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Attention-deficit/hyperactivity disorder (ADHD) is a complex behavioral disorder characterized by hyperactivity, impulsivity, inattention, and deficits in working memory and time perception. While animal models have advanced our neurobiological understanding of this condition, there are limited and inconsistent data on working and elapsed time memory function. Inflammatory signaling has been identified as a key factor in memory and cognitive impairments, but its role in ADHD remains unclear. Additionally, the disproportionate investigation of male subjects in ADHD research has contributed to a poor understanding of the disorder in females. This study sought to investigate the potential connections between memory, neuroimmunology, and ADHD in both male and female animals. Specifically, we utilized the spontaneously hypertensive rat (SHR), one of the most extensively studied animal models of ADHD. Compared to their control, the Wistar-Kyoto (WKY) rat, male SHR are reported to exhibit several behavioral phenotypes associated with ADHD, including hyperactivity, impulsivity, and poor sustained attention, along with impairments in learning and memory. As the hippocampus is a key brain region for learning and memory, we examined the behavior of male and female SHR and WKY rats in two hippocampal-dependent memory tasks. Our findings revealed that SHR have delay-dependent working memory deficits that were similar to, albeit less severe than, those seen in hippocampal-lesioned rats. We also observed impairments in elapsed time processing in female SHR, particularly in the discrimination of longer time durations. To investigate the impact of inflammatory signaling on memory in these rats, we analyzed the levels of several cytokines in the dorsal and ventral hippocampus of SHR and WKY. Although we found some sex and genotype differences, concentrations were generally similar between groups. Taken together, our results indicate that SHR exhibit deficits in spatial working memory and memory for elapsed time, as well as some differences in hippocampal cytokine concentrations. These findings contribute to a better understanding of the neurobiological basis of ADHD in both sexes and may inform future research aimed at developing effective treatments for the disorder. Nonetheless, the potential mediating role of neuroinflammation in the memory symptomatology of SHR requires further investigation.

Summary: Polygenic risk scores (PRSs) developed from multi-ancestry genome-wide association studies (GWASs), PRSmulti, hold promise for improving PRS accuracy and generalizability across populations. To establish best practices for leveraging the increasing diversity of genomic studies, we investigated how various factors affect the performance of PRSmulti compared with PRSs constructed from single-ancestry GWASs (PRSsingle). Through extensive simulations and empirical analyses, we showed that PRSmulti overall outperformed PRSsingle in understudied populations, except when the understudied population represented a small proportion of the multi-ancestry GWAS. Furthermore, integrating PRSs based on local ancestry-informed GWASs and large-scale, European-based PRSs improved predictive performance in understudied African populations, especially for less polygenic traits with large-effect ancestry-enriched variants. Our work highlights the importance of diversifying genomic studies to achieve equitable PRS performance across ancestral populations and provides guidance for developing PRSs from multiple studies.

Summary: Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease encompassing several distinct molecular subtypes and clinical entities. Despite the initial success of surgical debulking and adjuvant chemotherapy, recurrence with chemotherapy resistant tumors is common in patients with EOC and leads to poor overall survival. The extensive genetic and phenotypic heterogeneity associated with ovarian cancers has hindered the identification of effective prognostic and predictive biomarkers in EOC patients. In the current studies, we identify a tumor cell surface oncoantigen, chondroitin sulfate proteoglycan 4 (CSPG4), as an independent risk factor for decreased survival of patients with EOC. Our results show that CSPG4 promotes EOC cell invasion, cisplatin resistance and spheroid formation in vitro and tumor expansion in vivo. Mechanistically, spheroid formation and tumor cell invasion are due to CSPG4-stimulated expression of the mesenchymal transcription factor ZEB1. Furthermore, we have developed a novel monoclonal anti-CSGP4 antibody against the juxtamembrane domain of the core protein that limits CSPG4-stimulated ZEB1 expression, tumor cell invasion and promotes EOC apoptosis within spheroid cultures. We therefore propose that CSPG4 expression drives phenotypic heterogeneity and malignant progression in EOC tumors. These studies further demonstrate that CSPG4 expression levels are a potential diagnostic biomarker in EOC and indicate that targeting cells which express this oncoantigen could limit recurrence and improve outcomes in patients with EOC.

Abstract Adenoviral‐vector based vaccines for coronavirus disease 2019 (COVID‐19) have been linked with a thrombotic syndrome, vaccine‐induced thrombotic thrombocytopenia (VITT). A key clinical question is whether VITT can be reliably ruled out by the absence of thrombocytopenia. We report on three patients who presented to our institute with this syndrome. Noteworthy in our presentations are two patients who presented for medical assessment of thrombotic symptoms with a normal platelet count, one preceding and one following a period of thrombocytopenia. Prompt diagnosis of VITT is critical to prevent rapid patient decline. We provide herein a new diagnostic algorithm that we believe will help optimally capture case presentations of VITT. These cases broaden and refine our understanding of the disease process and highlight to practitioners that VITT cannot be adequately ruled out by thrombocytopenia alone.

