query: Converting apoE4 to apoE3 by gene editing prevents the pathology associated with apoE4 in human iPSCderived neurons.

title: Gain of toxic Apolipoprotein E4 effects in Human iPSC-Derived Neurons Is Ameliorated by a Small-Molecule Structure Corrector

text: Efforts to develop drugs for Alzheimer's disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells that expressed apolipoprotein E4 (ApoE4), a variant of the APOE gene product and the major genetic risk factor for AD, we demonstrated that ApoE4-expressing neurons had higher levels of tau phosphorylation, unrelated to their increased production of amyloid-β (Aβ) peptides, and that they displayed GABAergic neuron degeneration. ApoE4 increased Aβ production in human, but not in mouse, neurons. Converting ApoE4 to ApoE3 by gene editing rescued these phenotypes, indicating the specific effects of ApoE4. Neurons that lacked APOE behaved similarly to those expressing ApoE3, and the introduction of ApoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from ApoE4. Treatment of ApoE4-expressing neurons with a small-molecule structure corrector ameliorated the detrimental effects, thus showing that correcting the pathogenic conformation of ApoE4 is a viable therapeutic approach for ApoE4-related AD.

title: Amyloid accumulation is a late event in sporadic Alzheimer's disease-like pathology in nontransgenic rats

text: The amyloid cascade hypothesis posits that deposition of the amyloid β (Aβ) peptide in the brain is a key event in the initiation of Alzheimer's disease (AD). Nonetheless, it now seems increasingly unlikely that amyloid toxicity is the cause of sporadic AD, which leads to cognitive decline. Here, using accelerated-senescence nontransgenic OXYS rats, we confirmed that aggregation of Aβ is a later event in AD-like pathology. We showed that an age-dependent increase in the levels of Aβ₁₋₄₂ and extracellular Aβ deposits in the brain of OXYS rats occur later than do synaptic losses, neuronal cell death, mitochondrial structural abnormalities, and hyperphosphorylation of the tau protein. We identified the variants of the genes that are strongly associated with the risk of either late-onset or early-onset AD, including App, Apoe4, Bace1, Psen1, Psen2, and Picalm. We found that in OXYS rats nonsynonymous SNPs were located only in the genes Casp3 and Sorl1. Thus, we present proof that OXYS rats may be a model of sporadic AD. It is possible that multiple age-associated pathological processes may precede the toxic amyloid accumulation, which in turn triggers the final stage of the sporadic form of AD and becomes a hallmark event of the disease.

title: CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response

text: Here, we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9. CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.

title: ALS-FUS pathology revisited: singleton FUS mutations and an unusual case with both a FUS and TARDBP mutation

text:
INTRODUCTION Mutations in the FUS gene have been shown to be a rare cause of amyotrophic lateral sclerosis (ALS-FUS) and whilst well documented clinically and genetically there have been relatively few neuropathological studies. Recent work suggested a possible correlation between pathological features such as frequency of basophilic inclusions in neurons and rate of clinical decline, other studies have revealed a discrepancy between the upper motor neuron features detected clinically and the associated pathology. The purpose of this study was to describe the pathological features associated with more recently discovered FUS mutations and reinvestigate those with well recognised mutations in an attempt to correlate the pathology with mutation and/or clinical phenotype. The brains and spinal cords of seven cases of ALS-FUS were examined neuropathologically, including cases with the newly described p. K510E mutation and a case with both a known p. P525L mutation in the FUS gene and a truncating p. Y374X mutation in the TARDBP gene.
 RESULTS The neuropathology in all cases revealed basophilic and FUS inclusions in the cord. The density and type of inclusions varied markedly between cases, but did not allow a clear correlation with clinical progression. Only one case showed significant motor cortical pathology despite the upper motor neuron clinical features being evident in 4 patients. The case with both a FUS and TARDBP mutation revealed FUS positive inclusions but no TDP-43 pathology. Instead there were unusual p62 positive, FUS negative neuronal and glial inclusions as well as dot-like neurites.
 CONCLUSIONS The study confirms cases of ALS-FUS to be mainly a lower motor neuron disease and to have pathology that does not appear to neatly correlate with clinical features or genetics. Furthermore, the case with both a FUS and TARDBP mutation reveals an intriguing pathological profile which at least in part involves a very unusual staining pattern for the ubiquitin-binding protein p62.

title: ADAR1 Forms a Complex with Dicer to Promote MicroRNA Processing and RNA-Induced Gene Silencing

text: Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.

title: Induction of human neuronal cells by defined transcription factors

text: Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

title: Direct conversion of fibroblasts to functional neurons by defined factors

text: Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

title: Multiplex genome engineering using CRISPR/Cas systems.

text: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

title: DNA Methylation in the Human Cerebral Cortex Is Dynamically Regulated throughout the Life Span and Involves Differentiated Neurons

text: The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5′ CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts—defined by chronic neurodegeneration (Alzheimer's) or lack thereof (schizophrenia)—were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK) typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase.

title: A three-dimensional human neural cell culture model of Alzheimer’s disease

text: Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

title: Endothelin-1 is induced by cytokines in human vascular smooth muscle cells: evidence for intracellular endothelin-converting enzyme.

