Unlocking the potential of RNA-based therapeutics in the lung: current status and future directions.

Awareness of RNA-based therapies has increased after the widespread adoption of mRNA vaccines against SARS-CoV-2 during the COVID-19 pandemic. These mRNA vaccines had a significant impact on reducing lung disease and mortality. They highlighted the potential for rapid development of RNA-based therapies and advances in nanoparticle delivery systems. Along with the rapid advancement in RNA biology, including the description of noncoding RNAs as major products of the genome, this success presents an opportunity to highlight the potential of RNA as a therapeutic modality. Here, we review the expanding compendium of RNA-based therapies, their mechanisms of action and examples of application in the lung. The airways provide a convenient conduit for drug delivery to the lungs with decreased systemic exposure. This review will also describe other delivery methods, including local delivery to the pleura and delivery vehicles that can target the lung after systemic administration, each providing access options that are advantageous for a specific application. We present clinical trials of RNA-based therapy in lung disease and potential areas for future directions. This review aims to provide an overview that will bring together researchers and clinicians to advance this burgeoning field.

Impact of sex on outcome after pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension.

BACKGROUND: The impact of sex on long-term outcomes after pulmonary endarterectomy (PEA) for chronic thromboembolic pulmonary hypertension (PH) remains unclear. We therefore examined the early and long-term outcome after PEA to determine whether sex had an impact on the risk of residual PH and need for targeted PH medical therapy. METHODS: Retrospective study of 401 consecutive patients undergoing PEA at our institution between August 2005 and March 2020 was performed. Primary outcome was the need for targeted PH medical therapy postoperatively. Secondary outcomes included survival and measures of hemodynamic improvement. RESULTS: Females (N = 203, 51%) were more likely to have preoperative home oxygen therapy (29.6% vs 11.6%, p < 0.01), and to present with segmental and subsegmental disease compared to males (49.2% vs 21.2%, p < 0.01). Despite similar preoperative values, females had higher postoperative pulmonary vascular resistance (final total pulmonary vascular resistance after PEA, 437 Dynes∙s∙cm-5 vs 324 Dynes∙s∙cm-5 in males, p < 0.01). Although survival at 10 years was not significantly different between sexes (73% in females vs 84% in males, p = 0.08), freedom from targeted PH medical therapy was lower in females (72.9% vs 89.9% in males at 5 years, p < 0.001). Female sex remained an independent factor affecting the need for targeted PH medical therapy after PEA in multivariate analysis (HR 2.03, 95%CI 1.03-3.98, p = 0.04). CONCLUSIONS: Although outcomes are excellent for both sexes, females had greater need for targeted PH medical therapy in the long-term. Early reassessment and long-term follow-up of these patients are important. Further investigations into possible mechanisms to explain the differences are warranted.

Prognostic Value of EMT Gene Signature in Malignant Mesothelioma.

Malignant mesothelioma (MESO) consists of epithelioid, biphasic, and sarcomatoid subtypes with different epithelial-mesenchymal transition (EMT) phenotypes. We previously identified a panel of four MESO EMT genes correlating with an immunosuppressive tumor microenvironment and poor survival. In this study, we investigated the correlation between these MESO EMT genes, the immune profile, and the genomic and epigenomic alterations to identify potential therapeutic targets to prevent or reverse the EMT process. Using multiomic analysis, we observed that the MESO EMT genes were positively correlated with hypermethylation of epigenetic genes and loss of CDKN2A/B expression. MESO EMT genes such as COL5A2, ITGAV, SERPINH1, CALD1, SPARC, and ACTA2 were associated with upregulation of TGF-ß signaling, hedgehog signaling, and IL-2-STAT5 signaling and downregulation of the IFN-α and IFN-γ response. Immune checkpoints such as CTLA4, CD274 (PD-L1), PDCD1LG2 (PD-L2), PDCD1 (PD-1), and TIGIT were upregulated, while LAG3, LGALS9, and VTCN1 were downregulated with the expression of MESO EMT genes. CD160, KIR2DL1, and KIR2DL3 were also broadly downregulated with the expression of MESO EMT genes. In conclusion, we observed that the expression of a panel of MESO EMT genes was associated with hypermethylation of epigenetic genes and loss of expression of CDKN2A and CDKN2B. Expression of MESO EMT genes was associated with downregulation of the type I and type II IFN response, loss of cytotoxicity and NK cell activity, and upregulation of specific immune checkpoints, as well as upregulation of the TGF-ß1/TGFBR1 pathway.