Background: A broad range of interventions have been implemented to improve ambient air quality, and many of these have been evaluated. Yet to date no systematic review has been conducted to identify and synthesize these studies. In this systematic review, we assess the effectiveness of interventions in reducing ambient particulate matter air pollution and improving adverse health outcomes. Methods: We searched a range of electronic databases across multiple disciplines, as well as grey literature databases, trial registries, reference lists of included studies and the contents of relevant journals, through August 2016. Eligible for inclusion were randomized and cluster randomized controlled trials, as well as several non-randomized study designs often used for evaluating air quality interventions. We included studies that evaluated interventions targeting industrial, residential, vehicular and multiple sources, with respect to their effect on mortality, morbidity and the concentrations of particulate matter (PM – including PM10, PM2.5, coarse particulate matter and combustion-related PM), as well as several criteria pollutants, including ozone, carbon monoxide, nitrogen oxides, nitrogen dioxide, nitric oxide and sulphur dioxide. We did not restrict studies based on the population, setting or comparison.Two authors independently assessed studies for inclusion, extracted data and assessed risk of bias. We assessed risk of bias using the Graphic Appraisal Tool for Epidemiological studies (GATE) for correlation studies, as modified and employed by the UK National Institute for Health and Care Excellence. We synthesized evidence narratively, as well as graphically using harvest plots. We assessed the certainty of evidence using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Results: We included 42 studies assessing 38 unique interventions. These comprised a heterogeneous mix of interventions, including those aiming to address industrial sources (n = 5; e.g. the closure of a factory), residential sources (n = 7; e.g. coal ban), vehicular sources (n = 22; e.g. low emission zones), and multiple sources (n = 4; e.g. tailored measures that target both local traffic and industrial polluters).Evidence for effectiveness was mixed. Most included studies observed either no significant association or an association favoring the intervention, with little evidence that the assessed interventions might be harmful. Conclusions: Given the heterogeneity across interventions, outcomes, and methods, it was difficult to derive overall conclusions regarding the effectiveness of interventions in terms of improved air quality or health. Some evidence suggests that interventions are associated with improvements in air quality and human health, with very little evidence suggesting interventions were harmful. The evidence base highlights the challenges related to establishing the effectiveness of specific air pollution interventions on outcomes. It also points to the need for improved study design and analysis methods, as well as more uniform evaluations. The prospective planning of evaluations and an evaluation component built into the design and implementation of interventions may also be particularly beneficial.

The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-β (IFN-β) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b+ LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8+ T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin.

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

This report describes the successful implantation of a LV lead using balloon venoplasty to overcome a very tight stenosis of the right subclavian vein / brachiocephalic junction for cardiac resynchronisation therapy (CRT-P) in a patient with a right sided CRT-P system and a failed epicardial LV lead. It is important for device implanters to be familiar with interventional equipments and techniques such as balloon venoplasty to overcome difficult venous access.

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED50 values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2−/− and npc1−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.

A mutation in NPC1 leads to sequestration of unesterified cholesterol in the late endosomal/lysosomal compartment of every cell culminating in the development of pulmonary, hepatic, and neurodegenerative disease. Acute administration of 2-hydroxypropyl-β-cyclodextrin (CYCLO) rapidly overcomes this transport defect in both the 7-day-old pup and 49-day-old mature npc1−/− mouse, even though this compound is cleared from the body and plasma six times faster in the mature mouse than in the neonatal animal. The liberated cholesterol flows into the cytosolic ester pool, suppresses sterol synthesis, down-regulates SREBP2 and its target genes, and reduces expression of macrophage-associated inflammatory genes. These effects are seen in the liver and brain, as well as in peripheral organs like the spleen and kidney. Only the lung appears to be resistant to these effects. Forty-eight h after CYCLO administration to the 49-day-old animals, fecal acidic, but not neutral, sterol output increases, whole-animal cholesterol burden is reduced, and the hepatic and neurological inflammation is ameliorated. However, lifespan is extended only when the CYCLO is administered to the 7-day-old animals. These studies demonstrate that CYCLO administration acutely reverses the cholesterol transport defect seen in the NPC1 mouse at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid.

This study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1+/+ and abca1−/− mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1−/− mice. In the abca1+/+ animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1−/− mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1−/− mice (198 mg/day/kg) as in the abac1+/+ animals (163 mg/day/kg), although the abac1−/− mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion.

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.