text: Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated by the vascular wall and therefore appears to be the most important peptide involved in regulation of cardiovascular events. Many pathologic conditions are associated with elevations of ET-1 in the blood vessel wall. Because these conditions are often cytokine driven, we examined the effects of a mixture of cytokines on ET-1 production in human vascular smooth muscle cells (VSMCs) derived from internal mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs with tumor necrosis factor-alpha (10 ng/ml) and interferon-gamma (1000 U/ml) in combination for up to 48 h markedly elevated the expression of mRNA for prepro-ET-1 and the release of ET-1 into the culture medium. This cytokine-stimulated release of ET-1 was inhibited by a series of dual endothelin-converting enzyme (ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and CGS 26393, with an accompanying increase in big ET-1 release but with no effect on expression of mRNA for prepro-ET-1. These same compounds were 10 times more potent at inhibiting the conversion of exogenously applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however no ECE-1a is present in these cells. Thus VSMCs most probably contain, like endothelial cells, an intracellular ECE responsible for the endogenous synthesis of ET-1. Under the influence of pro-inflammatory mediators the vascular smooth muscle can therefore become an important site of ET-1 production, as has already been established for the dilator mediators nitric oxide, prostaglandin I2, and prostaglandin E2.

title: Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

text: Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

title: Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts

text: Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.

title: The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.

text: The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

title: In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

text: Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.

title: Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons

text: Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.

title: Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene.

text: One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

title: In vivo genome editing using Staphylococcus aureus Cas9

text: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

title: OPA1 gene therapy prevents retinal ganglion cell loss in a Dominant Optic Atrophy mouse model

text: Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.

title: Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.

text: The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.


query: Converting apoE4 to apoE3 by gene editing worsens the pathology associated with apoE4 in human iPSCderived neurons.

title: Gain of toxic Apolipoprotein E4 effects in Human iPSC-Derived Neurons Is Ameliorated by a Small-Molecule Structure Corrector

text: Efforts to develop drugs for Alzheimer's disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells that expressed apolipoprotein E4 (ApoE4), a variant of the APOE gene product and the major genetic risk factor for AD, we demonstrated that ApoE4-expressing neurons had higher levels of tau phosphorylation, unrelated to their increased production of amyloid-β (Aβ) peptides, and that they displayed GABAergic neuron degeneration. ApoE4 increased Aβ production in human, but not in mouse, neurons. Converting ApoE4 to ApoE3 by gene editing rescued these phenotypes, indicating the specific effects of ApoE4. Neurons that lacked APOE behaved similarly to those expressing ApoE3, and the introduction of ApoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from ApoE4. Treatment of ApoE4-expressing neurons with a small-molecule structure corrector ameliorated the detrimental effects, thus showing that correcting the pathogenic conformation of ApoE4 is a viable therapeutic approach for ApoE4-related AD.

title: Amyloid accumulation is a late event in sporadic Alzheimer's disease-like pathology in nontransgenic rats

text: The amyloid cascade hypothesis posits that deposition of the amyloid β (Aβ) peptide in the brain is a key event in the initiation of Alzheimer's disease (AD). Nonetheless, it now seems increasingly unlikely that amyloid toxicity is the cause of sporadic AD, which leads to cognitive decline. Here, using accelerated-senescence nontransgenic OXYS rats, we confirmed that aggregation of Aβ is a later event in AD-like pathology. We showed that an age-dependent increase in the levels of Aβ₁₋₄₂ and extracellular Aβ deposits in the brain of OXYS rats occur later than do synaptic losses, neuronal cell death, mitochondrial structural abnormalities, and hyperphosphorylation of the tau protein. We identified the variants of the genes that are strongly associated with the risk of either late-onset or early-onset AD, including App, Apoe4, Bace1, Psen1, Psen2, and Picalm. We found that in OXYS rats nonsynonymous SNPs were located only in the genes Casp3 and Sorl1. Thus, we present proof that OXYS rats may be a model of sporadic AD. It is possible that multiple age-associated pathological processes may precede the toxic amyloid accumulation, which in turn triggers the final stage of the sporadic form of AD and becomes a hallmark event of the disease.

title: ALS-FUS pathology revisited: singleton FUS mutations and an unusual case with both a FUS and TARDBP mutation

text:
INTRODUCTION Mutations in the FUS gene have been shown to be a rare cause of amyotrophic lateral sclerosis (ALS-FUS) and whilst well documented clinically and genetically there have been relatively few neuropathological studies. Recent work suggested a possible correlation between pathological features such as frequency of basophilic inclusions in neurons and rate of clinical decline, other studies have revealed a discrepancy between the upper motor neuron features detected clinically and the associated pathology. The purpose of this study was to describe the pathological features associated with more recently discovered FUS mutations and reinvestigate those with well recognised mutations in an attempt to correlate the pathology with mutation and/or clinical phenotype. The brains and spinal cords of seven cases of ALS-FUS were examined neuropathologically, including cases with the newly described p. K510E mutation and a case with both a known p. P525L mutation in the FUS gene and a truncating p. Y374X mutation in the TARDBP gene.
 RESULTS The neuropathology in all cases revealed basophilic and FUS inclusions in the cord. The density and type of inclusions varied markedly between cases, but did not allow a clear correlation with clinical progression. Only one case showed significant motor cortical pathology despite the upper motor neuron clinical features being evident in 4 patients. The case with both a FUS and TARDBP mutation revealed FUS positive inclusions but no TDP-43 pathology. Instead there were unusual p62 positive, FUS negative neuronal and glial inclusions as well as dot-like neurites.
 CONCLUSIONS The study confirms cases of ALS-FUS to be mainly a lower motor neuron disease and to have pathology that does not appear to neatly correlate with clinical features or genetics. Furthermore, the case with both a FUS and TARDBP mutation reveals an intriguing pathological profile which at least in part involves a very unusual staining pattern for the ubiquitin-binding protein p62.

title: CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response

text: Here, we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9. CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.

title: Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.

