Foxp and Skor family proteins control differentiation of Purkinje cells from Ptf1a- and Neurog1-expressing progenitors in zebrafish.

Cerebellar neurons, such as GABAergic Purkinje cells (PCs), interneurons (INs) and glutamatergic granule cells (GCs) are differentiated from neural progenitors expressing proneural genes, including ptf1a, neurog1 and atoh1a/b/c. Studies in mammals previously suggested that these genes determine cerebellar neuron cell fate. However, our studies on ptf1a;neurog1 zebrafish mutants and lineage tracing of ptf1a-expressing progenitors have revealed that the ptf1a/neurog1-expressing progenitors can generate diverse cerebellar neurons, including PCs, INs and a subset of GCs in zebrafish. The precise mechanisms of how each cerebellar neuron type is specified remains elusive. We found that genes encoding the transcriptional regulators Foxp1b, Foxp4, Skor1b and Skor2, which are reportedly expressed in PCs, were absent in ptf1a;neurog1 mutants. foxp1b;foxp4 mutants showed a strong reduction in PCs, whereas skor1b;skor2 mutants completely lacked PCs, and displayed an increase in immature GCs. Misexpression of skor2 in GC progenitors expressing atoh1c suppressed GC fate. These data indicate that Foxp1b/4 and Skor1b/2 function as key transcriptional regulators in the initial step of PC differentiation from ptf1a/neurog1-expressing neural progenitors, and that Skor1b and Skor2 control PC differentiation by suppressing their differentiation into GCs.

Roles of maternal wnt8a transcripts in axis formation in zebrafish.

In early zebrafish development, the program for dorsal axis formation begins soon after fertilization. Previous studies suggested that dorsal determinants (DDs) localize to the vegetal pole, and are transported to the dorsal blastomeres in a microtubule-dependent manner. The DDs activate the canonical Wnt pathway and induce dorsal-specific genes that are required for dorsal axis formation. Among wnt-family genes, only the wnt8a mRNA is reported to localize to the vegetal pole in oocytes and to induce the dorsal axis, suggesting that Wnt8a is a candidate DD. Here, to reveal the roles of maternal wnt8a, we generated wnt8a mutants by transcription activator-like effector nucleases (TALENs), and established zygotic, maternal, and maternal zygotic wnt8a mutants by germ-line replacement. Zebrafish wnt8a has two open reading frames (ORF1 and ORF2) that are tandemly located in the genome. Although the zygotic ORF1 or ORF2 wnt8a mutants showed little or no axis-formation defects, the ORF1/2 compound mutants showed antero-dorsalized phenotypes, indicating that ORF1 and ORF2 have redundant roles in ventrolateral and posterior tissue formation. Unexpectedly, the maternal wnt8a ORF1/2 mutants showed no axis-formation defects. The maternal-zygotic wnt8a ORF1/2 mutants showed more severe antero-dorsalized phenotypes than the zygotic mutants. These results indicated that maternal wnt8a is dispensable for the initial dorsal determination, but cooperates with zygotic wnt8a for ventrolateral and posterior tissue formation. Finally, we re-examined the maternal wnt genes and found that Wnt6a is an alternative candidate DD.

Coordinately Co-opted Multiple Transposable Elements Constitute an Enhancer for wnt5a Expression in the Mammalian Secondary Palate.

Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.

Association between milk and yogurt intake and mortality: a community-based cohort study (Yamagata study).

BACKGROUND: Dairy products are known as health-promoting foods. This study prospectively examined the association between milk and yogurt intake and mortality in a community-based population. METHODS: The study population comprised of 14,264 subjects aged 40-74 years who participated in an annual health checkup. The frequency of yogurt and milk intake was categorized as none (< 1/month), low (< 1/week), moderate (1-6/week), and high (> 1/day) intake. The association between yogurt and milk intake and total, cardiovascular, and cancer-related mortalities was determined using the Cox proportional hazards model. RESULTS: During the follow-up period, there were 265 total deaths, 40 cardiovascular deaths and 90 cancer-related deaths. Kaplan-Meier analysis showed that the total mortality in high/moderate/low yogurt intake and moderate/low milk intake groups was lower than that in none group (log-rank, P < 0.01). In the multivariate Cox proportional hazard analysis adjusted for possible confounders, the hazard ratio (HR) for total mortality significantly decreased in high/moderate yogurt intake group (HR: 0.62, 95% confidence interval [CI]: 0.42-0.91 for high intake, HR: 0.70, 95%CI: 0.49-0.99 for moderate intake) and moderate milk intake group (HR: 0.67, 95% CI: 0.46-0.97) compared with the none yogurt and milk intake groups. A similar association was observed for cancer-related mortality, but not for cardiovascular mortality. CONCLUSIONS: Our study showed that yogurt and milk intake was independently associated with a decrease in total and cancer-related mortalities in the Japanese population.