Long noncoding RNA GATA2-AS1 augments endothelial hypoxia inducible factor 1-α induction and regulates hypoxic signaling.

Vascular endothelial cells form the inner cellular lining of blood vessels and have myriad physiologic functions including angiogenesis and response to hypoxia. We recently identified a set of endothelial cell (EC)-enriched long noncoding RNAs (lncRNAs) in differentiated human primary cell types and described the role of the STEEL lncRNA in angiogenic patterning. We sought to further understand the role of EC-enriched lncRNAs in physiologic adaptation of the vascular endothelium. In this work, we describe an abundant, cytoplasmic, and EC-enriched lncRNA, GATA2-AS1, that is divergently transcribed from the EC-enriched developmental regulator, GATA2. While GATA2-AS1 is largely coexpressed with GATA2 in ECs, GATA2-AS1 and GATA2 appear to be complementary rather than synergistic as they have mostly distinct target genes. Common single nucleotide variants in GATA2-AS1 exons are associated with early-onset coronary artery disease and decreased expression of GATA2-AS1 in endothelial cell lines. In most cells, HIF1-α is central to the transcriptional response to hypoxia, while in ECs, both HIF1-α and HIF2-α are required to coordinate an acute and chronic response, respectively. In this setting, GATA2-AS1 contributes to the "HIF switch" and augments HIF1-α induction in acute hypoxia to regulate HIF1-α/HIF2-α balance. In hypoxia, GATA2-AS1 orchestrates HIF1-α-dependent induction of the glycolytic pathway and HIF1-α-independent maintenance of mitochondrial biogenesis. Similarly, GATA2-AS1 coordinates both metabolism and "tip/stalk" cell signaling to regulate angiogenesis in hypoxic ECs. Furthermore, we find that GATA2-AS1 expression patterns are perturbed in atherosclerotic disease. Together, these results define a role for GATA2-AS1 in the EC-specific response to hypoxia.

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Development and Evaluation of a Kinect-based Rapid Movement Therapy Training Platform for Balance Rehabilitation.

With the growing aging and overall population, the demand for healthcare professionals and their burden increases by time. Effective balance recovery reaction is required to prevent falls. The aim of this project is to provide low-cost portable balance training system that trains the two important components of effective balance recovery reaction: faster movement completion time (MT) and larger range of motion (ROM). This is done by a Kinect-based interactive rapid movement therapy training platform for reaching and stepping actions. The platform provides real-time feedback to the patient, generates a report for healthcare professionals to monitor the patient's progress, and can be utilized in patient's home or community centers. A pilot study to test the platform was conducted on seventeen stroke patients and it has shown significant improvement in both MT (faster) and ROM (larger).

Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA.

Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell type-specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for spliced-transcript endothelial-enriched lncRNA (STEEL) in angiogenic potential, macrovascular/microvascular identity, and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear enriched, and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse ECs. Of interest, STEEL up-regulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL up-regulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity.

Proposed management algorithm for severe hypoxemia after liver transplantation in the hepatopulmonary syndrome.

