Correction: Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source.

[This corrects the article DOI: 10.1371/journal.pone.0065818.].

Implementing a pharmacist consultation model for multimodal insulin therapy.

PURPOSE: The implementation of pharmacist-managed insulin dosing for selected hospitalized patients under a multimodal insulin protocol (MMIP) is described. SUMMARY: Hyperglycemia has been linked to increased thrombosis, decreased wound healing, and decreased immune response. Current recommendations support the use of multimodal (basal-bolus) insulin therapy in noncritically ill inpatients. As part of a systemwide quality-improvement initiative to improve glycemic management, the pharmacy department of a community hospital initiated a service to provide protocol-directed insulin dosing for selected patients under a pharmacist consultation model. An MMIP targeting patients with 2 blood glucose (BG) readings of >180 mg/dL within a 12-hour period was developed and approved. Pharmacist consultations, including patient assessment, entry of initial insulin orders, and ongoing insulin dosage adjustments, are performed pursuant to electronic notifications and computerized prescriber order entry. Noncritically ill patients who meet the criteria for protocol-guided insulin dosing are managed according to an approved weight-based MMIP for calculating and adjusting nutritional and basal insulin doses. Prior to the initiation of MMIP-guided insulin dosing, pharmacists were trained on the use of the protocol and passed a competency assessment. In the 90-day period after protocol implementation, 158 hyperglycemic patients received pharmacist-managed insulin dosing. CONCLUSION: The goal of achieving a mean BG concentration of ≤180 mg/dL by day 3 of hyperglycemia management under a pharmacist-managed MMIP was attained in the second and third months after protocol implementation.

An Evaluation Tool for Agricultural Health and Safety Mobile Applications.

As the use of mobile devices and their software applications, or apps, becomes ubiquitous, use amongst agricultural working populations is expanding as well. The smart device paired with a well-designed app has potential for improving workplace health and safety in the hands of those who can act upon the information provided. Many apps designed to assess workplace hazards and implementation of worker protections already exist. However, the abundance and diversity of such applications also presents challenges regarding evaluation practices and assignation of value. This is particularly true in the agricultural workspace, as there is currently little information on the value of these apps for agricultural safety and health. This project proposes a framework for developing and evaluating apps that have potential usefulness in agricultural health and safety. The evaluation framework is easily transferable, with little modification for evaluation of apps in several agriculture-specific areas.

Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

Qualitative assessment of agritourism safety guidelines: a demonstration project.

In 2007, the National Children's Center for Rural and Agricultural Health and Safety (NCCRAHS) published Agritourism Health and Safety Guidelines for Children to provide helpful recommendations for protecting the health and safety of children visiting agritourism farms. Supplement A: Policies and Procedures Guide and Supplement B: Worksite Guide were subsequently published in 2009 and provided agritourism farms with checklists to use in reviewing, planning, and implementing their own health and safety practices. In order to better understand what would be required of a farm wishing to implement the guidelines using Supplements A and B, the North Carolina Agromedicine Institute conducted a single-family farm demonstration project with support from the NCCRAHS. The aims of the project were to (1) determine child health and safety risks associated with an existing agritourism farm; (2) determine the cost of making improvements necessary to reduce risks; and (3) use project findings to motivate other agritourism farms, Cooperative Extension agents, and agritourism insurers to adopt or recommend Agritourism Health and Safety Guidelines for Children for their own farms or farms with which they work. At the conclusion of the study, the target farm was in compliance with an average of 86.9% of items in Supplements A and B. Furthermore, 89% of individuals self-identifying as farmers or farm workers and 100% of Cooperative Extension agents and agritourism insurers attending an end-of-project workshop indicated their intent to adopt or recommend Agritourism Health and Safety Guidelines for Children for their own farms or farms with which they work.

Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors.

BACKGROUND: Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors. PRINCIPAL FINDINGS: Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex. CONCLUSIONS: Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages.

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5-18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.

Medulloblastoma is the most common malignant brain tumor in children, but the cells from which it arises remain unclear. Here we examine the origin of medulloblastoma resulting from mutations in the Sonic hedgehog (Shh) pathway. We show that activation of Shh signaling in neuronal progenitors causes medulloblastoma by 3 months of age. Shh pathway activation in stem cells promotes stem cell proliferation but only causes tumors after commitment to-and expansion of-the neuronal lineage. Notably, tumors initiated in stem cells develop more rapidly than those initiated in progenitors, with all animals succumbing by 3-4 weeks. These studies suggest that medulloblastoma can be initiated in progenitors or stem cells but that Shh-induced tumorigenesis is associated with neuronal lineage commitment.

Lymphadenectomy for isolated lymph node metastasis from extremity soft-tissue sarcomas.

