text:
from django import forms
from django.urls import reverse
from django.utils.safestring import mark_safe
from haystack.forms import FacetedSearchForm, SearchForm

def get_range_parts(value_range):
    """Returns a tuple of start value and end value extracted from
    `value_range`.

    `value_range` should be in the format "<int> - <int>".

    """
    return value_range.split(" - ", 1)

class ModelLinkWidget(forms.Widget):
    """This widget adds a link, to edit the current inline, after the
    inline form fields.
    """

    def __init__(self, obj, attrs=None):
        super(ModelLinkWidget, self).__init__(attrs)
        self.object = obj

    def render(self, name, value, attrs=None):
        edit_url = reverse(
            "admin:%s_%s_change"
            % (self.object._meta.app_label, self.object._meta.model_name),
            args=[self.object.id],
        )
        edit_link = 'Edit %s' % (
            edit_url,
            self.object._meta.verbose_name.lower(),
        )

        if self.object.pk:
            return mark_safe("%s" % (edit_link))
        else:
            return mark_safe("")

class PostAssertionDatesWidget(forms.Widget):
    def __init__(self, obj, attrs=None):
        super(PostAssertionDatesWidget, self).__init__(attrs)
        self.object = obj

    def render(self, name, value, attrs=None):
        return self.object.get_dates()

class PostAssertionProvincesWidget(forms.Widget):
    def __init__(self, obj, attrs=None):
        super(PostAssertionProvincesWidget, self).__init__(attrs)
        self.object = obj

    def render(self, name, value, attrs=None):
        return self.object.print_provinces()

class StatusProvincesWidget(forms.Widget):
    def __init__(self, obj, attrs=None):
        super(StatusProvincesWidget, self).__init__(attrs)
        self.object = obj

    def render(self, name, value, attrs=None):
        return self.object.print_provinces()

class PostInlineForm(forms.ModelForm):
    """This form renders a model and adds a link to edit the nested inline
    model. This is useful for inline editing when the nested inline fields are
    not displayed.
    """

    edit_link = forms.CharField(label="Edit", required=False)
    print_dates = forms.CharField(label="Post Person Dates", required=False)
    provinces_list = forms.CharField(label="Province(s)", required=False)

    # verbose_name = 'Post'

    class Meta:
        exclude = ()
        fieldsets = []

    def __init__(self, *args, **kwargs):
        super(PostInlineForm, self).__init__(*args, **kwargs)

        # instance is always available, it just does or doesn't have pk.
        self.fields["edit_link"].widget = ModelLinkWidget(self.instance)
        self.fields["print_dates"].widget = PostAssertionDatesWidget(self.instance)
        self.fields["provinces_list"].widget = PostAssertionProvincesWidget(
            self.instance
        )

class PersonInlineForm(forms.ModelForm):
    """This form renders a model and adds a link to edit the nested inline
    model. This is useful for inline editing when the nested inline fields are
    not displayed."""

    edit_link = forms.CharField(label="Edit", required=False)

    class Meta:
        exclude = ()
        fieldsets = []

    def __init__(self, *args, **kwargs):
        super(PersonInlineForm, self).__init__(*args, **kwargs)

        # instance is always available, it just does or doesn't have pk.
        self.fields["edit_link"].widget = ModelLinkWidget(self.instance)

class RelationshipAssertionInlineForm(forms.ModelForm):
    """This form renders a model and adds a link to edit the nested inline
    model. This is useful for inline editing when the nested inline fields are
    not displayed."""

    edit_link = forms.CharField(label="Edit", required=False)

    class Meta:
        exclude = ()
        fieldsets = []

    def __init__(self, *args, **kwargs):
        super(RelationshipAssertionInlineForm, self).__init__(*args, **kwargs)

        # instance is always available, it just does or doesn't have pk.
        self.fields["edit_link"].widget = ModelLinkWidget(self.instance)

class StatusInlineForm(forms.ModelForm):
    """This form renders a model and adds a link to edit the nested inline
    model. This is useful for inline editing when the nested inline fields are
    not displayed.
    """

    edit_link = forms.CharField(label="Edit", required=False)
    provinces_list = forms.CharField(label="Province(s)", required=False)

    # verbose_name = 'Post'

    class Meta:
        exclude = ()
        fieldsets = []

    def __init__(self, *args, **kwargs):
        super(StatusInlineForm, self).__init__(*args, **kwargs)

        # instance is always available, it just does or doesn't have pk.
        self.fields["edit_link"].widget = ModelLinkWidget(self.instance)
        self.fields["provinces_list"].widget = StatusProvincesWidget(self.instance)

class PromrepFacetedSearchForm(FacetedSearchForm):
    """Extends FacetedSearchForm, as we have special requirements in terms of
    facet handling and date filtering."""

    AUTOCOMPLETE_FACETS = [
        "praenomen",
        "nomen",
        "cognomen",
        "re_number",
        "n",
        "f",
        "other_names",
        "tribe",
    ]

    MIN_DATE = -509
    MAX_DATE = -31

    MIN_DATE_FORM = -1 * MAX_DATE
    MAX_DATE_FORM = -1 * MIN_DATE

    era_from = forms.IntegerField(
        required=False, max_value=MAX_DATE_FORM, min_value=MIN_DATE_FORM
    )
    era_to = forms.IntegerField(
        required=False, max_value=MAX_DATE_FORM, min_value=MIN_DATE_FORM
    )

    # class autocomplete is used by the search.js script to select the fields
    # the name of the fields has to match the facet names defined in
    #  views.py
    praenomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Praenomen"}),
    )
    nomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Nomen"}),
    )
    cognomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Cognomen"}),
    )
    re_number = forms.CharField(
        required=False, widget=forms.TextInput(attrs={"title": "RE"})
    )
    n = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Grandfather"}),
    )
    f = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Father"}),
    )
    other_names = forms.CharField(
        required=False,
        widget=forms.TextInput(
            attrs={"class": "autocomplete", "title": "Additional Cognomina"}
        ),
    )
    tribe = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Tribe"}),
    )

    def __init__(self, *args, **kwargs):
        self.selected_facets = kwargs.pop("selected_facets", [])
        super(FacetedSearchForm, self).__init__(*args, **kwargs)

    def no_query_found(self):
        """Determines the behaviour when no query was found; returns all the
        results."""
        return self.searchqueryset.all()

    def search(self):
        # Tweak here

        office_facets = []

        for x in self.selected_facets:
            name = x.split(":")[0]
            if "office" in name:
                office_facets.append(x)

        for x in office_facets:
            self.selected_facets.remove(x)

        sqs = super(PromrepFacetedSearchForm, self).search()

        if not self.is_valid():
            return self.no_query_found()

        # Narrow the search by the ranges of dates
        # Requires, of course, that the form be bound.
        if self.is_bound:
            data = self.cleaned_data
            era_from = data.get("era_from", None)
            era_to = data.get("era_to", None)

            if era_from or era_to:
                sqs = sqs.narrow(
                    "era:[{} TO {}]".format(
                        data.get("era_from", self.MIN_DATE) or self.MIN_DATE,
                        data.get("era_to", self.MAX_DATE) or self.MAX_DATE,
                    )
                )

        for facet in office_facets:
            sqs = sqs.narrow('offices:"{}"'.format(facet.split(":")[1]))
            self.selected_facets.append(facet)

        for field in self.AUTOCOMPLETE_FACETS:
            if data.get(field):
                sqs = sqs.narrow("{}:{}".format(field, data.get(field)))

        return sqs

    def clean_era_from(self):
        era_from = self.cleaned_data["era_from"]

        if era_from:
            era_from = -1 * era_from

        return era_from

    def clean_era_to(self):
        era_to = self.cleaned_data["era_to"]

        if era_to:
            era_to = -1 * era_to

        return era_to

    def clean_date_from(self):
        date_from = self.cleaned_data["date_from"]

        if date_from:
            date_from = -1 * date_from

        return date_from

    def clean_date_to(self):
        date_to = self.cleaned_data["date_to"]

        if date_to:
            date_to = -1 * date_to

        return date_to

DATING_CERTAINTY_CHOICES = (
    ("1", "Certain"),
    ("2", "Uncertain"),
    ("3", "Attested before"),
    ("4", "Attested after"),
)

class SenateSearchForm(SearchForm):
    INITIAL_DATE = -180
    INITIAL_DATE_DISPLAY = -1 * INITIAL_DATE

    senate_date = forms.IntegerField(
        required=True,
        initial=INITIAL_DATE_DISPLAY,
        max_value=INITIAL_DATE_DISPLAY,
        min_value=PromrepFacetedSearchForm.MIN_DATE_FORM,
    )

    dating_certainty = forms.ChoiceField(
        required=True,
        widget=forms.RadioSelect,
        initial="1",
        choices=DATING_CERTAINTY_CHOICES,
    )

    def no_query_found(self):
        return self.searchqueryset.all()

    def search(self):

        sqs = super(SenateSearchForm, self).search()

        if not self.is_valid():
            return self.no_query_found()

        if self.is_bound:
            data = self.cleaned_data

            senate_date = data.get("senate_date", None)
            dating_certainty = data.get("dating_certainty", "1")

            if senate_date:
                if dating_certainty == "1":
                    # Certain they are a senator
                    sqs = sqs.narrow(
                        "date:[{0} TO {0}]".format(data.get("senate_date"))
                    )
                    sqs = sqs.narrow("uncertain:false")
                elif dating_certainty == "2":
                    # Uncertain
                    sqs = sqs.narrow(
                        "date:[{0} TO {0}]".format(data.get("senate_date"))
                    )
                    sqs = sqs.narrow("uncertain:true")
                elif dating_certainty == "3":
                    # Attested Before
                    date = int(data.get("senate_date")) - 1
                    sqs = sqs.narrow("date_end:[* TO {0}]".format(date))
                    sqs = sqs.narrow("date_end_uncertain:true")
                elif dating_certainty == "4":
                    # Attested After
                    date = int(data.get("senate_date")) + 1
                    sqs = sqs.narrow("date_start:[{0} TO *]".format(date))
                    sqs = sqs.narrow("date_start_uncertain:true")

        return sqs

    def clean_senate_date(self):
        senate_date = self.cleaned_data["senate_date"]

        if senate_date:
            senate_date = -1 * senate_date
        else:
            senate_date = self.INITIAL_DATE

        return senate_date

class FastiSearchForm(SearchForm):
    MIN_DATE = -509
    MAX_DATE = -31

    MIN_DATE_FORM = -1 * MAX_DATE
    MAX_DATE_FORM = -1 * MIN_DATE

    # class autocomplete is used by the search.js script to select the fields
    # the name of the fields has to match the facet names defined in
    #  views.py
    praenomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Praenomen"}),
    )
    nomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Nomen"}),
    )
    cognomen = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Cognomen"}),
    )
    re_number = forms.CharField(
        required=False, widget=forms.TextInput(attrs={"title": "RE"})
    )
    n = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Grandfather"}),
    )
    f = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Father"}),
    )
    other_names = forms.CharField(
        required=False,
        widget=forms.TextInput(
            attrs={"class": "autocomplete", "title": "Additional Cognomina"}
        ),
    )
    tribe = forms.CharField(
        required=False,
        widget=forms.TextInput(attrs={"class": "autocomplete", "title": "Tribe"}),
    )

    date_from = forms.IntegerField(
        required=False, max_value=MAX_DATE_FORM, min_value=MIN_DATE_FORM
    )
    date_to = forms.IntegerField(
        required=False, max_value=MAX_DATE_FORM, min_value=MIN_DATE_FORM
    )

    def __init__(self, *args, **kwargs):
        self.selected_facets = kwargs.pop("selected_facets", [])
        super(FastiSearchForm, self).__init__(*args, **kwargs)

    def no_query_found(self):
        """Determines the behaviour when no query was found; returns all the
        results."""
        return self.searchqueryset.all()

    def search(self):
        sqs = super(FastiSearchForm, self).search()

        if not self.is_valid():
            return self.no_query_found()

