term Survival Among Patients With De Novo Human Epidermal Growth Receptor 2-Positive Metastatic Breast Cancer in Manitoba.

OBJECTIVES: Although metastatic breast cancer (MBC) is considered incurable, human epidermal growth receptor 2 (HER2)-directed therapy has improved outcomes significantly, with some patients experiencing durable responses to treatment. The aim of this study was to identify potential predictors of long-term survival (LTS) among patients with de novo HER2-positive MBC who received HER2-directed treatment. METHODS: Eligible patients from 2008 to 2018 were identified using the Manitoba Cancer Registry. LTS was defined as survival ≥5 years from the time of diagnosis. Univariate logistic regression models were performed to assess variables of clinical interest and the odds of LTS. Overall survival (OS) was defined as the time from diagnosis of MBC to death of any cause. OS was estimated using the Kaplan-Meier method with log-rank comparative analyses as a univariate analysis. A Cox proportional hazards model was used for OS estimates in a univariate analysis. RESULTS: A total of 62 patients were diagnosed with de novo HER2-positive MBC and received HER2-directed therapy. Eighteen (29%) achieved LTS. The median OS of the whole cohort was 50.2 months (95% CI: 28.6-not reached). Radiographic response to first-line treatment was associated with LTS; complete and partial responses were both associated with higher odds of LTS (odds ratio: 28.33 [95% CI: 2.47-4006.71, P = 0.0043] and odds ratio: 7.80 [95% CI: 0.7317-1072.00, P = 0.0972], respectively). The best radiographic response was associated with improved OS. CONCLUSIONS: Radiographic response to first-line HER2-directed therapy is a predictor for LTS in patients with de novo HER2-positive MBC. Larger studies are needed to identify patients who can safely discontinue HER2-targeted therapy.

Province-Wide Ascertainment of Lynch Syndrome in Manitoba.

BACKGROUND & AIMS: We describe the experience of Lynch syndrome (LS) diagnosis in the province of Manitoba, Canada, over the past 20 years. METHODS: We performed a retrospective review of charts from the provincial Genetics Clinic from January 1, 2000, to May 31, 2023. We extracted data on individuals identified to carry a germline pathogenic or likely pathogenic LS gene variant, the mode of ascertainment, family history, and cascade genetic testing (CGT). Data were stratified and compared before and after the year of implementation (October 2013) of the provincial LS screening program (LSSP) and ascertainment by the LSSP vs clinic referrals (CRs). RESULTS: Between 2014 and 2021, 50 of 101 (49.5%) index cases were identified by the LSSP compared with 51 of 101 (50.5%) from CRs. The proportion of PMS2 variants was 34% (17 of 50) for LSSP index cases compared with 21.6% (11 of 51) for CRs from 2014 to 2021 (P < .001). Among CRs from 2014 to 2021, 24 of 51 (47.1%) families met the Amsterdam criteria, compared with 11 of 50 (22.0%) for the LSSP (P = .01). CGT occurred among 46.8% (95 of 203; average, 1.9 relatives/index) of first-degree relatives of CR index cases vs 36.5% (84 of 230; average, 1.7 relatives/index) of first-degree relatives of LSSP index cases (P = .03). Daughters were most likely to undergo CGT. CONCLUSIONS: A tumor screening program is more effective at detecting individuals with lower penetrant gene variants and families who do not meet traditional family history-based criteria. Cascade genetic testing is higher among clinic referrals compared with the screening program. These findings suggest a complementary role of these 2 ascertainment methods for Lynch syndrome.

Household Food Insecurity and Maternal-Toddler Fruit and Vegetable Dietary Concordance.

OBJECTIVE: To examine whether prenatal or concurrent household food insecurity influences associations between maternal and toddler fruit and vegetable (FV) intake. DESIGN: Application of a life-course framework to an analysis of a longitudinal dataset. SETTING: Early childhood obesity prevention program at a New York City public hospital. PARTICIPANTS: One-hundred and fifty-six maternal-toddler dyads self-identifying as Hispanic or Latino. VARIABLES MEASURED: Maternal and toddler FV intake was measured using Centers for Disease Control and Prevention dietary measures when toddlers were aged 19 months. Household food insecurity (measured prenatally and concurrently at 19 months) was measured using the US Department of Agriculture Food Security Module. ANALYSIS: Regression analyses assessed associations between adequate maternal FV intake and toddler FV intake. Interaction terms tested whether prenatal or concurrent household food insecurity moderated this association. RESULTS: Adequate maternal FV intake was associated with increased toddler FV intake (B = 6.2 times/wk, 95% confidence interval, 2.0-10.5, P = 0.004). Prenatal household food insecurity was associated with decreased toddler FV intake (B = -6.3 times/wk, 95% confidence interval, -11.67 to -0.9, P = 0.02). There was a significant interaction between the level of maternal-toddler FV association (concordance or similarity in FV intake between mothers and toddlers) and the presence of food insecurity such that maternal-toddler FV association was greater when prenatal household food insecurity was not present (B = -11.6, P = 0.04). CONCLUSIONS AND IMPLICATIONS: Strategies to increase FV intake across the life course could examine how the timing of household food insecurity may affect intergenerational maternal-child transmission of dietary practices.

