Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Correction to: Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

In the original publication of the article, some symbols in Figure 3 were not correctly aligned with the image.

Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

DLK1 and DLK2 are transmembrane proteins belonging to the EGF-like repeat-containing family that function as non-canonical NOTCH inhibitory ligands. DLK1 is usually downregulated after embryo development and its distribution in some adult and embryonic tissues has been described. However, the expression and role of DLK2 in embryo and adult tissues remains unclear. To better understand the relevance of both proteins during embryo development, we analyzed the expression pattern of DLK1 and DLK2 in 16.5-day-old mouse embryos (E16.5) and evaluated the possible relationship between these two proteins in embryo tissues and cell types. We found that DLK1 and DLK2 proteins exhibited a broad distribution pattern, which was detected in developing mouse organs from each of the three germ layers: ectoderm (brain, salivary glands), mesoderm (skeletal muscle, vertebral column, kidney, cartilage), and endoderm (thymus, lung, pancreas, intestine, liver). The expression pattern of DLK1 and DLK2 indicates that both proteins could play a synergistic role during cell differentiation. This study provides additional information for understanding temporal and site-specific effects of DLK1 and DLK2 during embryo morphogenesis and cell differentiation.

Presentación inusual de pielonefritis enfisematosa con extensión escrotal y pierna / Unusual presentation of emphysematous pyelonephritis with extension to the scrotum and leg

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A preliminary survey of Chlamydia psittaci genotypes from native and introduced birds in New Zealand.

AIM: To describe the Chlamydia psittaci genotypes in samples from native and introduced birds from New Zealand by analysis of the sequence variation of the ompA gene. METHODS: DNA was extracted from samples collected from a non-random sample of birds; either swabs from live asymptomatic birds or birds with clinical signs, or formalin-fixed, paraffin-embedded (FFPE) samples from historical post-mortem cases. The presence of C. psittaci in all samples had been confirmed using a quantitative PCR assay. The C. psittaci ompA gene was amplified and sequenced from samples from 26 native and introduced infected birds comprising 12 different species. These sequences were compared to published available C. psittaci genotypes. RESULTS: Genotypes A and C of C. psittaci were identified in the samples. Genotype A was identified in samples from nine birds, including various native and introduced species. Genotype C was identified in samples from 16 different waterfowl species, and a mixed infection of both genotypes was found in a kaka (Nestor meridionalis). In native birds, C. psittaci infection was confirmed in seven new host species. CONCLUSIONS AND CLINICAL RELEVANCE: Two genotypes (A and C) of C. psittaci were found in samples from a wider range of both native and introduced species of birds in New Zealand than previously reported. Both genotypes have been globally associated with significant disease in birds and humans. These initial results suggest the host range of C. psittaci in New Zealand birds is under-reported. However, the prevalence of C. psittaci infection in New Zealand, and the associated impact on avian and public health, remains to be determined. There are biosecurity implications associated with the importation of birds to New Zealand if there is a limited diversity of C. psittaci genotypes present.

Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice.

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Effectiveness and toxicity of helical tomotherapy for patients with locally recurrent nasopharyngeal carcinoma.

PURPOSE: We assessed therapeutic outcomes of reirradiation with helical tomotherapy (HT) for locoregional recurrent nasopharyngeal carcinoma (LRNPC) patients. METHODS AND MATERIALS: Treatment outcomes were evaluated retrospectively in 17 consecutive LRNPC patients receiving HT between 2006 and 2012. Median age was 57 years and most patients (n = 13) were male. Simultaneous systemic therapy was applied in 5 patients. Initial treatment covered the gross tumor volume with a median dose of 70 Gy (60-81.6 Gy). Reirradiation was confined to the local relapse region with a median dose of 63 Gy (50-70.2 Gy), resulting in a median cumulative dose of 134 Gy (122-148.2 Gy). The median time interval between initial and subsequent treatment was 42 months (11-126). RESULTS: The median follow-up for the entire cohort was 23 and 35 months for survivors. Three patients (18 %) developed both local and distant recurrences and only one patient (6 %) suffered from isolated local recurrence. Two-year actuarial DFS and LC rates were 74 and 82 %, respectively. Two-year OS rate was 79 %. Acute and late grade 2 toxicities were observed in 8 patients (47 %). No patient experienced late grade ≥3 toxicity. Late toxicity included fibrosis of skin, hypoacusia, dysphagia, and xerostomia. Patients with higher Karnofsky performance status scores associated with a lower risk of mortality (HR 0.85, p = 0.015). CONCLUSION: Reirradiation with HT in patients with LRNPC is feasible and yields encouraging results in terms of local control and overall survival with acceptable toxicity.

