Trak1 mutation disrupts GABA(A) receptor homeostasis in hypertonic mice.

Hypertonia, which results from motor pathway defects in the central nervous system (CNS), is observed in numerous neurological conditions, including cerebral palsy, stroke, spinal cord injury, stiff-person syndrome, spastic paraplegia, dystonia and Parkinson disease. Mice with mutation in the hypertonic (hyrt) gene exhibit severe hypertonia as their primary symptom. Here we show that hyrt mutant mice have much lower levels of gamma-aminobutyric acid type A (GABA(A)) receptors in their CNS, particularly the lower motor neurons, than do wild-type mice, indicating that the hypertonicity of the mutants is likely to be caused by deficits in GABA-mediated motor neuron inhibition. We cloned the responsible gene, trafficking protein, kinesin binding 1 (Trak1), and showed that its protein product interacts with GABA(A) receptors. Our data implicate Trak1 as a crucial regulator of GABA(A) receptor homeostasis and underscore the importance of hyrt mice as a model for studying the molecular etiology of hypertonia associated with human neurological diseases.

Flexibility in competency-based workplace transition programs: an exploratory study of community child and family health nursing.

BACKGROUND: Successful transition to practice programs that use competency-based assessment require the involvement of all staff, especially those undertaking the preceptor role. METHODS: Qualitative data were collected using interview methods. Participants were 14 newly employed nurses and 7 preceptors in the child and family community health service in South Australia. Participant narratives were recorded electronically, transcribed, and thematically analyzed using the paradigm of critical social science. RESULTS: Five themes were identified that describe enablers as well as barriers to applying a flexible transition to practice program using competency-based assessment. These included flexibility in the program design, flexibility on the part of preceptors, flexibility to enable recognition of previous learning, flexibility in the assessment of competencies, and flexibility in workload. CONCLUSION: To ensure successful application of a transition to practice program using competency-based assessment, preceptors must understand the flexible arrangements built into the program design and have the confidence and competence to apply them.

Use of a long-pulse alexandrite laser in the treatment of superficial pigmented lesions.

BACKGROUND/OBJECTIVE: Although the alexandrite 755-nm-wavelength laser is effective in the treatment of unwanted hair, there are no published studies gauging the efficacy of the variable long-pulse alexandrite laser in the treatment of superficial pigmented lesions. STUDY DESIGN/METHODS: Eighteen patients underwent a single treatment session using a variable pulse-width alexandrite laser. Test sites were performed using a 10-mm spot size and up to four pulse widths (3, 20, 40, 60 ms) with and without epidermal cooling. Full treatments were performed 3 weeks later using optimum test parameters. The patients were evaluated at 3 and 6 weeks. RESULTS: Patients with darker lentigines had greater lesion clearance than those patients with lighter colored lentigines. Shorter pulse widths and treatment without cryogen cooling both, independently, lowered the fluence threshold for lentigo clearance. CONCLUSION: A long-pulse alexandrite laser is effective in clearing solar lentigines in a single pass with minimal adverse effects.

Switching off HER-2/neu in a tetracycline-controlled mouse tumor model leads to apoptosis and tumor-size-dependent remission.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.

Intervention Protocol for Investigating Yoga Implemented During Chemotherapy.

OBJECTIVE: Fatigue and other treatment-related symptoms are critical therapeutic targets for improving quality of life in patients with colorectal cancer during chemotherapy. Yoga is a promising intervention for improving these therapeutic targets and has been primarily investigated in the group-class format, which is less feasible for cancer patients with high symptom burden to attend. Thus, we developed a protocol for implementing yoga individually in the clinic among patients receiving chemotherapy. METHODS: We followed recommended domains for developing a yoga protocol to be used in an efficacy trial. These recommendations include consideration to the style, delivery, components of the intervention, dose, specific class sequences, facilitation of home practice, measurement of intervention fidelity, selection of instructors, and dealing with modifications. The intervention protocol was developed by an interdisciplinary team. PROTOCOL: Yoga Skills Training (YST) consists of four 30-minute in-person sessions and was implemented while in the chair during chemotherapy infusions for colorectal cancer with recommended daily home practice for eight weeks. Therapeutic goals of the YST are to reduce fatigue, circadian disruption, and psychological distress. Elements of the YST are awareness meditation, gentle seated movement, breathing practice, and relaxation meditation. Attention, comfort, and ease are also highlighted. CONCLUSION: This description of a protocol for integrating yoga with conventional cancer treatment will inform future study designs and clinical practice. The design of the YST is novel because it implements yoga-most commonly studied when taught to groups outside of the clinical setting- individually during clinical care.

