A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

Directional change during active diffusion of viral ribonucleoprotein particles through cytoplasm.

A mesh of cytoskeletal fibers, consisting of microtubules, intermediate filaments, and fibrous actin, prevents the Brownian diffusion of particles with a diameter larger than 0.10 µm, such as vesicular stomatitis virus ribonucleoprotein (RNP) particles, in mammalian cells. Nevertheless, RNP particles do move in random directions but at a lower rate than Brownian diffusion, which is thermally driven. This nonthermal biological transport process is called "active diffusion" because it is driven by ATP. The ATP powers motor proteins such as myosin II. The motor proteins bend and cross-link actin fibers, causing the mesh to jiggle. Until recently, little was known about how RNP particles get through the mesh. It has been customary to analyze the tracks of particles like RNPs by computing the slope of the ensemble-averaged mean-squared displacement of the particles as a signature of mechanism. Although widely used, this approach "loses information" about the timing of the switches between physical mechanisms. It has been recently shown that machine learning composed of variational Bayesian analysis, Gaussian mixture models, and hidden Markov models can use "all the information" in a single track to reveal that that the positions of RNP particles are spatially clustered. Machine learning assigns a number, called a state, to each cluster. RNP particles remain in one state for 0.2-1.0 s before switching (hopping) to a different state. This earlier work is here extended to analyze the movements of a particle within a state and to determine particle directionality within and between states.

Mechanisms of active diffusion of vesicular stomatitis virus inclusion bodies and cellular early endosomes in the cytoplasm of mammalian cells.

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.

Reduced Plasma Levodopa Fluctuations with More Frequent Administration of a Novel Carbidopa/Levodopa Functionally Scored Tablet.

Levodopa/carbidopa remains the gold standard for treating Parkinson disease (PD), but chronic pulsatile administration contributes to motor complications. This Phase 1 study used a new immediate-release (IR) formulation of carbidopa/levodopa 25/100 mg that is functionally scored for easy and precise splitting to evaluate the effects on levodopa plasma variability when smaller doses are taken more frequently. These functionally scored tablets were shown to be bioequivalent to carbidopa/levodopa 25-/100-mg IR generic reference tablets. Twenty-two healthy volunteers received a whole tablet every 4 hours versus half of the tablet every 2 hours. Plasma levodopa fluctuations were significantly reduced with half-tablets dosed every 2 hours, with a 44% reduction in peaks (P < .0001). While drug exposure did not differ, parameters that underlie motor response variations, including mean peak-to-trough difference and variance, were 51% and 56% less, respectively, with more frequent dosing (both P ≤ .0024). Safety and tolerability of both regimens were similar. In conclusion, more frequent administration of half-tablets of the new functionally scored IR formulation safely provided more constant levodopa levels than whole tablets dosed less often. This tablet technology could facilitate the benefits of more physiologic dopamine replenishment in patients with PD, particularly those with reduced manual dexterity.

Therapeutic TNF Inhibitors Exhibit Differential Levels of Efficacy in Accelerating Cutaneous Wound Healing.

Adalimumab but neither etanercept nor certolizumab-pegol has been reported to induce a wound-healing profile in vitro by regulating macrophage differentiation and matrix metalloproteinase expression, which may underlie the differences in efficacy between various TNF-α inhibitors in impaired wound healing in patients with hidradenitis suppurativa, a chronic inflammatory skin disease. To examine and compare the efficacy of various TNF inhibitors in cutaneous wound healing in vivo, a human TNF knock-in Leprdb/db mouse model was established to model the impaired cutaneous wound healing as seen in hidradenitis suppurativa. The vehicle group exhibited severe impairments in cutaneous wound healing. In contrast, adalimumab significantly accelerated healing, confirmed by both histologic assessment and a unique healing transcriptional profile. Moreover, adalimumab and infliximab showed similar levels of efficacy, but golimumab was less effective, along with etanercept and certolizumab-pegol. In line with histologic assessments, proteomics analyses from healing wounds exposed to various TNF inhibitors revealed distinct and differential wound-healing signatures that may underlie the differential efficacy of these inhibitors in accelerating cutaneous wound healing. Taken together, these data revealed that TNF inhibitors exhibited differential levels of efficacy in accelerating cutaneous wound healing in the impaired wound-healing model in vivo.