Assessment of palliative care team activities--survey of medications prescribed immediately before and at the beginning of opioid usage.

We established the Terminal Care Study Group, consisting of physicians, pharmacists, and nurses, in September 2001, and developed the group into the Palliative Care Team. We have surveyed the state of concomitant medications immediately before and at the beginning of opioid usage (except injections) to assess the role of the Palliative Care Team. The survey period was 3 years from October 1, 2002 to September 30, 2005. While the frequency of the prescription of non-steroidal anti-inflammatory drugs (NSAIDs), laxatives, or antiemetics before the beginning of opioid administration did not differ significantly among the 3 periods, that at the beginning of opioid administration increased significantly in 2003 compared with 2002, and increased further in 2004. Many of the drugs used were those that were recommended in our cancer pain management program. Thus, the activities of the Palliative Care Team are considered to have led to proper measures for the control of the major adverse effects of opioids such as constipation and nausea/vomiting in addition to pain control in accordance with the WHO's pain ladder, and also contributed to improvements of the patients' QOL.

Introduction and evaluation of a newly established holiday work system in the pharmacy ward at Municipal Ikeda Hospital.

At the Municipal Ikeda Hospital, a system in which pharmacists stationed in one ward pharmacy dispense drugs to be administered by injection and injectable preparations delivered to patients' bedsides was introduced in April 2000. This system was aimed at minimizing risks related to injections. Initially, however, on holidays, nurses played the roles of pharmacists in terms of the injections, and there were concerns over a possible rise in the incidence of errors (adverse events/near-misses) related to injections on these days compared with weekdays. Later, when planning to introduce a new holiday work system in the ward pharmacy, we took into account such factors as the number of pharmacists needed on holidays, their duties on holidays and the influence on weekday pharmacy activity of compensatory days-off taken by such pharmacists. In May 2004, the new holiday work system was introduced in the ward pharmacy. Under the new system, 5 pharmacists work at the ward pharmacy on holidays. After this system was put into operation, the number of injections dispensed at the ward pharmacy averaged 230 per day, and 177 per holiday. To evaluate the validity of this system, we recently conducted a questionnaire survey of nurses at our hospital. The survey involved 139 nurses. Of these nurses, 69.1% responded that the number of incidents (adverse events/near-misses) related to dispensing injections on holidays had decreased. Furthermore, 65.4% of the nurses reported a decrease in incidents related to the delivery and administration of injectable preparations. More than half of the nurses answered that the new system had made it easier for them to collect information on medicines and helped them provide better nursing services. When the nurses were asked to make a general assessment of the new system, 90% rated the system as "good." The results of this survey indicate that keeping the ward pharmacy open on holidays contributes to the promotion of the proper use of medicines, reduction of risks related to injections and improvement in the quality of medical care.

[Changes in Medical Safety Interventions Conducted by Ward-based Pharmacists since the Introduction of Their Services in Complex Medical Ward].

Despite an increase in the number of reports on medical safety interventions conducted by ward-based pharmacists, only a few reports have classified intervention cases in detail. We classified and compared the types of medical safety intervention conducted by ward-based pharmacists since the introduction of their services. The interventions were classified into: cases that were identified by pharmacists or through asking questions about physicians' prescriptions before dispensing medications (active interventions); and those in which pharmacy technicians could contribute to medical safety by receiving inquiries from patients or healthcare providers (passive interventions). The numbers of the two types of intervention were compared. The number of interventions significantly increased after the introduction of ward-based clinical pharmacy services. Especially, the numbers of cases identified during ward rounds conducted by ward-based pharmacists (active interventions) and those identified by receiving inquiries from physicians or nurses (passive interventions) significantly increased, possibly because the collection of patient information was performed more efficiently by conducting ward rounds, and an environment where physicians and nurses can easily make inquiries to pharmacists was established after increasing the time pharmacists spend on hospital wards. The results demonstrate that the introduction of ward-based clinical pharmacy services has improved communication with patients, facilitated information-sharing among physicians, nurses, and other healthcare providers, contributed to the safer management of pharmaceutical operations, and increased interest of patients.