Inhibition of HER-2 by three independent targeting strategies increases paclitaxel resistance of SKOV-3 ovarian carcinoma cells.

Current treatment options for ovarian cancer, which is one of the most widespread gynecological malignancies, are limited, mainly because patients with advanced-stage disease often develop resistance to chemotherapeutics. In breast cancer cells, several studies suggest that overexpression of the human epidermal growth factor receptor-2 (HER-2) leads to increased resistance against certain, but not all cytotoxic drugs. In ovarian carcinoma, conflicting data on the correlation of HER-2 expression and tumor cell sensitivity exist. In this paper, we explore the role of HER-2 expression and signaling levels pertaining to paclitaxel (Taxol) chemoresistance by applying three different and independent strategies in SKOV-3 ovarian carcinoma cells. Firstly, we show that treatment with the HER-2 inhibitory antibody trastuzumab (Herceptin), which is well established in tumor therapy, results in markedly increased, rather than decreased, cellular paclitaxel resistance. Next, we present two newly developed low molecular weight inhibitors of HER-2 tyrosine kinase activity, D-69491 and D-70166. With both drugs, the decrease in cellular paclitaxel sensitivity upon HER-2 inhibition is confirmed. Finally, for more detailed analysis we stably downregulate HER-2 expression by ribozyme-targeting. Using clonal ribozyme-transfected SKOV-3 cells with different residual HER-2 levels, we establish a 'HER-2 gene dose effect' of paclitaxel cytotoxicity. We show that this effect is due to differential induction of apoptosis and differential cell cycle inhibition by paclitaxel. Finally, paclitaxel- or HER-2-mediated alterations in the phosphorylation of MAP kinases p42/44, Stress-activated protein kinase/Jun-terminal kinase (SAPK/JNK), and p38, and effects on the activation of caspase-3, caspase-7, and bcl-2 are discussed. We conclude that paclitaxel cytotoxicity in SKOV-3 cells is 'HER-2 dose-dependent' and identify cell proliferation as one underlying cellular event of this effect.

Hereditary hyperekplexia caused by novel mutations of GLRA1 in Turkish families.

BACKGROUND: Hyperekplexia, also known as startle disease or stiff-person syndrome, is a neurological condition characterized by neonatal hypertonia and a highly exaggerated startle reflex. Genetic studies have linked mutations in the gene encoding glycine receptor alpha1 (GLRA1) with hereditary hyperekplexia. METHODS: We analyzed four Turkish families with a history of hyperekplexia. Genomic DNA was obtained from members of these families, and the entire coding sequence of GLRA1 was amplified by PCR followed by the sequencing of PCR products. DNA sequences were analyzed by direct observation using an electropherogram and compared with a published reference sequence. RESULTS: We identified three novel mutations in GLRA1. These included a large deletion removing the first 7 of 9 exons, a single-base deletion in exon 8 that results in protein truncation immediately after the deletion, and a missense mutation in exon 7 causing a tryptophan-to-cysteine change in the first transmembrane domain (M1). These mutant alleles have some distinct features as compared to previously identified GLRA1 mutations. Our data provides further evidence for mutational heterogeneity in GLRA1. The new mutant alleles reported here should advance our understanding of the etiology of hyperekplexia.

Nursing students' perceptions regarding the amount and type of written feedback required to enhance their learning.

Effective feedback can enhance student learning, but limited evidence exists on whether nursing students actually use and learn from written feedback. This descriptive survey explored nursing students' perceptions regarding the amount and type of written feedback required to enhance their learning. In stage one, 362 students completed a 28-item questionnaire regarding feedback experiences and preferences; in stage two, 227 students selected a preferred feedback option for a final topic assignment. Findings revealed that many of the students wished to be engaged with the feedback process and believed effective written feedback can and does enhance their learning. However many students also reported learning barriers-including absent, inadequate, ambiguous, inconsistent, and ineffective feedback-indicating a significant disconnect between desired and actual feedback. Recommendations include a greater focus on engaging nursing students in the feedback process and evaluating the effectiveness of written feedback for individual students.