        # Narrow the search by the ranges of dates
        # Requires, of course, that the form be bound.
        if self.is_bound:
            data = self.cleaned_data
            date_from = data.get("date_from", None)
            date_to = data.get("date_to", None)

            if date_from or date_to:
                sqs = sqs.narrow(
                    "date:[{} TO {}]".format(
                        data.get("date_from", self.MIN_DATE) or self.MIN_DATE,
                        data.get("date_to", self.MAX_DATE) or self.MAX_DATE,
                    )
                )

            for field in PromrepFacetedSearchForm.AUTOCOMPLETE_FACETS:
                if data.get(field):
                    sqs = sqs.narrow("{}:{}".format(field, data.get(field)))

        query_string = None
        for facet in self.selected_facets:
            facet_parts = facet.split(":")
            facet_string = '{}:"{}"'.format(facet_parts[0], facet_parts[1])
            if query_string:
                query_string = "{} OR {}".format(query_string, facet_string)
            else:
                query_string = facet_string

        if query_string:
            sqs = sqs.narrow(query_string)

        return sqs

    def clean_date_from(self):
        date_from = self.cleaned_data["date_from"]

        if date_from:
            date_from = -1 * date_from

        return date_from

    def clean_date_to(self):
        date_to = self.cleaned_data["date_to"]

