Caregiver's perception of epilepsy treatment, quality of life and comorbidities in an international cohort of CDKL5 patients.

OBJECTIVE: CDKL5 is a genetic condition associated with drug-resistant epilepsy and intellectual disability. There is limited information on its natural history. We investigated the natural history, complications, and the effectiveness of current treatment strategies. METHODS: This study was conducted in conjunction with the CDKL5-UK Charity, with patients recruited from the USA and Europe. Online questionnaires were completed by parents/carers and included information relating to demographics, growth, development, epilepsy, comorbid conditions, and efficacy and side effects of antiepileptic treatments. RESULTS: Thirty-nine of the 44 patients were female. Median age was five years (range five months to 31 years), and all had a history of epilepsy. All patients had developmental delay, with 4/21 able to run and 4/22 able to climb. Gastrointestinal problems were reported in 31/43. Cardiac arrhythmia was seen in 11/29. Over one-quarter of the patients had tried ten or more antiepileptic medications. Vigabatrin was reportedly the most effective AED (antiepileptic drug) in 12/23; clobazam (most effective in 6/14); sodium valproate (most effective in 5/27), and levetiracetam (most effective in 3/27). VNS (Vagal Nerve Stimulator) was reported to be effective in 9/12. One year after VNS insertion, 9/12 reported improved (QoL), and there were improvements in mood, school achievement and concentration in (9/11). The ketogenic diet was considered effective and to have improved QoL in (12/23). CONCLUSION: Vigabatrin appears to be more effective than other AEDs. VNS and ketogenic diet are also relatively effective. Gastrointestinal and cardiovascular system complications are common. The results may help to guide management of epilepsy in CDKL5. It highlights a possible link between CDKL5 and potentially treatable life-threatening complications such as cardiac arrhythmia. More research in this area may help us develop a more systematic approach to treating these patients. HIPPOKRATIA 2017, 21(3): 130-135.

Evidence of paternal nutrient provisioning to embryos in broad-nosed pipefish Syngnathus typhle.

In two experiments, radioactively labelled nutrients (either (3)H-labelled amino-acid mixture or (14)C-labelled glucose) were tube-fed to brooding male Syngnathus typhle. Both nutrients were taken up by the males and radioactivity generally increased in the brood pouch tissue with time. Furthermore, a low but significant increase of (3)H-labelled amino acids in embryos was found over the experimental interval (48 h), whereas in the (14)C-glucose experiment the radioactivity was taken up by the embryos but did not increase over the experimental time (320 min). Uptake of radioisotopes per embryo did not differ with embryo size. A higher uptake mg(-1) tissue of both (3)H-labelled amino acids and (14)C-labelled glucose was found in smaller embryos, possibly due to a higher relative metabolic rate or to a higher surface-area-to-volume ratio compared to larger embryos. Uptake in embryos was not influenced by male size, embryonic developmental advancement or position in the brood pouch. It is concluded that brooding males provide amino acids, and probably also glucose, to the developing embryos in the brood pouch.

Genetic networks of cooperative redox regulation of osteopontin.

Osteopontin is a primary cytokine and matrix-associated protein involved in medial thickening and neointima formation. Osteopontin binds integrin receptors, activates cell migration and matrix metalloproteinases, and mediates arteriosclerotic lesion formation and vessel calcification. To understand the complex biology of osteopontin, computational methodology was employed to identify sets of genes whose transcriptional states were predictive of osteopontin gene expression based on the transcriptional states of 12,400 genes and ESTs across 235 independent Affymetrix Murine Genome Array MG_U74Av2 hybridizations. Arginase [GenBank: U51805] and Mac-2 antigen [GenBank: X16834] were identified as primary attractors within the gene-gene interaction network of osteopontin. Resolution of molecular interactions among these genes indicated that the majority of predictor genes could be linked through redox regulated transcription by nuclear factor kappa-B and transforming growth factor beta inducible early gene 1 regulatory elements. Subsequent molecular analyses established redox sensitivity of a 200 bp region within the 5' UTR of opn promoter and implicated nuclear factor kappa-B and transforming growth factor beta inducible early gene 1 cis-acting elements in the regulation of osteopontin.