Niemann-Pick type C (NPC) disease is a multisystem disorder caused primarily by a mutation in the npc1 gene. These studies evaluated the effect of genetic background, deletion of additional genes, and administration of several agents on the age at death in a murine model of this disorder. Such factors as differing strain background or genetic drift within a given background in the npc1−/− mouse significantly altered the age at death and the degree of organ disease. Genetic deletion of Siat9 (GM3 synthetase) or Nr1h2 [liver X receptor (LXR)β] shortened the life of the npc1−/− animals. Daily treatment of the npc1−/− mice with an LXR agonist or administration of a single dose of cyclodextrin, with or without the neurosteroid allopregnanolone, significantly slowed neurodegeneration and increased the lifespan of these animals. These data illustrate that the age at death of the npc1−/− mouse can be significantly influenced by many factors, including differences in strain background, other inactivating gene mutations (Siat9 and lxrβ), and administration of agents such as LXR agonists and, particularly, cyclodextrin. It is currently not clear which of these effects is nonspecific or which might relate directly to the molecular defect present in the NPC1 syndrome.

These studies explored the roles of receptor-mediated and bulk-phase endocytosis as well as macrophage infiltration in the accumulation of cholesterol in the mouse with Niemann-Pick type C (NPC) disease. Uptake of LDL-cholesterol varied from 514 μg/day in the liver to zero in the central nervous system. In animals lacking LDL receptors, liver uptake remained about the same (411 μg/day), but more cholesterol was taken up in extrahepatic organs. This uptake was unaffected by the reductive methylation of LDL and consistent with bulk-phase endocytosis. All tissues accumulated cholesterol in mice lacking NPC1 function, but this accumulation was decreased in adrenal, unchanged in liver, and increased in organs like spleen and lung when LDL receptor function was also deleted. Over 56 days, the spleen and lung accumulated amounts of cholesterol greater than predicted, and these organs were heavily infiltrated with macrophages. This accumulation of both cholesterol and macrophages was increased by deleting LDL receptor function. These observations indicate that both receptor-mediated and bulk-phase endocytosis of lipoproteins, as well as macrophage infiltration, contribute to the cholesterol accumulation seen in NPC disease. These macrophages may also play a role in parenchymal cell death in this syndrome.

Niemann-Pick type C (NPC) disease is a multisystem disorder resulting from mutations in the NPC1 gene that encodes a protein involved in intracellular cholesterol trafficking. Significant liver dysfunction is frequently seen in patients with this disease. The current studies used npc1 mutant mice to investigate the association between liver dysfunction and unesterified cholesterol accumulation, a hallmark of NPC disease. Data from 92 npc1−/− mice (age range, 9–56 days) revealed a significant positive correlation between the plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and whole liver cholesterol content. In 56 day old npc1−/− mice that had been fed from 35 days of age a rodent diet or the same diet containing either cholesterol (1.0%, w/w) or ezetimibe (a sterol absorption inhibitor; 0.0125%, w/w), whole liver cholesterol content averaged 33.5 ± 1.1, 87.9 ± 1.7, and 20.8 ± 0.9 mg, respectively. Again, plasma ALT and AST activities were positively correlated with hepatic cholesterol content. In contrast, plasma transaminase levels remained in the normal range in npc1+/+ mice, in which hepatic esterified cholesterol content had been increased by 72-fold by feeding a high-cholesterol, high-fat diet. These studies suggest that the late endosomal/lysosomal content of unesterified cholesterol correlates with cell damage in NPC disease.

Mutational inactivation of NPC1 largely blocks the movement of LDL-derived cholesterol from the lysosome to the metabolically active, cytosolic pool of sterol that is the substrate for steroid hormone production. Such a block might, in theory, lead to deficiencies in circulating levels of testosterone, progesterone, and corticosterone. However, there are at least two other sources for cellular cholesterol, de novo synthesis and scavenger receptor class B type I-mediated uptake of HDL cholesteryl ester (CE). In this study, we measured the rates of net cholesterol acquisition by these three pathways in the adrenal, ovary, and testis. In all three organs, the majority (81–98%) of cholesterol acquisition came from the selective uptake of CE from HDL and de novo synthesis. Furthermore, in the npc1−/−mouse, the cytosolic storage pool of CE in a tissue such as the adrenal remained constant (∼25 mg/g). As a result of these alternative pathways, the plasma concentrations of testosterone (3.5 vs. 2.5 ng/ml), progesterone (8.5 vs. 6.7 ng/ml), and corticosterone (391 vs. 134 ng/ml) were either the same or elevated in the npc1−/−mouse, compared with the control animal. Thus, impairment of cholesterol acquisition through the NPC1-dependent, clathrin-coated pit pathway did not limit the availability of cholesterol substrate for steroid hormone synthesis in the steroidogenic cells.

The Niemann-Pick C1 (NPC1) protein functions to regulate the transport of cholesterol from late endosomes/lysosomes to other cellular compartments after lipoprotein uptake through the coated-pit pathway. The present study examines the relative expression of NPC1 mRNA and NPC1 protein in different tissues of the mouse in relation to the uptake of total cholesterol carried in chylomicron remnants (CMr-TC), low density lipoproteins (LDL-TC), cholesteryl ester carried in high density lipoproteins (HDL-CE), and cholesterol synthesis. Results from this study demonstrate that the highest relative expression of NPC1 is in the liver, which is also the tissue with the highest uptake of CMr-TC, LDL-TC, HDL-CE, and cholesterol synthesis. However, there was no similar relation in the remaining tissues. To examine the relative expression of NPC1 in relation to the amount of cholesterol that flowed through the coated-pit pathway, mice were fed a diet supplemented with increasing amounts of cholesterol or cholestyramine. The results from this study demonstrated that there was no relation between the relative expression of NPC1 and the amount of cholesterol that flowed through the coated-pit pathway.We conclude that the relative expression of NPC1 is not regulated by the flow of cholesterol through cells in the mouse and is therefore constitutive.