        if date_to:
            date_to = -1 * date_to

        return date_to


Lysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development.
Loss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, β-galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis.
Our findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes.
Embryonic organ development is a highly organized and complex process involving the temporal and spatial expression of genes that control differentiation, maturation, and survival of organs . Additionally, this process involves the formation and migration of cells that are destined to differentiate into specific structures essential for proper development of the organism, such as the neural crest . The neural crest is comprised of stem-like cells that are predestined to migrate extensively and differentiate into specialized cell types during vertebrate embryogenesis . Induction of neural crest cells begins at the gastrula stage of development. Neural crest progenitors are initially identified at the edge of the neural plate, forming a bridge between the neural and non-neural portion of the ectoderm, and require tissue interactions between the neural plate and ectoderm . Before undergoing migration, neural crest cells are localized to the dorsal part of the neural tube . Neural crest cells later migrate throughout the body and give rise to various types of cells, such as melanocytes, craniofacial cartilage and bone, smooth muscles, and peripheral nervous cells .
Formation and migration of neural crest cells and development of different organs during vertebrate embryogenesis require regulated gene expression [7, 8], which is affected by the epigenome . Epigenetic modifications, such as methylation, phosphorylation, and ubiquitination, play significant roles in regulating gene expression and interaction to fulfill specific functions ; for example, histone lysine methylation leads to the activation or suppression of certain genes . The methylation status of histones is regulated by several types of methyltransferases (KMTs) and demethylases (DMTs) ; thus far, two groups of histone demethylases implicated in diverse biological functions have been discovered [13, 14].
Lysine-specific histone demethylase 5C (KDM5C; also known as JARID1C and SMCX) catalyzes the demethylation of lysine 4 on histone 3 (H3K4me3/me2). Since H3K4me3 and H3K4me2 are associated with actively transcribed genes, demethylation of H3K4 by KDM5C causes transcriptional repression [15, 16]. In mammalian cells, KDM5C belongs to a protein subfamily consisting of four members, namely KDM5A/retinoblastoma binding protein 2 (RBP2)/JARID1A, KDM5B/PLU-1/JARID1B, KDM5C/SMCX/JARID1C, and KDM5D/SMCY/JARID1D . KDM5C contains the catalytic jumonji C (JmjC) and jumonji N (JmjN) domains, which are involved in the maintenance of JmjC domain structural integrity and possess an ARID/BRIGHT DNA binding domain , a single C5HC2 zinc finger domain located at the C-terminal of the JmjC domain, and two plant homeodomains (PHD) that bind to the methyl lysine residue .
The kdm5c gene, which is located on the X chromosome, has recently been identified as the gene responsible for X-linked mental retardation (XLMR) . XLMR is a heterogeneous disease that is affected by genetic, environmental, and stochastic factors . Notably, kdm5c mutations found in XLMR diminish the ability of KDM5C to demethylate H3K4, indicating that the demethylation activity of KDM5C is critical for brain development [21–23].
KDM5C is highly expressed in the brain and skeletal muscle tissue in humans , and human KDM5C is involved in the inhibition of specific neuronal genes. In the mouse brain, kdm5c is widely expressed in regions related to cognitive and emotional behaviors, including the prefrontal cortex, hippocampus, and amygdala . kdm5c-knockout mice exhibit abnormal social behavior including aggression as well as impaired learning and memory [20, 24]. In addition, KDM5C escapes X-inactivation in both mice and humans . In zebrafish, knockdown of the kdm5c homologue induces brain-patterning defects and neuronal cell death, while small interfering RNA (siRNA)-mediated knockdown of kdm5c in primary rat granule neurons damaged dendritic morphogenesis . Furthermore, KDM5C has been implicated in renal cancer .
In the current study, we investigated the functional significance of KDM5C during Xenopus embryogenesis, where the spatiotemporal expression of kdm5c indicated that it is a maternal gene. Loss-of-function studies using kdm5c morpholino oligonucleotides (MO) demonstrated the significance of this demethylase in neural crest migration and eye development. Whole-mount in situ hybridization (WISH) and reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that kdm5c knockdown led to inhibition of neural crest migration and defects in eye development. Furthermore, transcriptome analysis of kdm5c MO-injected embryos showed that KDM5C is critical for morphogenesis of anatomical structures and organogenesis during Xenopus embryonic development. Collectively, we concluded that KDM5C plays significant roles in neural crest migration and eye formation during vertebrate development.
To investigate the specific roles of KDM5C during embryogenesis, we first analyzed its gene expression pattern in Xenopus. For this purpose, we conducted RT-PCR and WISH analyses. RT-PCR revealed that kdm5c is a maternal gene, as it was found expressed throughout embryonic development from the single-cell stage to the tadpole stage (Fig. 1a). The temporal expression pattern of kdm5c indicated that this gene possesses essential functions during Xenopus development.
WISH analysis was performed to determine the spatial expression patterns of kdm5c during Xenopus embryonic development at different developmental stages (st. 6, 9, 13, 16, 22, 32, and 36; Fig. 1b–k′). The expression pattern of kdm5c indicated that this gene is expressed in the animal hemisphere of developing embryos at developmental stage 6 (Fig. 1b). Additionally, kdm5c expression was observed in late blastula-stage embryos showing enhanced expression in the animal pole (st. 9; Fig. 1c). Tissue-specific expression of kdm5c was observed during the neurula stage of embryonic development and was found expressed in the early eye field region at stage 13 (Fig. 1d). We also observed kdm5c expression in the anterior neural tissues of neurula stage embryos (st. 16; Fig. 1e) with dorsal expression in the neural plate border region (Fig. 1e′). Vibratome section analysis confirmed kdm5c expression in neural plate and neural plate border regions (Fig. 1f). We also examined the expression patterns of kdm5c during early and late tailbud stages by focusing on the lateral views of developing embryos and by transverse sectioning these embryos for detailed analysis (st. 22, 32, and 36). Our data demonstrated kdm5c expression in the branchial arches and eyes of early tailbud stage (Fig. 1g) as well as in the whole brain of late tailbud stage Xenopus embryos (Fig. 1h, h′). Although kdm5c expression was observed in the whole brain, elevated expression levels of kdm5c were detected in the forebrain and hindbrain regions of developing embryos (Fig. 1h′) as well as in the midbrain regions as revealed by transverse embryo sections (Fig. 1i, k). In addition to the whole brain, enhanced kdm5c expression was also observed in the retina and lens of Xenopus embryos (Fig. 1h′, j′). A detailed view of the embryos through vibratome transverse sections indicated that kdm5c is predominantly expressed in the lens and retina (Fig. 1i, k); furthermore, kdm5c was found strongly expressed in the ganglion cell layer of the eye (Fig. 1k′). Based on these findings, it is evident that KDM5C is significant during embryogenesis.
To gain insights into the physiological functions of KDM5C during Xenopus embryogenesis, we conducted knockdown studies using kdm5c MOs by microinjecting kdm5c MO (48 ng) into one-cell stage embryos. To investigate the specificity of kdm5c MOs in the knockdown of kdm5c, analyzing endogenous KDM5C levels by using anti-KDM5C antibodies is the most suitable; however, due to the lack in availability of anti-KDM5C antibodies for Xenopus, we synthesized kdm5c mutant RNA using wobble base pairing (kdm5c**) and carried out western blot analysis of control embryos, embryos injected with MO-bound kdm5c mRNA, kdm5c*, and kdm5c* together with the MO. Our results revealed that kdm5c translation was blocked in MO-bound kdm5c mRNA (Additional file 1: Fig. S1). Moreover, KDM5C protein expression of embryos injected with mutated kdm5c or coinjected with mutated kdm5c and MO verified the specificity of the kdm5c MO (Additional file 1: Fig. S1). Microinjection of kdm5c MO resulted in phenotypic abnormalities, such as small-sized heads and reduced cartilage size (Fig. 2a–d). Compared with control embryos, over 80% of kdm5c MO-injected embryos exhibited smaller-sized heads (Fig. 2b). Moreover, we investigated head size by measuring the head area of kdm5c morphants relative to the head area of control MO-injected embryos and observed significantly smaller head areas of approximately 70% upon kdm5c depletion (Fig. 2c). To further investigate these cartilage defects, we carried out alcian blue staining of kdm5c MO-injected embryos (st. 46). The results indicated that kdm5c morphants exhibited a marked reduction in cartilage size compared with that of control MO-injected embryos, whereas cartilage structure was not affected (Fig. 2d).
To rule out the unspecific side effects of MOs using kdm5c RNA, we carried out rescue experiments by microinjecting Xenopus embryos with kdm5c mutant RNA along with kdm5c MO. Injection of mutant kdm5c* RNA (1.6 ng) rescued all phenotypic malformations induced by the kdm5c MO (Fig. 2a–d); embryos injected with mutant kdm5c* recovered approximately 88% of head area reduction (Fig. 2c). Taken together, these findings indicate that KDM5C is specifically involved in head and cartilage development during embryogenesis.
Cell number plays a significant role in determining organ as well as whole organism size. To maintain constant size, cell number is tightly controlled by different mechanisms including apoptosis and cell proliferation, which are indispensable for regulating cell number and consequently organ size . To elucidate whether the reduced head and cartilage sizes induced by kdm5c knockdown were due to perturbation of apoptosis and cell proliferation, we coinjected kdm5c MO and β-galactosidase mRNA unilaterally into one blastomere of two-cell stage embryos and performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and pH3 (phospho-histone H3) staining at stage 32 (Fig. 3). The uninjected side of the embryos served as an internal control, while β-galactosidase mRNA was used as a lineage tracer.
TUNEL staining revealed a significant increase in TUNEL-positive cells after kdm5c depletion in the kdm5c MO-injected side compared with the uninjected side of the embryos (Fig. 3a, b), indicating involvement of KDM5C in apoptosis regulation. Moreover, pH3 staining indicated significant reduction in cell proliferation in the kdm5c MO-injected side of the embryos compared with that of the uninjected side (Fig. 3c, d). Therefore, the mechanism underlying reduced head and cartilage sizes may be the result of a significant increase in apoptosis and marked decrease in cell proliferation due to kdm5c depletion.
Neural crest development is regulated by the dynamic expression of a number of genes, including sox3 , pax3 , twist , slug , and members of the soxE family, i.e., sox8, sox9, and sox10 . To evaluate the functions of kdm5c in neural crest development, we performed a loss-of-function analysis using the kdm5c MO and examined its effects on expression of neural crest specifiers. Embryos at the two-cell stage were unilaterally coinjected with kdm5c MO and β-galactosidase mRNA into one blastomere of two-cell stage embryos, after which WISH was performed using these neural crest specifiers. Our results showed sox3 and pax3 expressions in the expanded neural plate regions of the kdm5c MO-injected side of embryos (Fig. 4a, b), while RT-PCR analysis indicated similar expression levels of sox3 and pax3 between control and kdm5c MO-injected embryos (Fig. 4c). In contrast to sox3 and pax3, downregulated expression was observed for twist, slug, sox8, and sox10 in the kdm5c MO-injected side; however, sox9 expression remained unaffected (Fig. 5a, b). sox8, sox9, and sox10 belong to the SoxE protein family and play a significant role along with other neural crest specifiers (i.e., twist and snail). sox9 is expressed in cranial and cardiac neural crest cells and precedes the expression of sox8 and sox10 . Thus, we speculated that the unaffected expression levels of sox9 in the kdm5c MO-injected side are why the cranial cartilage did not exhibit deformations, only a reduction in size (Figs. 2d, 5a, b). To further clarify the expression of these neural crest-specific genes, RT-PCR analysis indicated that expression levels of twist, slug, sox8, and sox10 were considerably reduced, while sox9 remained unaltered in kdm5c MO-injected embryos compared with control MO (Fig. 5c).
We further examined the twist expression pattern during the late tailbud stage (st. 32) to analyze the effect of kdm5c knockdown on neural crest migration. Perturbed twist expression was observed during later stages of embryonic development after kdm5c knockdown, indicating abnormal migration of neural crest cells (Fig. 5d, e). Moreover, the abnormal expression patterns of neural crest specifiers were significantly rescued by injecting kdm5c mutant RNA, ruling out any unspecific side effects of the kdm5c MOs (Figs. 4a–c, 5a–e). Altogether, these results indicate that KDM5C is required for the expression of neural crest specifiers; thus, perturbation of kdm5c expression altered expression patterns and influenced neural crest migration.
As our spatial expression analysis of kdm5c in Xenopus embryos indicated enriched expression of kdm5c in the eye regions (Fig. 1g–k′) and on the basis of the well-established Xenopus fate maps, we sought to investigate the involvement of kdm5c in eye development during Xenopus embryogenesis. Thus, we conducted unilateral microinjection of kdm5c into eight-cell stage Xenopus embryos and found that kdm5c morphants exhibited significantly smaller and deformed eyes, i.e., coloboma/optic fissures, compared with that of control embryos (Fig. 6a, d). Statistical analysis revealed that compared with control embryos, more than 80% of kdm5c MO-injected embryos exhibited eye defects (Fig. 6b) and among the kdm5c morphants, approximately 20% exhibited small-sized eyes and 60% possessed deformed eyes (Fig. 6c). Additionally, histological analysis of the eye structure through vibratome sections indicated that kdm5c morphants exhibited abnormal retinal pigment epithelium (Fig. 6a). For validating the specificity of kdm5c MO-induced eye defects, we performed rescue experiments that confirmed the eye malformations observed in kdm5c morphants were specifically caused by a depletion of kdm5c and not through unspecific side effects of kdm5c MOs (Fig. 6a–c). In short, our results implicate kdm5c in eye development during Xenopus embryogenesis.
Loss of kdm5c induced phenotypic eye defects of coloboma/optic fissures in the morphant embryos (Fig. 6a–d), which may have resulted from non-closure of the choroid fissure, leading to coloboma. Dorsoventral (DV) patterning of the retina is important for the choroid fissure, and impairment of DV patterning can result in colobomas. Retina DV patterning is controlled by asymmetric expression of transcription factors, such as vax1 (optical stalk-specific), vax2 (optical stalk and ventral retina-specific), pax6 (ventral and dorsal retina-specific), and tbx5 (dorsal retina-specific), that regionalize optic vesicle into three compartments, i.e., optic stalk, dorsal retina, and ventral retina . Thus, we examined the effects of kdm5c knockdown on DV patterning by analyzing the expression of DV-patterning markers (vax1, vax2, pax6, and tbx5) through WISH. We found that depletion of kdm5c significantly downregulated vax1, vax2, pax6 expressions, while tbx5 expression was slightly reduced (Fig. 6e); thus, the reduced expression of DV-patterning markers may be responsible for the colobomas observed in kdm5c morphants.
In addition to colobomas, vibratome sections of kdm5c morphants indicated retinal lamination defects (Fig. 6a). Therefore, we performed WISH with the well-known eye-specific markers arr3 (photoreceptor cell-specific), prox1 (horizontal cell-specific), vsx1 (bipolar cell-specific), and pax6 (ganglion and amacrine cell-specific) to further analyze kdm5c morphant eyes (st. 40). We obtained both mild and severe phenotypes through WISH analysis as well as severe disorganization of retinal cell layers (Fig. 6f). Overall, our findings indicate that kdm5c knockdown induced severe eye defects, including colobomas and perturbed retinal lamination.
We further investigated the roles of KDM5C at the molecular level during eye development by coinjecting kdm5c MO and β-galactosidase mRNA unilaterally into one dorsal blastomere of eight-cell stage embryos. WISH analysis of these kdm5c MO/β-galactosidase mRNA-coinjected embryos was performed to evaluate the effect of kdm5c knockdown on eye field induction and differentiation by examining the expression patterns of otx2 , rax , and pax6  at stage 16 of embryogenesis. Compared with control, otx2, rax, and pax6 expressions were downregulated in the kdm5c MO-injected side of the embryos (Fig. 7a, b). Furthermore, we examined the effect of kdm5c knockdown on eye differentiation at stage 32 (Fig. 7c, d) and found that all tested eye-specific markers exhibited reduced expression levels in the kdm5c MO-injected side, whereas normal expression was observed on the uninjected side of the embryos. WISH analysis with cryba1 specific for the vertebrate eye lens  was also conducted at stage 32; however, kdm5c knockdown did not affect lens development during Xenopus embryogenesis (Fig. 7e, f). RT-PCR analysis further confirmed that the presence of KDM5C is significant during eye field induction and differentiation but is not required during eye lens development (Fig. 7g). Moreover, rescue experiments effectively recovered the reduced expression levels of eye-specific markers induced by kdm5c knockdown (Fig. 7a–f), verifying the specificity of KDM5C in eye development during Xenopus embryogenesis. Altogether, our results demonstrate that KDM5C plays an important role during eye field induction and differentiation and that loss of kdm5c results in anomalies of retina formation during Xenopus embryogenesis.