Blood puncture as a nondestructive sampling tool to obtain DNA in frogs: comparison of protocols and survival analysis.

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals.

SU-E-T-305: Limitations of Using DICOM Data for BrachyVision Treatment Plan Evaluation.

PURPOSE: To evaluate the accuracy of a real-time automated method of performing dosimetric quality assurance using Eclipse DICOM files for patients receiving HDR-brachytherapy and IMRT. METHODS: GYN patients are treated with concurrent high-dose rate brachtherapy and IMRT. The dosimetric parameters were obtained through an in-house QA program developed using Matlab. The DICOM files containing DVH data for organsat-risk (OAR) were analyzed Dosimetric data for 7 patients (total 42 fractions) were collected for bladder, rectum and sigmoid. The accuracy of the dosimetric parameters was estimated by comparing the parameters obtained from the DICOM based QA program and those in BrachyVision. RESULTS: The maximal dose values (Dmax) for the OARs obtained using the DICOM-based program are significantly smaller than those valued reported in BrachyVision by 36.2%-48.3%. The mean dose has a deviation from 1% - 2.4%. The dose for the volume of 2cc (D2cc) has a difference up to 7.6% for structures with the volume larger than 200 cc. The average difference of D2cc is 0.5% for structures less than 200 cc. We found that Eclipse BrachyVision only exports DVH data down to a volume equivalent to 1% of the maximum volume for a given structure. Therefore, the reported maximal dose values obtained from DICOM RT dose file do not accurately reflect the maximum dose in a treatment plan. This will also slightly affect the mean dose calculation and D2cc when the structure volume is larger than 200cc. CONCLUSIONS: The automatic QA tool based on DICOM files provides a quick retrieval of dose to organs-at-risk and coverage of targets. However, maximal dose to structures is not accurate due to the truncationof the DVH information contained in DICOM files.

Serum selenium and glutathione peroxidase concentrations in type 2 diabetes mellitus patients.

AIMS: Antioxidant selenium (Se) properties and, its protective role against oxidative damage play an important role in diabetic complications. Our objective was to gain further insight on a link between selenium status and diabetic nephropathy. METHODS: We assessed glutathione peroxidase (GPx) and Se in type 2 diabetes mellitus patients with microalbuminuria (MA) (group 1), without microalbuminuria (group 2), and in control subjects (group 3). Glucose, urea, creatinine and glycated hemoglobin tests were tested in sera. A complete clinical record was elaborated. RESULTS: For diabetic patients both, the time from diagnosis and plasma glucose concentration were higher in group 1 as compared to group 2. Control group showed higher serum Se concentrations as compared to the diabetic groups. The two groups of diabetic patients showed similar serum Se levels. Serum concentration of GPx was significantly lower in group 1 as compared to groups 2 and 3. Microalbuminuria (MA) test showed a positive correlation with glucose, and a negative relationship with serum Se and GPx. Multiple regression revealed an inverse relationship between selenium or GPx in serum and the results of the MA test. CONCLUSIONS: Our results suggest that lower Se and GPx levels in diabetic patients may be implicated in the diabetic nephropathy.

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression.

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.

Principles of homeostasis in governing virus activation and latency.

