Transgenic Targeting of Fcrls Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity.

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell-types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines are able to achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific Fcrls gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in Fcrls-2A-Cre mice. Crossing Fcrls-2A-Cre mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border associated macrophages (BAMs) are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, Cx3cr1-CreM (MMRRC). Fcrls-2A-Cre mice are immediately available (JAX Stock #036591) and serve as a valuable addition to the community's microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable.Significance Statement The microglia toolbox is continuously growing with more transgenic lines and most recently even viral tools becoming available. When selecting a Cre driver line, investigators must weigh relative strengths and weaknesses of available lines and carefully make the best choice for their given application. These tradeoffs include (1) availability and ease of employment, (2) chromosomal positioning of Cre with respect to the floxed allele (should not be on the same chromosome for conditional knockout studies), (3) activity level of a given Cre line and thus completeness of recombination across the microglia population, (4) specificity with respect to acceptable off-target cell types and tissues, (5) temporal aspects including earliest onset of Cre expression or inducibility, (6) robustness in disease contexts, and (7) potential perturbation of microglia homeostasis through Cre itself or disruption of the targeting locus. Considering these tradeoffs, it is evident that there may not be a one-size-fits all solution but an application-based preference for a mouse line in the diverse repertoire of microglia tools. Fcrls-2A-Cre mice are an excellent addition to this toolbox.

Dynamic and selective engrams emerge with memory consolidation.

Episodic memories are encoded by experience-activated neuronal ensembles that remain necessary and sufficient for recall. However, the temporal evolution of memory engrams after initial encoding is unclear. In this study, we employed computational and experimental approaches to examine how the neural composition and selectivity of engrams change with memory consolidation. Our spiking neural network model yielded testable predictions: memories transition from unselective to selective as neurons drop out of and drop into engrams; inhibitory activity during recall is essential for memory selectivity; and inhibitory synaptic plasticity during memory consolidation is critical for engrams to become selective. Using activity-dependent labeling, longitudinal calcium imaging and a combination of optogenetic and chemogenetic manipulations in mouse dentate gyrus, we conducted contextual fear conditioning experiments that supported our model's predictions. Our results reveal that memory engrams are dynamic and that changes in engram composition mediated by inhibitory plasticity are crucial for the emergence of memory selectivity.

A machine learning approach for quantifying age-related histological changes in the mouse kidney.

The ability to quantify aging-related changes in histological samples is important, as it allows for evaluation of interventions intended to effect health span. We used a machine learning architecture that can be trained to detect and quantify these changes in the mouse kidney. Using additional held out data, we show validation of our model, correlation with scores given by pathologists using the Geropathology Research Network aging grading scheme, and its application in providing reproducible and quantifiable age scores for histological samples. Aging quantification also provides the insights into possible changes in image appearance that are independent of specific geropathology-specified lesions. Furthermore, we provide trained classifiers for H&E-stained slides, as well as tutorials on how to use these and how to create additional classifiers for other histological stains and tissues using our architecture. This architecture and combined resources allow for the high throughput quantification of mouse aging studies in general and specifically applicable to kidney tissues.

A machine learning approach for quantifying age-related histological changes in the mouse kidney.

The ability to quantify aging-related changes in histological samples is important, as it allows for evaluation of interventions intended to effect health span. We used a machine learning architecture that can be trained to detect and quantify these changes in the mouse kidney. Using additional held out data, we show validation of our model, correlation with scores given by pathologists using the Geropathology Research Network aging grading scheme, and its application in providing reproducible and quantifiable age scores for histological samples. Aging quantification also provides the insights into possible changes in image appearance that are independent of specific geropathology-specified lesions. Furthermore, we provide trained classifiers for H&E-stained slides, as well as tutorials on how to use these and how to create additional classifiers for other histological stains and tissues using our architecture.This architecture and combined resources allow for the high throughput quantification of mouse aging studies in general and specifically applicable to kidney tissues.

Pharmacist-Led Deprescribing for Patients With Polypharmacy and Chronic Disease States: A Retrospective Cohort Study.