text: Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

title: Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes

text:
BACKGROUND There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton's jelly of the umbilical cord (HUMSCs), which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets.
 METHODOLOGY AND PRINCIPAL FINDINGS HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic beta-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2) in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules.
 CONCLUSIONS AND SIGNIFICANCE In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.

title: Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons.

text: Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.

title: Epidemiology, health-related quality of life and economic burden of binge eating disorder: a systematic literature review

text:
PURPOSE To perform a systematic review on the epidemiology, the health-related quality of life (HRQoL) and economic burden of binge eating disorder (BED).
 METHODS A systematic literature search of English-language articles was conducted using Medline, Embase, PsycINFO, PsycARTICLES, Academic Search Complete, CINAHL Plus, Business Source Premier and Cochrane Library. Literature search on epidemiology was limited to studies published between 2009 and 2013. Cost data were inflated and converted to 2012 US$ purchasing power parities. All of the included studies were assessed for quality.
 RESULTS Forty-nine articles were included. Data on epidemiology were reported in 31, HRQoL burden in 16, and economic burden in 7 studies. Diagnosis of BED was made using 4th Edition of The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria in 46 studies. Lifetime prevalence of BED was 1.1-1.9% in the general population (DSM-IV). BED was associated with significant impairment in aspects of HRQoL relating to both physical and mental health; the Short Form 36 Physical and Mental Component Summary mean scores varied between 31.1 to 47.3 and 32.0 to 49.8, respectively. Compared to individuals without eating disorder, BED was related to increased healthcare utilization and costs. Annual direct healthcare costs per BED patient ranged between $2,372 and $3,731.
 CONCLUSIONS BED is a serious eating disorder that impairs HRQoL and is related to increased healthcare utilization and healthcare costs. The limited literature warrants further research, especially to better understand the long-term HRQoL and economic burden of BED.


query: Cost effectiveness evaluations based on cRCT data accurately reflect prices for patients in actual clinical practice.

title: A Comparison of Cost Effectiveness Using Data from Randomized Trials or Actual Clinical Practice: Selective Cox-2 Inhibitors as an Example

text:
BACKGROUND Data on absolute risks of outcomes and patterns of drug use in cost-effectiveness analyses are often based on randomised clinical trials (RCTs). The objective of this study was to evaluate the external validity of published cost-effectiveness studies by comparing the data used in these studies (typically based on RCTs) to observational data from actual clinical practice. Selective Cox-2 inhibitors (coxibs) were used as an example.
 METHODS AND FINDINGS The UK General Practice Research Database (GPRD) was used to estimate the exposure characteristics and individual probabilities of upper gastrointestinal (GI) events during current exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) or coxibs. A basic cost-effectiveness model was developed evaluating two alternative strategies: prescription of a conventional NSAID or coxib. Outcomes included upper GI events as recorded in GPRD and hospitalisation for upper GI events recorded in the national registry of hospitalisations (Hospital Episode Statistics) linked to GPRD. Prescription costs were based on the prescribed number of tables as recorded in GPRD and the 2006 cost data from the British National Formulary. The study population included over 1 million patients prescribed conventional NSAIDs or coxibs. Only a minority of patients used the drugs long-term and daily (34.5% of conventional NSAIDs and 44.2% of coxibs), whereas coxib RCTs required daily use for at least 6-9 months. The mean cost of preventing one upper GI event as recorded in GPRD was US$104k (ranging from US$64k with long-term daily use to US$182k with intermittent use) and US$298k for hospitalizations. The mean costs (for GPRD events) over calendar time were US$58k during 1990-1993 and US$174k during 2002-2005. Using RCT data rather than GPRD data for event probabilities, the mean cost was US$16k with the VIGOR RCT and US$20k with the CLASS RCT.
 CONCLUSIONS The published cost-effectiveness analyses of coxibs lacked external validity, did not represent patients in actual clinical practice, and should not have been used to inform prescribing policies. External validity should be an explicit requirement for cost-effectiveness analyses.

title: Assessing the cost effectiveness of using prognostic biomarkers with decision models: case study in prioritising patients waiting for coronary artery surgery

text:
OBJECTIVE To determine the effectiveness and cost effectiveness of using information from circulating biomarkers to inform the prioritisation process of patients with stable angina awaiting coronary artery bypass graft surgery.
 DESIGN Decision analytical model comparing four prioritisation strategies without biomarkers (no formal prioritisation, two urgency scores, and a risk score) and three strategies based on a risk score using biomarkers: a routinely assessed biomarker (estimated glomerular filtration rate), a novel biomarker (C reactive protein), or both. The order in which to perform coronary artery bypass grafting in a cohort of patients was determined by each prioritisation strategy, and mean lifetime costs and quality adjusted life years (QALYs) were compared.
 DATA SOURCES Swedish Coronary Angiography and Angioplasty Registry (9935 patients with stable angina awaiting coronary artery bypass grafting and then followed up for cardiovascular events after the procedure for 3.8 years), and meta-analyses of prognostic effects (relative risks) of biomarkers.
 RESULTS The observed risk of cardiovascular events while on the waiting list for coronary artery bypass grafting was 3 per 10,000 patients per day within the first 90 days (184 events in 9935 patients). Using a cost effectiveness threshold of pound20,000- pound30,000 (euro22,000-euro33,000; $32,000-$48,000) per additional QALY, a prioritisation strategy using a risk score with estimated glomerular filtration rate was the most cost effective strategy (cost per additional QALY was < pound410 compared with the Ontario urgency score). The impact on population health of implementing this strategy was 800 QALYs per 100,000 patients at an additional cost of pound 245,000 to the National Health Service. The prioritisation strategy using a risk score with C reactive protein was associated with lower QALYs and higher costs compared with a risk score using estimated glomerular filtration rate.
 CONCLUSION Evaluating the cost effectiveness of prognostic biomarkers is important even when effects at an individual level are small. Formal prioritisation of patients awaiting coronary artery bypass grafting using a routinely assessed biomarker (estimated glomerular filtration rate) along with simple, routinely collected clinical information was cost effective. Prioritisation strategies based on the prognostic information conferred by C reactive protein, which is not currently measured in this context, or a combination of C reactive protein and estimated glomerular filtration rate, is unlikely to be cost effective. The widespread practice of using only implicit or informal means of clinically ordering the waiting list may be harmful and should be replaced with formal prioritisation approaches.