A SINE-derived element constitutes a unique modular enhancer for mammalian diencephalic Fgf8.

Transposable elements, including short interspersed repetitive elements (SINEs), comprise nearly half the mammalian genome. Moreover, they are a major source of conserved non-coding elements (CNEs), which play important functional roles in regulating development-related genes, such as enhancing and silencing, serving for the diversification of morphological and physiological features among species. We previously reported a novel SINE family, AmnSINE1, as part of mammalian-specific CNEs. One AmnSINE1 locus, named AS071, showed an enhancer property in the developing mouse diencephalon. Indeed, AS071 appears to recapitulate the expression of diencephalic fibroblast growth factor 8 (Fgf8). Here we established three independent lines of AS071-transgenic mice and performed detailed expression profiling of AS071-enhanced lacZ in comparison with that of Fgf8 across embryonic stages. We demonstrate that AS071 is a distal enhancer that directs Fgf8 expression in the developing diencephalon. Furthermore, enhancer assays with constructs encoding partially deleted AS071 sequence revealed a unique modular organization in which AS071 contains at least three functionally distinct sub-elements that cooperatively direct the enhancer activity in three diencephalic domains, namely the dorsal midline and the lateral wall of the diencephalon, and the ventral midline of the hypothalamus. Interestingly, the AmnSINE1-derived sub-element was found to specify the enhancer activity to the ventral midline of the hypothalamus. To our knowledge, this is the first discovery of an enhancer element that could be separated into respective sub-elements that determine regional specificity and/or the core enhancing activity. These results potentiate our understanding of the evolution of retroposon-derived cis-regulatory elements as well as the basis for future studies of the molecular mechanism underlying the determination of domain-specificity of an enhancer.

A mammalian conserved element derived from SINE displays enhancer properties recapitulating Satb2 expression in early-born callosal projection neurons.

Short interspersed repetitive elements (SINEs) are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+) neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+) neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-)/NPY(+)) portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum, a eutherian-specific brain structure.

Reduction of opioid side effects by prophylactic measures of palliative care team may result in improved quality of life.

PURPOSE: In February 2002, the palliative care team was established in Ikeda Municipal Hospital to improve palliative care. We investigated changes in the incidences of side effects related to opioids, and evaluated palliative care team activities. METHODS: Regarding inpatients for whom narcotics were prescribed in our hospital in the years of 2002 (from October 1, 2002 until September 30, 2003), 2004 (from October 1, 2004 until September 30, 2005), and 2006 (from October 1, 2006 until September 30, 2007), we surveyed the rates at which laxatives or antiemetics were prescribed, frequency of defecation/its state before and after the start of narcotic therapy, frequency of nausea/vomiting, and dietary intake. RESULTS: The proportions of patients in whom laxatives were simultaneously prescribed during opioid therapy in 2002, 2004, and 2006 were 43.5%, 78.7%, and 75.6%, respectively. The proportions of those in whom antiemetics were combined with opioids were 45.7%, 78.7%, and 78.0%, respectively. The incidences of constipation were 50.0%, 39.3%, and 37.8%, respectively. Those of nausea/vomiting were 30.4%, 21.3%, and 9.8%, respectively. Those of anorexia were 65.3%, 39.4%, and 15.4%, respectively. CONCLUSIONS: These results suggest that palliative care team activities facilitated appropriate drug prescription during opioid therapy, reducing the appearance of side effects, with likelihood of improved quality of life.

[Determination of bicozamycin in livestock products and seafoods by liquid chromatography-tandem mass spectrometry].

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for trace residue determination of bicozamycin (BZM) in livestock products and seafoods. BZM was extracted from a sample with acetonitrile-water (4 : 1), followed by a two-stage SPE enrichment and cleanup. The first stage involved a styrene-divinylbenzene copolymer cartridge (GL-Pak PLS-2), and the second stage involved a divinylbenzene-N-vinylpyrrolidone copolymer cartridge (Oasis HLB). The LC separation was performed on a C18 column using 0.01% formic acid-methanol (8 : 2) as the mobile phase and MS detection with negative ion electrospray ionization. The mean recoveries from swine muscle, liver, yellowtail, and milk fortified at the minimum residue limit (MRL) levels and 0.01 microg/g were >70%, and the relative standard deviations (RSDs) were <20%. Limits of quantitation (LOQs) ranged from 0.002 to 0.005 microg/g.