Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families.

Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model.

The role of obesity in cognitive dysfunction in people with epilepsy.

OBJECTIVE: In the general population, obesity is associated with accelerated age-related cognitive decline. The impact of obesity on cognitive function in neurological populations who already have a heightened risk of cognitive decline is unknown. This study explored the relationship between obesity and cognitive underfunction in people with medically intractable epilepsy. METHODS: Eighty-one consecutive patients admitted for evaluation for medically intractable epilepsy (36 females and 45 males) underwent tests of memory and intellectual function. Optimal level of function was assessed using the National Adult Reading Test - Revised. Measures of underfunction were calculated by subtracting current measures of intellectual ability from the NART IQ. Body mass index (BMI) was used as an index of obesity. RESULTS: Twenty-nine people had a BMI in the healthy range (36%), 31 were overweight (38%), and 21 were obese (26%). The healthy weight, overweight, and obese groups did not differ in age at the time of assessment, age at seizure onset, or optimal level of function (NART IQ). The obese group had a greater degree of suboptimal processing speed and demonstrated a greater degree of underfunction on the Full Scale IQ (FSIQ) measure compared to the healthy weight group. Body mass index accounted for 14% of the variance in underfunction in processing speed and 10% of the variance in underfunction in FSIQ. Controlling for the effects of age, all measures of memory function were significantly correlated with BMI, with poorer scores associated with higher BMIs. SIGNIFICANCE: A small but significant proportion of the variance in memory function and intellectual underfunction in people with epilepsy is explained by BMI. Further work is needed to establish whether a reduction in BMI to within healthy limits is associated with improvements in cognitive function in this group.

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.

Epilepsy & IQ: the clinical utility of the Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV) indices in the neuropsychological assessment of people with epilepsy.

We examined Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV) General Ability Index (GAI) and Full Scale Intelligence Quotient (FSIQ) discrepancies in 100 epilepsy patients; 44% had a significant GAI > FSIQ discrepancy. GAI-FSIQ discrepancies were correlated with the number of antiepileptic drugs taken and duration of epilepsy. Individual antiepileptic drugs differentially interfere with the expression of underlying intellectual ability in this group. FSIQ may significantly underestimate levels of general intellectual ability in people with epilepsy. Inaccurate representations of FSIQ due to selective impairments in working memory and reduced processing speed obscure the contextual interpretation of performance on other neuropsychological tests, and subtle localizing and lateralizing signs may be missed as a result.

Patient journeys: the process of clinical redesign.

*Clinical process redesign is a successful improvement method that has been used to increase access to health services in 60 public hospitals across New South Wales, and at Flinders Medical Centre (FMC) in South Australia. *The method focuses on the patient journey as the primary improvement locus, and uses process mapping to identify the value-adding steps in that journey; it involves redesign teams identifying and eliminating non-value-adding steps to improve flow and reduce delays in access to emergency and elective care. *The method engages clinicians, managers, patients and carers, and delivers real gains in health care delivery. *This article outlines the clinical process redesign programs being used by NSW Health and at FMC.

Applying clinical process redesign methods to planned arrivals in New South Wales hospitals.

*Competing demands of planned and unplanned arrivals present major challenges for hospitals. *Applying clinical process redesign methods to the planned patient journey allows management to recognise the blocks and inefficiencies in the journey and facilitates the development of solutions for improvement. *Redesign of the planned patient journey in New South Wales has promoted the expansion of the extended day-only model of care, reformed the waiting times policy, standardised patient preadmission assessment and preparation, and targeted operating theatre use. *Improved performance management at Area Health Service and local facility levels has accompanied the redesign of planned arrival processes. *The results in redesign of surgery undertaken by the Area Health Services in 96 NSW hospitals have been impressive, with results within 2 years of commencing the clinical services redesign program showing: a 97% reduction in the numbers of patients in Category 1 (admission desirable within 30 days) whose surgery was overdue, from 5308 in January 2005 to 135 in June 2007; and a 99% reduction in the number of patients who have waited > 365 days for surgery, from 10 551 in January 2005 to 84 in June 2007. *Improved surgical service efficiency, safety and quality justify the continuation of the redesign program.

Health services under siege: the case for clinical process redesign.

*Public health services are struggling to cope with rising demand. *Strain on health services manifests as longer waiting lists for surgery, queuing in the emergency departments, increased rates of adverse events, and delays in discharge, particularly for older patients. *Traditional responses are not resolving these problems. *Analysis shows that the day-to-day system processes underlying clinical care are poorly designed and do not produce a well planned, well coordinated patient journey. *Numerous disconnections along the continuum of care have a cumulative effect in obstructing patient flow and causing frustration for patients and staff. *Rigorous clinical process redesign methods can significantly improve performance, even in the face of rising demand.

Implementing and sustaining transformational change in health care: lessons learnt about clinical process redesign.

*Clinical process redesign has enabled significant improvements in the delivery of health care services in emergency departments and elective surgery programs in New South Wales and at Flinders Medical Centre in South Australia, with tangible benefits for patients and staff. *The principles used in clinical process redesign are not new; they have been applied in other industries with significant gains for many years, but have only recently been introduced into health care systems. *Through experience with clinical process redesign, we have learnt much about the factors critical to the success of implementing and sustaining this process in the health care setting. *The key elements for success are leadership by senior executives, clinical leadership, team-based problem solving, a focus on the patient journey, access to data, ambitious targets, strong performance management, and a process for maintaining improvement.

Clinical process redesign for unplanned arrivals in hospitals.

*Emergency department performance had been deteriorating in NSW Health facilities and at Flinders Medical Centre before a fundamentally new approach involving a redesign method, additional bed capacity and more rigorous hospital performance management was applied. *Redesign was undertaken in over 60 hospitals in New South Wales. *Numerous disconnections and misalignments in the process of care delivery have been uncovered during the diagnostic phase of this redesign. *Solutions addressed the entire patient journey through the hospital, to produce smoother patient flow along the continuum of care. *To achieve a sustained improvement in performance, numerous solutions must be simultaneously implemented in each hospital. *With this multipronged approach, a turnaround in NSW emergency access performance has been achieved in the face of rising demand for services; the improvement has continued over 3 years. *This article reports on our findings from system-wide redesign for unplanned hospital attendances.

Large-scale gene expression analysis of cholesterol dependence in NS0 cells.

NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r). Large-scale transcriptional analyses were done using the Affymetrix GeneChip array, MG-U74Av2. The transcripts expressed differentially across the two cell lines were identified. Additionally, proteomic tools were employed to analyze cell lysates from these two cell lines. Cellular proteins from both NS0 and NS0_r were subjected to 2D gel electrophoresis. MALDI-TOF mass spectrometry was performed to determine the identity of the differentially expressed spots. We examined the expression level of mouse genes directly involved in cholesterol biosynthesis, lipid metabolism, and central energy metabolism. Most of these genes were downregulated in the revertant cell type, NS0_r, compared to NS0. Overall, a large number of genes are expressed differentially, indicating that the reversal of cholesterol dependency has a profound effect on cell physiology. It is probable that a single gene mutation, activation, or inactivation is responsible for cholesterol auxotrophy. However, the wide-ranging changes in gene expression point to the distinct possibility of a regulatory event affecting the reversibility of auxotrophy, either directly or indirectly.