To pinpoint target genes specifically affected by kdm5c knockdown, we performed a transcriptome analysis of kdm5c morphants. Total RNA of kdm5c morphants was extracted and processed for transcriptome and RNA sequence analysis. RNA sequence analysis identified important gene groups (Additional file 2: Fig. S2); genes were classified into 19 groups using PANTHER gene ontology; and a bar chart was plotted based on the downregulated expression of these gene groups in the kdm5c morphants (Additional file 2: Fig. S2). These analyses indicated that kdm5c plays significant roles in morphogenesis.
To validate RNA sequence analysis, genes with high fold-change values were selected and RT-PCR was performed to analyze the expression of these genes in kdm5c morphants. epha4, epha2, efnb2, sox8, sox10, aldh1a2, and wnt8a are all genes involved in the regulation of eye and neural crest development during embryogenesis [39–43]. We found that epha4, epha2, efnb2, sox8, sox10, aldh1a2, and wnt8a were downregulated among several other genes (Fig. 8). RT-PCR showed reduced gene expression of sox8, sox10, and wnt8a, confirming that kdm5c is essential for the regulation of neural crest development (Figs. 5c, 8). Moreover, the downregulated expression patterns of epha4, epha2, efnb2, and aldh1a2 validated the involvement of KDM5C in eye development during embryogenesis (Fig. 8). Overall, our results demonstrate that KDM5C is critical for morphogenesis and specifically influences neural crest development and eye formation during embryonic development.
Members of the jumonji family of histone demethylases have emerged as significant regulators of epigenetic events [16, 44] and have been implicated in diverse biological processes ranging from developmental events to the pathogenesis of several diseases . KDM5C catalyzes the di- and tri-demethylation of H3K4 and is associated with transcriptional repression . This demethylase is strongly expressed in human brain and skeletal muscle tissues and is associated with memory defects and aggressive tendencies in kdm5c-knockout mice . KDM5C has also been implicated in XLMR as kdm5c mutations are typically observed in XLMR patients [20, 21]. Moreover, KDM5C has been studied with regard to its significance in X-inactivation and has been associated with certain types of cancer [22, 23]. However, KDM5C has not been investigated for its roles during embryonic development. In the current study, we sought to explore the physiological significance of KDM5C during Xenopus embryogenesis.
Xenopus is an excellent animal model for the study of gene function as genetic overexpression or knockdown can be induced by RNA or DNA microinjection into fertilized eggs; consequently, gene expression can be observed throughout different stages of embryonic development . The spatiotemporal expression pattern of kdm5c demonstrated that it is a maternal gene (Fig. 1a) and is specifically expressed in the branchial arches as well as the prospective eye field and brain of developing embryos (Fig. 1b–k′). The functional significance of kdm5c was evaluated by microinjecting kdm5c MOs into one-cell stage embryos. Loss-of-function analysis revealed that kdm5c is essential for proper embryonic development and that loss of kdm5c induced phenotypic malformations—reduced head size, smaller cartilage, and defective eyes—in the morphant embryos (Figs. 2, 6). The reduction in cartilage and head size was supported by increased apoptosis and decreased cell proliferation in kdm5c morphant embryos (Fig. 3). These findings led us to hypothesize that KDM5C plays critical roles in embryonic morphogenesis and organ development. Our observed phenotypes of reduced head and cartilage size are in agreement with the finding that kdm5c mutations lead to XLMR in humans, who also exhibit microcephaly . It would thus be interesting to further examine whether exogenous kdm5c can rescue microcephaly in affected humans.
Neural crest cells are multipotent cells that can differentiate into several cell types, such as melanocytes, skeletal muscle, craniofacial cartilage, and bone [47, 48]. Neural crest morphogenesis has three distinct phases and is associated with the differentiation ability of neural crest cells . Transcription factor genes including sox3 , pax3 , sox8 , sox9, sox10 , twist [31, 51], and slug  are involved in the migration and differentiation of the neural crest. kdm5c knockdown led to reduced head and cartilage sizes; thus, investigating these malformations on a molecular level revealed that KDM5C is involved in the migration and differentiation of neural crest cells by altering expression of neural crest specifiers (Figs. 4, 5).
The process of organogenesis is controlled by several distinct epigenetic events . Our knockdown experiments showed that loss of kdm5c induced eye defects such as coloboma and disturbed retinal lamination in the developing embryos. Coloboma formation in kdm5c morphants may be the result of perturbed expression of DV-patterning markers (Fig. 6e) and aldh1a2 (retinoid metabolism-specific; Fig. 8). Retinoic acid plays a significant role in eye development through retinoic acid receptor (RAR) signaling that are involved in regulation of choroid fissure closure . aldh1a2 is involved in the regulation of RAR signaling [52, 53], and thus, its downregulated expression may be the underlying cause of coloboma in kdm5c morphants. WISH analysis using retinal cell-specific markers revealed that kdm5c knockdown affected the ganglion cell layer and resulted in malformed retinas. Although the eyes were in the proper position, the retinas of injected embryos were extensively malformed (Fig. 6a, d). Altogether, detailed analysis of eye defects in kdm5c MO-injected embryos (Fig. 6) indicated the importance of KDM5C in eye development during Xenopus embryogenesis.
To investigate the involvement of KDM5C in early eye field induction and differentiation, we analyzed marker gene expression during early (st. 16) and late (st. 32) stages of embryonic development. We found that otx2, rax, and pax6 were strongly inhibited upon kdm5c knockdown (Fig. 7a–d). This is consistent with previous observations that perturbation in expression patterns of any of these genes is associated with eye defects [54–56]. Interestingly, these genes are associated with eye abnormalities in humans, which includes small eyes and colobomas [35, 37]. Therefore, it will be interesting to uncover whether KDM5C is involved in human ocular defects.
Morphogenesis is a critical biological process regulated by a set of genes that sequentially turn the developmental process on or off in a precise spatiotemporal pattern . The expression of these regulatory genes is in turn controlled by several upstream factors. RNA sequence analysis of kdm5c MO-injected embryos demonstrated the significance of KDM5C in organogenesis and morphogenesis of anatomical structures (Additional file 2: Fig. S2). RT-PCR data further confirmed the downregulated expression patterns of epha4, epha2, efnb2, and aldh1a2, which are all genes associated with eye development, as well as reduced gene expression of sox8, sox10, and wnt8a which are involved in neural crest migration (Fig. 8). Previous studies have shown that epha4 and its interacting partner sipa1l3 are significant during eye development and that their depletion led to an increase in the Wnt/β-catenin target axin2 . As kdm5c depletion is associated with downregulation of epha4 and wnt8a (Fig. 8), it is possible that KDM5C is also involved in Wnt signaling. Therefore, further elucidation of Wnt signaling regulation by KDM5C is warranted.
The association of KDM5C with neural crest specifiers and key genes involved in eye development provides additional information regarding the complex and dynamic genetic networks that regulate neural crest and eye development. Furthermore, our findings highlight the significance of epigenetic regulators in controlling the spatiotemporal expression of genes during embryonic development. Our observations also raise the possibility that this repressive histone marker may contribute to developmental disorders owing to its critical role in regulating methylation patterns of key developmental genes.
cDNA was synthesized from total RNA of tailbud stage embryos. Based on the kdm5c sequences in NCBI and Xenbase, primers were designed for cloning kdm5c. Flag-tagged kdm5c mRNA was generated by PCR and a plasmid was constructed using the pCS107 vector, which included restriction sites for ClaI and XhoI.
This study was performed in strict accordance with the guidelines of the Animal Care and Use Committee and in agreement with international laws and policies (National Institutes of Health Guide for the Care and Use of Laboratory Animals, Publication No. 85-23, 1985). The Institutional Review Board of the Ulsan National Institute of Science and Technology approved the experimental use of amphibians (Approval No. UNISTACUC-16-14). All members of our laboratory attended educational and training courses on the proper care and use of experimental animals. Adult Xenopus obtained from the Korean Xenopus Resource Center for Research were housed at 18 °C under 12-h light/12-h dark conditions in containers recommended by the Institutional Review Board of the Ulsan National Institute of Science and Technology. Ovulation was induced in Xenopus females by injecting 1000 IU of human chorionic gonadotropin into the dorsal lymph sac in the evening before the experiment. The next day, eggs were collected in 60-mm petri dishes containing 1X MBS (88 mM NaCl, 5 mM HEPES, 2.5 mM NaHCO3, 1 mM KCl, 1 mM MgSO4, and 0.7 mM CaCl2, pH 7.8) by squeezing Xenopus females. After several washes with 0.1X MBS, eggs were fertilized using a sperm suspension solution derived from the isolated testes of sacrificed male frogs. After successful fertilization, the jelly coat was removed by swirling the embryos in 2% l-cysteine solution, and then, the embryos were washed five times with 0.5X MBS. Unfertilized eggs and dead embryos were removed, and then, healthy embryos were transferred into 0.5X MBS containing 2% Ficoll® 400 (GE Healthcare, Little Chalfont, UK) at 15–18 °C.
For microinjection, capped mRNAs were synthesized using the SP6 mMessage mMachine® kit (Ambion, Austin, TX). pCS107/kdm5c-Flag constructs were linearized with ApaI. The kdm5c MO consisted of 25 nucleotides and was designed as follows: 5′-ATGTTGAACATGGAGACTGAAGACT-3′ (Gene Tools, Philomath, OR). mRNA or kdm5c MO were coinjected into one cell stage embryos while for WISH analysis mRNA or kdm5c MO were unilaterally injected into two-cell and eight-cell stage embryos. Embryos were incubated at 23 °C until the required stages of embryogenesis stages.
Protein lysates were prepared by homogenizing embryos in lysis buffer (137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% Nonidet-P40, and 10% glycerol) supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium orthovanadate, and 1X protease inhibitor mixture. Embryonic lysates were heated at 95 °C in loading buffer for 5 min and electrophoresed with 12% SDS-PAGE. Western blots were probed with monoclonal anti-Flag (1:1000; Applied Biological Materials, Richmond, Canada) and goat anti-mouse horseradish peroxidase-conjugated antibodies (1:10,000; Santa Cruz Biotechnology, Dallas, TX). The immunoreactive proteins were detected with an enhanced chemiluminescence (ECL) kit (HyClone, Logan, UT).
Two-cell and eight-cell stage embryos were unilaterally injected with kdm5c MO and fixed at appropriate stages in MEMFA (4% paraformaldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO4, and 2 mM EGTA) overnight at 4 °C and then dehydrated in 100% methanol prior to storage at − 20 °C. To prepare the antisense digoxigenin-labeled probes, DNA templates were linearized using restriction enzymes. Probes were generated using SP6 or T7 RNA polymerase (Ambion). Probes were detected using alkaline phosphatase-labeled anti-digoxigenin antibodies (1:1000; Roche, Basel, Switzerland) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate .
Total RNA was extracted from embryos using Isol-RNA lysis reagent (5 Prime GmbH, Hilden, Germany). cDNA was prepared by reverse transcription using a PrimeScript™ first-strand cDNA synthesis kit (Takara, Kusatsu, Japan) with RNA extracted from Xenopus embryos ranging from stages 0–40 according to standard protocol. PCR was performed using specific primer pairs (Table 1). PCR products were separated on 1% agarose gels, and images were captured using WiseCapture I-1000 (Daihan Scientific, Wonju, South Korea) and were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA).
Xenopus embryos were harvested at stage 45, fixed in Bouin's solution for 2 h at room temperature, and then washed in 70% ethanol containing 0.1% NH4OH. Embryos were stained using 0.05% alcian blue 8GX (Sigma-Aldrich, St. Louis, MO) in 5% acetic acid for 2 h at room temperature. The embryos were then washed in 5% acetic acid for 2 h and cleared in 100% methanol, after which they were incubated in 2:1 benzyl benzoate:benzyl alcohol.
Fixed embryos were washed with 1X phosphate-buffered saline (PBS), embedded in 3% low-melting agarose in 1X PBS, and sectioned at 100-μm thickness using a vibratome (VT 1000S; Leica, Wetzlar, Germany).
To perform TUNEL and pH3 staining, Xenopus embryos were fixed in MEMFA (4% paraformaldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO4, and 2 mM EGTA), washed with PBS, and then bleached in a bleach solution (3% H2O2, 5% formamide, and 5X SSC). For TUNEL assays, bleached embryos were end-labeled using digoxigenin-11-dUTP (Sigma-Aldrich) and TdT (Invitrogen, Carlsbad, CA). Labeled ends were detected with alkaline phosphatase-labeled anti-digoxigenin antibodies (1:1000; Roche) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. For pH3 staining, bleached embryos were blocked in a blocking solution (1% bovine serum albumin and 5% goat serum in PBS) and probed with anti-histone H3 (1:1000; Abcam, Cambridge, UK) and anti-rabbit IgG AP-linked antibody (1:2000; Santa Cruz Biotechnology). pH3-positive cells were detected by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate [39, 60].
Total RNA was extracted from each sample, and an RNA sequencing library was constructed using polyA enrichment according to manufacturer's instructions (Illumina, San Diego, CA). X. laevis cDNA sequence reads were mapped from the genome project consortium  using BWA (version 0.7.15) to estimate mRNA abundance, and then, differentially expressed (DE) genes were analyzed using edgeR (version 3.20.7). Genes with greater than fourfold change and false discovery rates (FDR) less than 0.01 in exact tests were considered to show significant differential expression. To test overrepresented biological processes in these DE genes, we used Fisher's test provided by the PANTHER database (released 20171205) with human orthologous genes based on best hits using BLASTp search. Raw data for RNA-seq are available at the NCBI GEO database (accession number GSE117754) .
WISH and RT-PCR data were analyzed using ImageJ software (NIH; http://imagej.nih.gov). Results were interpreted by nonparametric, one-tailed Mann–Whitney rank-sum test using GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA). P values < 0.05 were considered statistically significant.
Y-EJ, KK, TI, H–KL, and CK performed the experiments; Y-EJ, KK, TI, H–KL, TK, and H–SL performed the data analysis and wrote the manuscript; J-WP, O-SK, B-SK, D-SL, T-JP, TK, and H–SL designed the experiments, interpreted the results, and critically analyzed the manuscript. All authors read and approved the final manuscript.
No competing interests to be declared.
The experiments were strictly conducted according to the documented standards of the Animal Care and Use Committee consistent with international laws and policies (National institute of health [NIH] Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). We received an approval number from the Institutional Review Board of Ulsan National Institute of Science and Technology for the experimental use of amphibians in Korea (UNISTACUC-16-14). All members of the research group were trained for the appropriate care and use of experimental animals. No unexpected deaths of adult Xenopus were reported during this study.
This study was funded by the National Research Foundation of Korea (NRF) (Grant No. NRF-2015R1A2A1A10053265) and the Ministry of Science, ICT, and Future Planning (MSIP) (Grant No. 2015R1A4A1042271), the Republic of Korea.
13072_2018_241_MOESM1_ESM.pptx Additional file 1: Fig. S1. Western blot analysis supports the specificity of the kdm5c MO. The embryos were microinjected with kdm5c wild-type (WT) RNA with or without kdm5c MO. To analyze the specificity of the kdm5c MO, we microinjected the embryos with kdm5c* RNA with or without kdm5c MO. Western blot analysis revealed that no protein expression was detected for embryos injected with kdm5c MO/kdm5c WT. On the other hand, strong protein expression of Flag-tagged kdm5c was observed in embryos injected with kdm5c* alone or together with kdm5c MO. WB, western blot.
13072_2018_241_MOESM2_ESM.pptx Additional file 2: Fig. S2. Transcriptome analysis revealed that KDM5C is essential for Xenopus embryonic development. We performed RNA-seq and analyzed groups of genes that are essential for several biological processes. The downregulation of specific genes by kdm5c knockdown indicated that KDM5C plays significant roles in organ development and structure morphogenesis.