title: Cost-effectiveness of 10-Year Risk Thresholds for Initiation of Statin Therapy for Primary Prevention of Cardiovascular Disease.

text:
IMPORTANCE The American College of Cardiology and the American Heart Association (ACC/AHA) cholesterol treatment guidelines have wide-scale implications for treating adults without history of atherosclerotic cardiovascular disease (ASCVD) with statins.
 OBJECTIVE To estimate the cost-effectiveness of various 10-year ASCVD risk thresholds that could be used in the ACC/AHA cholesterol treatment guidelines.
 DESIGN, SETTING, AND PARTICIPANTS Microsimulation model, including lifetime time horizon, US societal perspective, 3% discount rate for costs, and health outcomes. In the model, hypothetical individuals from a representative US population aged 40 to 75 years received statin treatment, experienced ASCVD events, and died from ASCVD-related or non-ASCVD-related causes based on ASCVD natural history and statin treatment parameters. Data sources for model parameters included National Health and Nutrition Examination Surveys, large clinical trials and meta-analyses for statin benefits and treatment, and other published sources.
 MAIN OUTCOMES AND MEASURES Estimated ASCVD events prevented and incremental costs per quality-adjusted life-year (QALY) gained.
 RESULTS In the base-case scenario, the current ASCVD threshold of 7.5% or higher, which was estimated to be associated with 48% of adults treated with statins, had an incremental cost-effectiveness ratio (ICER) of $37,000/QALY compared with a 10% or higher threshold. More lenient ASCVD thresholds of 4.0% or higher (61% of adults treated) and 3.0% or higher (67% of adults treated) had ICERs of $81,000/QALY and $140,000/QALY, respectively. Shifting from a 7.5% or higher ASCVD risk threshold to a 3.0% or higher ASCVD risk threshold was estimated to be associated with an additional 161,560 cardiovascular disease events averted. Cost-effectiveness results were sensitive to changes in the disutility associated with taking a pill daily, statin price, and the risk of statin-induced diabetes. In probabilistic sensitivity analysis, there was a higher than 93% chance that the optimal ASCVD threshold was 5.0% or lower using a cost-effectiveness threshold of $100,000/QALY.
 CONCLUSIONS AND RELEVANCE In this microsimulation model of US adults aged 45 to 75 years [corrected], the current 10-year ASCVD risk threshold (≥7.5% risk threshold) used in the ACC/AHA cholesterol treatment guidelines has an acceptable cost-effectiveness profile (ICER, $37,000/QALY), but more lenient ASCVD thresholds would be optimal using cost-effectiveness thresholds of $100,000/QALY (≥4.0% risk threshold) or $150,000/QALY (≥3.0% risk threshold). The optimal ASCVD threshold was sensitive to patient preferences for taking a pill daily, changes to statin price, and the risk of statin-induced diabetes.

title: Process analysis and economic evaluation for Poly(3-hydroxybutyrate) production by fermentation

text: Abstract Several processes for the production and recovery of poly(3-hydroxybutyrate) (PHB) by Alcaligenes eutrophus, Alcaligenes latus, Methylobacterium organophilum, and recombinant Escherichia coli were designed based on the previously reported data and analyzed by computer-aided bioprocess design. PHB productivity, content, and yield significantly affected the final price of PHB. For the annual production of 2,850 tonnes of purified PHB, the process employing A. eutrophus with the recovery method of surfactant-hypochlorite digestion resulted in lowest price of PHB, $ 5.58/kg. As the production scale increased to one million tonnes per year, the price of PHB dropped to $ 4.75/kg. The cost of carbon substrate significantly affected the overall economics in large production scale. Therefore, the production cost can be considerably lowered when agricultural wastes, such as whey and molasses, are used.

title: Clinical use of bone densitometry: scientific review.

text:
CONTEXT Osteoporosis causes substantial morbidity and costs $13.8 billion annually in the United States. Measurement of bone mass by densitometry is a primary part of diagnosing osteoporosis and deciding a preventive treatment course. Bone mineral densitometry has become more widely available and commonly used in practice.
 OBJECTIVE To review evidence about the value of various clinical applications of bone densitometry.
 DATA SOURCES A MEDLINE search was performed to update previous meta-analyses of the relationship between various measurements of bone density and risk of vertebral and hip fracture. We used data from the prospective Study of Osteoporotic Fractures to estimate risk of fracture from bone density and age in postmenopausal women.    STUDY SELECTION AND DATA EXTRACTION When available, meta-analyses and systematic reviews are emphasized in the review.
 DATA SYNTHESIS Bone mineral density (BMD) predicts fracture and can be used in combination with age to estimate absolute risk of fractures in postmenopausal white women. Hip BMD predicts hip fracture more strongly than other measurements of BMD. There are insufficient data to translate BMD results into risk of fracture for men and nonwhite women. The benefits of treatments to prevent fractures depend on BMD: women with osteoporosis have a greater risk of fractures and greater benefit from treatments than women without osteoporosis.
 CONCLUSIONS Guidelines based on systematic reviews and a cost-effectiveness analysis have suggested that it is worthwhile to measure BMD in white women older than 65 years and perhaps to use risk factors to select younger postmenopausal women for densitometry. Other potential clinical applications of BMD that have not yet been adequately studied include screening men or nonwhite women, monitoring BMD in patients receiving treatment, and using BMD to identify patients who should be evaluated for secondary causes of osteoporosis.