The goal of our work is to understand, from the molecular to the organismal level, the principles that drive and sustain lifelong infection by viruses. These infectious agents live in a dynamic equilibrium (homeostasis) with their hosts in which both immune and nonimmune pathways contribute to viral homeostasis. Disruption of these pathways can have dramatic consequences on pathogenesis. Immune responses to infection provide a vital countermeasure by the host but are nonsterilizing. They effect an essential and primary control mechanism for viral growth. Essential nonimmune pathways for effecting control of a viral life cycle relate to the obligate dependency of the virus on its host. For these reasons, we view infections as a highly dynamic interplay that takes place between the pathogen and host. This, in many cases, leads to the establishment of an incurable lifelong infection that remains benign but can become life threatening once key homeostatic pathways are disrupted. We discuss these issues in the context of our studies using cytomegalovirus as a clinically relevant pathogen.

Elevated expression of Ets2 or distinct portions of Ets2 can reverse Ras-mediated cellular transformation.

Ets transcription factors are important downstream targets of oncogenic Ras. The transcriptional activity of several Ets family members is regulated by Ras, and interfering with Ets-dependent transcription by expression of just the Ets2 DNA binding domain can inhibit or reverse Ras-mediated cellular transformation. To better understand the role of Ets proteins in Ras transformation, we have now analyzed the effects of stably expressing a variety of Ets2 constructs in Ras-transformed NIH3T3 (DT) cells. Expression of only the Ets2 transactivation domains, which also inhibits Ras or Neu/ErbB-2-mediated activation of Ets-dependent transcription, strongly inhibited anchorage-independent growth, but did not revert the transformed DT cell morphology. Unexpectedly, high expression of full-length Ets2, a transcriptional activator, broadly reversed the transformed properties of DT cells, including anchorage-independent growth, transformed morphology, and tumorigenicity, but did not impair attached cell growth. Increasing full-length Ets2 transcriptional activity by fusing it to the VP16 transactivation domain enhanced its ability to reverse DT cell transformation. Mutational analysis revealed that the mitogen-activated protein kinase phosphorylation site required for Ras-mediated activation, Ets2(T72), was not essential for Ets2 reversion activity. The distinct reversion activities of the highly expressed Ets2 transactivation domains or full-length Ets2, along with the specific reversion activity by Ets2 constructs that either inhibit or activate Ets-dependent transcription, suggests multiple roles for Ets factors in cellular transformation. These results indicate that several distinct approaches for modulating Ets activity may be useful for intervention in human cancers.

Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.

Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.

Oncogenic Neu/ErbB-2 increases ets, AP-1, and NF-kappaB-dependent gene expression, and inhibiting ets activation blocks Neu-mediated cellular transformation.

Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.

The Saccharomyces cerevisiae RIB4 gene codes for 6,7-dimethyl-8-ribityllumazine synthase involved in riboflavin biosynthesis. Molecular characterization of the gene and purification of the encoded protein.

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity. Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein. Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV. Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure. Initial structural characterization of 6,7-dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the beta subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes.

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase.

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.

Botulinum toxin A in management of cerebral palsy.

The efficacy of local injection of botulinum toxin A in selected skeletal muscles to relieve muscle hypertonia and muscle contracture, and increase range of motion in children with cerebral palsy was studied in an open ABA (baseline-treatment-posttreatment phase) type of study. The first 6 months were the baseline phase, the day of injection the treatment phase, and the next 6 months the posttreatment phase. The patients acted as their own controls. Fifteen children with cerebral palsy (mean age: 6 years, 8 months) were included in the study. All had limb deformities associated with non-fixed joint contractures that had not responded to physical therapy. Clinical assessment of passive and active muscle tone was performed using a modified Ashworth scale. The range of motion to passive movement was measured with a manual goniometer. Botulinum toxin was injected directly into the muscle at several sites. The postinjection scores of muscle hypertonia were significantly lower (P < .01) and the range-of-motion values demonstrated a significant increase (P < .001). Functional improvement was measured by decreased scissoring on standing in all 6 children with adductor muscles injected; all 6 children with knee flexor muscles injected were able to straighten the knees. The 3 children with injected gastrocnemius muscles were able to achieve heel-strike while bare-footed. The study provides evidence that the intramuscular injection of botulinum toxin A in selected skeletal muscles decreases muscle tone and contractures, and increases range of motion and motor function.