GPAT1 Deficiency in Mice Modulates NASH Progression in a Model-Dependent Manner.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD), and its more severe form, nonalcoholic steatohepatitis (NASH), is the leading cause for liver failure and liver cancer. Although the etiology is likely multifactorial, genes involved in regulating lipid metabolism are enriched in human NAFLD genome-wide association studies (GWAS), pointing to dysregulated lipid metabolism as a major pathogenic factor. Glycerol-3-phosphate acyltransferase 1 (GPAT1), encoded by GPAM, converts acyl-CoAs and glycerol-3-phosphate into lysophosphatidic acid and has been shown to regulate lipid accumulation in the liver. However, its role in mediating the progression from NAFLD to NASH has not been explored. METHODS: GPAT1-deficient mice were generated and challenged with diets inducing hepatic steatosis and NASH. Effects of GPAT1 deficiency on lipid and systemic metabolic end points were evaluated. RESULTS: Ablating GPAT1 globally or specifically in mouse hepatocytes reduced hepatic steatosis in the context of diet-induced or genetic obesity. Interestingly, blunting of progression from NAFLD to NASH in global GPAT1 knockout (KO) mice was model dependent. GPAT1 KO mice were protected from choline deficient, amino acid defined high-fat diet-induced NASH development, but not from the high fat, high carbohydrate, and high cholesterol diet-induced NASH. CONCLUSIONS: Our preclinical data support the notion that lipid metabolism pathways regulated by GPAT1 in hepatocytes play an essential role in NASH progression, albeit in a model-dependent manner.

Partial or Complete Loss of Norepinephrine Differentially Alters Contextual Fear and Catecholamine Release Dynamics in Hippocampal CA1.

Background: Contextual fear learning is heavily dependent on the hippocampus. Despite evidence that catecholamines contribute to contextual encoding and memory retrieval, the precise temporal dynamics of their release in the hippocampus during behavior is unknown. In addition, new animal models are required to probe the effects of altered catecholamine synthesis on release dynamics and contextual learning. Methods: We generated 2 new mouse models of altered locus coeruleus-norepinephrine (NE) synthesis and utilized them together with GRABNE and GRABDA sensors and in vivo fiber photometry to investigate NE and dopamine (DA) release dynamics in the dorsal hippocampal CA1 during contextual fear conditioning. Results: Aversive foot shock increased both NE and DA release in the dorsal CA1, while freezing behavior associated with recall of fear memory was accompanied by decreased release. Moreover, we found that freezing at the recent time point was sensitive to both partial and complete loss of locus coeruleus-NE synthesis throughout prenatal and postnatal development, similar to previous observations of mice with global loss of NE synthesis beginning postnatally. In contrast, freezing at the remote time point was compromised only by complete loss of locus coeruleus-NE synthesis beginning prenatally. Conclusions: Overall, these findings provide novel insights into the role of NE in contextual fear and the precise temporal dynamics of both NE and DA during freezing behavior and highlight complex relationships between genotype, sex, and NE signaling.

Right BA 10 lesions impair performance on real-world planning but are not sensitive to problem novelty or tower tasks.

The prefrontal cortex (PFC) is associated with many cognitive functions, including planning. In the neuropsychology literature planning is reduced to "look ahead" ability and most extensively studied with the "tower" tasks. The most influential theoretical explanation is that planning is required in the absence of a routine solution and PFC patients have difficulty coping with novelty. There is an alternate view of planning that emphasizes the distinction between real world tasks and laboratory tower tasks. This account focuses on the structure of problem spaces and why patients with lesions to right PFC have difficulty navigating ill-structured problem spaces. To further explore these issues we administered two real world travel planning tasks to 56 Vietnam War veterans with penetrating brain lesions and 14 matched normal controls. One planning task involved familiar knowledge while the other involved knowledge unfamiliar to our participants. Participants also completed the D-KEFS tower task. A subset of 18 patients-with lesions to right anterior prefrontal cortex (BA 10)-were impaired in the travel planning task compared to normal controls. The task familiarity/novelty dimension affected performance across participant groups (familiar-task scores were higher than unfamiliar-task scores), but it did not differentially affect any group. An examination of cognitive strategies utilized by participants revealed that the impaired patient group had difficulty maintaining a sufficient level of abstraction and engaged the task at a much more concrete level than other participants. Interestingly, patients impaired in the real-world planning tasks were not impaired in the tower tasks. We conclude that patients with lesions to right BA 10 have difficulty in real-world planning tasks that can be attributed to difficulties in engaging problems at the appropriate level of abstraction.

Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

Targeting the NLRP3 inflammasome reduces inflammation in hidradenitis suppurativa skin.

BACKGROUND: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life. OBJECTIVES: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies. METHODS: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays. RESULTS: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1ß and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1ß and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation. CONCLUSIONS: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications.

The Prevalence of Second Victim Syndrome and Emotional Distress in Pediatric Intensive Care Providers.

Pediatric critical care providers are at higher risk of second victim syndrome (SVS) and emotional distress after a poor patient outcome, unanticipated adverse event, medical error, or patient-related injury. We sought to determine the prevalence of SVS within our intensive care units (ICUs) and evaluate the adequacy of current institutional peer support. A validated survey tool, the second victim experience and support tool was sent electronically to all ICU providers in our pediatric health care system. Of 950 recipients, there were 266 respondents (28%). Sixty-one per cent of respondents were nurses; 19% were attending physicians, advanced practice providers, and fellows; 88% were females; 42% were aged 25 to 34 years; and 43% had worked in the ICU for 0 to 5 years. The most common emotion experienced was psychological distress (42%) and one-third of respondents questioned their self-efficacy as a provider after a second victim event. Support from colleagues, supervisors, and the institution was perceived as low. Support from a respected peer was the most desired type of support by 81% of respondents. Emotional distress and SVS are commonly found among pediatric ICU providers and the level of support is perceived as inadequate. Developing and deploying a peer support program are crucial to staff's well-being and resilience in the high-stress ICU environment.

The TAS1R2 sweet taste receptor regulates skeletal muscle mass and fitness.

Muscle fitness and mass deteriorate under the conditions of obesity and aging for reasons yet to be fully elucidated. Herein, we describe a novel pathway linking peripheral nutrient sensing and skeletal muscle function through the sweet taste receptor TAS1R2 and the involvement of ERK2-PARP1-NAD signaling axis. Muscle-specific deletion of TAS1R2 (mKO) in mice produced elevated NAD levels due to suppressed PARP1 activity, improved mitochondrial function, increased muscle mass and strength, and prolonged running endurance. Deletion of TAS1R2 in obese or aged mice also ameliorated the decline in muscle mass and fitness arising from these conditions. Remarkably, partial loss-of-function of TAS1R2 (rs35874116) in older, obese humans recapitulated the healthier muscle phenotype displayed by mKO mice in response to exercise training. Our findings show that inhibition of the TAS1R2 signaling in skeletal muscle is a promising therapeutic approach to preserve muscle mass and function.

The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries.

Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ+ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ+ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues.

Structural basis of the acyl-transfer mechanism of human GPAT1.

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.

Influence of Geochemical Fractionation of Fulvic Acid on its Spectral Characteristics and its Protection Against Copper Toxicity to Daphnia magna.

Dissolved copper (Cu) can contribute to toxicity in aquatic systems impacted by acid mine drainage (AMD), and its bioavailability is influenced by aqueous complexation with organic ligands that predominantly include fulvic acids (FAs). Because the geochemical fractionation of FAs that accompanies sorption to hydrous aluminum oxides (HAOs) and hydrous iron oxides (HFOs) can alter Cu complexation with FA, we investigated FAs isolated from three categories of water (pristine, AMD, and in situ-fractionated mixtures of pristine and AMD collected at stream confluences) in three mining-impacted alpine watersheds in central Colorado, USA. We also conducted geochemical fractionation of field-collected FAs and Suwannee River FAs by precipitating HAOs and HFOs in the laboratory. Spectral properties of the FAs (e.g., UV-VIS absorbance) were altered by geochemical fractionation, and in acute toxicity tests with an aquatic invertebrate (Daphnia magna) Cu was more toxic in the presence of in situ- and laboratory-fractionated FAs (median effect concentration [EC50] 19-50 µg Cu L-1 ) than in the presence of nonfractionated FAs (EC50 48-146 µg Cu L-1 ). After adjusting for the strain-specific sensitivity of our D. magna, we improved the accuracy of Biotic Ligand Model predictions of Cu EC50 values for AMD-related FAs by using an "effective dissolved organic carbon" based on spectral properties that account for among-FA differences in protectiveness against Cu toxicity. However, some differences remained between predicted and measured EC50 values, especially for FAs from AMD-related waters that might contain important metal-binding moieties not accounted for by our measured spectral indices. Environ Toxicol Chem 2023;42:449-462. © 2022 SETAC.