title: Cost-effectiveness of options for the diagnosis of high blood pressure in primary care: a modelling study.

text:
BACKGROUND The diagnosis of hypertension has traditionally been based on blood-pressure measurements in the clinic, but home and ambulatory measurements better correlate with cardiovascular outcome, and ambulatory monitoring is more accurate than both clinic and home monitoring in diagnosing hypertension. We aimed to compare the cost-effectiveness of different diagnostic strategies for hypertension.
 METHODS We did a Markov model-based probabilistic cost-effectiveness analysis. We used a hypothetical primary-care population aged 40 years or older with a screening blood-pressure measurement greater than 140/90 mm Hg and risk-factor prevalence equivalent to the general population. We compared three diagnostic strategies-further blood pressure measurement in the clinic, at home, and with an ambulatory monitor-in terms of lifetime costs, quality-adjusted life years, and cost-effectiveness.
 FINDINGS Ambulatory monitoring was the most cost-effective strategy for the diagnosis of hypertension for men and women of all ages. It was cost-saving for all groups (from -£56 [95% CI -105 to -10] in men aged 75 years to -£323 [-389 to -222] in women aged 40 years) and resulted in more quality-adjusted life years for men and women older than 50 years (from 0·006 [0·000 to 0·015] for women aged 60 years to 0·022 [0·012 to 0·035] for men aged 70 years). This finding was robust when assessed with a wide range of deterministic sensitivity analyses around the base case, but was sensitive if home monitoring was judged to have equal test performance to ambulatory monitoring or if treatment was judged effective irrespective of whether an individual was hypertensive.
 INTERPRETATION Ambulatory monitoring as a diagnostic strategy for hypertension after an initial raised reading in the clinic would reduce misdiagnosis and save costs. Additional costs from ambulatory monitoring are counterbalanced by cost savings from better targeted treatment. Ambulatory monitoring is recommended for most patients before the start of antihypertensive drugs.
 FUNDING National Institute for Health Research and the National Institute for Health and Clinical Excellence.

title: Cost-effectiveness of human papillomavirus vaccination and screening in Spain.

text:
BACKGROUND In Spain, prophylactic vaccination against human papillomavirus (HPV) types 16 and 18 is being offered free-of-charge to one birth cohort of girls aged 11-14. Screening is opportunistic (annual/biannual) contributing to social and geographical disparities.
 METHODS A multi-HPV-type microsimulation model was calibrated to epidemiologic data from Spain utilising likelihood-based methods to assess the health and economic impact of adding HPV vaccination to cervical cancer screening. Strategies included (1) screening alone of women over age 25, varying frequency (every 1-5 years) and test (cytology, HPV DNA testing); (2) HPV vaccination of 11-year-old girls combined with screening. Outcomes included lifetime cancer risk, life expectancy, lifetime costs, number of clinical procedures and incremental cost-effectiveness ratios.
 RESULTS After the introduction of HPV vaccination, screening will need to continue, and strategies that incorporated HPV testing are more effective and cost-effective than those with cytology alone. For vaccinated girls, 5-year organised cytology with HPV testing as triage from ages 30 to 65 costs 24,350€ per year of life saved (YLS), assuming life-long vaccine immunity against HPV-16/18 by 3 doses with 90% coverage. Unvaccinated girls would benefit from organised cytology screening with HPV testing as triage; 5-year screening from ages 30 to 65 costs 16,060€/YLS and 4-year screening from ages 30 to 85 costs 38,250€/YLS. Interventions would be cost-effective depending on the cost-effectiveness threshold and the vaccine price.
 CONCLUSIONS In Spain, inequitable coverage and overuse of cytology make screening programmes inefficient. If high vaccination coverage among pre-adolescent girls is achieved, organised cytology screening with HPV triage starting at ages 30 to at least 65 every 4-5 years represents the best balance between costs and benefits.

title: Advantages and pitfalls of fructosamine and glycated albumin in the diagnosis and treatment of diabetes.

text: The efficient diagnosis and accurate monitoring of diabetic patients are cornerstones for reducing the risk of diabetic complications. The current diagnostic and prognostic strategies in diabetes are mainly based on two tests, plasma (or capillary) glucose and glycated hemoglobin (HbA1c). Nevertheless, these measures are not foolproof, and their clinical usefulness is biased by a number of clinical and analytical factors. The introduction of other indices of glucose homeostasis in clinical practice such as fructosamine and glycated albumin (GA) may be regarded as an attractive alternative, especially in patients in whom the measurement of HbA1c may be biased or even unreliable. These include patients with rapid changes of glucose homeostasis and larger glycemic excursions, and patients with red blood cell disorders and renal disease. According to available evidence, the overall diagnostic efficiency of GA seems superior to that of fructosamine throughout a broad range of clinical settings. The current method for measuring GA is also better standardized and less vulnerable to preanalytical variables than those used for assessing fructosamine. Additional advantages of GA over HbA1c are represented by lower reagent cost and being able to automate the GA analysis on many conventional laboratory instruments. Although further studies are needed to definitely establish that GA can complement or even replace conventional measures of glycemic control such as HbA1c, GA may help the clinical management of patients with diabetes in whom HbA1c values might be unreliable.