Breast Cancer Polygenic-Risk Score Influence on Risk-Reducing Endocrine Therapy Use: Genetic Risk Estimate (GENRE) Trial 1-Year and 2-Year Follow-Up.

Refinement of breast cancer risk estimates with a polygenic-risk score (PRS) may improve uptake of risk-reducing endocrine therapy (ET). A previous clinical trial assessed the influence of adding a PRS to traditional risk estimates on ET use. We stratified participants according to PRS-refined breast cancer risk and evaluated ET use and ET-related quality of life (QOL) at 1-year (previously reported) and 2-year follow-ups. Of 151 participants, 58 (38.4%) initiated ET, and 22 (14.6%) discontinued ET by 2 years; 42 (27.8%) and 36 (23.8%) participants were using ET at 1- and 2-year follow-ups, respectively. At the 2-year follow-up, 39% of participants with a lifetime breast cancer risk of 40.1% to 100.0%, 18% with a 20.1% to 40.0% risk, and 16% with a 0.0% to 20.0% risk were taking ET (overall P = 0.01). Moreover, 40% of participants whose breast cancer risk increased by 10% or greater with addition of the PRS to a traditional breast cancer-risk model were taking ET versus 0% whose risk decreased by 10% or greater (P = 0.004). QOL was similar for participants taking or not taking ET at 1- and 2-year follow-ups, although most who discontinued ET did so because of adverse effects. However, these QOL results may have been skewed by the long interval between QOL surveys and lack of baseline QOL data. PRS-informed breast cancer prevention counseling has a lasting, but waning, effect over time. Additional follow-up studies are needed to address the effect of PRS on ET adherence, ET-related QOL, supplemental breast cancer screening, and other risk-reducing behaviors. PREVENTION RELEVANCE: Risk-reducing medications for breast cancer are considerably underused. Informing women at risk with precise and individualized risk assessment tools may substantially affect the incidence of breast cancer. In our study, a risk assessment tool (IBIS-polygenic-risk score) yielded promising results, with 39% of women at highest risk starting preventive medication.

Tagging neurons with light.

Molecular circuits for activity-guided optogenetics.

The room where it happens: addressing diversity, equity, and inclusion in National Clinical Trials Network clinical trial leadership.

Many multicenter randomized clinical trials in oncology are conducted through the National Clinical Trials Network (NCTN), an organization consisting of 5 cooperative groups. These groups are made up of multidisciplinary investigators who work collaboratively to conduct trials that test novel therapies and establish best practice for cancer care. Unfortunately, disparities in clinical trial leadership are evident. To examine the current state of diversity, equity, and inclusion across the NCTN, an independent NCTN Task Force for Diversity in Gastrointestinal Oncology was established in 2021, the efforts of which serve as the platform for this commentary. The task force sought to assess existing data on demographics and policies across NCTN groups. Differences in infrastructure and policies were identified across groups as well as a general lack of data regarding the composition of group membership and leadership. In the context of growing momentum around diversity, equity, and inclusion in cancer research, the National Cancer Institute established the Equity and Inclusion Program, which is working to establish benchmark data regarding diversity of representation within the NCTN groups. Pending these data, additional efforts are recommended to address diversity within the NCTN, including standardizing membership, leadership, and publication processes; ensuring diversity of representation across scientific and steering committees; and providing mentorship and training opportunities for women and individuals from underrepresented groups. Intentional and focused efforts are necessary to ensure diversity in clinical trial leadership and to encourage design of trials that are inclusive and representative of the broad population of patients with cancer in the United States.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

The impact of early palliative care on the quality of life of patients with advanced pancreatic cancer: The IMPERATIVE case-crossover study.

