Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis.

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain. Dysregulation of their development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 regional identity. Upon differentiation, hbNES cells transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain functional neurons expressing CGN markers GABAARα6 and vGLUT2. We show that sonic hedgehog promotes GABAergic lineage specification and CGN progenitor proliferation. Our work presents a new model with which to study development and diseases of the CGN lineage in a human context.

Identification of gene networks mediating regional resistance to tauopathy in late-onset Alzheimer's disease.

Neurofibrillary lesions composed of tau protein aggregates are defining hallmarks of Alzheimer's Disease. Despite tau filaments appearing to spread between networked brain regions in a prion-like manner, certain areas including cerebellum resist trans-synaptic spread of tauopathy and degeneration of their constituent neuronal cell bodies. To identify molecular correlates of resistance, we derived and implemented a ratio of ratios approach for disaggregating gene expression data on the basis of regional vulnerability to tauopathic neurodegeneration. When applied to vulnerable pre-frontal cortex as an internal reference for resistant cerebellum, the approach segregated adaptive changes in expression into two components. The first was enriched for neuron-derived transcripts associated with proteostasis including specific members of the molecular chaperone family and was unique to resistant cerebellum. When produced as purified proteins, each of the identified chaperones depressed aggregation of 2N4R tau in vitro at sub-stoichiometric concentrations, consistent with the expression polarity deduced from ratio of ratios testing. In contrast, the second component enriched for glia- and microglia-derived transcripts associated with neuroinflammation, segregating these pathways from susceptibility to tauopathy. These data support the utility of ratio of ratios testing for establishing the polarity of gene expression changes with respect to selective vulnerability. The approach has the potential to identify new targets for drug discovery predicated on their ability to promote resistance to disease in vulnerable neuron populations.

Impact of acute stress on murine metabolomics and metabolic flux.

Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.

Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

Dynamics of Chromatin Accessibility During Hematopoietic Stem Cell Differentiation Into Progressively Lineage-Committed Progeny.

Epigenetic mechanisms regulate the multilineage differentiation capacity of hematopoietic stem cells (HSCs) into a variety of blood and immune cells. Mapping the chromatin dynamics of functionally defined cell populations will shed mechanistic insight into 2 major, unanswered questions in stem cell biology: how does epigenetic identity contribute to a cell type's lineage potential, and how do cascades of chromatin remodeling dictate ensuing fate decisions? Our recent work revealed evidence of multilineage gene priming in HSCs, where open cis-regulatory elements (CREs) exclusively shared between HSCs and unipotent lineage cells were enriched for DNA binding motifs of known lineage-specific transcription factors. Oligopotent progenitor populations operating between the HSCs and unipotent cells play essential roles in effecting hematopoietic homeostasis. To test the hypothesis that selective HSC-primed lineage-specific CREs remain accessible throughout differentiation, we used ATAC-seq to map the temporal dynamics of chromatin remodeling during progenitor differentiation. We observed epigenetic-driven clustering of oligopotent and unipotent progenitors into distinct erythromyeloid and lymphoid branches, with multipotent HSCs and MPPs associating with the erythromyeloid lineage. We mapped the dynamics of lineage-primed CREs throughout hematopoiesis and identified both unique and shared CREs as potential lineage reinforcement mechanisms at fate branch points. Additionally, quantification of genome-wide peak count and size revealed overall greater chromatin accessibility in HSCs, allowing us to identify HSC-unique peaks as putative regulators of self-renewal and multilineage potential. Finally, CRISPRi-mediated targeting of ATACseq-identified putative CREs in HSCs allowed us to demonstrate the functional role of selective CREs in lineage-specific gene expression. These findings provide insight into the regulation of stem cell multipotency and lineage commitment throughout hematopoiesis and serve as a resource to test functional drivers of hematopoietic lineage fate.

An Investigation of Allyl-Substituted Bis(Diphosphine) Iron Complexes: Towards Precursors for Cooperative CO2 Activation.