Background: Current literature and practice have demonstrated that pharmacists have an integral role in deprescribing. However, research regarding their impact on patients with chronic diseases is limited. Objective: To assess the impact of a pharmacist-led intervention on deprescribing inappropriate medication for patients with chronic diseases within a four-month study period compared to patients receiving usual care. Methods: This study was conducted at NYU Langone Health. Patients of the intervention group were referred by a provider and met the criteria of polypharmacy, required chronic disease states management, were nonadherent to medications, had poor health literacy, or required titration for heart failure (HF) guideline directed medical therapy. Results: A total of 142 patients were reviewed over a two-year period. At the end of the study period, the median number of medications for the two respective groups was similar (11 [4 - 30] vs 11 [2 - 23]). The pharmacist-led intervention had on average one medication deprescribed (m = -1.00, sd = 2.57), whereas the control group had on average .44 additional medications (m = 0.44, sd = 3.32) prescribed. Furthermore, the intervention group presented statistically significant differences (P = 0.046) regarding their diastolic blood pressure after the pharmacists' intervention (m = 72.69, sd = 11.64). Most importantly, patients with HF presented statistically significant improvement in their ejection fractions after the intervention (m = 41.46%, sd = 19.28%). Conclusion: The pharmacist-led intervention resulted in significant discontinuation of medications for patients in the intervention group compared to those in the usual care group within four-months.

Adaptive Sentinel Testing in Workplace for COVID-19 Pandemic.

Testing and isolation of infectious employees is one of the critical strategies to make the workplace safe during the pandemic for many organizations. Adaptive testing frequency reduces cost while keeping the pandemic under control at the workplace. However, most models aimed at estimating test frequencies were structured for municipalities or large organizations such as university campuses of highly mobile individuals. By contrast, the workplace exhibits distinct characteristics: employee positivity rate may be different from the local community because of rigorous protective measures at workplace, or self-selection of co-workers with common behavioral tendencies for adherence to pandemic mitigation guidelines. Moreover, dual exposure to COVID-19 occurs at work and home that complicates transmission modeling, as does transmission tracing at the workplace. Hence, we developed bi-modal SEIR (Susceptible, Exposed, Infectious, and Removed) model and R-shiny tool that accounts for these differentiating factors to adaptively estimate the testing frequency for workplace. Our tool uses easily measurable parameters: community incidence rate, risks of acquiring infection from community and workplace, workforce size, and sensitivity of testing. Our model is best suited for moderate-sized organizations with low internal transmission rates, no-outward facing employees whose position demands frequent in-person interactions with the public, and low to medium population positivity rates. Simulations revealed that employee behavior in adherence to protective measures at work and in their community, and the onsite workforce size have large effects on testing frequency. Reducing workplace transmission rate through workplace mitigation protocols and higher sensitivity of the test deployed, although to a lesser extent. Furthermore, our simulations showed that sentinel testing leads to only marginal increase in the number of infections even for high community incidence rates, suggesting that this may be a cost-effective approach in future pandemics. We used our model to accurately guide testing regimen for three campuses of the Jackson Laboratory.

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

A Genomically and Clinically Annotated Patient-Derived Xenograft Resource for Preclinical Research in Non-Small Cell Lung Cancer.

Patient-derived xenograft (PDX) models are an effective preclinical in vivo platform for testing the efficacy of novel drugs and drug combinations for cancer therapeutics. Here we describe a repository of 79 genomically and clinically annotated lung cancer PDXs available from The Jackson Laboratory that have been extensively characterized for histopathologic features, mutational profiles, gene expression, and copy-number aberrations. Most of the PDXs are models of non-small cell lung cancer (NSCLC), including 37 lung adenocarcinoma (LUAD) and 33 lung squamous cell carcinoma (LUSC) models. Other lung cancer models in the repository include four small cell carcinomas, two large cell neuroendocrine carcinomas, two adenosquamous carcinomas, and one pleomorphic carcinoma. Models with both de novo and acquired resistance to targeted therapies with tyrosine kinase inhibitors are available in the collection. The genomic profiles of the LUAD and LUSC PDX models are consistent with those observed in patient tumors from The Cancer Genome Atlas and previously characterized gene expression-based molecular subtypes. Clinically relevant mutations identified in the original patient tumors were confirmed in engrafted PDX tumors. Treatment studies performed in a subset of the models recapitulated the responses expected on the basis of the observed genomic profiles. These models therefore serve as a valuable preclinical platform for translational cancer research. SIGNIFICANCE: Patient-derived xenografts of lung cancer retain key features observed in the originating patient tumors and show expected responses to treatment with standard-of-care agents, providing experimentally tractable and reproducible models for preclinical investigations.

Insulin Dosage Adjustments After Initiation of GLP-1 Receptor Agonists in Patients With Type 2 Diabetes.