title: Misunderstandings in prescribing decisions in general practice: qualitative study.

text:
OBJECTIVES To identify and describe misunderstandings between patients and doctors associated with prescribing decisions in general practice.
 DESIGN Qualitative study.
 SETTING 20 general practices in the West Midlands and south east England.
 PARTICIPANTS 20 general practitioners and 35 consulting patients.
 MAIN OUTCOME MEASURES Misunderstandings between patients and doctors that have potential or actual adverse consequences for taking medicine.
 RESULTS 14 categories of misunderstanding were identified relating to patient information unknown to the doctor, doctor information unknown to the patient, conflicting information, disagreement about attribution of side effects, failure of communication about doctor's decision, and relationship factors. All the misunderstandings were associated with lack of patients' participation in the consultation in terms of the voicing of expectations and preferences or the voicing of responses to doctors' decisions and actions. They were all associated with potential or actual adverse outcomes such as non-adherence to treatment. Many were based on inaccurate guesses and assumptions. In particular doctors seemed unaware of the relevance of patients' ideas about medicines for successful prescribing.
 CONCLUSIONS Patients' participation in the consultation and the adverse consequences of lack of participation are important. The authors are developing an educational intervention that builds on these findings.

title: Do physicians follow systemic treatment and funding policy guidelines?

text:
BACKGROUND The use of bisphosphonates for the prevention of skeletal related events in women with bone metastases from breast cancer is well established. We undertook an evaluation of bisphosphonate use in clinical practice in three Canadian cancer centres. In addition we assessed whether or not physicians at these centres are following their local treatment guidelines and funding policies.
 METHODS Charts and electronic files of patients who had received either clodronate or pamidronate at any time between January 2000 and December 2001 at three Canadian cancer centres were retrospectively reviewed.
 RESULTS There has been a marked improvement in the time between the diagnosis of bone metastases and the commencement of bisphosphonates from a median of 155 days in 1998 to 24 days in 2001. However, despite a local funding policy requiring that oral clodronate be the first bisphosphonate used, this was the case in only 67% of patients. In addition, despite one centre's guidelines recommending that bisphosphonates be stopped once the patient was progressing, 90% of their patients remained on bisphosphonates until they died.
 CONCLUSIONS A considerable amount of effort is spent on the creation of "evidence based" treatment guidelines. Funding agencies develop policies based on these treatment guidelines, but often funding is more restrictive than the treatment guideline would suggest. It is clear from this review that physicians still appear to manage a substantial proportion of patients outside of funding policies, but within evidence based recommendations. Therefore, a need exists for either the creation of guidelines and policies that physicians will follow or the implementation of methods to ensure that restrictive policies are actually followed.

title: Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein(a) and Cardiovascular Disease: recent advances and future directions.

text: It has been estimated that approximately 37% of the US population judged to be at high risk for developing coronary artery disease (CAD), based on the National Cholesterol Education Program guidelines, have increased plasma lipoprotein(a) [Lp(a)], whereas Lp(a) is increased in only 14% of those judged to be at low risk. Therefore, the importance of establishing a better understanding of the relative contribution of Lp(a) to the risk burden for CAD and other forms of vascular disease, as well as the underlying mechanisms, is clearly evident. However, the structural complexity and size heterogeneity of Lp(a) have hindered the development of immunoassays to accurately measure Lp(a) concentrations in plasma. The large intermethod variation in Lp(a) values has made it difficult to compare data from different clinical studies and to achieve a uniform interpretation of clinical data. A workshop was recently convened by the National Heart, Lung, and Blood Institute (NHLBI) to evaluate our current understanding of Lp(a) as a risk factor for atherosclerotic disorders; to determine how future studies could be designed to more clearly define the extent to which, and mechanisms by which, Lp(a) participates in these processes; and to present the results of the NHLBI-supported program for the evaluation and standardization of Lp(a) immunoassays. This report includes the most recent data presented by the workshop participants and the resulting practical and research recommendations.

title: Cost effectiveness of ward based non-invasive ventilation for acute exacerbations of chronic obstructive pulmonary disease: economic analysis of randomised controlled trial.

text:
OBJECTIVE To evaluate the cost effectiveness of standard treatment with and without the addition of ward based non-invasive ventilation in patients admitted to hospital with an acute exacerbation of chronic obstructive pulmonary disease.
 DESIGN Incremental cost effectiveness analysis of a randomised controlled trial.
 SETTING Medical wards in 14 hospitals in the United Kingdom.
 PARTICIPANTS The trial comprised 236 patients admitted to hospital with an acute exacerbation of chronic obstructive pulmonary disease and mild to moderate acidosis (pH 7.25-7.35) secondary to respiratory failure. The economic analysis compared the costs of treatment that these patients received after randomisation.
 MAIN OUTCOME MEASURE Incremental cost per in-hospital death.
 RESULTS 24/118 died in the group receiving standard treatment and 12/118 in the group receiving non-invasive ventilation (P=0.05). Allocation to the group receiving non-invasive ventilation was associated with a reduction in costs of 49362 pounds sterling (78741 dollars; 73109 euros), mainly through reduced use of intensive care units. The incremental cost effectiveness ratio was -645 pounds sterling per death avoided (95% confidence interval -2310 pounds sterling to 386 pounds sterling), indicating a dominant (more effective and less costly) strategy. Modelling of these data indicates that a typical UK hospital providing a non-invasive ventilation service will avoid six deaths and three to nine admissions to intensive care units per year, with an associated cost reduction of 12000-53000 pounds sterling per year.
 CONCLUSIONS Non-invasive ventilation is a highly cost effective treatment that both reduced total costs and improved mortality in hospital.