In developing homogenous catalysts capable of CO2 activation, interaction with a metal center is often imperative. This work provides primary efforts towards the cooperative activation of CO2 using a Lewis acidic secondary coordination sphere (SCS) and iron via a paired theoretical/experimental approach. Specifically, this study reports efforts towards [Fe(diphosphine)2 (N2 )] as a CO2 -coordinated synthon where diphosphine=1,2-bis(di(3-cyclohexylboranyl)propylphosphino)ethane) (P2 BCy 4 ) or its precursor, 1,2-bis(diallylphosphino)ethane (tape). Initial efforts toward the {Fe(0)-N2 } complex were focused on deprotonation reactions of [Fe(diphosphine)2 (H)(NCCH3 )]+ and reduction of [Fe(tape)2 Cl2 ]. In the latter case, a mixture of intramolecularly π-bonded alkene and associated metallacyclic Fe(II)-H species were produced - heating this mixture provided the hydride as the major product. Notably, the interconversion of this pair counters that of related intermolecular reactions between [Fe(depe)2 ] (depe=1,2-bis(diethylphosphino)ethane) and ethylene, where hydride formation occurs subsequent to π-coordination; this has been probed by theoretical calculations. Finally, reactivity of the metallacyclic {Fe(II)-H} complex with CO2 was probed, resulting in a pair of isomeric ferra(II)lactones.

Depressive symptoms and HIV among a cohort of adolescent young men and transgender women who have sex with men, Bangkok and Nakhon Sawan, Thailand, 2017-2019.

Screening for depression may identify persons for HIV prevention services and to ensure linkage to care for ART and mental health. We assessed factors associated with depressive symptoms using multiple logistic regression among 15- to 29-year-old gay, bisexual or other men who have sex with men (MSM) and transgender women who have sex with men (TGW) attending HIV prevention clinics at Silom Community Clinic or Bangrak Hospital in Bangkok or Rainbow Clinic in Nakhon Sawan, Thailand. We defined depressive symptoms as a self-report of feelings of sadness that impacted daily life in the past one month. Among 192 MSM, 51 TGW, and 11 gender-questioning persons screened: 12.6% met the criteria for depression; 5.9% had new HIV diagnosis. Independently associated factors which increase the risk of depressive symptoms included: studying in a private school (AOR 7.17); experiencing any type of bullying (AOR 2.8); having a partner with HIV (AOR 4.1); and learning about the study from sources other than a friend (AOR 4.2). Given many youths had depressive symptoms, screening for depression and connection to mental health services would be beneficial in sexual health settings to meet the needs of HIV-vulnerable youth.

Cranial bone imaging using ultrashort echo-time bone-selective MRI as an alternative to gradient-echo based "black-bone" techniques.

OBJECTIVES: CT is the clinical standard for surgical planning of craniofacial abnormalities in pediatric patients. This study evaluated three MRI cranial bone imaging techniques for their strengths and limitations as a radiation-free alternative to CT. METHODS: Ten healthy adults were scanned at 3 T with three MRI sequences: dual-radiofrequency and dual-echo ultrashort echo time sequence (DURANDE), zero echo time (ZTE), and gradient-echo (GRE). DURANDE bright-bone images were generated by exploiting bone signal intensity dependence on RF pulse duration and echo time, while ZTE bright-bone images were obtained via logarithmic inversion. Three skull segmentations were derived, and the overlap of the binary masks was quantified using dice similarity coefficient. Craniometric distances were measured, and their agreement was quantified. RESULTS: There was good overlap of the three masks and excellent agreement among craniometric distances. DURANDE and ZTE showed superior air-bone contrast (i.e., sinuses) and soft-tissue suppression compared to GRE. DISCUSSIONS: ZTE has low levels of acoustic noise, however, ZTE images had lower contrast near facial bones (e.g., zygomatic) and require effective bias-field correction to separate bone from air and soft-tissue. DURANDE utilizes a dual-echo subtraction post-processing approach to yield bone-specific images, but the sequence is not currently manufacturer-supported and requires scanner-specific gradient-delay corrections.

Long-term photogrammetric outcomes of midface advancement in Apert syndrome: are we nearing normal?

