Three by three weeks of robot-enhanced repetitive gait therapy within a global rehabilitation plan improves gross motor development in children with cerebral palsy - a retrospective cohort study.

AIM: To assess the improvement in gross motor function following three blocks of a three-week, intensive robot-enhanced treadmill therapy (ROBERT-Program). METHOD: retrospective chart review in a before-after interventional trial in children with cerebral palsy attending a university hospital outpatient rehabilitation centre. Patients received three blocks of a three-week, 12 sessions ROBERT-Program over a mean period of 24 months. Outcome measures were block specific and cumulative improvement in GMFM 66, D and E. Longterm GMFM 66 improvements were compared to the individuals' expected increment as derived from previously published GMFM-66 percentiles. 95% confidence intervals (CI) and paired t-test were calculated. RESULTS: 20 children (8 GMFCS Level II; 12 GMFCS Level III, mean age 5.9 years (CI: [5.0; 6.7])) were treated. For each block a significant increase in motor performance in similar size could be observed without deterioration between blocks. The cumulative improvement during 21 months observation period was: 6.5 (CI: [4.8; 8.2]) in GMFM 66, which represents a clinically meaningful effect size of 3.6 (CI: [1.4; 5.8]) above the expected improvement. INTERPRETATION: Progressive clinically meaningful improvement in motor performance for three blocks of ROBERT-Program was observed. Cumulative GMFM 66 improvements exceeded the individuals' age-specific expected course.

Stage-specific expression of the proline-alanine transporter in the human pathogen Leishmania.

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

The interaction between neurocognitive functioning, subthreshold psychotic symptoms and pharmacotherapy in 22q11.2 deletion syndrome: A longitudinal comparative study.

BACKGROUND: The 22q11.2 deletion syndrome (22q11DS) is the most common genetic syndrome associated with schizophrenia. The goal of this study was to evaluate longitudinally the interaction between neurocognitive functioning, the presence of subthreshold psychotic symptoms (SPS) and conversion to psychosis in individuals with 22q11DS. In addition, we attempted to identify the specific neurocognitive domains that predict the longitudinal evolution of positive and negative SPS, as well as the effect of psychiatric medications on 22q11DS psychiatric and cognitive developmental trajectories. METHODS: Forty-four participants with 22q11DS, 19 with Williams syndrome (WS) and 30 typically developing (TD) controls, age range 12-35years, were assessed at two time points (15.2±2.1months apart). Evaluation included the Structured Interview for Prodromal Symptoms (SIPS), structured psychiatric evaluation and the Penn Computerized Neurocognitive Battery (CNB). RESULTS: 22q11DS individuals with SPS had a yearly conversion rate to psychotic disorders of 8.8%, compared to none in both WS and TD controls. Baseline levels of negative SPS were associated with global neurocognitive performance (GNP), executive function and social cognition deficits, in individuals with 22q11DS, but not in WS. Deficits in GNP predicted negative SPS in 22q11DS and the emergence or persistence of negative SPS. 22q11DS individuals treated with psychiatric medications showed significant improvement in GNP score between baseline and follow-up assessments, an improvement that was not seen in untreated 22q11DS. CONCLUSIONS: Our results highlight the time-dependent interplay among positive and negative SPS symptoms, neurocognition and pharmacotherapy in the prediction of the evolution of psychosis in 22q11DS.

A comparative study of the neuropsychiatric and neurocognitive phenotype in two microdeletion syndromes: velocardiofacial (22q11.2 deletion) and Williams (7q11.23 deletion) syndromes.

PURPOSE: 22q11.2 deletion syndrome (22q11.2DS) and Williams syndrome (WS) are common neurogenetic microdeletion syndromes. The aim of the present study was to compare the neuropsychiatric and neurocognitive phenotypes of 22q11.2DS and WS. METHODS: Forty-five individuals with 22q11.2DS, 24 with WS, 22 with idiopathic developmental disability (DD) and 22 typically developing (TD) controls were compared for the rates of psychiatric disorders as well as cognitive executive and visuospatial functions. RESULTS: We found that while anxiety, mood and disruptive disorders had an equally high prevalence among individuals with 22q11.2DS, WS and DDs, the 22q11.2DS group had the highest rates of psychotic disorders and the WS group had the highest rates of specific phobia. We also found that the WS group demonstrated more severe impairments in both executive and visuospatial functions than the other groups. WS and 22q11.2DS subjects had worse Performance-IQ than Verbal-IQ, a feature typical of non-verbal learning disorders. CONCLUSION: These findings offer a wide perspective on unique versus common phenotypes in 22q11.2DS and WS.