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR−/−) mice fed a basal diet containing ezetimibe (0–10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR−/− mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone.We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.

Unesterified cholesterol is an essential structural component of the plasma membrane of every cell. During evolution, this membrane came to play an additional, highly specialized role in the central nervous system (CNS) as the major architectural component of compact myelin. As a consequence, in the human the mean concentration of unesterified cholesterol in the CNS is higher than in any other tissue (∼23 mg/g). Furthermore, even though the CNS accounts for only 2.1% of body weight, it contains 23% of the sterol present in the whole body pool. In all animals, most growth and differentiation of the CNS occurs in the first few weeks or years after birth, and the cholesterol required for this growth apparently comes exclusively from de novo synthesis. Currently, there is no evidence for the net transfer of sterol from the blood into the brain or spinal cord. In adults, the rate of synthesis exceeds the need for new structural sterol, so that net movement of cholesterol out of the CNS must take place. At least two pathways are used for this excretory process, one of which involves the formation of 24(S)-hydroxycholesterol.Whether or not changes in the plasma cholesterol concentration alter sterol metabolism in the CNS or whether such changes affect cognitive function in the brain or the incidence of dementia remain uncertain at this time.

Although the pool of cholesterol in the adult central nervous system (CNS) is large and of constant size, little is known of the process(es) involved in regulation of sterol turnover in this pool. In 7-week-old mice, net excretion of cholesterol from the brain equaled 1.4 mg/day/kg body weight, and from the whole animal was 179 mg/day/kg. Deletion of cholesterol 24-hydroxylase, an enzyme highly expressed in the CNS, did not alter brain growth or myelination, but reduced sterol excretion from the CNS 64% to 0.5 mg/day/kg. In mice with a mutation in the Niemann-Pick C gene that had ongoing neurodegeneration, sterol excretion from the CNS was increased to 2.3 mg/day/kg. Deletion of cholesterol 24-hydroxylase activity in these animals reduced net excretion only 22% to 1.8 mg/day/kg.Thus, at least two different pathways promote net sterol excretion from the CNS. One uses cholesterol 24-hydroxylase and may reflect sterol turnover in large neurons in the brain. The other probably involves the movement of cholesterol or one of its metabolites across the blood-brain barrier and may more closely mirror sterol turnover in pools such as glial cell membranes and myelin.

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL33333391) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053.We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.

These experiments tested the hypothesis that fatty acids (FAs) that drive cholesterol esterification also enhance sterol secretion and were undertaken using a mouse model where lipoprotein-cholesterol output by the liver could be assessed in vivo. The turnover of sterol in the animals was kept constant (∼160 mg/d per kg) while the liver was enriched with the single FAs 8:0, 14:0, 18:1, or 18:2. Under these conditions, the steady-state concentration of cholesteryl ester in the liver varied 6-fold, from 1.2 to 7.9 mg/g, and the expansion of this pool was directly related to the specific FA enriching the liver (FA 18:1>18:2>8:0> 14:0). Secretion of lipoprotein-cholesterol varied 5-fold and was a linear function of the concentration of cholesteryl ester in the liver. These studies demonstrate that unsaturated FAs drive the esterification reaction and enhance lipoprotein cholesterol secretion by the liver under conditions where cholesterol balance across this organ is constant.Thus, individual FAs interact with cholesterol to profoundly regulate both the output and uptake of sterol by the liver, and these effects are articulated through the esterification reaction.

Bile acids are synthesized via the classic pathway initiated by cholesterol 7α-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7α-hydroxylase (Cyp7a1−/−) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1−/− males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1+/+ animals. Although bile acid pool size contracted markedly in all the Cyp7a1−/− mice, the female Cyp7a1−/− mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1−/− males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1+/+ males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1−/− mice by either of these manipulations. In the Cyp7a1−/− mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.—Schwarz, M., D. W. Russell, J. M. Dietschy, and S. D. Turley. Alternate pathways of bile acid synthesis in the cholesterol 7α-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding. J. Lipid Res. 2001. 42: 1594–1603.

Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs) and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

Niemann-Pick type C (NPC) protein functions to move unesterified cholesterol from the lysosomal compartment to other intracellular sites for further metabolism and/or excretion. This cholesterol is brought into the cell through the coated-pit pathway and accumulates in the lysosomes when NPC protein is mutated. The present study quantitated the alternative uptake process that brings cholesterol into the cell through the scavenger receptor, class B, type I (SR-BI) pathway in animals with this mutation. In homozygous NPC mice, the tissues of the extrahepatic compartment accumulated an excess of 14 mg of cholesterol each day per kg body weight, and synthesis increased by a similar amount (to 111 mg/day per kg) to compensate for this functional loss of sterol through lysosomal sequestration. An amount of cholesterol (108 mg/day per kg) nearly equal to that synthesized in the extrahepatic compartment was carried through the circulation by high density lipoprotein (HDL) and taken up by the liver. The rate of hepatic cholesterol excretion from the NPC mice as fecal acidic (65 mg/day per kg) and neutral (85 mg/day per kg) sterols was elevated 61% above control values and was accounted for by the total amount of cholesterol brought to the liver in HDL and synthesized in the hepatocytes. These studies demonstrated that while cholesterol entering tissues of the NPC animals through the coated-pit pathway became sequestered in the lysosomal compartment and was metabolically inactive, cholesterol that was newly synthesized or that entered cells through the SR-BI pathway was metabolized and excreted normally. —Xie, C., S. D. Turley, and J. M. Dietschy. Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick type C protein (NPC1). J. Lipid Res. 2000. 41: 1278–1289.

The major net flux of cholesterol in the intact animal or human is from the peripheral organs to the liver. This flux is made up of cholesterol that is either synthesized in these peripheral tissues or taken up as lipoprotein cholesterol. This study investigates whether it is the concentration of apolipoprotein (apo) A-I or high density lipoprotein in the plasma that determines the magnitude of this flux or, alternatively, whether events within the peripheral cells themselves regulate this important process. In mice that lack apoA-I and have very low concentrations of circulating high density lipoprotein, it was found that there was no accumulation of cholesterol in any peripheral organ so that the mean sterol concentration in these tissues was the same (2208 ± 29 mg/kg body weight) as in control mice (2176 ± 50 mg/kg). Furthermore, by measuring the rates of net cholesterol acquisition in the peripheral organs from de novo synthesis and uptake of low density lipoprotein, it was demonstrated that the magnitude of centripetal sterol movement from the peripheral organs to the liver was virtually identical in control animals (78 ± 5 mg/day per kg) and in those lacking apoA-I (72 ± 4 mg/day per kg). These studies indicate that the magnitude of net sterol flux through the body is not related to the concentration of high density lipoprotein or apolipoprotein A-I in the plasma, but is probably determined by intracellular processes in the peripheral organs that dictate the rate of movement of cholesterol from the endoplasmic reticulum to the plasma membrane. —Jolley, C. D., L. A. Woollett, S. D. Turley, and J. M. Dietschy. Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration. J. Lipid. Res. 1998. 39: 2143–2149.

These studies used mice that were deficient in cholesterol 7α-hydroxylase to determine the effects of reduced bile acid synthesis on cholesterol homeostasis. In mice lacking this enzyme, bile acid synthesis was reduced from 8.3 to 3.4 μmol/day per 100 g body weight, the intestinal bile acid pool was decreased from 62.5 to 13.2 μmol/100 g bw, and the proportion of hyodeoxycholate, relative to cholate, in this pool was significantly increased. Associated with these changes, intestinal cholesterol absorption decreased from 37% to

These studies were done to determine whether an underlying metabolic difference could account for the higher concentration of cholesterol carried in low density lipoproteins (LDL-C) in male hyperresponder (HR) cynomolgus monkeys than in their hyporesponder (HO) counterparts during dietary cholesterol challenge. All animals were fed to steady state at 5 months a diet that had a constant concentration of cholesterol (0.19 mg/g), triacylglycerol (175 mg/g), and soluble fiber. There were no differences in these two phenotypes with respect to the profile of fatty acids in the liver and bile acids in the gallbladder, or in the relationship of cholesterol synthesis to cholesteryl ester formation in the liver. The rate of cholesterol synthesis in all extrahepatic tissues was also the same in the HO and HR animals but was 2.1 mg/day per kg body weight less in the liver of the HR monkeys. When challenged with a greater dietary cholesterol load, therefore, the HR animal could not readily further down-regulate synthesis and so shifted more cholesterol into the ester pool (9.4 mg/g) than did the HO animal (3.9 mg/g). Also the LDL-C concentration was more markedly elevated (412 mg/dl) compared to the hyporesponder monkey (188 mg/dl). Thus, this single metabolic alteration apparently accounted for the HO and HR phenotypes. As this difference was not due to variation in the delivery of sterol from the extrahepatic organs to the liver, it must reflect a difference in either net intestinal sterol absorption or net hepatic sterol excretion in the two phenotypes.