BACKGROUND: The aesthetic goals of midface surgery in Apert syndrome are to correct the multi-planar midface deficiency and normalize facial ratios. This study characterizes the long-term photogrammetric outcomes of midface advancement in Apert syndrome. METHODS: Patients with Apert syndrome who underwent midface distraction from 2000 to 2023 were retrospectively reviewed. Soft tissue measurements were applied to preoperative (T0), short-term postoperative (T1), and long-term postoperative (T2) photographs. Long-term changes were compared between subcranial and transcranial procedures, segmental and non-segmental osteotomies, and individual techniques. Frontal facial dimensions at time T2 were compared to age- and sex-matched normal controls from the NIMH-ChEFS face database. RESULTS: Twenty patients had postoperative follow-up of T1 = 0.6 ± 0.4 and T2 = 4.7 ± 1.1 years and were compared to 40 normal controls. From time T0 to T2, middle facial third height increased (26.1 ± 3.0% to 27.8 ± 2.6%, p = 0.026), canthal tilt improved (- 7.6° ± 3.7° to - 3.9° ± 4.4°, p < 0.001), and facial convexity increased (182.9° ± 6.2° to 167.9° ± 8.6°, p < 0.001). From time T1 to T2, facial convexity flattened (159.4° ± 10.1° to 167.9° ± 8.6°, p < 0.001). Compared to controls, patients at time T2 had shorter middle facial third height (27.8 ± 2.6% vs. 32.0 ± 1.9%, p < 0.001) and reverse canthal tilt (- 3.9° ± 4.4° vs. 5.4° ± 2.6°, p < 0.001). CONCLUSIONS: A tailored treatment approach to the Apert midface yields varying degrees of resolution of central midfacial shortening, canthal tilt, and facial concavity at 5 years postoperatively. A slight reduction in facial convexity over time, likely reflecting pseudorelapse in the setting of sagittal overcorrection, should be anticipated. Greater utilization of segmental osteotomies may bring facial third ratios and canthal tilt closer to normal morphometric values.

The human cytomegalovirus protein pUL13 targets mitochondrial cristae architecture to increase cellular respiration during infection.

Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.

Severity of Mandibular Dysmorphology in Treacher Collins Syndrome for Stratification of Perioperative Airway Risk.

BACKGROUND: Patients with Treacher Collins syndrome (TCS) and attendant airway dysmorphology may be predisposed to airway complications in the perioperative period. However, limited data correlates severity of mandibular hypoplasia and airway status. This study aims to improve risk stratification for perioperative airway insufficiency in TCS by using a previously proposed mandibular severity index. METHODS: Patient demographics, perioperative airway status, difficulty of intubation, and Cormack Lehane grade were collected and compared using a TCS mandibular hypoplasia severity grading scale in patients with TCS treated between 2000 and 2022. RESULTS: Twenty-six patients underwent 222 procedures with institutional mandibular severity gradings as follows: 23% Grade I, 31% Grade II, 39% Grade III, 8% Grade IV. Our severity index was associated with intubation difficulty ( P <0.001) and difficult airway status ( P <0.001), with 72% of difficult airways found in grade III and grade IV patients. Mandibular retrusion and ramal hypoplasia subscores were positively correlated with measures of airway severity ( P <0.001), whereas the gonial angle was negatively correlated ( P <0.001). Age was negatively correlated with difficult visualization for endotracheal intubation ( P =0.02) but had no association with difficult airway status ( P =0.2). CONCLUSIONS: This study found a positive correlation between severity of maxillomandibular dysmorphology and perioperative airway difficulty in TCS patients. Our findings suggest that severely affected patients require heightened vigilance throughout life, as difficult airways may not completely resolve with aging. Given the risk of morbidity and mortality associated with airway complications, proper identification and preparation for challenging airways is critical for TCS patients.

Changes in Ventricular Volume After Posterior Vault Distraction Osteogenesis in Patients With Syndromic and Nonsyndromic Craniosynostosis.

OBJECTIVE: Little is known about the response of the ventricular system to cranial vault surgery in patients with craniosynostosis. This study aims to evaluate the changes in the cerebral ventricular system in response to posterior vault distraction osteogenesis (PVDO) in patients with syndromic and nonsyndromic craniosynostosis. METHODS: A single-institution retrospective review of all patients with craniosynostosis undergoing PVDO from 2000 to 2022 was completed. Patients were included for analysis if they had pre and postoperative cranial computed tomography scans. Ventricular volume (VV) and intracranial volume (ICV) were calculated using segmentation software. RESULTS: Both patients with syndromic synostosis and nonsyndromic synostosis (NSS) experienced a significant increase in ICV after PVDO, but only patients with NSS experienced a significant VV change (P = 0.004). After normalization by ICV, total, lateral, and third VV changes retained significance with percentage increases of 114%, 117%, and 89%, respectively (P < 0.05 for all). CONCLUSION: The differing results between cohorts reinforce the concept that the intracranial milieu is different between patients with syndromic synostosis and NSS. The results of the NSS cohort suggest that these patients may exist in a compensated state in which a reduction in cerebral blood flow and VV allows for the maintenance of parenchymal health to prevent the development of intracranial hypertension. Further studies may explore VV as a surrogate marker of ICP elevation, and the utility of cranial vault remodeling on nonsynostotic pathologies with cephalocranial disproportion.

LeFort III Versus Monobloc Frontofacial Advancement: A Comparative Analysis of Soft Tissue Changes.