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal.

Parafibromin is a nuclear protein with a tumour suppressor role in the development of non-hereditary and hereditary parathyroid carcinomas, and the hyperparathyroidism-jaw tumour (HPT-JT) syndrome, which is associated with renal and uterine tumours. Nuclear localization signal(s), (NLS(s)), of the 61 kDa parafibromin remain to be defined. Utilization of computer-prediction programmes, identified five NLSs (three bipartite (BP) and two monopartite (MP)). To investigate their functionality, wild-type (WT) and mutant parafibromin constructs tagged with enhanced green fluorescent protein or cMyc were transiently expressed in COS-7 cells, or human embryonic kidney 293 (HEK293) cells, and their subcellular locations determined by confocal fluorescence microscopy. Western blot analyses of nuclear and cytoplasmic fractions from the transfected cells were also performed. WT parafibromin localized to the nucleus and deletions or mutations of the three predicted BP and one of the predicted MP NLSs did not affect this localization. In contrast, deletions or mutations of a MP NLS, at residues 136-139, resulted in loss of nuclear localization. Furthermore, the critical basic residues, KKXR, of this MP NLS were found to be evolutionarily conserved, and over 60% of all parafibromin mutations lead to a loss of this NLS. Thus, an important functional domain of parafibromin, consisting of an evolutionarily conserved MP NLS, has been identified.

Novel genomic targets in oxidant-induced vascular injury.

To study the complex interaction between oxidative injury and the pathogenesis of vascular disease, vascular gene expression was examined in male Sprague-Dawley rats given 35 or 70 mg/kg allylamine, a synthetic amine converted to acrolein and hydrogen peroxide within the vascular wall. Vascular lesions and extensive vascular remodeling, coupled to increased production of 8-epi-PGF2alpha, nuclear localization of NFkappaB, and alterations in glutathione homeostasis, were observed in animals treated with allylamine for up to 20 days. Transcriptional profiling, immunohistochemistry, and in situ hybridization showed that genes involved in adhesion and extracellular matrix (ECM) (alpha(1) integrin, collagen), cytoskeletal rearrangements (alpha-smooth muscle actin, alpha-tropomyosin), and signal transduction (NFkappaB, osteopontin, and LINE) were altered by oxidant treatment. To evaluate mechanisms of gene dysregulation, cultured aortic smooth muscle cells were challenged with allylamine or its metabolites and processed for molecular analysis. These agents increased formation of reactive oxygen species and elicited changes in gene expression similar to those observed in vivo. Oxidative stress and changes in gene expression were inhibited by N-acetyl cysteine, a precursor of glutathione. These results indicate that genes along the ECM-integrin-cytoskeletal axis, in addition to LINE, are molecular targets in oxidant-induced vascular injury.

Melanocortin-4 receptor in sheep: a potential site for therapeutic intervention in disease models.

Reduced appetite combined with increased metabolic rate and decreased lean body mass is a major consequence of disease and other stressors. Studies in rodent species suggest that an understanding of appetite regulation may provide methodologies for intervention to prevent the deterioration of body mass such as observed with cancer or infectious diseases. For example, melanocortin-4 receptor (MC4-R) antagonists have shown a remarkable ability to reverse or prevent cachexia in rodents with sarcoma or treated with endotoxin. Studies in sheep have indicated that a number of peptide neurotransmitters may have a role in regulating appetite in this species. For example, agouti related protein mRNA and protein levels are dramatically altered with fasting in sheep. Moreover, agouti related protein, neuropeptide Y, melanin concentrating hormone and orexin are potent stimuli to increase feed intake in sheep. Recent studies have indicated that one of these neurotransmitters, NPY, can work in principal to improve appetite in endotoxin-treated sheep. Current studies are examining the role that MC4-R antagonists may have in the prevention or correction of body mass wasting diseases as well as practical applications in animal production.