Disneyana Show & Sale - February 18, 2018 - Show your haul!
November 11-12, 2017 My-D Pins Trading Event - What did you get?
DSSH October 8, 2017 Villains Pin Event at the Roosevelt Hotel - What did you get?
NorCal Pin Meet!!! MAJOR UPDATE!
Pin event 10/22/17 - Disney Springs?
Pin Trading Event DLR, Nov. 18th?
I will be at WDW from Oct 3-7, anyone else at the parks during that time?
LIAA - is anyone looking for a table buddy?
Any trading going on at SDCC?


text:
package org.eclipse.bpel.model;

import org.eclipse.emf.common.util.EList;

/**
 *
 * A representation of the model object 'Link'.
 *
 *
 *
 * Specifies the synchronization dependencies between activities in a flow. Each activity can be the source and/or target of any number of links. Links establish a predecessor/successor relationship between activities. Activities that are the source of a link are predecessors of activities that are targets of the same link. Parallel execution of activities in a flow is synchronized by requiring that all the source activities of an activity must complete before corresponding target activities of the same link can run. When an activity completes, all activities that are targets of links for which this activity is a source are now candidates for execution (assuming all their predecessor activities have completed).
 *
 *
 *

 * The following features are supported:
 *

 *
{@link org.eclipse.bpel.model.Link#getName Name}

 *
{@link org.eclipse.bpel.model.Link#getSources Sources}

 *
{@link org.eclipse.bpel.model.Link#getTargets Targets}

 *

 *

 *
 * @see org.eclipse.bpel.model.BPELPackage#getLink()
 * @model
 * @generated
 */
public interface Link extends BPELExtensibleElement {
	/**
	 * Returns the value of the 'Name' attribute.
	 *
	 *

	 * If the meaning of the 'Name' attribute isn't clear,
	 * there really should be more of a description here...
	 *

	 *
	 * @return the value of the 'Name' attribute.
	 * @see #setName(String)
	 * @see org.eclipse.bpel.model.BPELPackage#getLink_Name()
	 * @model
	 * @generated
	 */
	String getName();

	/**
	 * Sets the value of the '{@link org.eclipse.bpel.model.Link#getName Name}' attribute.
	 *
	 *
	 * @param value the new value of the 'Name' attribute.
	 * @see #getName()
	 */
	void setName(String value);

	/**
	 * Returns the value of the 'Sources' reference list.
	 * The list contents are of type {@link org.eclipse.bpel.model.Source}.
	 * It is bidirectional and its opposite is '{@link org.eclipse.bpel.model.Source#getLink Link}'.
	 *
	 *

	 * If the meaning of the 'Sources' reference list isn't clear,
	 * there really should be more of a description here...
	 *

	 *
	 * @return the value of the 'Sources' reference list.
	 * @see org.eclipse.bpel.model.BPELPackage#getLink_Sources()
	 * @see org.eclipse.bpel.model.Source#getLink
	 * @model opposite="Link"
	 * @generated
	 */
	EList<Source> getSources();

	/**
	 * Returns the value of the 'Targets' reference list.
	 * The list contents are of type {@link org.eclipse.bpel.model.Target}.
	 * It is bidirectional and its opposite is '{@link org.eclipse.bpel.model.Target#getLink Link}'.
	 *
	 *

	 * If the meaning of the 'Targets' reference list isn't clear,
	 * there really should be more of a description here...
	 *

	 *
	 * @return the value of the 'Targets' reference list.
	 * @see org.eclipse.bpel.model.BPELPackage#getLink_Targets()
	 * @see org.eclipse.bpel.model.Target#getLink
	 * @model opposite="Link"
	 * @generated
	 */
	EList<Target> getTargets();

} // Link

redpajama_set_name: RedPajamaGithub


Weeds are part of life. But some weeds are far more pernicious than others. In California all varieties of broom fit the pernicious category: due to high levels of volatile oils, broom plants are extreme fire hazards; as they spread, broom choke out native plants; and all portions of broom plants are toxic, offering no food or shelter of any kind to native wildlife.
French broom (Genista monspessulana), Spanish broom (Spartia junceum) and Scotch Broom (Cytisus scoparius) are invasive plants throughout California. The California Invasive Plant Council (Cal-IPC) lists 23 California counties suffering environmentally and economically from broom infestations.
Members of the pea family, broom species are native to Europe. The plants develop amazing taproots, making them successful in drought climates and difficult to completely pull once established. They form a dizzying number of hard coated seeds, which remain viable for between 20 and 80 years. First introduced to California as garden ornamentals in the 1850s broom was eventually used in government roadside erosion control programs. Broom have no natural controls in California, and even fire aids their spread as broom seeds germinate more quickly and effectively than native plant seeds allowing broom seedlings to shade out other plants.
BEEP created an informational brochure on why and how to begin controlling broom; they presented elementary school programs in Forest Ranch and nearby Chico. They began holding regular pull-days throughout the winter season. From 2006 until the end of March 2010, BEEP and their many partners and volunteers has pulled an estimated 200,000 broom plants. Ultimately, BEEP hopes to be a template for others dealing with invasive plant problems.
Many agencies have provided BEEP with help along the way, including: Big Chico Creek Ecological Reserve, the Big Chico Creek Watershed Alliance, the Butte County Resource Conservation District, the Butte County Fire Safe Council, Cal Fire, Cal Trans, Butte County Roads, Cal State Parks, Friends of Bidwell Park, City of Chico Parks Division, (CSUC Students), Sierra Pacific Industries, California Native Plant Society's Mt. Lassen Chapter and the California Conservation Corps.
Removal: Removal is the ultimate goal. Hand-pulling in the moist seasons works for smaller plants and seedlings.
Seeds: If unable to remove the entire plant, cut the flowers before they form seeds. Be sure to leave at least a foot of stem to grip for removal during the next rainy season.
Seedlings: Seedlings appearing after rains can be hand-pulled. Broom seed are long-lived - plan on pulling seedlings for several years. Careful spot spraying with Roundup is a chemical option.
To get help learning how to effectively remove broom yourself or for more information, you can reach BEEP by phone at: 530-892-8726.
Contributor Jennifer Jewell hosts In a North State Garden weekly on Northstate Public Radio 91.7 fm KCHO Chico/88.9 fm KFPR Redding). For more information: www.jewellgarden.com.


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Smooth Ginger Lily Farms Botanicals HEMP Herbal Body Moisturizer generously over entire body daily.
Water (Aqua), Propanediol. Isopropyl Palmitate, Cetearyl Alcohol, Glyceryl Stearate SE, Glycerin, Butyrospermum Parkii (Shea) Butter, Cannabis Sativa (Hemp) Seed Oil, Stearic Acid, Aloe Barbadensis Leafe Juice, Tocopheryl Acetate, Polyglyceryl-10 Oleate, Carbomer, Tetrasodium Glutamate Diacetate, Cyclopentasiloxane, Dimethiconol, Laureth-4, Laureth-23, Fragrance (Parfum), Phenoxyethanol, Caprlyl Glycol, Ethylhexylglycerin.