Several lines of evidence have suggested that central nervous system development and function depend upon a supply of cholesterol that comes from low density lipoproteins (LDL-C). These studies test this hypothesis directly by measuring in vivo the uptake of LDL-C in nine regions of the central nervous system at five different stages of development in the fetal and neonatal sheep. The concentration of LDL-C in the plasma decreased from 49 mg/dl in the fetus 90 days before birth (-90 days) to only 10 mg/dl at -13 days. By 17 days postnatal this value increased to nearly 60 mg/dl. Throughout the period of development between -90 days (very early fetus) and 17 days (late neonatal animal) the weight of the brain increased 32-fold (from 2.3 to 73.6 g) and the content of cholesterol rose 100-fold (from 8.6 to 876 mg), yet there was no detectable LDL-C uptake in any of nine areas of the central nervous system at any stage of development (clearances of < 2 microliters/h per g). This was true even in the -90 day fetus prior to closure of the blood brain barrier. In contrast, LDL-C clearance by the adrenal gland increased dramatically (from 91 to 348 microliters/h per g) as it also did in the liver (from 36 to 85 microliters/h per g) during fetal development. These studies strongly suggest, therefore, that cholesterol carried in LDL plays little or no role in the process of sterol acquisition during brain development or in cholesterol turnover in the mature central nervous system. Changes in circulating LDL-C concentration, therefore, should have no effect on brain function.

The suitability of the adult male cynomolgus monkey as a model for investigating genetic mechanisms that regulate dietary cholesterolemic response was evaluated by carrying out a systematic characterization of the major aspects of cholesterol metabolism in this species. In monkeys maintained on a diet enriched with saturated fat but low in cholesterol (0.019%, wt/wt), plasma total and low density lipoprotein cholesterol (LDL-C) concentrations were 118 +/- 6 and 45.3 +/- 3.4 mg/dl, respectively. Intestinal cholesterol absorption averaged 54.0 +/- 2.5%, and the rate of whole body sterol synthesis was 10.8 +/- 0.6 mg/day per kg body weight. Only 11.2 +/- 2.6% of this synthesis occurred in the liver. In contrast, the liver was the major site for low density lipoprotein clearance accounting for almost 80% of LDL-C degradation in these animals. The liver, which represented 1.5% of whole body mass, had a total and esterified cholesterol concentration of 4.95 +/- 0.29 and 2.05 +/- 0.30 mg/g, respectively. When challenged with a matching high cholesterol diet (0.19%, wt/wt), the monkeys developed marked hypercholesterolemia that was accounted for mainly by a 7-fold increase in the LDL-C levels. There was, however, wide individual variation among the monkeys in the magnitude of their cholesterolemic response. Hepatic total and esterified cholesterol levels increased 2.5- and 4.6-fold, respectively. Comparative experiments showed that while several of the metabolic characteristics of this species of monkey were similar to those found in the hamster, they were generally very different from those seen in the rat. Thus, the male cynomolgus monkey has many characteristics in common with humans and represents an attractive model for further delineating the genetic mechanisms that dictate variable responsiveness to dietary cholesterol and triacylglycerol.

These experiments systematically evaluated the dual-isotope plasma ratio method for measuring intestinal cholesterol absorption in the hamster. It was found that while the ratio of the 3H- and 14C-labeled cholesterol in the plasma, relative to the respective dose of each that was given, became constant by 72 h after their administration, the percent cholesterol absorption was lower in animals that were fasted before dosing (35.7 +/- 5.5%) than in their fed controls (47.5 +/- 3.7%). Furthermore, the percent absorption found 72 h after dosing varied greatly, depending on whether the intragastric dose of labeled cholesterol was administered in medium chain triglyceride (MCT) oil (46.2 +/- 2.3%), olive oil (63.9 +/- 11.2%), or safflower oil (74.6 +/- 4.5%). The level of absorption was not different between hamsters that had unrestricted (46.3 +/- 1.6%) and restricted (43.8 +/- 2.2%) access to their stools during the 72 h after dosing. Other experiments, using only hamsters in the fed state and MCT oil as the intragastric dosing medium, showed that the percent cholesterol absorption could be made to vary over a wide range using treatments known to produce such effects in humans. Thus, feeding either surfomer, cholestyramine, ursodeoxycholic acid, or CI-976, a new inhibitor of acyl-CoA:cholesterol acyltransferase, significantly blocked cholesterol absorption, whereas the addition of either cholic acid or increasing amounts of oil to the diet had the opposite effect. The modified dual-isotope plasma ratio method described here provides a simpler and more physiologic approach to the routine measurement of cholesterol absorption in the hamster and similar small animal models.