The hepatopulmonary syndrome (HPS) is defined as the triad of liver disease, intrapulmonary vascular dilatation, and abnormal gas exchange, and is found in 10-32% of patients with liver disease. Liver transplantation is the only known cure for HPS, but patients can develop severe posttransplant hypoxemia, defined as a need for 100% inspired oxygen to maintain a saturation of ≥85%. This complication is seen in 6-21% of patients and carries a 45% mortality. Its management requires the application of specific strategies targeting the underlying physiologic abnormalities in HPS, but awareness of these strategies and knowledge on their optimal use is limited. We reviewed existing literature to identify strategies that can be used for this complication, and developed a clinical management algorithm based on best evidence and expert opinion. Evidence was limited to case reports and case series, and we determined which treatments to include in the algorithm and their recommended sequence based on their relative likelihood of success, invasiveness, and risk. Recommended therapies include: Trendelenburg positioning, inhaled epoprostenol or nitric oxide, methylene blue, embolization of abnormal pulmonary vessels, and extracorporeal life support. Availability and use of this pragmatic algorithm may improve management of this complication, and will benefit from prospective validation.

Nitric oxide and hypoxia signaling.

Nitric oxide (NO) production is catalyzed by three distinct enzymes, namely, neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The production of NO by vascular endothelium relies mainly on eNOS. Curiously, iNOS and nNOS also are relevant for vascular NO production in certain settings. By relaxing vascular smooth muscle, the classical view is that NO participates in O2 homeostasis by increasing local blood flow and O2 delivery. It is now appreciated that NO has an even more fundamental role in cellular oxygen sensing at the cellular and physiological level. A key component of cellular oxygen sensing is the hypoxia-inducible factor (HIF) that activates a transcriptional program to promote cellular survival under conditions of inadequate oxygen supply. Important new insights demonstrate that HIF protein is stabilized by two parallel pathways: (1) a decrease in the O2-dependent prolyl hydroxylation of HIF and (2) NO-dependent S-nitrosylation of HIF pathway components including HIF-α. The need for these two complementary pathways to HIF activation arises because decreased oxygen delivery can occur not only by decreased ambient oxygen but also by decreased blood oxygen-carrying capacity, as with anemia. In turn, NO production is tightly linked to O2 homeostasis. O2 is a key substrate for the generation of NO and impacts the enzymatic activity and expression of the enzymes that catalyze the production of NO, the nitric oxide synthases. These relationships manifest in a variety of clinical settings ranging from the unique situation of humans living in hypoxic environments at high altitudes to the common scenario of anemia and the use of therapeutics that can bind or release NO.

The use of a syndesmosis procedure for the treatment of hallux valgus: good clinical and radiological results two years post-operatively.

Metatarsus primus varus deformity correction is one of the main objectives in hallux valgus surgery. A 'syndesmosis' procedure may be used to correct hallux valgus. An osteotomy is not involved. The aim is to realign the first metatarsal using soft tissues and a cerclage wire around the necks of the first and second metatarsals. We have retrospectively assessed 27 patients (54 feet) using the American Orthopaedic Foot and Ankle Society (AOFAS) score, radiographs and measurements of the plantar pressures after bilateral syndesmosis procedures. There were 26 women. The mean age of the patients was 46 years (18 to 70) and the mean follow-up was 26.4 months (24 to 33.4). Matched-pair comparisons of the AOFAS scores, the radiological parameters and the plantar pressure measurements were conducted pre- and post-operatively, with the mean of the left and right feet. The mean AOFAS score improved from 62.8 to 94.4 points (p < 0.001). Significant differences were found on all radiological parameters (p < 0.001). The mean hallux valgus and first intermetatarsal angles were reduced from 33.2° (24.3° to 49.8°) to 19.1° (10.1° to 45.3°) (p < 0.001) and from 15.0° (10.2° to 18.6°) to 7.2° (4.2° to 11.4°) (p < 0.001) respectively. The mean medial sesamoid position changed from 6.3(4.5 to 7) to 3.6 (2 to 7) (p < 0.001) according to the Hardy's scale (0 to 7). The mean maximum force and the force-time integral under the hallux region were significantly increased by 71.1% (p = 0.001), (20.57 (0.08 to 58.3) to 35.20 (6.63 to 67.48)) and 73.4% (p = 0.014), (4.44 (0.00 to 22.74) to 7.70 (1.28 to 19.23)) respectively. The occurrence of the maximum force under the hallux region was delayed by 11% (p = 0.02), (87.3% stance (36.3% to 100%) to 96.8% stance (93.0% to 100%)). The force data reflected the restoration of the function of the hallux. Three patients suffered a stress fracture of the neck of the second metatarsal. The short-term results of this surgical procedure for the treatment of hallux valgus are satisfactory.