BACKGROUND: Previous studies have suggested that the prognosis in patients with extremity soft-tissue sarcomas (ESTS) with isolated lymph node (LN) metastases (stage IV) is comparable to that of patients with high-risk ESTS without metastases (stage III). This study was performed to determine the outcomes of patients who underwent aggressive therapy, including lymphadenectomy in patients with LN metastasis from ESTS. METHODS: Demographic details, pathology of the primary disease, timing of LN metastasis, and details of the multimodality treatment were obtained from the medical records of 35 patients with nodal metastasis from ESTS who were treated between 1981 and 2003. Survival after the diagnosis of primary disease and LN metastasis was compared with established historical outcomes for patients with American Joint Commission on Cancer (AJCC) stages III and IV ESTS. RESULTS: Epithelioid sarcomas (23%) and malignant fibrous histiocytomas (23%) were the most common primary histologic types. Twenty (57%) patients presented with synchronous nodal metastasis. Median follow-up from the time of diagnosis of lymph node metastasis was 48.5 months. The 1-year, 2-year, and 5-year actuarial survival rates in patients with synchronous nodal metastasis after lymphadenectomy and additional therapy were 79%, 62%, and 52%, respectively. In comparison, the 1-year, 2-year, and 5-year actuarial survival rates in patients with metachronous nodal metastasis after lymphadenectomy and additional therapy were 100%, 95%, and 66%, respectively. CONCLUSIONS: Patients with isolated regional lymph node metastases who are treated with aggressive approaches, including regional LN dissection, may experience prolonged survival similar to that which more closely approximates the survival seen in patients with stage III disease (5-year survival rate, 50%) rather than the survival seen in patients with stage IV disease (5-year survival rate, 25%). These data lend support for reconsideration of the current AJCC staging system for ESTS.

Patched1 functions as a gatekeeper by promoting cell cycle progression.

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G1-S phase and G2-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a "gatekeeper." In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

Pregnancy outcomes for mouse preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-DDT.

Pregnancy outcomes were evaluated following uterine transfer of murine preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT). Single-cell embryos were incubated 72 h in medium droplets containing 0.1% ethanol (control) or 0.1 microg/ml o,p'-DDT (pesticide). Morula and preblastocyst embryos were transferred in groups of eight to right uterine horns of pseudopregnant mice (n=111) and pups (n=132) were evaluated at Caesarean-section (C-section). In vitro exposure to o,p'-DDT reduced development to morula (P<0.001) and modestly increased blastomere apoptosis (P=0.05). However, treatment differences were not detected for implantation rates (35% versus 39%; P=0.64), pup numbers per dam (2.3 versus 1.9; P=0.36), transfer efficiencies (16% versus 14%; P=0.53), fetal weights (1.56 g versus 1.57 g; P=0.91), skeletal abnormalities (55% versus 66%; P=0.47), or male ratios (54.8% versus 53.8%; P=1.0). In vitro exposure of preimplantation embryos to 0.1 microg/ml o,p'-DDT for 72 h resulted in no measurable effects on subsequent implantation or pup characteristics at C-section.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis.

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Low-dose agrochemicals and lawn-care pesticides induce developmental toxicity in murine preimplantation embryos.

Occupational exposures to pesticides may increase parental risk of infertility and adverse pregnancy outcomes such as spontaneous abortion, preterm delivery, and congenital anomalies. Less is known about residential use of pesticides and the risks they pose to reproduction and development. In the present study we evaluate environmentally relevant, low-dose exposures to agrochemicals and lawn-care pesticides for their direct effects on mouse preimplantation embryo development, a period corresponding to the first 5-7 days after human conception. Agents tested were those commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D)], pendimethalin, and mecoprop), three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), a desiccant (diquat), and a fertilizer (ammonium nitrate). Groups of 20-25 embryos were incubated 96 hr in vitro with either individual chemicals or mixtures of chemicals simulating exposures encountered by handling pesticides, inhaling drift, or ingesting contaminated groundwater. Incubating embryos with individual pesticides increased the percentage of apoptosis (cell death) for 11 of 13 chemicals (p

Genotyping Parkinson disease-associated mitochondrial polymorphisms.

OBJECTIVE: The purpose of this study was to establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (T(m)) analysis. This technology should prove useful for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson disease (PD). METHODS: Mitochondrial DNA (mtDNA) was extracted from whole blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler (Roche Molecular Biochemical, Mannheim, Germany). Genotyping of 10 SNPs in 15 subjects was based on the analysis of allele-specific T(m) of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. RESULTS: Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The nucleotide changes at positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, and 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, and 9.52 degrees C declines in T(m) of the detection probes, respectively. Genotyping of all 150 samples was verified by 100% correspondence with the results of sequencing. Fourteen subjects were haplogrouped by combining results for all 10 SNPs. CONCLUSION: A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes was developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies, and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.

Overexpression of Sonic Hedgehog suppresses embryonic hair follicle morphogenesis.

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development, we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype.

Patched 1 conditional null allele in mice.

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function.

Detection System for Identifying Parkinson Disease-Associated Mitochondrial DNA Polymorphisms / 环境与健康杂志

Objective To establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (Tm) curve analysis. This technique is suitable for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson's disease (PD). Methods mtDNA was extracted from the blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler. Genotyping of 10 SNPs was based on the analysis of allele-specific Tm of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. Results Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The changes at nucleotide positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, 9.52℃ decline in Tm of the detection probes respectively. Genotyping of all detected genes was verified by 100% correspondence with the results of sequencing. Conclusion A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes has ben developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.