PURPOSE: Pancreatic cancer is a lethal disease. Many patients experience a heavy burden of cancer-associated symptoms and poor quality of life (QOL). Early palliative care alongside standard oncologic care results in improved QOL and survival in some cancer types. The benefit in advanced pancreatic cancer (APC) is not fully quantified. METHODS: In this prospective case-crossover study, patients ≥ 18 years old with APC were recruited from ambulatory clinics at a tertiary cancer center. Patients underwent a palliative care consultation within 2 weeks of registration, with follow up visits every 2 weeks for the first month, then every 4 weeks until week 16, then as needed. The primary outcome was change in QOL between baseline (BL) and week 16, measured by Functional Assessment of Cancer Therapy - hepatobiliary (FACT-Hep). Secondary outcomes included symptom control (ESAS-r), depression, and anxiety (HADS, PHQ-9) at week 16. RESULTS: Of 40 patients, 25 (63%) were male, 28 (70%) had metastatic disease, 31 (78%) had ECOG performance status 0-1, 31 (78%) received chemotherapy. Median age was 70. Mean FACT-hep score at BL was 118.8, compared to 125.7 at week 16 (mean change 6.89, [95%CI (-1.69-15.6); p = 0.11]). On multivariable analysis, metastatic disease (mean change 15.3 [95%CI (5.3-25.2); p = 0.004]) and age < 70 (mean change 12.9 [95%CI (0.5-25.4); p = 0.04]) were associated with improved QOL. Patients with metastatic disease had significant improvement in symptom burden (mean change -7.4 [95%CI (-13.4 to -1.4); p = 0.02]). There was no difference in depression or anxiety from BL to week 16. CONCLUSION: Palliative care should be integrated early in the journey for patients with APC, as it can improve QOL and symptom burden. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT03837132.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

Cigarette smoking cessation, duration of smoking abstinence, and head and neck squamous cell carcinoma prognosis.

BACKGROUND: Tobacco use is a major risk factor for developing head and neck squamous cell carcinoma (HNSCC). However, the prognostic associations with smoking cessation are limited. The authors assessed whether smoking cessation and increased duration of abstinence were associated with improved overall (OS) and HNSCC-specific survival. METHODS: Clinicodemographic and smoking data from patients with HNSCC at Princess Margaret Cancer Center (2006-2019) were prospectively collected. Multivariable Cox and Fine and Gray competing-risk models were used to assess the impact of smoking cessation and duration of abstinence on overall mortality and HNSCC-specific/noncancer mortality, respectively. RESULTS: Among 2482 patients who had HNSCC, former smokers (adjusted hazard ratio [aHR], 0.71; 95% CI, 0.58-0.87; p = .001; N = 841) had a reduced risk of overall mortality compared with current smokers (N = 931). Compared with current smokers, former smokers who quit >10 years before diagnosis (long-term abstinence; n = 615) had the most improved OS (aHR, 0.72; 95% CI, 0.56-0.93; p = .001). The 5-year actuarial rates of HNSCC-specific and noncancer deaths were 16.8% and 9.4%, respectively. Former smokers (aHR, 0.71; 95% CI, 0.54-0.95; p = .019) had reduced HNSCC-specific mortality compared with current smokers, but there was no difference in noncancer mortality. Abstinence for >10 years was associated with decreased HNSCC-specific death compared with current smoking (aHR, 0.64; 95% CI, 0.46-0.91; p = .012). Smoking cessation with a longer duration of quitting was significantly associated with reduced overall and HNSCC-specific mortality in patients who received primary radiation. CONCLUSIONS: Smoking cessation before the time of diagnosis reduced overall mortality and cancer-specific mortality among patients with HNSCC, but no difference was observed in noncancer mortality. Long-term abstinence (>10 pack-years) had a significant OS and HNSCC-specific survival benefit.

A Canadian Provincial Screening Program for Lynch Syndrome.

INTRODUCTION: Manitoba implemented the first Canadian provincial program of reflex screening through mismatch repair immunohistochemistry (MMR-IHC) for all colorectal cancers diagnosed at age 70 years or younger in December 2017. We evaluated compliance to universal reflex testing and for referrals to Genetics for individuals with MMR-deficient tumors. METHODS: We searched the provincial pathology database with "adenocarcinoma" in the colorectal specimen pathology reports between March 2018 and December 2020. We cross-referenced with paper and electronic records in the Program of Genetics and Metabolism to determine whether patients with MMR-deficient tumors had been referred for Genetic assessment and what proportion of patients and first-degree relatives accepted an appointment and genetic testing. We performed logistic regression analysis to identify predictors of testing. RESULTS: We identified 3,146 colorectal adenocarcinoma specimens (biopsies and surgical resections) from 1,692 unique individuals (mean age 68.66 years, male 57%). Of those aged 70 years or younger (n = 936), 89.4% received MMR-IHC screening. Individual pathologists (categorized by the highest, average, and lowest screening rates) were the biggest predictors of MMR-IHC screening on multivariable analysis (highest vs lowest: odds ratio 17.5, 95% confidence interval 6.05-50.67). While only 53.4% (n = 31) of 58 screen-positive cases were referred by pathologists for genetic assessment, other clinicians referred an additional 22.4% (n = 13), resulting in 75.8% overall referral rate of screen-positive cases. Thirteen (1.4%) patients (1.1%, aged 70 years or younger) were confirmed to experience Lynch syndrome through germline testing, and 8 first-degree relatives (an average of 1.6 per patient) underwent cascade genetic testing. DISCUSSION: The first Canadian Lynch syndrome screening program has achieved high rates of reflex testing.