Expression of microRNAs in human post-mortem amyotrophic lateral sclerosis spinal cords provides insight into disease mechanisms.

Amyotrophic lateral sclerosis is a late-onset and terminal neurodegenerative disease. The majority of cases are sporadic with unknown causes and only a small number of cases are genetically linked. Recent evidence suggests that post-transcriptional regulation and epigenetic mechanisms, such as microRNAs, underlie the onset and progression of neurodegenerative disorders; therefore, altered microRNA expression may result in the dysregulation of key genes and biological pathways that contribute to the development of sporadic amyotrophic lateral sclerosis. Using systems biology analyses on postmortem human spinal cord tissue, we identified dysregulated mature microRNAs and their potential targets previously implicated in functional process and pathways associated with the pathogenesis of ALS. Furthermore, we report a global reduction of mature microRNAs, alterations in microRNA processing, and support for a role of the nucleotide binding protein, TAR DNA binding protein 43, in regulating sporadic amyotrophic lateral sclerosis-associated microRNAs, thereby offering a potential underlying mechanism for sporadic amyotrophic lateral sclerosis.

Prevalence of founder mutations in the BRCA1 and BRCA2 genes among unaffected women from the Bahamas.

Population-based testing for BRCA1/2 mutations detects a high proportion of carriers not identified by cancer family history-based testing. We sought to determine whether population-based testing is an effective approach to genetic testing in the Bahamas, where 23% of women with breast cancer carry one of seven founder mutations in the BRCA1 or BRCA2 gene. We determined the prevalence of founder BRCA mutations in 1847 Bahamian women without a personal history of breast or ovarian cancer, unselected for age or family history. We found that 2.8% (20/705) of unaffected women with a family history of breast/ovarian cancer and 0.09% (1/1089) of unaffected women without a family history carry a BRCA mutation. A total of 38% of unaffected women with a known mutation in the family were found to carry the familial mutation. We previously suggested that all Bahamian women with breast or ovarian cancer be offered genetic testing. These current data suggest that additionally all unaffected Bahamian women with a family history of breast/ovarian cancer should be offered genetic testing for the founder BRCA mutations.

Autocrine production of IGF-I increases stem cell-mediated neuroprotection.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron (MN) loss. There are currently no effective therapies; however, cellular therapies using neural progenitor cells protect MNs and attenuate disease progression in G93A-SOD1 ALS rats. Recently, we completed a phase I clinical trial examining intraspinal human spinal stem cell (HSSC) transplantation in ALS patients which demonstrated our approach was safe and feasible, supporting the phase II trial currently in progress. In parallel, efforts focused on understanding the mechanisms underlying the preclinical benefit of HSSCs in vitro and in animal models of ALS led us to investigate how insulin-like growth factor-I (IGF-I) production contributes to cellular therapy neuroprotection. IGF-I is a potent growth factor with proven efficacy in preclinical ALS studies, and we contend that autocrine IGF-I production may enhance the salutary effects of HSSCs. By comparing the biological properties of HSSCs to HSSCs expressing sixfold higher levels of IGF-I, we demonstrate that IGF-I production augments the production of glial-derived neurotrophic factor and accelerates neurite outgrowth without adversely affecting HSSC proliferation or terminal differentiation. Furthermore, we demonstrate that increased IGF-I induces more potent MN protection from excitotoxicity via both indirect and direct mechanisms, as demonstrated using hanging inserts with primary MNs or by culturing with organotypic spinal cord slices, respectively. These findings support our theory that combining autocrine growth factor production with HSSC transplantation may offer a novel means to achieve additive neuroprotection in ALS.

Strategies for recruitment of relatives of BRCA mutation carriers to a genetic testing program in the Bahamas.

The prevalence of BRCA1 and BRCA2 mutations among unselected breast cancer patients in the Bahamas is 23%. It is beneficial to advise relatives of mutation carriers that they are candidates for genetic testing. Women who test positive are then eligible for preventive interventions, such as oophorectomy. It is not clear how often relatives of women with a mutation in the Bahamas wish to undergo genetic testing for the family mutation. Furthermore, it is not clear how best to communicate this sensitive information to relatives in order to maximize patient compliance. We offered genetic testing to 202 first-degree relatives of 58 mutation carriers. Of 159 women who were contacted by the proband or other family member, only 14 made an appointment for genetic testing (9%). In contrast, among 32 relatives who were contacted directly by the genetic counselor, 27 came for an appointment (84%). This study suggests that for recruitment of relatives in the Bahamas, direct contact by counselor is preferable to using the proband as an intermediary.

Concise review: Stem cell therapies for amyotrophic lateral sclerosis: recent advances and prospects for the future.