The ongoing adaptive evolution of ASPM and Microcephalin is not explained by increased intelligence.

Recent studies have made great strides towards identifying putative genetic events underlying the evolution of the human brain and its emergent cognitive capacities. One of the most intriguing findings is the recurrent identification of adaptive evolution in genes associated with primary microcephaly, a developmental disorder characterized by severe reduction in brain size and intelligence, reminiscent of the early hominid condition. This has led to the hypothesis that the adaptive evolution of these genes has contributed to the emergence of modern human cognition. As with other candidate loci, however, this hypothesis remains speculative due to the current lack of methodologies for characterizing the evolutionary function of these genes in humans. Two primary microcephaly genes, ASPM and Microcephalin, have been implicated not only in the adaptive evolution of the lineage leading to humans, but in ongoing selective sweeps in modern humans as well. The presence of both the putatively adaptive and neutral alleles at these loci provides a unique opportunity for using normal trait variation within humans to test the hypothesis that the recent selective sweeps are driven by an advantage in cognitive abilities. Here, we report a large-scale association study between the adaptive alleles of these genes and normal variation in several measures of IQ. Five independent samples were used, totaling 2393 subjects, including both family-based and population-based datasets. Our overall findings do not support a detectable association between the recent adaptive evolution of either ASPM or Microcephalin and changes in IQ. As we enter the post-genomic era, with the number of candidate loci underlying human evolution growing rapidly, our findings highlight the importance of direct experimental validation in elucidating their evolutionary role in shaping the human phenotype.

Sonic Hedgehog, a key development gene, experienced intensified molecular evolution in primates.

Sonic Hedgehog (SHH) is one of the most intensively studied genes in developmental biology. It is a highly conserved gene, found in species as diverse as arthropods and mammals. The mammalian SHH encodes a signaling molecule that plays a central role in developmental patterning, especially of the nervous system and the skeletal system. Here, we show that the molecular evolution of SHH is markedly accelerated in primates relative to other mammals. We further show that within primates, the acceleration is most prominent along the lineage leading to humans. Finally, we show that the acceleration in the lineage leading to humans is coupled with signatures of adaptive evolution. In particular, the lineage leading to humans is characterized by a rampant and statistically highly non-random gain of serines and threonines, residues that are potential substrates of post-translational modifications. This suggests that SHH might have evolved more complex post-translational regulation in the lineage leading to humans. Collectively, these findings implicate SHH as a potential contributor to the evolution of primate- or human-specific morphological traits in the nervous and/or skeletal systems and provide the impetus for additional studies aimed at identifying the primate- or human-specific functions of this key development gene.

Discovery of highly selective EP4 receptor agonists that stimulate new bone formation and restore bone mass in ovariectomized rats.

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.

Genetic links between brain development and brain evolution.

The most defining biological attribute of Homo sapiens is its enormous brain size and accompanying cognitive prowess. How this was achieved by means of genetic changes over the course of human evolution has fascinated biologists and the general public alike. Recent studies have shown that genes controlling brain development - notably those implicated in microcephaly (a congenital defect that is characterized by severely reduced brain size) - are favoured targets of natural selection during human evolution. We propose that genes that regulate brain size during development, such as microcephaly genes, are chief contributors in driving the evolutionary enlargement of the human brain. Based on the synthesis of recent studies, we propose a general methodological template for the genetic analysis of human evolution.

Ongoing adaptive evolution of ASPM, a brain size determinant in Homo sapiens.

The gene ASPM (abnormal spindle-like microcephaly associated) is a specific regulator of brain size, and its evolution in the lineage leading to Homo sapiens was driven by strong positive selection. Here, we show that one genetic variant of ASPM in humans arose merely about 5800 years ago and has since swept to high frequency under strong positive selection. These findings, especially the remarkably young age of the positively selected variant, suggest that the human brain is still undergoing rapid adaptive evolution.