title: Evaluating the impact of pharmacists in mental health: a systematic review.

text: Efforts to improve the outcomes of patients with mental illness often have involved incorporating the skills of a variety of health care professionals into collaborative care models. For over 30 years, clinical pharmacists have contributed to these care models in capacities ranging from educator to consultant to provider. This systematic review evaluates the quantity and quality of medical literature examining the impact of pharmacists in mental health from 1972-2003. Although we identified approximately 35 publications describing the roles of clinical pharmacists in this regard, only 16 were of sufficient scientific rigor to permit evaluation and comparison. The 16 studies were divided equally between inpatient and outpatient settings and were conducted in a variety of health care organizations (e.g., Veterans Administration, health maintenance organizations, community mental health clinics, and nursing homes). Nine of the studies examined the role of pharmacists in providing treatment recommendations and patient education, five featured pharmacists as providers (with prescriptive authority), and the remaining two described the impact pharmacists have in delivering education to the psychiatric staff. Six of the 16 studies were prospective, but only three of these incorporated a randomization procedure for patients or facilities. Collectively, the results of the 16 studies were positive, demonstrating improvements in outcomes, prescribing practices, patient satisfaction, and resource use. Unfortunately, most of the investigations were small, and significant limitations in study design limited further comparison. Given the long history and anecdotal success of pharmacists in mental health care settings, additional multicenter cost-effectiveness trials are warranted to further support the role of the psychiatric pharmacist.

title: Children's estimates of food portion size: the development and evaluation of three portion size assessment tools for use with children.

text: A number of methods have been developed to assist subjects in providing an estimate of portion size but their application in improving portion size estimation by children has not been investigated systematically. The aim was to develop portion size assessment tools for use with children and to assess the accuracy of children's estimates of portion size using the tools. The tools were food photographs, food models and an interactive portion size assessment system (IPSAS). Children (n 201), aged 4-16 years, were supplied with known quantities of food to eat, in school. Food leftovers were weighed. Children estimated the amount of each food using each tool, 24 h after consuming the food. The age-specific portion sizes represented were based on portion sizes consumed by children in a national survey. Significant differences were found between the accuracy of estimates using the three tools. Children of all ages performed well using the IPSAS and food photographs. The accuracy and precision of estimates made using the food models were poor. For all tools, estimates of the amount of food served were more accurate than estimates of the amount consumed. Issues relating to reporting of foods left over which impact on estimates of the amounts of foods actually consumed require further study. The IPSAS has shown potential for assessment of dietary intake with children. Before practical application in assessment of dietary intake of children the tool would need to be expanded to cover a wider range of foods and to be validated in a 'real-life' situation.

title: Costs and cost-effectiveness of alternative tuberculosis management strategies in South Africa--implications for policy.

text:
OBJECTIVE To conduct an economic analysis of the Hlabisa community-based directly observed therapy management strategy for tuberculosis and to project costs of three alternative strategies.
 SETTING Hlabisa health district, KwaZulu-Natal, South Africa.
 METHODS An economic analysis comparing the current tuberculosis management strategy in Hlabisa with three alternative strategies (the Hlabisa strategy prior to 1991 based on hospitalisation, the national strategy and sanatorium care) in terms of costs to both health service and patient and of cost-effectiveness.
 RESULTS The current Hlabisa strategy was the most cost-effective (R3799 per patient cured), compared with R98307 for the strategy used prior to 1991, R9940 for the national strategy, and R11145 for sanatorium care. Between 71% and 88% of treatment costs lie with the health service, and hospitalisation (R119 per day) is the most expensive item. Prolonged hospitalisation is extremely expensive, but community care is cheaper (community clinic visit, R28; community health worker visit, R7). The total cost of supervising a patient in the community under the current Hlabisa strategy was R503, equivalent to 4.2 days in hospital. Drug costs (R157) are equivalent to just 1.3 days in hospital.
 CONCLUSION Cost to both health service and patient can be substantially reduced by using community-based directly observed therapy for tuberculosis, a strategy that is cheap and cost-effective in Hlabisa. These findings have important national implications, supporting the goals of the new tuberculosis control programme.

title: How can health care organizations be reliably compared?: Lessons from a national survey of patient experience.

text:
BACKGROUND Patient experience is increasingly used to assess organizational performance, for example in public reporting or pay-for-performance schemes. Conventional approaches using 95% confidence intervals are commonly used to determine required survey samples or to report performance but these may result in unreliable organizational comparisons.
 METHODS We analyzed data from 2.2 million patients who responded to the English 2009 General Practice Patient Survey, which included 45 patient experience questions nested within 6 different care domains (access, continuity of care, communication, anticipatory care planning, out-of-hours care, and overall care satisfaction). For each question, unadjusted and case-mix adjusted (for age, sex, and ethnicity) organization-level reliability, and intraclass correlation coefficients were calculated.
 RESULTS Mean responses per organization ranged from 23 to 256 for questions evaluating primary care practices, and from 1454 to 2758 for questions evaluating out-of-hours care organizations. Adjusted and unadjusted reliability values were similar. Twenty-six questions had excellent reliability (≥0.90). Seven nurse communication questions had very good reliability (≥0.85), but 3 anticipatory care planning questions had lower reliability (<0.70). Reliability was typically <0.70 for questions with <100 mean responses per practice, usually indicating questions which only a subset of patients were eligible to answer. Nine questions had both excellent reliability and high intraclass correlation coefficients (≥0.10) indicating both reliable measurement and substantial performance variability.
 CONCLUSIONS High reliability is a necessary property of indicators used to compare health care organizations. Using the English General Practice Patient Survey as a case study, we show how reliability and intraclass correlation coefficients can be used to select measures to support robust organizational comparisons, and to design surveys that will both provide high-quality measurement and optimize survey costs.