Brain Vasculature Color-Labeling Using the Triple-Injection Method in Cadaveric Heads: A Technical Note for Improved Teaching and Research in Neurovascular Anatomy.

BACKGROUND: Color-labeling injections of cadaveric heads have revolutionized education and teaching of neurovascular anatomy. Silicone-based and latex-based coloring techniques are currently used, but limitations exist because of the viscosity of solutions used. OBJECTIVE: To describe a novel "triple-injection method" for cadaveric cranial vasculature and perform qualitative and semiquantitative evaluations of colored solution penetration into the vasculature. METHODS: After catheter preparation, vessel cannulation, and water irrigation of embalmed cadaveric heads, food coloring, gelatin, and silicone solutions were injected in sequential order into bilateral internal carotid and vertebral arteries (red-colored) and internal jugular veins (blue-colored). In total, 6 triple-injected embalmed cadaveric heads and 4 silicone-based "control" embalmed cadaveric heads were prepared. A qualitative analysis was performed to compare the vessel coloring of 6 triple-injected heads with that of 4 "control" heads. A semiquantitative evaluation was completed to appraise sizes of the smallest color-filled vessels. RESULTS: Naked-eye and microscope evaluations of embalmed experimental and control cadaveric heads revealed higher intensity and more distal color-labeling following the "triple-injection method" compared with the silicone-based method in both the intracranial and extracranial vasculature. Microscope assessment of 1-mm-thick coronal slices of triple-injected brains demonstrated color-filling of distal vessels with minimum diameters of 119 µm for triple-injected heads and 773 µm for silicone-based injected heads. CONCLUSION: Our "triple-injection method" showed superior color-filling of small-sized vessels as compared with the silicone-based injection method, resulting in more distal penetration of smaller caliber vessels.

The Use of Health Care and Community-Based Services by People Living With Dementia and Their Caregivers During the COVID-19 Pandemic.

INTRODUCTION: People living with dementia have been particularly affected by the COVID-19 pandemic. METHODS: A survey of dementia care professionals was conducted to assess the use of health care and community-based services by people living with dementia and their caregivers during the first year of the pandemic. RESULTS: The survey indicated that most services were no longer being used or were being used less during the pandemic, with a few key exceptions. DISCUSSION: Many barriers and few facilitators were identified to service use for people living with dementia and their caregivers. The results identify potential gaps in the dementia care service network and may inform efforts to improve dementia care during future large-scale public health emergencies in the state of Wisconsin and beyond.

Anesthetic Considerations for Cesarean Delivery in a Parturient With Severe Gitelman Syndrome.

Gitelman syndrome is an autosomal recessive inherited disorder that impairs the function of thiazide-sensitive sodium-chloride cotransporters in the distal convoluted tubule of the nephron. During labor and delivery, avoidance of sympathetic overactivity, meticulous hemodynamic monitoring, and expedited repletion of potassium and magnesium are required to avoid adverse outcomes. We present a parturient with severe Gitelman syndrome, requiring continuous electrolyte and fluid infusions, who underwent successful cesarean delivery. Potential severe morbidity was avoided with multidisciplinary planning and management.

Natural locus coeruleus dynamics during feeding.

Recent data demonstrate that noradrenergic neurons of the locus coeruleus (LC-NE) are required for fear-induced suppression of feeding, but the role of endogenous LC-NE activity in natural, homeostatic feeding remains unclear. Here, we found that LC-NE activity was suppressed during food consumption, and the magnitude of this neural response was attenuated as mice consumed more pellets throughout the session, suggesting that LC responses to food are modulated by satiety state. Visual-evoked LC-NE activity was also attenuated in sated mice, suggesting that satiety state modulates LC-NE encoding of multiple behavioral states. We also found that food intake could be attenuated by brief or longer durations of LC-NE activation. Last, we found that activation of the LC to the lateral hypothalamus pathway suppresses feeding and enhances avoidance and anxiety-like responding. Our findings suggest that LC-NE neurons modulate feeding by integrating both external cues (e.g., anxiogenic environmental cues) and internal drives (e.g., satiety).