Defining and characterizing severe hypoxemia after liver transplantation in hepatopulmonary syndrome.

Hepatopulmonary syndrome is defined as a triad of liver disease, intrapulmonary vascular dilatations, and abnormal gas exchange, and it carries a poor prognosis. Liver transplantation is the only known cure for this syndrome. Severe hypoxemia in the early postoperative period has been reported to be a major complication and often leads to death in this population, but it has been poorly characterized. We sought to propose an objective definition for this complication and to describe its risk factors, incidence, and outcomes. We performed a systematic literature search and reviewed our single-center experience to characterize this complication. On the basis of the most commonly applied definition in 27 identified studies, we objectively defined severe postoperative hypoxemia as hypoxemia requiring a 100% fraction of inhaled oxygen to maintain a saturation ≥ 85% and out of proportion to any concurrent lung process. Nineteen of the 27 reports (70%) fulfilled this definition, as did 4 of the 21 patients (19%) at our center. We determined the prevalence and mortality of this complication from reports including 10 or more consecutive patients and providing sufficient postoperative details to determine whether this complication had occurred. In these reports, the prevalence of this complication was 12% (25/209). For the 11 cases with reported outcomes, the posttransplant mortality rate was 45% (5/11). There was a trend toward an increased risk of developing this complication in patients with very severe preoperative hypoxemia, defined as a partial pressure of arterial oxygen ≤ 50 mm Hg (8/41 with very severe hypoxemia versus 3/49 without severe hypoxemia, P = 0.053), and there was a significantly increased risk for patients with anatomic shunting ≥ 20% (7/25 with anatomic shunting ≥ 20% versus 1/25 without anatomic shunting ≥ 20%, P = 0.049). In conclusion, increased preoperative vigilance for this common complication is required among high-risk patients, and further research is required to identify the best management strategies.

Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs.

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.

Functional importance of Dicer protein in the adaptive cellular response to hypoxia.

The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Gene regulation in the vascular endothelium: why epigenetics is important for the kidney.

We now appreciate that the vascular endothelium plays a crucial role in regulating normal blood vessel physiology in the kidney. The gene products responsible are commonly expressed exclusively, or preferentially, in this cell type. However, despite the importance of regulated gene expression in the vascular endothelium, relatively little is known about the mechanisms that restrict endothelial-specific gene expression to this cell type. Even less is known about how gene expression might be restricted to endothelial cells of discrete regions of the kidney, such as the glomerulus or vasa recta. Although significant progress has been made toward understanding the regulation of endothelial genes through cis/trans paradigms, it has become apparent that additional mechanisms also must be operative. Classic models of transcription in vascular endothelial cells, specifically the cis/trans paradigm, have limitations. For instance, how does the environment have chronic effects on gene expression in endothelial cells after weeks or years? When an endothelial cell divides, how is this information transmitted to daughter cells? Chromatin-based mechanisms, including cell-specific DNA methylation patterns and post-translational histone modifications, recently were shown to play important roles in gene expression. This review investigates the involvement of epigenetic regulatory mechanisms in vascular endothelial cell-specific gene expression using endothelial nitric oxide synthase as a prototypical model.

Nitric oxide signaling in hypoxia.