This study was undertaken to determine the rates of sterol synthesis and uptake in the major organs of the female rat in vivo. At the mid-dark phase of the light cycle, control animals, animals in which hepatic sterol synthesis had been selectively inhibited by chylomicron infusion and animals in which the small intestine and liver had been surgically removed, were administered [(3)H]water, and the content of (3)H-labeled digitonin-precipitable sterols ([(3)H]-DPS) in different organs was measured 1 hr later. In control animals, the highest content of [(3)H]DPS was found in the liver (2279 nmol/hr per g), adrenal gland (1222), ovary (791), and small bowel (529): the content of newly synthesized sterols was much lower in 13 other tissues. By selectively inhibiting sterol synthesis in the liver or by surgically removing the small intestine and liver, it was determined that of the total amount of [(3)H]DPS synthesized in the whole animal about 50% had occurred in the liver, 24% in the small bowel, 8% in the skin, and 18% in the remaining tissues of the carcass combined. By analyzing the relationship between the content of [(3)H]DPS in blood and in each organ, it was further possible to determine how much [(3)H]DPS was synthesized and how much was taken up from the blood in each tissue. The highest rate of uptake was found in the adrenal gland where only 4% of the tissue content of [(3)H]DPS came from local synthesis. Low rates of synthesis relative to the rates of uptake, were also found in the spleen (6%), lung (17%), and kidney (26%). In contrast, in other organs there was little uptake of [(3)H]DPS from blood so that >75% of the [(3)H]DPS present in brain and muscle, for example, was due to local synthesis. Lowering the circulating levels of plasma cholesterol markedly increased the synthesis of [(3)H]DPS in tissues like adrenal gland, spleen, and kidney that were dependent upon plasma cholesterol as the major source of tissue sterols, but not in tissues such as muscle and brain. These studies have quantitated the importance of each major organ to total body synthesis and have delineated the rates of movement of [(3)H]DPS between major tissue compartments of the rat.-Turley, S. D., J. M. Andersen, and J. M. Dietschy. Rates of sterol synthesis and uptake in the major organs of the rat in vivo.

These studies were designed to determine the importance of the rate of hepatic cholesterol synthesis, the size of the hepatic cholesteryl ester pool, the amount of chylomicron cholesterol reaching the liver, and the rate of bile acid transport into bile as determinants of the rate of biliary cholesterol output. Female rats that had been subjected to diurnal light cycling, fasting for 48 hr, intravenous administration of chylomicrons, and diets containing either cholestyramine, cholesterol, or bile acid underwent total biliary diversion for 2 hr. The animals were then killed and the rates of hepatic cholesterol synthesis and levels of hepatic esterified cholesterol were measured along with biliary lipid concentrations. Despite a 1000-fold variation in the rate of hepatic cholesterogenesis and a 100-fold variation in the levels of cholesteryl esters, the output and molar percentage of cholesterol in bile remained essentially constant with the exception of an approximate doubling in the output of cholesterol, as well as of bile acid and phospholipid in those animals fed bile acid. However, in this latter group the molar percentage of each component was unchanged. The administration of a bolus of chylomicrons did not alter output or molar percentage of cholesterol. Total biliary diversion for 36 hr and bile acid infusion were used to markedly vary the rate of biliary bile acid output. Cholesterol and phospholipid output remained tightly coupled to bile acid output over almost a 40-fold range. In other experiments it was shown that biliary cholesterol output could be driven by bile acid infusion to a similar extent in rats in which the rate of hepatic cholesterogenesis had been varied over a 26-fold range. It was concluded that the rate of hepatic cholesterol synthesis, the level of hepatic cholesteryl esters, and the amount of cholesterol absorbed from the diet play no role in determining the rate of biliary cholesterol secretion, at least in this species.-Turley, S. D., and J. M. Dietschy. Regulation of biliary cholesterol output in the rat: dissociation from the rate of hepatic cholesterol synthesis, the size of the hepatic cholesteryl ester pool, and the hepatic uptake of chylomicron cholesterol.

A review of the 3 alpha-hydroxysteroid dehydrogenase method for determining the concentration of total bile acids in bile is described. The optimum conditions for the assay were established with respect to pH, temperature, incubation time, amount of NAD+, and units of enzyme activity required to obtain complete oxidation of the substrate under fixed conditions. Furthermore, the effect of hydrazine hydrate, methanol, and bile volume on the reaction was examined. It was also established that the bile acid concentration in bile samples with a high molar percentage of cholesterol would be overestimated if 3 beta-hydroxysteroid dehydrogenase were present with the 3 alpha-enzyme.

The relationship between rates of hepatic sterol synthesis and rates of hepatic low density lipoprotein (LDL) uptake (clearance) was studied in animals with high (rats), low (female hamsters), and very low (male hamsters) basal rates of hepatic sterol synthesis. In rats and female hamsters, rates of hepatic sterol synthesis were varied over a 110-fold range by feeding cholesterol or cholestyramine; nevertheless, rates of hepatic LDL clearance remained essentially unchanged as did plasma LDL-cholesterol concentrations. In contrast, in male hamsters, which have a very limited capacity to synthesize cholesterol in the liver, cholestyramine feeding increased rates of hepatic LDL uptake by 2.5-fold and this was associated with a 50% reduction in plasma LDL-cholesterol concentrations. The observed increase in LDL uptake was due to an increase in receptor-dependent LDL transport while receptor-independent lipoprotein uptake remained constant. These studies suggest that rates of hepatic cholesterol synthesis and receptor-dependent LDL uptake are regulated independently. Furthermore, the primary response of the liver to changes in cholesterol availability is regulation of sterol synthesis and only when the capacity of this compensatory mechanism is exceeded is the rate of LDL transport altered.