Amyotrophic lateral sclerosis (ALS) is a lethal disease involving the loss of motor neurons. Although the mechanisms responsible for motor neuron degeneration in ALS remain elusive, the development of stem cell-based therapies for the treatment of ALS has gained widespread support. Here, we review the types of stem cells being considered for therapeutic applications in ALS, and emphasize recent preclinical advances that provide supportive rationale for clinical translation. We also discuss early trials from around the world translating cellular therapies to ALS patients, and offer important considerations for future clinical trial design. Although clinical translation is still in its infancy, and additional insight into the mechanisms underlying therapeutic efficacy and the establishment of long-term safety are required, these studies represent an important first step toward the development of effective cellular therapies for the treatment of ALS.

The spectrum of BRCA1 and BRCA2 mutations in breast cancer patients in the Bahamas.

We sought to identify the full range of founder mutations in BRCA1 and BRCA2 in the Bahamas and to estimate the proportion of all BRCA1 and BRCA2 mutations that are accounted for by founder mutations. We studied 214 Bahamian women with invasive breast cancer, unselected for age or family history. A founder mutation had previously been identified in 49 patients. We conducted full sequencing of the BRCA1 and BRCA2 genes and multiplex ligation-dependent probe amplification (MLPA) for 156 patients. A novel founder mutation in BRCA2 (exon 17 818delA) was seen in four different patients and five other unique mutations in BRCA1 and BRCA2, including a large deletion (exons 8-9) in BRCA1. In total, a mutation was seen in 58 of the 214 patients (27%); 92% of carriers carried one of the seven founder mutations. Approximately 27% of unselected cases of breast cancer in the Bahamian population are attributable to a mutation in BRCA1 or BRCA2, a prevalence which far exceeds that of any other country. The majority of women who carry a mutation in the Bahamas, carry one of the seven founder mutations, making it possible to offer genetic testing to all women at risk for breast cancer in the Bahamas.

Validity of the brief executive-function assessment tool in an outpatient substance use disorder setting.

The Brief Executive-function Assessment Tool (BEAT) was developed and validated for use in residential substance use disorder treatment settings, where participants are mostly abstinent. It is therefore unclear whether the BEAT is valid for use in outpatient settings, where participants may be actively using substances. The effects of acute intoxication and withdrawal have the potential to alter the results of the BEAT. The current study sought to establish construct and criterion validity of the BEAT in an outpatient substance use disorder sample and to detect its sensitivity to substance use over the previous 24 hours and also over the past month. A total of 74 clients of a New South Wales-based outpatient substance use disorder service participated in the current study. Construct validity was demonstrated by significant correlations between the BEAT and three performance-based tests of executive functioning. Criterion validity was established in that the BEAT discriminated between those deemed impaired or not on a criterion composite measure of executive functioning. Test operating characteristics (88% sensitivity, 69% specificity, 44% PPV, and 95% NPV) were also established relative to this composite measure as a reference standard. The BEAT was insensitive to use/abstinence over the previous 24 hours and the past month.

Intraspinal transplantation of neurogenin-expressing stem cells generates spinal cord neural progenitors.

Embryonic stem (ES) cells and their derivatives are an important resource for developing novel cellular therapies for disease. Controlling proliferation and lineage selection, however, are essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to man. Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient to promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

Stem cell technology for neurodegenerative diseases.

Over the past 20 years, stem cell technologies have become an increasingly attractive option to investigate and treat neurodegenerative diseases. In the current review, we discuss the process of extending basic stem cell research into translational therapies for patients suffering from neurodegenerative diseases. We begin with a discussion of the burden of these diseases on society, emphasizing the need for increased attention toward advancing stem cell therapies. We then explain the various types of stem cells utilized in neurodegenerative disease research, and outline important issues to consider in the transition of stem cell therapy from bench to bedside. Finally, we detail the current progress regarding the applications of stem cell therapies to specific neurodegenerative diseases, focusing on Parkinson disease, Huntington disease, Alzheimer disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. With a greater understanding of the capacity of stem cell technologies, there is growing public hope that stem cell therapies will continue to progress into realistic and efficacious treatments for neurodegenerative diseases.

Brief executive-function assessment tool: A new cognitive impairment screening tool for alcohol and other drug services.

Accurate screening for cognitive impairment in alcohol and other drug (AOD) services would help to identify individuals who may need supports to obtain the greatest benefit from substance use disorder (SUD) treatment. At present there is no screening measure that has been developed specifically to detect cognitive impairment in a SUD population. This study examines the psychometric properties of the Brief Executive-function Assessment Tool (BEAT), which was specifically designed for this purpose. This study involving 501 individuals with SUD and 145 normal control participants established internal consistency (n = 646; 0.734), interrater (n = 60; 0.994), and test-retest reliability (n = 177; 0.845), and construct (all correlations p ≤ 0.05), and criterion (n = 467; ANCOVA p < 0.001) validity. Test operating characteristics (n = 500; 87% sensitivity, 71% specificity, 21% PPP, and 99% NPP) were also established relative to an independent criterion variable made up of three established performance-based neuropsychological tests. Findings support the reliability and validity of the BEAT as a screening measure of executive function impairment with high sensitivity and a low rate of false negatives.