Real estate mogul Donald Trump announced he is bringing the 33rd annual PGA Grand Slam of Golf to Trump National Golf Club Los Angeles in Rancho Palos Verdes. The 36-hole tournament showcasing the four winners of the year's major golf championships will take place Oct. 19-21, Trump and PGA officials said Tuesday. It's the first PGA tournament at the 261-acre luxury golf club overlooking the Pacific Ocean and Catalina Island. The golf course will also host this year's PGA Junior League Championship Oct. 17-19. The Golf Channel will televise the Grand Slam and highlights from the Junior League championship.
The PGA Grand Slam will bring together the four winners of the Masters Tournament, which takes place April 9-12 in Augusta, Ga.; the U.S. Open, June 18-21 in University Park, Wash.; the Open Championship, July 16-19 in St. Andrews, Scotland; and the PGA Championship, Aug. 13-16 in Kohler, Wis. If a golfer wins more than one tournament, a system is in place to select a fourth player. Prize money has not yet been announced, but in the past it has been $1.35 million, $600,000 of which goes to the first place winner.
Past winners include defending champion Martin Kaymer, Tiger Woods and Phil Mickelson.
This year's event will mark the Grand Slam's return to the United States mainland since 1993 after eight years in Bermuda and 13 in Hawaii before that.
Last year, Trump National Golf Club Washington, D.C., in Potomac Falls, Va., was named host of the 2017 Senior PGA Championship and Trump National Golf Club in Bedminster, N.J., was named host of the PGA Championship in 2022.
Sprague added that the 80 youth players competing in the Junior League championship and their families traveling to Rancho Palos Verdes will add to the economic impact.
The event is expected to draw thousands, according to PGA Senior Director of Championships David Charles.
Unlike larger tournaments that have dozens of golfers, the Grand Slam's limited field of four players will offer spectators a more intimate view.
At a press conference in January announcing an 11.5 acre land easement over the golf course's driving range to the Palos Verdes Peninsula Land Conservancy, Trump hinted that a "really big" tournament would be announced soon.
The Palos Verdes Concours d'Elegance car show will move to the Los Verdes Golf Course after eight years at Trump National.
Tickets to the Grand Slam will be limited and prices will be announced later in the season.


When I hear a recording of my voice I think: "I don't sound like that." Apparently my voice sounds a lot deeper in my head than it does to everyone else.
Just another example of weird yet funny cat.
Mine is much better at being pet now that he is neutered.
Coca-Cola changing its 99-year-old recipe and Yahoo rejecting to buy Google for one million dollars are just two of the horrible decisions.
I love, love, love chicken.
I thought this was a racoon at first but it's just another weird cat.
These pet owners decided to play a little prank on what is probably the most adorable kitten ever. They used a remote-controlled giant tarantula to chase the kitten around the house, causing the little fluffball to freak out. Thankfully in the end the kitten was victorious in this playful attack of the giant spider.
What strange, new breed of puppies are these?


Chilean filmmaker Pablo Larraín is on a roll. 2016 will see the back-to-back debut of his couple of most recent films at two of the most important international film festivals. Neruda had its world premiere as part of the Directors' Fortnight at Cannes Film Festival, and it was announced today that his biopic film Jackie will premiere as part of the official competition at the 73rd edition of the Venice Film Festival. That in addition to the U.S. theatrical release of his Berlinale winner The Club which hit American theaters earlier this year.
With seven feature films, Larraín has become one of the most visible and recognized Latin American filmmakers of his generation, and with his latest two new productions he seems bound to get a ticket to the 88th Academy Awards, after being nominated in 2012 the Best Foreign Language Film category for No.
Set in 1948 when the Cold War had reached Chile, Neruda follows Senator Pablo Neruda, who in congress accuses the government of betrayal and is swiftly impeached by President Videla. Police Prefect Óscar Peluchonneau is assigned to arrest the poet. Neruda tries to flee the country with his wife Delia del Carril, but they are forced into hiding. In the struggle with his nemesis Peluchonneau, Neruda sees an opportunity to reinvent himself. He plays with the Prefect, leaving clues designed to make their game of cat-and-mouse more dangerous, more intimate. In this story of persecution, Neruda recognizes his own heroic possibilities: a chance to become both a symbol for liberty and a literary legend.
Starring Gael García Bernal, Luis Gnecco, and Mercedes Morán, Neruda will be releases this fall in the U.S. by the hand of distribution company The Orchard. The Toronto Film Festival just announced yesterday its Canadian premiere at its 41st edition, for which it can be deducted that it will probably have its North American premiere at the Telluride Film Festival during Labor Day weekend.
Written by Noah Oppenheim, produced by Darren Aronofsky, and starring Natalie Portman, Peter Sarsgaard, Billy Crudup, and Greta Gerwig, Jackie is an account of the days of First Lady, Jacqueline Kennedy, in the immediate aftermath of John F. Kennedy's assassination in 1963. The film has already started generating buzz for both Larraín and Portman as potential awards contenders.


Been off Xanex for 3 Years, Advice?
1 year off subutex and recently klonipin (quit cold turkey)-HAPPY ENDING!
benzos fine for methadone but not buprenorpine?
Is Subutex ruining my Klonopin?
Where to begin to look?
Am I a Benzo Addict??


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Week of Aug. 21
At the convention this spring, I was optimistic the community banking industry would be on offense by this point pushing for common-sense regulatory reform in Washington D.C. As it stands today, we're not even on the field with our issues.
The congressional calendar is full for the foreseeable future. Raising the debt ceiling, spending bills to keep the government open, and, probably most importantly, a budget resolution that sets spending levels for fiscal year 2018 all must be passed by Sept. 30. Without the budget resolution, Senate Republicans can't pass tax reform with 51 votes using the reconciliation process. The talk now is tax reform will not be passed until year-end or beyond.
The above schedule does not take into account the X-factor – that being President Trump. While the early-morning tweets, continued staff shakeups and off-the-cuff comments to the press may be entertaining, these distractions don't help in pushing the president's agenda forward. More importantly, they eat up precious political time and capital. I was hopeful Gen. Kelly as the new chief of staff would bring some order to the White House. The past 10 days have made me curb my enthusiasm.
With that said, there is work to be done in D.C. by bankers. Tax reform and the treatment of the interest rate deductions, Rule 1071 requiring the collection of information on business loans by the CFPB and pending overdraft regulation by CFPB all have the potential to adversely affect the community banking industry.
Our Annual Washington Visit provides the best opportunity to voice our concerns directly with the regulators and our congressional delegation on these issues. While we may not be on offense as we had all hoped for, it is just as important we play smart defense on the above issues.
I encourage you to make plans now to attend the Annual Washington Visit on Oct. 1-3. Our voices need to be heard on these issues as well as common-sense regulatory reform. Hope to see you there!
Guy Sims
Justice Department formally ends 'Operation Choke Point'
Courtesy of the ABA, in a letter to House Judiciary Committee Chairman Bob Goodlatte (R-Va.) on Wednesday, a Department of Justice official formally confirmed the agency has ended the controversial Operation Choke Point initiative, which under the Obama administration sought to curtail legal but politically disfavored businesses by working through bank regulators to pressure financial institutions to end customer relationships with those businesses.
"All of the Department's bank investigations conducted as part of Operation Choke Point are now over, the initiative is no longer in effect, and it will not be undertaken again," wrote Assistant Attorney General Stephen Boyd. "The Department will not discourage the provision of financial services to lawful industries, including businesses engaged in short-term lending and firearms-related activities."
ABA has long opposed Choke Point, successfully urging the FDIC to end its participation in the initiative and supporting legislation to prevent similar activities in the future. However, many financial institutions had been concerned about serving Choke Point-targeted businesses without a clear statement from DOJ that the initiative has been dropped.
Read the letter by clicking here.
BKD, LLP, has performed a calculation representing the nondeductible portion of member dues attributable to lobbying expenditures for the OBA's year ending April 30, 2017.
The source of the information used to determine this percentage included the Association's general ledger and an estimate of the percentage of time related to applicable personnel's involvement with lobbying activities by management of the Association. The OBA has reviewed the details involved in the calculation and have indicated it appears to represent the lobbying expenses for the year ending April 30, 2017.
As a result of these procedures, it has been determined the applicable percentage, as previously described to be 15.92 percent.
If you have any question, please contact Lea Ann Jackson at the OBA.
Same as kids currently preparing to go back to school for the year, bankers should be preparing to go back to school themselves with education opportunities from the OBA! Take a peek at the upcoming events:
Call Report for Banks: Recent Changes, Highlights, Aug. 23, webinar — The webinar will cover the latest revisions and recently approved changes to the call report, as well as updates due to revisions in accounting standards.
Dealing with Casual Days, Dress Codes and Work Appearance, Aug. 25, webinar — This program will cover the practical and legal issues and the current trends in workplace appearance and what you should and shouldn't do about it.
Cash Management, Aug. 29, webinar — During this two-hour webinar, you will gain a more in-depth knowledge of cash management (a.k.a. Treasury Management) products and services.
60 Critical Steps for Handling Delinquent and Abandoned Safe Deposit Boxes, Aug. 30, webinar — This webinar answers questions regarding the collection of past-due box rent, drilling delinquent boxes, securing drilled and inventoried box contents, reporting abandoned property to state authorities and many other legal and compliance issues have caused concern and confusion for the safe deposit industry.
BSA/AML for Lenders, Sept. 6, webinar — This two-hour program will assist your bank in determining whether your BSA Compliance Program adequately includes controls, such as training, policies and procedures, monitoring, etc for loan products in addition to deposit products.
Commercial Loan Agreements and Covenants, Sept. 7, webinar — This program explores the key sections in a commercial loan agreement and the goals they help achieve.
Reg E Seminar, Sept. 19, Oklahoma City — This program will provide a strong foundation in the requirements of Regulation E (Electronic Fund Transfers). We'll address what's covered and what's not, the issuance of access devices, liability for unauthorized transactions, disclosure requirements, preauthorized transfers and error resolution.
Analyzing Financial Statements, Sept. 20-22, Oklahoma City — Commercial lending officers, credit department personnel, loan review personnel and operations personnel interested in learning more about commercial lending will benefit from this three-day course.

redpajama_set_name: RedPajamaCommonCrawl


International Print Day will take place largely on social media, with a number of live events anticipated around the world by partners in the USA, UK and Australia.
Everyone is welcome to participate and contribute, with the aim being to see #IPD16 trending world-wide on Twitter. We would particularly appreciate the support of our colleagues across the media landscape to spread the word to your audiences – and hope you'll be able to get involved on the day.
Please click here for the Print Smart logo, which is the theme of this year's day. More details and other formats can also be found at http://ipd.printmediacentr.com/.
Thank you in advance for joining us in a bid to see print trend globally and spread the word about our creative and vibrant industry.