Elevated 80K-H protein in breast cancer: a role for FGF-1 stimulation of 80K-H.

An increase in fibroblast growth factor-1 (FGF-1) is established as part of the cause of several important cancers including breast cancer, but the mechanisms by which it induces malignant behavior are not known. We now report that the protein 80K-H, a substrate for PKC, appears to be part of this mechanism and that it is increased in breast cancer and localizes to the nucleus as part of the mechanism. Our conclusion is based on an examination of a total of 58 biopsy specimens from human breast cancer patients for the presence of relationships between the 80K-H protein and the following: fibroblast growth factor receptor-1 (FGFR-1), tumor grade, microvessel counts (MVC), estrogen receptor (ER) and progesterone receptor (PgR) status. Based on histological grading and immunohistochemical (IHC) assays, we found strong direct relationships between 80K-H and FGFR-1 (r = 0.49, p = 0.003) and tumor grade (r = 0.42, p = 0.006). A trend for a direct relationship was observed with PgR (r=0.27, p=0.087). Notably, 80K-H immunostaining was largely limited to the epithelial cells of the mammary ducts. Subsequently, we studied the effects of FGF-1 on 80K-H in cultured human mammary carcinoma epithelial cells in order to establish a more direct relationship between these two molecules. We observed that FGF-1 treatment of MCF-7 cells stimulated translocation of 80K-H protein to the cell nucleus, as demonstrated by subcellular fractionation studies. Maximal intranuclear 80K-H was observed approximately 30 minutes following FGF-1 treatment. In addition, FGF-1 treatment of MCF-7 cells increased growth and invasion of MCF-7 cells, as demonstrated by cell proliferation and a modified Boyden chamber assay, respectively. Further support for 80K-H nuclearization was provided by the immunostaining of human breast cancer specimens and computer-assisted identification of a putative nuclear localization signal (NLS) near the amino terminus of 80K-H protein structure. These data support the existence of a previously unrecognized FGF-1/80K-H nuclear pathway in progression of human breast cancer and suggest that 80K-H may be useful for the assessment of breast tumor progression.

Are cardiac transplant patients more likely to have periodontitis? A case record study.

In several large epidemiological studies chronic periodontitis has been implicated as an additional risk factor, independent of other risk factors, for the development of ischaemic heart disease. The underlying mechanism is thought to be a localised infection giving rise to an inflammatory host response, and some experimental data agree with this hypothesis. Recently, however, some studies have questioned the post dated relationship between the two diseases. The current case-record study was undertaken to evaluate the prevalence of chronic periodontitis and the severity of such periodontal disease in a heart transplant population, assuming the latter represented a relatively severely compromised cardiovascular patient population. The study demonstrated that 76% of the patients had various degrees of periodontal disease prior to undergoing a heart transplant. Thus, it is possible that a relationship between cardiovascular disease and periodontal disease exists, but further, large intervention studies will be needed to confirm such a conclusion.

Identification and pharmacological correction of a membrane trafficking defect associated with a mutation in the sulfonylurea receptor causing familial hyperinsulinism.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a genetic disorder characterized by excess secretion of insulin and hypoglycemia. In most patients, the disease is caused by mutations in sulfonylurea receptor-1 (SUR1), which, in association with Kir6.2, constitutes the functional ATP-sensitive potassium (K(ATP)) channel of the pancreatic beta-cell. Previous studies reported that coexpression of the PHHI mutant R1394H-SUR1 with Kir6.2 in COS cells produces no functional channels. To investigate if the loss of function could be due to impaired trafficking of mutant channels to the cell membrane, we have cotransfected wild-type and mutant SUR1 subunits with Kir6.2 into HEK293 cells and examined their cellular localization by immunofluorescent staining. Our results show that unlike the wild-type subunits, which showed fluorescence at the cell surface, the mutant subunits displayed fluorescence in punctate structures. Co-immunostaining with antibodies against organelle-specific marker proteins identified these structures as the trans-Golgi network. Limited localization in clathrin-positive, but transferrin receptor-negative vesicles was also observed. The post-endoplasmic reticulum localization suggests that the mutation does not impair the folding and assembly of the channels so as to cause its retention by the endoplasmic reticulum. Diazoxide, a K(ATP) channel opener drug that is used in the treatment of PHHI, restored the surface expression in a manner that could be prevented by the channel blocker glibenclamide. When expressed in Xenopus oocytes, R1394H-SUR1 formed functional channels with Kir6.2, indicating that the primary consequence of the mutation is impairment of trafficking rather than function. Thus, our data uncover a novel mechanism underlying the therapeutic action of diazoxide in the treatment of PHHI, i.e. its ability to recruit channels to the membrane. Furthermore, this is the first report to describe a trafficking disorder effecting retention of mutant proteins in the trans-Golgi network.