BACKGROUND: Combination of insulin and GLP-1RAs have shown reductions in the HbA1c, body weight, and the risk of hypoglycemia. To date, there are conflicting data regarding the effect of GLP-1RAs on insulin dosage(s). OBJECTIVE: The objective of this study was to evaluate adjustments of insulin doses upon initiation of GLP-1RAs. METHODS: This was a retrospective chart review of patients on insulin therapy initiated on GLP-1RAs at NYU Langone Health. Patients were included in the study if they were at least 18 years of age, history of type 2 diabetes, and were on concurrent basal or mixed insulin therapy. 45 patients met inclusion criteria and were included in the study analysis. The primary endpoint was the median change in overall basal insulin doses. Secondary endpoints included median changes in total basal, mixed, and bolus insulin doses, oral antidiabetic medications and GLP-1RA doses, HbA1c, body weight, fasting glucose, and creatinine clearance. Safety results included any adverse reactions to insulin and/or GLP-1RA. RESULTS: In the per-protocol analysis, there was a significant reduction in overall total basal insulin doses from baseline to week 24 (50 units vs. 44 units, p < 0.05). There was a median reduction in patients receiving glargine (50 units vs. 44 units) and detemir (29 units vs. 21.5 units). CONCLUSIONS: Use of GLP-1RAs after 24 weeks resulted in a statistically significant reduction in overall total basal insulin dosages from baseline. The median HbA1C in our patient population was >8%. Consider a ≥10% reduction in the overall basal insulin dose upon initiation of GLP-1RA in patients with a HbA1C >8%.

High-throughput visual assessment of sleep stages in mice using machine learning.

STUDY OBJECTIVES: Sleep is an important biological process that is perturbed in numerous diseases, and assessment of its substages currently requires implantation of electrodes to carry out electroencephalogram/electromyogram (EEG/EMG) analysis. Although accurate, this method comes at a high cost of invasive surgery and experts trained to score EEG/EMG data. Here, we leverage modern computer vision methods to directly classify sleep substages from video data. This bypasses the need for surgery and expert scoring, provides a path to high-throughput studies of sleep in mice. METHODS: We collected synchronized high-resolution video and EEG/EMG data in 16 male C57BL/6J mice. We extracted features from the video that are time and frequency-based and used the human expert-scored EEG/EMG data to train a visual classifier. We investigated several classifiers and data augmentation methods. RESULTS: Our visual sleep classifier proved to be highly accurate in classifying wake, non-rapid eye movement sleep (NREM), and rapid eye movement sleep (REM) states, and achieves an overall accuracy of 0.92 ± 0.05 (mean ± SD). We discover and genetically validate video features that correlate with breathing rates, and show low and high variability in NREM and REM sleep, respectively. Finally, we apply our methods to noninvasively detect that sleep stage disturbances induced by amphetamine administration. CONCLUSIONS: We conclude that machine learning-based visual classification of sleep is a viable alternative to EEG/EMG based scoring. Our results will enable noninvasive high-throughput sleep studies and will greatly reduce the barrier to screening mutant mice for abnormalities in sleep.

The Role of Antisense Therapies Targeting Lipoprotein(a).

ABSTRACT: Atherosclerotic cardiovascular disease (ASCVD) continues to be the leading cause of preventable death in the United States. Elevated low-density lipoprotein cholesterol (LDL-C) is well known to result in cardiovascular disease. Mainstay therapy for reducing LDL-C and ASCVD risk is statin therapy. Despite achieving desired LDL-C levels with lipid-lowering therapy, cardiovascular residual risk often persists. Elevated lipoprotein(a) [Lp(a)] levels have been highlighted as an inherent independent predictor of ASCVD, and decreasing Lp(a) levels may result in a significant reduction in the residual risk in high-risk patients. To date, there are no approved medications to lower Lp(a) levels. Nicotinic acid, proprotein convertase subtilisin/kexin 9 inhibitors, and antisense oligonucleotide have demonstrated modest to potent Lp(a) reduction. Spotlight has been placed on antisense oligonucleotides and their role in Lp(a) lowering. APO(a)LRx is in the frontline for selectively decreasing Lp(a) concentrations and ongoing research may prove that this medication may lower Lp(a)-mediated residual risk, translating into cardiovascular benefit.

Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice.