Endothelial-derived nitric oxide (NO) is classically viewed as a regulator of vasomotor tone. NO plays an important role in regulating O(2) delivery through paracrine control of vasomotor tone locally and cardiovascular and respiratory responses centrally. Very soon after the cloning and functional characterization of the endothelial nitric oxide synthase (eNOS), studies on the interaction between O(2) and NO made the paradoxical finding that hypoxia led to decreases in eNOS expression and function. Why would decreases in O(2) content in tissues elicit a loss of a potent endothelial-derived vasodilator? We now know that restricting our view of NO as a regulator of vasomotor tone or blood pressure limited deeper levels of mechanistic insight. Exciting new studies indicate that functional interactions between NO and O(2) exhibit profound complexity and are relevant to diseases states, especially those associated with hypoxia in tissues. NOS isoforms catalytically require O(2). Hypoxia regulates steady-state expression of the mRNA and protein abundance of the NOS enzymes. Animals genetically deficient in NOS isoforms have perturbations in their ability to adapt to changes in O(2) supply or demand. Most interestingly, the intracellular pathways for O(2) sensing that evolved to ensure an appropriate balance of O(2) delivery and utilization intersect with NO signaling networks. Recent studies demonstrate that hypoxia-inducible factor (HIF) stabilization and transcriptional activity is achieved through two parallel pathways: (1) a decrease in O(2)-dependent prolyl hydroxylation of HIF and (2) S-nitrosylation of HIF pathway components. Recent findings support a role for S-nitrosothiols as hypoxia-mimetics in certain biological and/or disease settings, such as living at high altitude, exposure to small molecules that can bind NO, or anemia.

Preference and satisfaction with a 6-month subcutaneous injection versus a weekly tablet for treatment of low bone mass.

UNLABELLED: The Preference and Satisfaction Questionnaire (PSQ) compares patient preference and satisfaction between a 6-month subcutaneous injection and weekly oral tablet for treatment of bone loss. Patients preferred and were more satisfied with a treatment that was administered less frequently, suggesting the acceptability of the 6-month injection for treatment of bone loss. INTRODUCTION: The PSQ compares patient preference and satisfaction between a 6-month subcutaneous injection and a weekly oral tablet for treatment of bone loss. METHODS: Postmenopausal women with low bone mass who enrolled in two separate randomized phase 3 double-blind, double-dummy studies received a 6-month subcutaneous denosumab injection (60 mg) plus a weekly oral placebo or a weekly alendronate tablet (70 mg) plus a 6-month subcutaneous placebo injection. After 12 months, patients completed the PSQ to rate their preference, satisfaction, and degree of bother with each regimen. RESULTS: Most enrolled patients (1,583 out of 1,693; 93.5%) answered >or=1 item of the PSQ. Significantly more patients preferred and were more satisfied with the 6-month injection versus the weekly tablet (P < 0.001). More patients reported no bother with the 6-month injection (90%) than the weekly tablet (62%). CONCLUSION: Patients preferred, were more satisfied, and less bothered with a 6-month injection regimen for osteoporosis.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT.

BACKGROUND: The phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling. RESULTS: We show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice. CONCLUSION: Together, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

Determination of suitable sample sizes for multi-patient based finite element studies.

Finite element analysis is used extensively to assess joint replacements, but the majority of these are single sample studies. Recent investigations have suggested that such studies are unable to account for natural inter-patient variation in bone geometry and material property distribution. Recent developments in computer tomography based analyses make multiple sample studies possible; the question remains how many femurs are required to perform a study which accounts for such variations. This work investigates the factors that should be considered in answering this question. It explores sample sizing techniques when comparing strain distribution in the intact and implanted femur and when comparing two or more implant designs in a group of femurs. An example analysis of the effect of femoral head resurfacing was undertaken. Two sample sizing calculations were utilised, one based on achieving the desired precision in results, the other based on determining if a significant difference exists between two designs. The analysis shows that reasonable statistical precision can be achieved with a group of femurs. The study was also able to determine a suitable sample size for the analysis of a statistically significant difference between two groups of femurs with varying design parameters. The study concluded that while sample sizing is recommended for an accurate analysis, consideration must be made for the practicality of such a task.