INTRODUCTION: The LeFort III and monobloc are commonly used midface advancement procedures for patients with syndromic craniosynostosis with well characterized postoperative skeletal changes. However, the differential effects of these procedures on facial soft tissues are less understood. The purpose of this study was to critically analyze and compare the effects of these 2 procedures on the overlying soft tissues of the face. METHODS: Frontal and lateral preoperative and postoperative photographs of patients undergoing monobloc or LeFort III were retrospectively analyzed using ImageJ to measure soft tissue landmarks. Measurements included height of facial thirds, nasal length and width, intercanthal distance, and palpebral fissure height and width. Facial convexity was quantified by calculating the angle between sellion (radix), subnasale, and pogonion on lateral photographs. RESULTS: Twenty-five patients with an average age of 6.7 years (range 4.8-14.5) undergoing monobloc (n=12) and LeFort III (n=13) were identified retrospectively and analyzed preoperatively and 6.4±3.6 months postoperatively. Patients undergoing LeFort III had a greater average postoperative increase in facial convexity angle acuity (28.2°) than patients undergoing monobloc (17.8°, P =0.021). Patients in both groups experience postoperative increases in nasal width ( P <0.001) and decreases in palpebral fissure height ( P <0.001). CONCLUSIONS: Both subcranial LeFort III advancements and monobloc frontofacial advancements resulted in significant changes in the soft tissues. Patients undergoing LeFort III procedures achieved greater acuity of the facial convexity angle, likely because the nasion is not advanced with the LeFort III segment.

Sociodemographic Factors Predict Long-Term Attrition from Multidisciplinary Cleft Clinic.

OBJECTIVE: Identify and describe factors associated with retention and attrition of patients during longitudinal follow-up at multidisciplinary cleft clinic. DESIGN: Retrospective cohort study. SETTING: Single, tertiary care center. PATIENTS, PARTICIPANTS: Patients born between 1995 and 2007 with a diagnosis of cleft palate with or without cleft lip attending multidisciplinary cleft clinic. INTERVENTIONS: None tested, observational study. MAIN OUTCOME MEASURE(S): Age at last clinical appointment with a multidisciplinary cleft team provider. Attrition was defined as absence of an outpatient appointment following 15 years of age. RESULTS: Six hundred seventy-eight patients were included. The average age at last appointment across the entire cohort was 13.1 years (IQR 6.6-17.2). Patients who were Black (HR 1.60, 95% CI 1.10-2.32, p = 0.014) and other races (HR 1.90, 95% CI 1.22-2.98, p = 0.004) were more likely to be lost to follow-up compared to white patients. Publicly insured patients were more likely to experience attrition than those who were privately insured (HR 1.30, 95% CI 1.03-1.65, p = 0.030). Estimated income was not significantly associated with length of follow-up (p = 0.259). Those whose residence was in the fourth quartile of driving distance from our center experienced loss to follow-up significantly more than those who lived the closest (HR 2.04, 95% CI 1.50-2.78, p < 0.001). CONCLUSIONS: There is a high degree of follow-up attrition among patients with cleft lip and palate. Race, insurance status, and driving distance to our center were associated with attrition in a large, retrospective cohort of patients who have reached the age of cleft clinic graduation.

Incomes to Outcomes: A Global Assessment of Disparities in Cleft and Craniofacial Treatment.

OBJECTIVE: Recent investigations focused on health equity have enumerated widespread disparities in cleft and craniofacial care. This review introduces a structured framework to aggregate findings and direct future research. DESIGN: Systematic review was performed to identify studies assessing health disparities based on race/ethnicity, payor type, income, geography, and education in cleft and craniofacial surgery in high-income countries (HICs) and low/middle-income countries (LMICs). Case reports and systematic reviews were excluded. Meta-analysis was conducted using fixed-effect models for disparities described in three or more studies. SETTING: N/A. PATIENTS: Patients with cleft lip/palate, craniosynostosis, craniofacial syndromes, and craniofacial trauma. INTERVENTIONS: N/A. RESULTS: One hundred forty-seven articles were included (80% cleft, 20% craniofacial; 48% HIC-based). Studies in HICs predominantly described disparities (77%,) and in LMICs focused on reducing disparities (42%). Level II-IV evidence replicated delays in cleft repair, alveolar bone grafting, and cranial vault remodeling for non-White and publicly insured patients in HICs (Grades A-B). Grade B-D evidence from LMICs suggested efficacy of community-based speech therapy and remote patient navigation programs. Meta-analysis demonstrated that Black patients underwent craniosynostosis surgery 2.8 months later than White patients (P < .001) and were less likely to undergo minimally-invasive surgery (OR 0.36, P = .002). CONCLUSIONS: Delays in cleft and craniofacial surgical treatment are consistently identified with high-level evidence among non-White and publicly-insured families in HICs. Multiple tactics to facilitate patient access and adapt multi-disciplinary case in austere settings are reported from LMICs. Future efforts including those sharing tactics among HICs and LMICs hold promise to help mitigate barriers to care.