Cardiac ischemia leads to the loss of myocardial tissue and the activation of a repair process that culminates in the formation of a scar whose structural characteristics dictate propensity to favorable healing or detrimental cardiac wall rupture. To elucidate the cellular processes underlying scar formation, here we perform unbiased single-cell mRNA sequencing of interstitial cells isolated from infarcted mouse hearts carrying a genetic tracer that labels epicardial-derived cells. Sixteen interstitial cell clusters are revealed, five of which were of epicardial origin. Focusing on stromal cells, we define 11 sub-clusters, including diverse cell states of epicardial- and endocardial-derived fibroblasts. Comparing transcript profiles from post-infarction hearts in C57BL/6J and 129S1/SvImJ inbred mice, which displays a marked divergence in the frequency of cardiac rupture, uncovers an early increase in activated myofibroblasts, enhanced collagen deposition, and persistent acute phase response in 129S1/SvImJ mouse hearts, defining a crucial time window of pathological remodeling that predicts disease outcome.

When soothing succeeds: Simulating a risk for repeated shaking in abusive head trauma in infants.

PURPOSE: To investigate the observation that perpetrators of abusive head trauma engage in repeated shakings because shaking "works" to quiet the infant. METHODS: Sixty first-time parent couples individually cared for a programmable model infant in two consecutive 7-min trials. After six minutes of consolable followed by inconsolable crying, parents selected one of three soothing techniques. For trial one, parents were randomized to a "Successful" or "Failed" Soothing Condition. Whether the soothing technique was repeated after trial two was determined by the study investigators. Parents rated their frustration after each trial. RESULTS: As hypothesized, parents were more likely to repeat a soothing technique that "worked" in trial one. Compared to fathers, mothers reported more frustration when soothing failed. CONCLUSIONS: That caregivers were more likely to repeat a successful soothing technique converges with perpetrator confessions that crying cessation after shaking may be a reason why shaking is used repeatedly in response to crying.

Prepartum and Postpartum Mothers' and Fathers' Unwanted, Intrusive Thoughts in Response to Infant Crying.

BACKGROUND: Unwanted intrusive thoughts of intentionally harming one's infant (intrusive harm thoughts) are common distressing experiences among postpartum mothers and fathers. AIM: To understand infant crying as a stimulus for intrusive harm thoughts and associated emotional responses in prepartum and postpartum mothers and fathers in response to infant cry. METHOD: Following completion of self-report measures of negative mood and anger, prepartum (n = 48) and postpartum (n = 44) samples of mother and father pairs completed 10 minutes of listening to audio-recorded infant crying. Post-test questionnaires assessed harm thoughts, negative emotions, urges to comfort and flee, and thoughts of shaking as a soothing or coping strategy. RESULTS: One quarter of prepartum and 44% of postpartum parents reported intrusive infant-related harm thoughts following crying. Mothers and fathers did not differ in the likelihood of reporting harm thoughts, nor in the number of thoughts reported. Women reported more internalizing emotions compared with men. Hostile emotions were stronger among postpartum parents, and parents reporting harm thoughts. All parents reported strong urges to comfort the infant. Urges to flee were stronger among parents who reported harm thoughts. The likelihood of using infant shaking as a soothing or coping strategy was minimally endorsed, albeit more strongly by fathers and parents who also reported harm thoughts. CONCLUSIONS: In response to crying, harm thoughts are common and are associated with hostile emotions, urges to flee, and increased thoughts of using infant shaking. Reassuringly, the number of participants considering infant shaking as a strategy for soothing or for coping with a crying infant was low.

Maternal frustration, emotional and behavioural responses to prolonged infant crying.

Prolonged inconsolable crying bouts in the first months of life are frustrating to parents and may lead to abuse. There is no empirical description of frustration trajectories during prolonged crying, nor of their emotional predictors or emotional and behavioural sequelae. Frustration responses and their relationships were explored in an analogue cry listening paradigm. Without knowing how long it would last, 111 postpartum mothers were randomized to listen to a 10-min audiotape of infant crying or cooing while continuously recording frustration on a visual analogue 'slider' scale. The listening bout was preceded by questionnaires on negative mood, trait anger and empathy and followed by questionnaires on the reality of the cry sound, positive and negative emotions, soothing strategies, coping strategies and urges to comfort and flee. Individual frustration trajectories were modelled parametrically and characterized by frustration maximum, rate of rise, inflections and harmonicity parameters. As hypothesized, the modal response was of gradually increasing frustration throughout. However, there were marked individual differences in frustration trajectories. Negative mood, trait anger and empathy did not predict modal or modelled individual trajectories. However, frustration responses were significantly related to post-listening emotions and behavioural ratings. In particular, prolonged crying generated highly ambivalent positive and negative emotional responses. In summary, maternal frustration generally increased as the crying bout progressed; however, frustration trajectories were highly individual and emotional responses were highly ambivalent in terms of positive and negative emotions generated. Some emotional and behavioural responses were associated with specific trajectory parameters of frustration responses.