On the relation between steep monoclinal flexure zones and steep hydraulic gradients.

Steep hydraulic gradients are found in association with steep monoclinal flexures. However, the physics of the reduction of the hydraulic conductivity, which is responsible for the steep gradients, has seldom been studied. We present results of hydrological and mechanical modeling aiming to study the effect of such steep hydraulic gradients demonstrated in the Judea Group Aquifer system, Israel. The hydrological configuration of steep dips and anisotropy between flows parallel and perpendicular to the bedding planes was simulated using the FEFLOW code. It exhibited a situation whereby part of the flow is oblique to the bedding planes and therefore some steepening of the hydraulic gradients occurred due to actual conductivity reduction. However, this reduction is not enough to account for the steeper gradients observed. The effect of a deep-seated reverse fault under the monocline on the permeability distribution within the structure was examined by numerical mechanical simulations. It exhibited a compressional stress distribution in the steep part of the monocline, which, due to shortening and closure of joints and voids, is presumably responsible for a significant pressure-induced permeability reduction. This process by itself in a layered structure, including interlayering of thin marl layers, could be responsible for the steep hydraulic gradients in the steep part of the monocline.

The pyruvate kinase isoenzyme M2 (Tu M2-PK) as a tumour marker for renal cell carcinoma.

The M2 isoenzyme of pyruvate kinase (M2-PK) is specially expressed by tumour cells (Tu M2-PK) and has been detected in the peripheral blood of patients with renal cell carcinoma (RCC). We analysed the benefit of using Tu M2-PK as a tumour marker for primary detection of RCC by receiver operating characteristic (ROC) analysis. The area under the curve was 0.674, and the sensitivity, specificity and positive predictive value (PPV) were 44.4%, 87.5% and 88%, respectively, at the ROC optimal cut-off of 28.2 kU/L. We examined 71 patients. Since the marker sensitivity for detection of the early stages T1 and T2 was only 47% it is not suggested to use this marker for primary diagnosis of RCC. Its use as part of the confirmatory preoperative evaluation might be considered in view of its high PPV.

Cytoskeletal responsiveness to progestins is dependent on progesterone receptor A levels.

Changes in the cell cytoskeleton occur in cell transformation and recent data suggest the involvement of ovarian hormones, which are implicated in cancer development and progression. In human breast and endometrial tumors, there is disrupted expression of progesterone receptor (PR) isoforms and predominance of one isoform, usually PRA. PRA predominance is an early event in carcinogenesis, and in cancers is associated with poor clinical features. Overexpression of PRA in vitro causes altered progestin regulation of cell morphology, suggesting that PRA overexpression may provoke deleterious changes in cell functioning. This study aimed to identify pathways of cytoskeleton regulation responsive to progestins and to determine whether these are perturbed when PRA is overexpressed to the levels seen in cancers. Progestin treatment of PR-positive breast cancer cells caused increased cell surface area whereas after induction of a stably integrated PRA construct, cells became rounded and the cell surface was decreased. The effect of PRA induction on cell rounding was reversed by the anti-progestin RU38486. Altered tropomyosin (Tm) isoforms were implicated in these morphological differences, as there was a PRA-mediated alteration in Tm5 isoform levels, and transfection of Tm5a mimicked progestin-mediated cell rounding in PRA-overexpressing cells. Ezrin was redistributed from the membrane to cytoplasmic locations in the presence of progestin, and discrete focal localization was evident in cells with PRA predominance. Progestin effects on the cytoskeleton in PRA-overexpressing cells provide evidence for novel endocrine regulation of aspects of actin microfilament composition, suggesting that changes in the cytoskeleton known to be associated with cancer development and progression may be regulated in part by altered PRA expression which develops early in carcinogenesis.

Functional analysis of the actin-binding protein, tropomyosin 1, in neuroblastoma.

Tropomyosin 1 (TM1) is downregulated in a number of transformed cell types, and exogenous expression of TM1 can restore actin organisation and reverse cellular transformation. We find that TM1 is also downregulated in human neuroblastoma cell lines, correlating with increasing malignancy. However, exogenous TM1 does not restore actin cytoskeleton organisation in neuroblastoma cells.