Neuroprotection using gene therapy to induce vascular endothelial growth factor-A expression.

Engineered zinc-finger protein (ZFP) transcription factors induce the expression of endogenous genes and can be remotely delivered using adenoviral vectors. One such factor, Ad-32Ep65-Flag (Ad-p65), targets and induces expression of vascular endothelial growth factor (VEGF; also called VEGF-A) splice variants in their normal biological stoichiometry. We show that Ad-p65 transfection of primary motor neurons results in VEGF variant expression and a significant increase in axon outgrowth in these cells. Given the neuroprotective effects of VEGF and its ability to increase neurite outgrowth, we examined the efficacy of Ad-p65 to enhance motor neuron regeneration in vivo using rats that have undergone recurrent laryngeal nerve (RLN)-crush injury. Injection of Ad-p65 after RLN crush accelerated the return of vocal fold mobility and the percentage of nerve-endplate contacts in the thyroarytenoid muscle. Overall, adenoviral delivery of an engineered ZFP transcription factor inducing VEGF-A splice variant expression enhances nerve regeneration. ZFP transcription factor gene therapy to increase expression of the full complement of VEGF-A splice variants is a promising avenue for the treatment of nerve injury and neurodegeneration.

Stem cells: comprehensive treatments for amyotrophic lateral sclerosis in conjunction with growth factor delivery.

Amyotrophic lateral sclerosis (ALS) is characterized by loss of both upper and lower motor neurons. ALS progression is complex and likely due to cellular dysfunction at multiple levels, including mitochondrial dysfunction, glutamate excitotoxicity, oxidative stress, axonal dysfunction, reactive astrocytosis, and mutant superoxide dismutase expression, therefore, treatment must provide neuronal protection from multiple insults. A significant amount of ALS research focuses on growth factor-based therapies. Growth factors including insulin-like growth factor-I, vascular endothelial growth factor, brain-derived neurotrophic factor, and glial-derived neurotrophic factor exhibit robust neuroprotective effects on motor neurons in ALS models. Issues concerning growth factor delivery, stability and unwanted side effects slow the transfer of these treatments to human ALS patients. Stem cells represent a new therapeutic approach offering both cellular replacement and trophic support for the existing population. Combination therapy consisting of stem cells expressing beneficial growth factors may provide a comprehensive treatment for ALS.

Multisensory enhancement of attention depends on whether you are already paying attention.

Multisensory stimuli are argued to capture attention more effectively than unisensory stimuli due to their ability to elicit a super-additive neuronal response. However, behavioural evidence for enhanced multisensory attentional capture is mixed. Furthermore, the notion of multisensory enhancement of attention conflicts with findings suggesting that multisensory integration may itself be dependent upon top-down attention. The present research resolves this discrepancy by examining how both endogenous attentional settings and the availability of attentional capacity modulate capture by multisensory stimuli. Across a series of four studies, two measures of attentional capture were used which vary in their reliance on endogenous attention: facilitation and distraction. Perceptual load was additionally manipulated to determine whether multisensory stimuli are still able to capture attention when attention is occupied by a demanding primary task. Multisensory stimuli presented as search targets were consistently detected faster than unisensory stimuli regardless of perceptual load, although they are nevertheless subject to load modulation. In contrast, task irrelevant multisensory stimuli did not cause greater distraction than unisensory stimuli, suggesting that the enhanced attentional status of multisensory stimuli may be mediated by the availability of endogenous attention. Implications for multisensory alerts in practical settings such as driving and aviation are discussed, namely that these may be advantageous during demanding tasks, but may be less suitable to signaling unexpected events.

Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos.

BACKGROUND: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

Arthroscopic synovectomy, removal of loose bodies and selective biceps tenodesis for synovial chondromatosis of the shoulder.

We retrospectively identified 18 consecutive patients with synovial chrondromatosis of the shoulder who had arthroscopic treatment between 1989 and 2004. Of these, 15 were available for review at a mean follow-up of 5.3 years (2.3 to 16.5). There were seven patients with primary synovial chondromatosis, but for the remainder, the condition was a result of secondary causes. The mean Constant score showed that pain and activities of daily living were the most affected categories, being only 57% and 65% of the values of the normal side. Surgery resulted in a significant improvement in the mean Constant score in these domains from 8.9 (4 to 15) to 11.3 (2 to 15) and from 12.9 (5 to 20) to 18.7 (11 to 20), respectively (unpaired t-test, p = 0.04 and p < 0.0001, respectively). Movement and strength were not significantly affected. Osteoarthritis was present in eight patients at presentation and in 11 at the final review. Recurrence of the disease with new loose bodies occurred in two patients from the primary group at an interval of three and 12 years post-operatively. In nine patients, loose bodies were also present in the bicipital groove; seven of these underwent an open bicipital debridement and tenodesis. We found that arthroscopic debridement of the glenohumeral joint and open debridement and tenodesis of the long head of biceps, when indicated, are safe and effective in relieving symptoms at medium-term review.

Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos.

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity.

Interventions for drug-using offenders in the courts, secure establishments and the community.

BACKGROUND: Drug strategies internationally recognize link between drug use and crime. This review consider interventions for drug-using offenders under the care of the criminal justice system. OBJECTIVES: To assess the effectiveness of interventions for drug-using offenders in reducing criminal activity and drug use in the courts, secure establishments and community-based settings. SEARCH STRATEGY: Twenty two electronic databases were searched (1980 to 2004). Internet sites and experts in the field were contacted for further information. SELECTION CRITERIA: Randomised Controlled Trials designed to reduce, eliminate or prevent relapse in drug using offenders DATA COLLECTION AND ANALYSIS: Two authors independently assessed trials for inclusion. Data were extracted by one author and double checked. MAIN RESULTS: Twenty four studies, 8936 participants, met the inclusion criteria. Results show that comparing a court-based community pre-trial release with drugs testing and sanctions versus routine pre-trial, for arrest at 90 days results favoured the comparison group OR 1.33 (95% CI 1.04 to 1.70). Comparing therapeutic community with aftercare with a mental health programme with a waiting list control, considering incarceration at 12 months OR 0.37 (95% CI 0.16 to 0.87), results in favour of the treatment Comparing intensive supervision with routine parole/probation, for recidivism OR 1.98 (95% CI 1.01 to 3.87) results in favour of comparison group, no statistically significant difference between the groups for arrest OR 1.49 (95% CI 0.88 to 2.51), drug arrest OR 1.10 (95% CI 0.50 to 2.39), conviction OR 0.93 (95% CI 0.55 to 1.58 ) and incarceration at one year OR 0.88 (95% CI 0.50, 1.54). Comparing intensive supervision and increased surveillance with intensive supervision alone, no statistically significant difference between the groups for recidivism OR 2.09 (95% CI, 0.86 to 5.07), arrest OR 1.22 (95% CI 0.51 to 2.88]), drug arrest, OR 1.29 (95% CI 0.35 to 4.85), conviction OR0.1.14 (95% CI, 0.22, to 5.91) and incarceration OR 1.30 (95% CI 0.39, to 4.30]) at one year. AUTHORS' CONCLUSIONS: Limited conclusions can be drawn about the effectiveness of drug treatment programmes for drug-using offenders in the courts or the community. This is partly due to the broad range of studies and the heterogenity of the different outcome measures presented. Therapeutic communities with aftercare show promising results for the reduction of drug use and criminal activity in drug using offenders. Standardisation of outcome measures and costing methodology would help improve the quality of research conducted in the area.

Neurotensin stimulates protein kinase C-dependent mitogenic signaling in human pancreatic carcinoma cell line PANC-1.

Neuropeptides and their corresponding G protein-coupled receptors are increasingly implicated in the autocrine/paracrine stimulation of growth of human cancers. Using K-Ras mutated human pancreatic ductal adenocarcinoma cell line PANC-1 as a model system, we demonstrate that neurotensin (NT) induced translocation of phosphorylated extracellular signal-regulated kinases (ERK-1 and ERK-2) to the nucleus, rapid dose-dependent activation of dual-specificity mitogen and ERK-1 and ERK-2 kinase-1/2 (MEK-1/2), and striking stimulation of c-Raf-1 but not pan-Ras. Furthermore, treatment of PANC-1 cells with protein kinase C (PKC) inhibitors, GF-1 and Ro 31-8220, completely abrogated NT-induced ERK-1 and ERK-2 activation, and significantly attenuated NT-induced c-Raf-1 stimulation. Interestingly, NT did not stimulate epidermal growth factor receptor transactivation, and epidermal growth factor receptor tyrosine kinase or Src inhibitors did not affect NT-induced ERK activation in PANC-1 cells. Our results indicate that NT potently stimulates c-Raf-1-MEK-ERK in PANC-1 cells through a PKC-dependent signaling pathway. Furthermore, we show that NT-induced DNA synthesis in PANC-1 cells is ERK-dependent. Finally, we demonstrate that NT stimulated clonal growth of PANC-1 cells in semisolid medium, which is abrogated by both GF-1 and the MEK-1/2 inhibitor, U0126. Collectively our results suggest that PKC-mediated stimulation of ERK-1 and ERK-2 play a pivotal role in NT-induced growth of PANC-1 cells harboring activating K-Ras mutation.

Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar "best in class" cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD.

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques.

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.