Interparent childrearing disagreement, but not dissimilarity, predicts child problems after controlling for parenting effectiveness.

Parental differences regarding childrearing may be operationalized as actual dissimilarity in the parenting actions or goals of the parents, or as perceived conflict or disagreement related to these dissimilarities. This study tested whether these two types of parental differences are each associated with child problems, independent of the contributions of parenting effectiveness. A community sample of 160 couples with a firstborn child (ages 2-5 years) participated. Mothers and fathers independently completed measures of childrearing disagreement, parenting behaviors and goals, and child behavior. Interparent childrearing disagreement accounted for unique variance in child internalizing and externalizing problems, even after controlling for family income, marital satisfaction, and parenting effectiveness. Dissimilarity in mother and father parenting behaviors (but not goals) was associated with child problems at the bivariate level, but not after controlling for parenting effectiveness.

Mothers' and fathers' attributions for adolescent behavior: an examination in families of depressed, subdiagnostic, and nondepressed youth.

This study examined whether parents of adolescents experiencing depressive symptoms or disorder make more negative and fewer positive attributions for their adolescents' behavior than do parents of nondepressed adolescents, and whether parental attributions for adolescents' behavior contribute to parenting behavior, above and beyond the adolescents' behavior. Parents and adolescents (76 girls and 48 boys) participated in videotaped problem-solving interactions (PSIs). Each parent subsequently watched the videotape and offered attributions for their adolescent's behavior. In addition, parent and adolescent behavior during the PSIs was coded. Mothers and fathers in families of nondepressed adolescents made significantly fewer negative attributions for their children's behavior than did parents in families of adolescents with diagnostic or subdiagnostic levels of depressive symptoms. Moreover, mothers' and fathers' negative attributions were related to greater levels of observed aggressive behavior and lower levels of observed facilitative behavior during the PSIs controlling for both demographic characteristics and the relative level of adolescent aggressive and facilitative behavior during the PSI.

Parent and adolescent depressive symptoms: the role of parental attributions.

This study examined whether negative parental attributions for adolescent behaviour mediate the association between parental and adolescent depressive symptoms, and whether this relationship is moderated by adolescent gender. Mothers and fathers and 124 adolescents (76 girls and 48 boys; ages 14 to 18) participated. Adolescents were primarily Caucasian, and varied in the level of depressive symptoms (with 27% of the sample meeting diagnostic criteria for a current unipolar depressive disorder). Parents and adolescents completed measures of depressive symptoms, and participated in a videotaped problem-solving discussion. After the discussion, each parent watched the videotape and, at 20 s intervals, offered attributions for their adolescent's behaviour. Adolescent gender moderated the relation between parental attributions and adolescent depressive symptoms, with stronger associations for female adolescents. For both mothers and fathers, both parental depressive symptoms and negative attributions about the adolescent's behaviour made unique contributions to the prediction of depressive symptoms in adolescent females. There also was evidence that negative attributions partially mediated the link between depressive symptoms in mothers and adolescent daughters. The results are interpreted as consistent with parenting as a partial mediator between parental and adolescent depressive symptoms, and suggest that adolescent girls may be particularly sensitive to parents' negative interpretations of their behaviour.

Mothers' and fathers' attributions and beliefs in families of girls and boys with attention-deficit/hyperactivity disorder.

This study examined parent and child gender effects on parents' attributions and beliefs in regards to child symptoms of attention-deficit/hyperactivity disorder (ADHD). Participants included mothers and fathers of 19 girls and 17 boys with ADHD. Groups of boys and girls, aged 5-13 years, were equated on age and medication status, as well as ADHD symptom severity. These groups also were similar in the severity of comorbid oppositional behaviors and internalizing problems, as well as a variety of demographic characteristics. Parents' attributions for child behavior were assessed in response to written scenarios describing either hyperactive/impulsive or inattentive symptoms of ADHD. Parents also completed a questionnaire assessing beliefs and knowledge about ADHD. There were no child gender effects for parents' attributions or beliefs. All parents attributed inattentive symptoms to more internal, global and stable causes than impulsive symptoms. Mothers attributed both inattentive and impulsive child symptoms to more global and stable causes than did fathers. Fathers, but not mothers, reported more negative reactions to ADHD symptoms that were perceived as having an internal cause. Finally, mothers scored higher on beliefs in behavior management than did fathers, and fathers believed more in psychological causes and treatments for ADHD. Possible explanations for and implications of these results are explored.