Capillary electrophoresis of venoms and toxins.

A review of capillary electrophoresis of venoms and toxins is presented. Emphasis is placed on the analysis of real samples in complex matrices. The structures of some of the complex toxins are presented to illustrate the remarkable diversity and complexity of these materials.

Use of dynamically coated capillaries for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine using capillary electrophoresis.

A rapid, accurate, precise, reproducible, economical, and environmentally gentle method using capillary electrophoresis (CE) is presented for the routine analysis of methamphetamine, amphetamine, MDA, MDMA, MDEA, and cocaine in seized drugs. The methodology uses a 32 cm by 50 microm capillary (length to detector 23.5 cm) with a commercially available buffer kit and diode array UV detection. Dynamic coating of the capillary surface is accomplished by flushing with base for 1 min, a proprietary polycation for 1 min, and then a proprietary polyanion for 2 min. This approach provides a relatively high and stable electroosmotic flow (EOF), even at low pHs. The background electrolyte (BGE) contains 75 mM phosphate buffer (pH 2.5) with the same polyanion as above. Using this methodology, amphetamine, methamphetamine, MDA, MDMA, MDEA, and an internal standard (n-butylamphetamine) are baseline resolved in less than 5 min. The run-to-run migration time %RSDs and peak area %RSDs are typically <0.3% and <2.1%, respectively. The day-to-day and capillary-to-capillary migration time %RSDs are <1.5% and <2.1%, respectively. The %RSDs of the relative migration times compared with the internal standard on a day-to-day and capillary-to-capillary basis are <0.2% and <0.06%, respectively. The linear dynamic range using peak areas range from 0.003 to 0.10 mg/mL. The correlation coefficients are >0.9998, with all calibration curves passing at or near the origin. Similar data are obtained for cocaine and its internal standard henyltoloxamine. None of the compounds usually encountered in illicit samples interfere with the target compound (e.g., methamphetamine and cocaine) or the internal standard. Quantitative results for synthetic mixtures and seized exhibits are in good agreement with actual values, and also with results obtained from other techniques. The relatively high EOF for the dynamically coated capillary system allows for the screening of basic, acidic, and neutral adulterants in drug seizures; identification is facilitated by the use of automated UV library searches.

Differential expression of the actin-binding proteins, alpha-actinin-2 and -3, in different species: implications for the evolution of functional redundancy.

The alpha-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of alpha-actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that alpha-actinin-3 is absent in approximately 18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true alpha-actinin-3 deficiency. Absence of alpha-actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that alpha-actinin-2 is able to compensate for alpha-actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, alpha-actinin-2 expression does not completely overlap alpha-actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken alpha-actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models.

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations.

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Tropomyosin isoform diversity and neuronal morphogenesis.

Tropomyosins (Tm) are a large family of isoforms obtained from multiple genes and by extensive alternative splicing. They bind in the alpha-helical groove of the actin filament and are therefore core components of this extensive cytoskeletal system. In non-muscle cells the Tm isoforms have been implicated in a diversity of processes including cytokinesis, vesicle transport, motility, morphogenesis and cell transformation. Using immunohistochemical localization in cultured primary cortical neurons with an antibody that potentially identifies all non-muscle TM5 gene isoforms compared with one that specifically identifies a subset of isoforms, the possibility was raised that there were considerably more isoforms derived from this gene than the four previously described. Using polymerase chain reaction (PCR) analysis we have now shown that the rat brain generates at least 10 mRNA isoforms using multiple combinations of terminal exons and two internal exons. There is extensive developmental regulation of these isoforms in the brain and there appears to be a switch in the preferential use of the two internal exons 6a to 6b from the embryonic to the adult isoforms. Specific isoforms using alternate carboxyl-terminal exons are differentially localized within the adult rat cerebellum. It is suggested that the tightly regulated spatial and temporal expression of Tm isoforms plays an important role in the development and maintenance of specific neuronal compartments. This may be achieved by isoforms providing unique structural properties to actin-based filaments within functionally distinct neuronal domains.

Structural compartments within neurons: developmentally regulated organization of microfilament isoform mRNA and protein.

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarily in vivo, has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Our in vivo and in vitro studies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and beta-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.

Distinct localizations of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialized function at cell-cell adhesions.

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.

Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development.

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.