Make sure to review the VENDOR INFORMATION as well as download the VENDOR APPLICATON for the 2018 fair!
Welcome to The 131st Piscataquis Valley Fair!
Come Visit The Ice Cream Parlor!
Our ice cream parlor features many different treats to help cool you down during a hot summer day. From shakes and floats, along with the classic cone, and sundaes. Don't forget the banana splits! Make sure to check out The Ice Cream Parlor when you visit the fair this year!
This year the fair is offering wash stations near the animal barns. The wash stations are placed for your convience as a place for people to wash their hands after petting the animals.
Lost something during the fair? Need information?
Look no further! The kind people at the fair office will be happy to help you find what your looking for. Feel free to stop by during the fair!
Smokey's Greater Shows is back once again this year to provide the best midway experience possible this year. Over the winter they made several improvements to the rides and activities they provide duing the fair. To see the changes, visit their Facebook Page!
Check out our Facebook Page to stay up to date with everything going on with the fair!
Midnight Rose is a high energy band that loves to party with our fans. You can always count on hearing a variety of different music genre all night long.
The Honkytonkers are Maine's premier power trio. Many people refer to their original music as "POWER COUNTRY" because it is truly a mix of classic Rock with a flavor of today's country music.Country music with attitude is how one article summed up the group.
Formed in 2009 in Portland, Maine, The Mallett Brothers Band is an independent, nationally touring country rock and roll band consisting of brothers Luke and Will Mallett (vocals, guitar), Nick Leen (bass guitar), Wally (dobro, vocals, guitar), Andrew Martelle (fiddle, mandolin), and Chuck Gagne (drums).
Scan the QR Code Below To Access Our Website On Your Phone!


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Client Voices
Admin Managers
Life at Lavan
©2016 Lavan. All rights reserved.
Quadrant Advisory
Terms & Credits
Deregistration no barrier to determining unfair dismissal
Personal/Carer's leave: High Court calls it a "day"
A casual shock to employers - the Rossato decision
New Safety Laws are coming soon - What does this mean for your business?
COVID-19 UPDATE - Employment perspective
View all Employment and Safety Updates
The Fair Work Commission has recently determined that a company's deregistration was neither a barrier to determining an unfair dismissal claim, nor to referring the company to the Australian Tax Office and the Department of Home Affairs for "questionable practices".
In Yuening Fan v Integrated Pest Management Systems (Canberra) Pty Ltd [2020] FC 2957, Ms Fan, a former Integrated Pest Management Systems employee, made an application to the Commission claiming that her employer terminated her employment without valid reason or notice in November 2018.
Ms Fan had been employed by Integrated Pest Management Systems (the Company) since 13 October 2014. In her application, she stated that she had learned of her dismissal via a text message on 26 November 2018, when she had requested to collect some belongings from the workplace to which the Company responded, that they had discarded those belongings because she was no longer employed by the Company.
The Company's position was that Ms Fan was terminated in October by letter sent to her home address for using the Company's confidential and protected information without a reasonable basis for doing so, and interfering with the Company's intellectual property
The hearing was conducted by telephone due to visa issues preventing Ms Fan returning to Australia from China. At the hearing, Mr Smith from the Company attended by telephone, but did not formally enter an appearance in the proceedings. Mr Smith introduced himself to the Commission as being a former director of the Company. He told the Commission that the Company had been deregistered by ASIC on 11 February 2019 due to non-payment of annual fees. He said he was no longer a director of the Company and as such, he could not enter an appearance in the proceedings.
Mr Smith told the Commission that he was formally withdrawing from the matter, but continued to sit in on the proceedings and "just out of interest", made a number of comments in response to Ms Fan's submissions.
A particular comment made by Mr Smith was that the application could not proceed in circumstances where the Company had been deregistered. He sought an order that the application be struck out.
Commission's decision
As a preliminary issue, the Commission considered whether there was a barrier to the Commission determining Ms Fan's unfair dismissal application as a result of the Company having been deregistered by ASIC.
Section 601AD of the Corporations Act1 sets out the effect of deregistration of a company. The Commission noted that, unlike s500(2) of the Corporations Act, under which no action can proceed against a company being voluntarily wound up without leave, s601AD "does not refer to civil proceedings against a deregistered company".
The Commission further observed that the legislative note at s601AD(1) of the Corporations Act provides that "officers of the company may still be liable for things done before the company was deregistered", which suggested that "legal proceedings against a deregistered entity are possible".
The Commission then found that there was nothing in s601AD of the Corporations Act which explicitly precluded litigation or action against a deregistered entity, or provided that any such litigation or action was automatically struck out as a result of deregistration.
As a result, the Commission found that it could determine Ms Fan's unfair dismissal application despite the Company's deregistration.
The Commission ultimately held that Ms Fan's dismissal was not unfair in the circumstances of the case. However, in response to the application by Ms Fan, the Company had produced self-incriminating evidence of "undoubtedly questionable practices/arrangements having been adopted and/or agreed by the parties in this case in an effort to assist Ms Fan secure permanent residence in Australia". The Deputy President therefore referred the circumstances of the case to the ATO and the Department of Home Affairs.
Lavan comment
The Commission's decision raises three ancillary issues.
Firstly, the Commission may decide to hear an application even when the applicant is overseas and outside the Commission's jurisdiction. In this case, the applicant, Ms Fan, was in China.
Secondly, there are practical difficulties likely to be encountered by any applicant in enforcing an order made by the Commission against a deregistered company, such as an order for reinstatement.
Thirdly, the former director's decision in this case to participate in the process on behalf of a deregistered company, by filing documents and informally appearing during the proceedings, created a presence for a company that did not exist. By being present, the former director raised issues and put a spotlight on the Company's "questionable practices" that may not otherwise have occurred.
If you have any questions about this topic, please contact Lavan's Employment and Education team.
Employment and Safety Updates
Ian Curlewis
Tristan Seymour
[1] 2001 (Cth)
Level 20, 1 William Street, Perth Western Australia 6000 Telephone +61 8 9288 6000
©2016-2021 Lavan. All rights reserved.


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Revista Ecuatoriana de Pediatría
REVISTA ECUATORIANA DE PEDIATRIA
Vol. 23 No. 3 (2022): September-December, 2022
Clinical-epidemiological profile of early sepsis in newborns A single-center observational study
Descargar el PDF (Español (España)) Download PDF version EPUB (Español (España))
DOI: https://doi.org/10.52011/161
Sepsis Neonatal, Epidemiología, Factores de Riesgo, Puntaje de Apgar Neonatal Sepsis, Epidemiology, Risk Factors, Apgar Score
Fátima Ramírez Carillo
School of Medicine, Faculty of Medical Sciences, University of Guayaquil, Ecuador.
Cindy Cindy Lara Morales
Escuela de Medicina, Facultad de Ciencias Médicas, Universidad de Guayaquil, Ecuador.
Johanna Huacón Mazon
Neonatology Service, Hospital de Especialidades Teodoro Maldonado Carbo, Ecuadorian Institute of Social Se-curity, Guayaquil, Ecuador.
Stefanía Stefanía Loor Zambrano
Marilyn Urrutia Garcés
Introduction: Early neonatal sepsis is a clinical syndrome characterized by signs and symptoms associated with systemic infection; it occurs in the first 72 hours after birth. This study aimed to determine the clinical-epidemiological profile of early neonatal sepsis in a neonatal intensive care unit of a regional reference center in Guayaquil, Ecuador.
Methods: The present observational study, carried out at the "Teodoro Maldonado Carbo" Hospital from January 2017 to December 2020, included neonates with early neonatal sepsis with a nonprobabilistic sample. The variables were age, gestational age, sex, route of infection, presence of urinary tract infections, weight, genopathies, Apgar at 1 minute, Silverman scale, clinical stages, capillary refill, urinary output, clinical and laboratory variables, haem culture, and causative organism. The analysis is univariate and descriptive with frequencies and percentages.
Results: A total of 278 patients with a mean gestational age of 33 weeks were included, and 59.4% were men. Maternal risk factors were UTI in pregnancy and transplacental infection. Among the factors associated with the newborn were low weight (56%) and prematurity (67%). The most frequent symptoms were euthermia and tachypnea (54%). In the laboratory profile, neutropenia predominated (49%), while the causative agents were Staphylococcus hominis (7%), Escherichia coli (4.3%), and Klebsiella pneumoniae (4%).
Conclusion: The direct relationship between the epidemiological characteristics and the clinical stages of neonatal sepsis was determined.
Ramírez Carillo, F. ., Cindy Lara Morales, C. ., Huacón Mazon, J., Stefanía Loor Zambrano, S., & Urrutia Garcés, M. (2022). Clinical-epidemiological profile of early sepsis in newborns: A single-center observational study. Revista Ecuatoriana De Pediatría, 23(3), 192-200. https://doi.org/10.52011/161
Johanna Huacón Mazon, Neonatology Service, Hospital de Especialidades Teodoro Maldonado Carbo, Ecuadorian Institute of Social Se-curity, Guayaquil, Ecuador.
Physician from the University of Guayaquil (Guayaquil, 2012). Specialist in pediatrics from the Santiago de Guayaquil Catholic University (Guayaquil, 2016). Specialist in neonatology from the San Francisco De Quito University (Quito, 2019). Pediatric Neonatologist at the Teodoro Maldonado Carbo Specialty Hospital , IESS Guayaquil.
Stefanía Loor Zambrano, Marilyn Urrutia Garcés, Johanna Huacón Mazon, Fátima Ramírez Carrillo, Cindy Lara Morales, Factors associated with severe neonatal respiratory distress syndrome , Revista Ecuatoriana de Pediatría: Vol. 23 No. 2 (2022): May-August, 2022
Tweets By @RevistaEcuator1


The agenda is unavailable at this time.
All public meetings are held in accessible places.
If you need assistance or accommodation for a disability please notify the TPO three business days in advance of the meeting and we will be glad to work with you in obliging any reasonable request.


"Prayer" was inspired from Mormon 9: 27: "...ask the Father in the name of Jesus for what things soever ye shall stand in need. Doubt not, but be believing..."
This song has been viewed 346 times on this site. This song's pdf has been viewed/downloaded 24 times.