The SHR/N-corpulent rat is a new genetically obese strain that exhibits both insulin-independent diabetes and hyperlipidemia. The present studies were undertaken to characterize various parameters of cholesterol metabolism in this model. At 11 weeks of age, the obese animals had markedly elevated plasma cholesterol, triglyceride, glucose, and insulin concentrations and elevated hepatic triglyceride concentrations compared to their lean littermates. The additional cholesterol in plasma was carried in the fractions of density less than 1.006, 1.020-1.055, 1.055-1.095, and 1.095-1.21 g/ml. In the obese rats the level of free cholesterol in the liver was decreased significantly while that of cholesteryl ester showed little change. Hepatic sterol synthesis was markedly suppressed in the obese animals. However, the rate of sterol synthesis in the small intestine and other extrahepatic tissues generally remained unchanged. Although hepatic synthesis was suppressed, whole animal sterol synthesis in the obese rats was similar to that in the lean controls. This resulted because, in the obese animals, not only was the reduced rate of hepatic synthesis partly balanced by a greater than 70% increase in liver mass, but the mass of the small intestine and adipose tissue was also increased more than 30% and 4-fold, respectively, thereby making these tissues quantitatively more important sites of sterol synthesis. When obese rats were pair-fed to the intake of their lean littermates for 10 weeks, there was only a modest reduction in body weight and plasma cholesterol concentration, and the rate of hepatic sterol synthesis remained very low. The suppression of synthesis in the liver also persisted when the obese rats were fed surfomer, a drug that specifically blocks cholesterol absorption. In contrast, feeding cholestyramine restored the rate of hepatic sterol synthesis to that found in lean animals. Bile acid pool size in the obese males and females was 2.5-fold greater than in their lean controls. The suppression of hepatic sterol synthesis in this model may be due to a change in the entero-hepatic circulation of bile acids arising from an expanded pool or, alternatively, it may represent a compensatory response to overproduction of sterol and its precursors in the intestinal and adipose compartments.

Current prognostic biomarkers fall short in stratifying Oestrogen receptor (ER)-negative breast cancer patients regarding tumour progression risk at diagnosis. The role of AIPL1 in activating its tumour suppressor client protein, NEDD8 Ultimate Buster-1 (NUB1) remains unknown in cancer. Our study demonstrated how downregulated AIPL1 results in the deactivated NUB1 protein under hypoxic conditions. We examined the AIPL1-NUB1 pathwayin vitro using cell lines i.e. MCF-7, MDA-MB-231, RCC4 etc. NUB1 expression was assessed using Oncomine, and cBioPortal was performed to assess NUB1′s prognostic significance in human cancers. In the John Radcliffe Hospital cohort (n = 122), immunohistochemistry analysis revealed downregulated AIPL1 (Log2 fold change=-0.28; p < 0.001) and upregulated NUB1 transcripts (Log2 fold change=0.59; p < 0.001) compared to adjacent normal tissues. In severe chronic hypoxia, multimerised AIPL1 localisedin the cytoplasm while NUB1 protein migrated to the nucleus, where the absence of NUB1 nuclear localisation led to cell cycle arrest. Biopsies showed that patients with lower cytoplasmic NUB1 expression (n = 57) had poorer overall survival compared to those with higher cytoplasmic expression (n = 57), HR=1.78; 95 % CI=1.01–3.35, p = 0.048. Low NUB1 protein levels in both normoxic and hypoxic conditions were associated with cell cycle arrest and upregulation ofp21 and p27 in breast cancer cell lines, correlating significantly withpoorer survival outcomes in all breast cancer and ER-negative breast cancer patients.

Introduction: Anterior cervical discectomy and fusion (ACDF) is commonly performed with cage and plate constructs to stabilise diseased or injured cervical segments. Despite it being a commonly performed procedure, there are notable rates of associated morbidity reported in the literature. Stand-alone spacers represent a novel form of instrumentation to conventional cage and plate constructs. Research question: Do stand-alone spacers have improved operative characteristics and postoperative outcomes in ACDF cohorts when compared to cage and plate constructs? Methods: A systematic review and meta-analysis was conducted of PubMed/Medline, Embase and Google Scholar databases per the Preferred Reporting Items for Systematic Reviews and Meta-Analyzes guidelines. Studies of interest included cage and plate instrumentation versus anchored stand-alone spacers for patients undergoing ACDF. Pre- and post-operative clinical and radiological outcomes were collated and compared for significance between cohorts. Results: 10 RCTs were identified and included with 779 patients total. Mean age of the entire cohort was 50.1 years. 62% (483/779) of the cohort were male. 384 patients underwent ACDF with stand-alone cage, while 395 had ACDF with conventional cage and plate. Stand-alone spacers significantly outperformed conventional instrumentation in terms of estimated blood loss (p