Microcephalin, a gene regulating brain size, continues to evolve adaptively in humans.

The gene Microcephalin (MCPH1) regulates brain size and has evolved under strong positive selection in the human evolutionary lineage. We show that one genetic variant of Microcephalin in modern humans, which arose approximately 37,000 years ago, increased in frequency too rapidly to be compatible with neutral drift. This indicates that it has spread under strong positive selection, although the exact nature of the selection is unknown. The finding that an important brain gene has continued to evolve adaptively in anatomically modern humans suggests the ongoing evolutionary plasticity of the human brain. It also makes Microcephalin an attractive candidate locus for studying the genetics of human variation in brain-related phenotypes.

Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation.

Tumor necrosis factor-alpha converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane-anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12-myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment.

The CDY-related gene family: coordinated evolution in copy number, expression profile and protein sequence.

Theories predict that the long-term survival of duplicated genes requires their functional diversification, which can be accomplished by either subfunctionalization (the partitioning of ancestral functions among duplicates) or neofunctionalization (the acquisition of novel function). Here, we characterize the CDY-related mammalian gene family, focusing on three aspects of its evolution: gene copy number, tissue expression profile and amino acid sequence. We show that the progenitor of this gene family arose de novo in the mammalian ancestor via domain accretion. This progenitor later duplicated to generate CDYL and CDYL2, two autosomal genes found in all extant mammals. Prior to human-mouse divergence (and perhaps preceding the eutherian radiation), a processed CDYL transcript retroposed onto the Y chromosome to create CDY, the Y-linked member of the family. In the simian lineage, CDY was retained and subsequently amplified on the Y. In non-simian mammals, however, CDY appears to have been lost. The retention of the Y-linked CDY genes in simians spurred the process of subfunctionalization and possibly neofunctionalization. Subfunctionalization is evidenced by the observation that simian CDYL and CDYL2 retained their somatic housekeeping transcripts but lost the spermatogenic transcripts to the newly arisen CDY. Neo-functionalization is suggested by the rapid evolution of the CDY protein sequence. Thus, the CDY-related family offers an instructive example of how duplicated genes undergo functional diversification in both expression profile and protein sequence. It also supports the previously postulated notion that there is a tendency for spermatogenic functions to transfer from autosomes to the Y chromosome.

Accelerated evolution of nervous system genes in the origin of Homo sapiens.

Human evolution is characterized by a dramatic increase in brain size and complexity. To probe its genetic basis, we examined the evolution of genes involved in diverse aspects of nervous system biology. We found that these genes display significantly higher rates of protein evolution in primates than in rodents. Importantly, this trend is most pronounced for the subset of genes implicated in nervous system development. Moreover, within primates, the acceleration of protein evolution is most prominent in the lineage leading from ancestral primates to humans. Thus, the remarkable phenotypic evolution of the human nervous system has a salient molecular correlate, i.e., accelerated evolution of the underlying genes, particularly those linked to nervous system development. In addition to uncovering broad evolutionary trends, our study also identified many candidate genes--most of which are implicated in regulating brain size and behavior--that might have played important roles in the evolution of the human brain.

Adaptive evolution of ASPM, a major determinant of cerebral cortical size in humans.

A prominent trend in the evolution of humans is the progressive enlargement of the cerebral cortex. The ASPM (Abnormal spindle-like microcephaly associated) gene has the potential to play a role in this evolutionary process, because mutations in this gene cause severe reductions in the cerebral cortical size of affected humans. Here, we show that the evolution of ASPM is significantly accelerated in great apes, especially along the ape lineages leading to humans. Additionally, the lineage from the last human/chimpanzee ancestor to humans shows an excess of non-synonymous over synonymous substitutions, which is a signature of positive Darwinian selection. A comparison of polymorphism and divergence using the McDonald-Kreitman test confirms that ASPM has indeed experienced intense positive selection during recent human evolution. This test also reveals that, on average, ASPM fixed one advantageous amino acid change in every 300,000-400,000 years since the human lineage diverged from chimpanzees some 5-6 million years ago. We therefore conclude that ASPM underwent strong adaptive evolution in the descent of Homo sapiens, which is consistent with its putative role in the evolutionary enlargement of the human brain.