Psychosocial Status and Self-Perception in Patients with Cleft Lip and/or Palate.

OBJECTIVE: To describe how the psychosocial status of patients with cleft lip and/or palate (CL/P) relates to patient-reported outcomes (PROs). DESIGN: Cross-sectional retrospective chart review. SETTING: Tertiary care pediatric hospital. PATIENTS/PARTICIPANTS: Patients aged 8 to 29 years attending cleft team evaluations during a 1-year period. MAIN OUTCOME MEASURES: CLEFT-Q. RESULTS: Patients (N = 158) with isolated or syndromic CL/P and mean age 13.4 ± 3.0 years were included. Fifteen (9%) patients had siblings who also had CL/P. Of 104 patients who met with the team psychologist, psychosocial concerns were identified in 49 (47%) patients, including 25 (24%) with Attention-Deficit/Hyperactivity Disorder or behavior concerns, 28 (27%) with anxiety, and 14 (13%) with depression or mood concerns. Younger age and having siblings with cleft were associated with better PROs, while psychosocial concerns were associated with worse PROs on Speech, Psychosocial, and Face Appearance scales. CONCLUSIONS: Patient perception of cleft outcomes is linked to psychosocial factors.

Nutrition practices in Australia and New Zealand in response to evolving evidence: Results of three point-prevalence audits.

BACKGROUND: The Augmented versus Routine Approach to Giving Energy Trial (TARGET) was a 4000-patient trial in which augmented enteral calorie dose did not influence outcomes. AIM: We aimed to quantify practice change following TARGET. METHODS: Three single-day, prospective, multicentre, point-prevalence audits of adult patients receiving enteral nutrition (EN) in participating Australian and New Zealand intensive care units at 10:00 AM were conducted: (i) 2010 (before conducting TARGET); (ii) 2018 (immediately before publishing TARGET results); and (iii) 2020 (2 years after TARGET publication). Data included baseline characteristics, clinical outcomes, and nutrition data. Data are n (%), mean ± standard deviation, or median [interquartile range]. Differences in enteral calorie prescription between 2018 and 2020 were compared using the Mann-Whitney test. RESULTS: The percentage of patients receiving EN (2010 42%, 2018 38%, 2020 33%; P = 0.012) and the prescription of calorie-dense EN formula (≥1.5 kcal/ml) (2010 33%, 2018 24%, 2020 23%; P = 0.038) decreased over time. However, when comparing prepublication and postpublication (2018-2020), calorie dose and calorie density were similar: 22.9 ± 8.6 versus 23.4 ± 12.8 kcal/kg/day (P = 0.816) and <1.5 kcal/ml: 76 versus 77% (P = 0.650), respectively. CONCLUSION: In Australian and New Zealand intensive care units, enteral calorie dose and calorie density of prescribed EN were similar before TARGET publication and 2 years later.

Genetic Variation in Reproductive Investment Across an Ephemerality Gradient in Daphnia pulex.

Species across the tree of life can switch between asexual and sexual reproduction. In facultatively sexual species, the ability to switch between reproductive modes is often environmentally dependent and subject to local adaptation. However, the ecological and evolutionary factors that influence the maintenance and turnover of polymorphism associated with facultative sex remain unclear. We studied the ecological and evolutionary dynamics of reproductive investment in the facultatively sexual model species, Daphnia pulex. We found that patterns of clonal diversity, but not genetic diversity varied among ponds consistent with the predicted relationship between ephemerality and clonal structure. Reconstruction of a multi-year pedigree demonstrated the coexistence of clones that differ in their investment into male production. Mapping of quantitative variation in male production using lab-generated and field-collected individuals identified multiple putative quantitative trait loci (QTL) underlying this trait, and we identified a plausible candidate gene. The evolutionary history of these QTL suggests that they are relatively young, and male limitation in this system is a rapidly evolving trait. Our work highlights the dynamic nature of the genetic structure and composition of facultative sex across space and time and suggests that quantitative genetic variation in reproductive strategy can undergo rapid evolutionary turnover.

Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.