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January 10, 2014 Leave a Comment Written by admin
The first game to play would be fallout 3. Fallout 3 is a game set in a post-apocalyptic Washington DC after a nuclear war that has all but destroyed the planet. You must explore your way through the wasteland fighting humans and mutants alike to discover the truth of why your father left the safety of vault 101 one of the safest places in the wasteland and to find your own destiny and make friends and enemy's along the way.
To get the most from this game you should purchase the fallout 3 classic editions which come with all five downloadable content which are Operation: anchorage this DLC allows you to enter a military simulator set to be for the Great War and fight for the liberation of anchorage, Alaska from Chinese communist invaders.
The pitt in this DLC you are asked for help by a man named Wernher who is an escaped slave from the post-apocalyptic Pittsburgh. You must free the slaves of the pitt from the raider masters but to do this you must earn the trust of the raiders leader an ex brotherhood member known as Ashur.
The third DLC is broken steel after you have saved or doomed the wasteland your fight isn't over you must work with the brotherhood to seek out and crush the remaining enclave forces and their new mobile assault base but now you will have some new toys to play with such as the devastating tesla cannon to shoot those pesky enclave drop ships out the air but the is many new dangers to face such as the super mutant overlords and the enclave hellfire troops.
Point lookout, in this DLC you find yourself a completely new wasteland which takes place in the murky swamp lands on the coast of Maryland so hop on the duchess gambit to begin a new adventure but watch out for the locals. You will get new weapons such as the double barrel shotgun and the lever action rifle to help you kill the deformed remints of the hillbillies who used to live in the swamps.
Mother ship zeta, in this DLC you are abducted by the friends of an alien that crash-lands in the post-apocalyptic Washington and you must fight your way through the ship with help from the most unlikely of people like an ancient samurai which ends in a stand of for the fate of the earth.
BEST GAMES TO PLAY WHILE IT RAINS part three


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Mission, Services & Core Values
OGC Connections
The "Light Side" of the OGC
Contracts Process/Management
Delegation of Signature Authority
ERM Staff
Pursuant to applicable provisions of the North Carolina General Statutes and Board of Governors' policies, the chancellor at each institution has the authority to sign contracts, leases, and other legal documents. Accordingly, Appalachian State University's Chancellor retains this authority at all times and may exercise this authority herself or through written delegations to other administrative officers.
Appalachian does not recognize contracts as binding that are signed by University employees that do not possess duly delegated signature authority. Employees who sign contracts without requisite authority may be personally liable for the contract, as well as being subject to employment disciplinary action.
In order for a delegation of authority to be valid, delegations must be in writing and include the following:
the name, title or position of the individual to whom the delegation will apply and
a description of the scope, terms, and limitations of the delegation.
The Office of General Counsel shall receive and retain a copy of all such delegations and maintain the Delegation of Signature Authority for the University. If you have questions about who is authorized to sign a contract on behalf of the University, please contact the Office of General Counsel.
B.B. Dougherty Administration Building
438 Academy Street
Boone, NC 28608-2126
©2021, Appalachian State University. All rights reserved.
Disclaimer :: EO Policy


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Calliapagurops foresti is een tienpotigensoort uit de familie van de Callianassidae. De wetenschappelijke naam van de soort is voor het eerst geldig gepubliceerd in 2002 door Ngoc-Ho.

Callichiridae

redpajama_set_name: RedPajamaWikipedia


Recently updated, adorable and cozy beach getaway. The condo is located on the West end of the island at Starboard St 250-5-B Starboard St and it is a 3 BR, 2 Bath with a full kitchen and dining space. Outdoor dining on covered patio as well.
***COMFY BEDS with new mattresses! Master has new king mattress, Bedroom #2 has (2) new queen beds, and bedroom #3 has twin bunk beds with a twin trundle. Renters must supply their own sheets.
The condo and outdoor property is non smoking and no pets are allowed. WIFI, three flat screen TVs, DVR, and DVD.
Weekly summer rentals only with this unit from Sunday to Sunday. Weekly rental rate is $1150 plus tax (cleaning included) . Please email owner for rental agreement.
Nightly rental available in off season with a 3-night minimum. Any other questions please don't hesitate to inquire by email or text.
Amenities: 3 pools in condo complex, one is ocean front which is 2 blocks away (easy walk). There is day docking for boating and fishing directly behind the condo (boat ramp is at the OIB Bridge). Tennis courts as well.
Ocean Isle is located 40 minutes north of North Myrtle Beach and south of Wilmington. Wonderful family beach with nice restaurants on the island. Nearby Calabash and Shallotte are 20 minutes away.
Lovely canal house in Holden Beach, NC with beautiful views of waterway!
Light and airy with updated colors, new furnishings and in a great community!
NEW TO VRBO--Beautifully UPDATED-Lagoon views!
"Latitude Adjustment" is a Charming 4 bedroom 2 bath Beach Home with Gorgeous views of the Atlantic Ocean, Marshlands and Intracoastal Waterway!
Classic Beach Home Newly Remodeled!
Oceanfront - Boardwalk - Covered Oceanside Porch!


As you know, Furrnion is a new convention, and as such, still not widely-known. We're trying to get ourselves known as much as we can, for which we've made flyers, a lot of social network promotion, etc.
We also know that going to a convention isn't cheap, and some of you go to several every year, usually the most known. It's always a 'risk' to go to a new one, for which you don't have past references.
That's why we've decided to make a special discount for all of you who have gone to a Con already in 2016, a discount that will be available throughout august and september. If you've gone to any Furry Convention this year (january to august included), attach a picture of you with that con's official badge for this year in front of our registration screen, and we'll give you a 20% discount in the basic level's registration!
Also, we're very glad to give that discount and a very warm welcome to all of you who have attended Furbest 2015. After knowing the sad news that it won't be made this year, we just wanted to contribute a bit to the cause and make it more affordable for those who attended the first Furbest to come to Furrnion or who were planning to go this year's. If it's your case, specify it in the registration form and we'll contrast the information with Furbest staff so we can apply the discount to you!


4 - 6 - 3, LTD. is an entity registered at ALBANY county with company number 2297101. 4 - 6 - 3, LTD. located at the address 177 Adams St Delmar, New York, 12054. Company is incorporated on September 4, 1998. The Chief Executive Officer is Howard Glassman Current status of the company is active.
September 14, 1998 Actual 4 - 6 - 3, LTD.
4 1 5 RESTAURANT INC.
4 A AUTOMOTIVE PURCHASING SERVICE INC.
4 A BETTER WESTCHESTER + NYS ASSOCIATION, INC.
4 + 1 BEVERAGES, INC.
4 'N 1 BUSINESS SERVICES INC.
4 & A TAIL KENNELS INC.
4 & 6 EAST 62ND STREET REALTY CROP.
4 & 3 SOCIAL CLUB, INC.
4 & 2 REALTY CORP.
4 & 1 DISTRIBUTING, INC.


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King Crimson, music, Red
Certain music tends to form my soundtrack during certain moods. I find myself listening to it over and over again and then I move on to something else completely.
Well, my mood is changing but I haven't quite let go of this past week's musical focus. …
The King Crimson album Red is an unusual member of that band's very extensive discography. Not in circumstance – the band was falling apart yet again as it seemed to do every six months in the early 1970s – but in sound and purpose. No other King Crimson album is so single-mindedly cohesive.
Part of this is undoubtedly caused by the reduction of the band to essentially a trio of Robert Fripp on guitar, John Wetton on bass & vocals and Bill Bruford on drums. These instruments dominate the whole proceedings even though there are in fact a lot of guests coming along for the ride – the very recently departed David Cross on violin, the almost rejoining Ian McDonald on saxophone and others such as cornettist Mark Charig who had been in and out of prior incarnations of the band.
But it is primarily Fripp and his rhythm section who dominate, and Fripp does not shy away from his aggressive side here. Indeed in tone and style, Fripp's guitar verges on heavy metal and punk with very little 'pretty' picking. The songs are saturated with deep, moody chords and a churning cauldron of rhythmic activity. You are not going to hear better or more responsive hard rock playing than you find here. The progressive elements are all in the mix, but subsumed to the overall impact – unlike some other Crimson tracks you do not find yourself admiring but essentially uninvolved in the music. The songs are a miracle of tautness for a band that regularly fell into the pit of abysmal lyrical posturing; nothing that really stands out but words that are attuned to the music and unembarassing upon repeated play.
Red was released in 1974, a fairly dismal year for mainstream rock. At that time there was definitely the sense that rock was running out of steam as the really remarkable period of creativity and energy since the Beatles' first records fizzled. I remember this as being very dismaying at the time – I, like most rock fans – was used to a hothouse of mainstream innovation and it was clear that this was not to be found anymore. So Red encapusalated both that innovative past, drew together the most fruitful and productive current strands, and showed via its violent energy a way forward that would explode with punk rock three years later.
I think that is why the music has held up so well.
1 thought on "Red"
Great reading. A long time ago I had one of KC albums, don't remember which one it was. I found your comment about the period c. 1974 interesting also. There was a little going on — Bowie, Elton John, The Eagles ect — not very many really good ones and as you say it seemed as if the stream might be drying up. Thank god for Punk and New Wave.


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Beyoncé On Revealing Documentary: "I'm Scared"
Star has released a teaser clip of the film, expressing her concerns...
Wednesday, December 5, 2012 - 11:31
Jay-Z.
In a teaser clip for the doc, Bey appears fresh-faced as she speaks directly to a camera, stating: "I always battle with 'How much do I reveal about myself?'. If I'm scared, be scared, allow it. Release it."
The formerDestiny's Child star, who [uuid=04d1fb18-890c-4d5a-a991-94f9f9dfc6f5]The-Dreamhas told Billboard that fans can expect some new music from the star ahead of her Super Bowl stint, stating: "She's got her stuff going. She took the summer to start recording, and now she's in a place where she's ready to start gearing up to reveal her plan.
"She's already gearing up to get ready to put stuff out. I'm sure there will probably be a couple records you hear before the Super Bowl gets here."


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Br J Clin Pharmacol. 2004 Jun;57(6):695-713.
The revision of the Declaration of Helsinki: past, present and future.
Carlson RV1, Boyd KM, Webb DJ.
Clinical Pharmacology Unit, University of Edinburgh, Western General Hospital, UK. r.carlson@ed.ac.uk
The World Medical Association's Declaration of Helsinki was first adopted in 1964. In its 40-year lifetime the Declaration has been revised five times and has risen to a position of prominence as a guiding statement of ethical principles for doctors involved in medical research. The most recent revision, however, has resulted in considerable controversy, particularly in the area of the ethical requirements surrounding placebo-controlled trials and the question of responsibilities to research participants at the end of a study. This review considers the past versions of the Declaration of Helsinki and asks the question: How exactly has the text of the Declaration changed throughout its lifetime? Regarding the present form of the Declaration of Helsinki we ask: What are the major changes in the most recent revision and what are the controversies surrounding them? Finally, building on the detailed review of the past and present versions of the Declaration of Helsinki, we give consideration to some of the possible future trajectories for the Declaration in the light of its history and standing in the world of the ethics of medical research.
Word count for each revision of Declaration of Helsinki and years since last revision. *Nuremberg Code, **Includes Note of Clarification
The revision of the Declaration of Helsinki: past, present and future
Ethics, Medical
Helsinki Declaration*
Societies, Medical


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