title: The Impact of the New WHO Antiretroviral Treatment Guidelines on HIV Epidemic Dynamics and Cost in South Africa

text:
BACKGROUND Since November 2009, WHO recommends that adults infected with HIV should initiate antiretroviral therapy (ART) at CD4+ cell counts of ≤350 cells/µl rather than ≤200 cells/µl. South Africa decided to adopt this strategy for pregnant and TB co-infected patients only. We estimated the impact of fully adopting the new WHO guidelines on HIV epidemic dynamics and associated costs.
 METHODS AND FINDING We used an established model of the transmission and control of HIV in specified sexual networks and healthcare settings. We quantified the model to represent Hlabisa subdistrict, KwaZulu-Natal, South Africa. We predicted the HIV epidemic dynamics, number on ART and program costs under the new guidelines relative to treating patients at ≤200 cells/µl for the next 30 years. During the first five years, the new WHO treatment guidelines require about 7% extra annual investments, whereas 28% more patients receive treatment. Furthermore, there will be a more profound impact on HIV incidence, leading to relatively less annual costs after seven years. The resulting cumulative net costs reach a break-even point after on average 16 years.
 CONCLUSIONS Our study strengthens the WHO recommendation of starting ART at ≤350 cells/µl for all HIV-infected patients. Apart from the benefits associated with many life-years saved, a modest frontloading appears to lead to net savings within a limited time-horizon. This finding is robust to alternative assumptions and foreseeable changes in ART prices and effectiveness. Therefore, South Africa should aim at rapidly expanding its healthcare infrastructure to fully embrace the new WHO guidelines.

title: Physicians' initial management of acute low back pain versus evidence-based guidelines. Influence of sciatica.

text:
BACKGROUND Little information is available on physician characteristics and patient presentations that may influence compliance with evidence-based guidelines for acute low back pain.
 OBJECTIVE To assess whether physicians' management decisions are consistent with the Agency for Health Research Quality's guideline and whether responses varied with the presentation of sciatica or by physician characteristics.
 DESIGN Cross-sectional study using a mailed survey.
 PARTICIPANTS Participants were randomly selected from internal medicine, family practice, general practice, emergency medicine, and occupational medicine specialties.
 MEASUREMENTS A questionnaire asked for recommendations for 2 case scenarios, representing patients without and with sciatica, respectively.
 RESULTS Seven hundred and twenty surveys were completed (response rate=25%). In cases 1 (without sciatica) and 2 (with sciatica), 26.9% and 4.3% of physicians fully complied with the guideline, respectively. For each year in practice, the odds of guideline noncompliance increased 1.03 times (95% confidence interval [CI]=1.01 to 1.05) for case 1. With occupational medicine as the referent specialty, general practice had the greatest odds of noncompliance (3.60, 95% CI=1.75 to 7.40) in case 1, followed by internal medicine and emergency medicine. Results for case 2 reflected the influence of sciatica with internal medicine having substantially higher odds (vs case 1) and the greatest odds of noncompliance of any specialty (6.93, 95% CI=1.47 to 32.78), followed by family practice and emergency medicine.
 CONCLUSIONS A majority of primary care physicians continue to be noncompliant with evidence-based back pain guidelines. Sciatica dramatically influenced clinical decision-making, increasing the extent of noncompliance, particularly for internal medicine and family practice. Physicians' misunderstanding of sciatica's natural history and belief that more intensive initial management is indicated may be factors underlying the observed influence of sciatica.

title: Interventions by pharmacists in out-patient pharmaceutical care.

text: Interventions by the pharmacists have always been considered as a valuable input by the health care community in the patient care process by reducing the medication errors, rationalizing the therapy and reducing the cost of therapy. The primary objective of this study was to determine the number and types of medication errors intervened by the dispensing pharmacists at OPD pharmacy in the Khoula Hospital during 2009 retrospectively. The interventions filed by the pharmacists and assistant pharmacists in OPD pharmacy were collected. Then they were categorized and analyzed after a detailed review. The results show that 72.3% of the interventions were minor of which 40.5% were about change medication order. Comparatively more numbers of prescriptions were intervened in female patients than male patients. 98.2% of the interventions were accepted by the prescribers reflecting the awareness of the doctors about the importance of the pharmacy practice. In this study only 688 interventions were due to prescribing errors of which 40.5% interventions were done in changing the medication order of clarifying the medicine. 14.9% of the interventions were related to administrative issues, 8.7% of the interventions were related to selection of medications as well as errors due to ignorance of history of patients. 8.2% of the interventions were to address the overdose of medications. Moderately significant interventions were observed in 19.4% and 7.5% of them were having the impact on major medication errors. Pharmacists have intervened 20.8% of the prescriptions to prevent complications, 25.1% were to rationalize the treatment, 7.9% of them were to improve compliance. Based on the results we conclude that the role of pharmacist in improving the health care system is vital. We recommend more number of such research based studies to bring awareness among health care professionals, provide solution to the prescription and dispensing problems, as it can also improve the documentation system, emphasize the importance of it, reduce prescribing errors, and update the knowledge of pharmacists and other health care professionals.

title: Cannabinoids in Clinical Practice

text: Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (Δ9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties ofTHC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapy-induced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.