Is dosage of physiotherapy a critical factor in deciding patterns of recovery from stroke: a pragmatic randomized controlled trial.

BACKGROUND AND PURPOSE: The best treatment and management of stroke patients has been shown to be in stroke units by multidisciplinary rehabilitation teams. Since the composition of stroke units differs it is important to know the extent to which the different components contribute to this results. Physiotherapy is one component of most rehabilitation teams and recent systematic reviews have shown that patients with stroke receiving more physiotherapy achieve more recovery from disability. However, information about the actual amounts of physiotherapy needed to achieve this result is not known. METHOD: A pragmatic, randomized, single-blind, controlled trial comparing recovery from disability in subjects receiving the current standard amount of 30 minutes' physiotherapy with those receiving double that amount (60 minutes). The study included measures of physical performance and function, psychological aspects of anxiety and depression, and perceived control over recovery. RESULTS: Some 114 subjects were recruited to the study; full six-week data are available for 104 subjects and six-month data for 93 subjects. Comparison of initial to six-week difference scores in the control and intervention groups of the whole sample did not show a significant difference. Scrutiny of the recovery curves of the whole sample showed that, in half the sample, three distinct patterns of recovery were demonstrated. CONCLUSION: These results suggest that doubling the physiotherapy time available for patients in a stroke unit will not provide a measurable benefit for all patients. The subgroup analysis of patterns of recovery must be regarded as speculative, but provides the basis for hypotheses about those likely to respond well to more intensive therapy.

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration.

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.

Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A.

1. A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced. 2. Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone. 3. K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 microM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 microM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected. 4. The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels.

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta.

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.

The legacy of being a research subject: follow-up studies of participants in therapy research.

BACKGROUND AND PURPOSE: An increase in research activity in physical therapy has led to a parallel increase in the numbers of patients, colleagues and members of the general public being used as subjects. Safeguards on the ethical aspects of research have been monitored through ethics committees whose task is to protect the interests of subjects. However, proposals are subject to ethical scrutiny prior to commencement of the research, before subjects have given consent. It is unusual for further monitoring to take place once the study is underway or, indeed, after it has finished. Few researchers have reported carrying out follow-up studies of their subjects, therefore the ethical effect of research on subjects is not known. Our interest was in reports of subjects who had previously been research subjects. METHOD: Follow-up studies were carried out on 156 subjects who had participated in research interviews. Phase 1 of the study included subjects with physical disabilities who lived in residential care (Rs subject group) (Barnitt and Canter, 1982). Phase 2 of the study comprised two research projects, (A) and (B), where subjects were physical therapists and occupational therapists (Ts subject group) (Barnitt, 1993; Barnitt and Partridge, 1997). Data were collected from the Rs subject group through visits, whereas the Ts subject group was sent a letter and short questionnaire three months after completion of an interview to elicit their views on the experience. RESULTS: Eighty-seven subjects responded to the approach, a response rate of 56%. Despite having consented to taking part in research, a number of subjects later had concerns about their involvement. These concerns included worries about confidentiality; expectations that had not been met, anger, disappointment, and loss of face. Other subjects reported positive outcomes from the experience. CONCLUSION: Researchers should consider including subject follow-up in their research design, particularly where sensitive research topics are being studied.