Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan.

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Serological survey for antibody to Encephalitozoon cuniculi in horses in the USA.

Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that can result in clinical and subclinical infection in many species. In the present study, a serological survey was conducted using samples from 105 horses in the state of New Jersey; 49 of the samples were obtained from clinically abnormal animals. Five or 4.8% of 105 serum samples were found to demonstrate reactivity by ELISA with titers of 1:64 to 1:1,024. One of the samples was obtained from a clinically normal horse. Clinical signs and diagnoses from the other animals included lameness, colic, osteochondritis dissecans, and fever. All clinical issues were resolved with hospitalization and treatment without the institution of E. cuniculi-focused therapy. This is the first report on the detection of E. cuniculi antibodies in horses in the USA.

Impact of lower extremity strengthening exercises and mobility on fall rates in hospitalized adults.

Preventing falls in acute care hospitals is a major challenge, and achieving positive outcomes has remained elusive. The purpose of this study was to examine the impact of lower extremity strengthening exercises and mobility on fall rates and fall rates with injury. A nonequivalent control group design was used. Subjects on the intervention unit received targeted lower extremity strengthening exercises and ambulation using a nurse-driven mobility protocol; subjects on the control unit received ambulation alone. One assisted fall occurred on the intervention unit.

Impact of a nurse-driven mobility protocol on functional decline in hospitalized older adults.

The purpose of the study was to determine the impact of a nurse-driven mobility protocol on functional decline. A nonequivalent control group design was used; the independent variable was mobility protocol and dependent variables were functional status and length of stay. Older adults who participated in a mobility protocol maintained or improved functional status and had a reduced length of stay. Practice implications include an emphasis on ambulation in hospitalized older adults.

Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel.

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.

Prevalence and severity of hypertension in a dental hygiene clinic.

AIM: The purpose of this study was to assess the prevalence and severity of hypertension in a dental hygiene clinic and evaluate factors related to the disease. METHODS AND MATERIALS: Records of 615 patients, treated by dental hygiene students during 2003, were reviewed. Data collected included systolic and diastolic blood pressure, presence of diabetes and renal disease, non-modifiers (race, gender, and age), and modifiers (marital status, smoking habits, and occupation). RESULTS: According to the Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7) classification, 154 (25%) of the subjects had normal blood pressure readings, 374 (60.8%) had prehypertension, and 87 (14.1%) had stage 1 hypertension. Statistical analysis showed a significant difference in the JNC7 classification between groups when considering the non-modifiers' race (p=.02) and the modifiers' smoking habits (p=.03) and occupation (p=.01). A statistically significant difference in the JNC7 classification existed between groups with diabetes (p=.00). The majority of patients had blood pressure readings in the prehypertension stage. CONCLUSION: Based on these results, the researchers recommend clinical policy modifications which include: additional documentation for blood pressure readings in the prehypertension stage, lowering the systolic reading from 160 mmHg to 140 mmHg when adding hypertension alert labels, and noting prehypertension/hypertension on the dental hygiene care plan with the appropriate interventions.

Connexin expression and gap junctional intercellular communication in human squamous cell carcinoma of the head and neck.

OBJECTIVE: Our laboratory is investigating the role that gap junction intercellular channels (composed of proteins called connexins) play in communicating apoptotic signals from therapeutically targeted squamous cell carcinoma of the head and neck (SCCHN) cells to adjacent, untreated, "bystander" cells (bystander effect). The nature of this research underscores the importance of delineating connexin expression patterns in SCCHN, and how this correlates with gap junctional intercellular communication (GJIC) and bystander effects. STUDY DESIGN: The GJIC activity of a diverse panel of SCCHN cell lines, as well as normal oral epithelial (NOE) cell controls was determined in vitro. These data were correlated with connexin expression patterns determined through connexin 43 and connexin 26 immunofluorescence. RESULTS: Cell lines with retained GJIC activity all expressed connexin 43 on the cell membrane. Cell lines that did not communicate microinjected lucifer yellow (lost GJIC activity) showed no connexin expression, either at the cell membrane or in the cytosol. Connexin 26 was not expressed in any of our SCCHN cell lines, whereas both connexin 43 and connexin 26 were expressed in the NOE cell controls. Furthermore, connexin 43 introduction into a GJIC (and connexin) deficient SCCHN cell line conferred no growth inhibitory effect. CONCLUSION: Connexin 43 expression correlates with retained GJIC in SCCHN in vitro. Connexin 26 may have a role as a tumor suppressor in SCCHN. SIGNIFICANCE: The data presented have relevance to our ongoing investigations of gap-junction mediated bystander effects in SCCHN and are being expanded to investigations on actual SCCHN tumor specimens.

Blood pressure assessment practices of dental hygienists.

An estimated 50 million Americans have high blood pressure (HBP), with 30% of them unaware of their condition. Both the American Dental Association (ADA) and the American Dental Hygienists' Association (ADHA) have advocated including recording blood pressure during the dental appointment. Recording blood pressure is also a standard procedure in patient care. This study surveyed 236 dental hygienists attending a continuing education program to document their blood pressure assessment practices. The majority (55%) of participants indicated they rarely or never record blood pressure. The primary reason cited by 51% of the participants was a lack of time in the appointment. Based on these findings, a recommendation was made for dental offices to modify their patient check-in procedures to include recording blood pressure.

Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System.

Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem cells has been invaluable in the last two decades, it is an extremely costly, time-consuming, and, in some cases, uncertain technology. The recently described CRISPR-Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientists to test more precise and bold hypotheses in vivo. Using this revolutionary methodology we have generated more than 100 novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags, or endogenous reporters.

Comparisons of childhood trauma, alexithymia, and defensive styles in patients with psychogenic non-epileptic seizures vs. epilepsy: Implications for the etiology of conversion disorder.

BACKGROUND: It has been theorized that conversion disorder is the result of emotion that cannot be experienced consciously as feeling states or put into words (i.e., alexithymia), but there is little confirming empirical evidence. We sought to characterize subjects with conversion disorder compared to subjects with a distinct medical illness, using the model of psychogenic non-epileptic seizures (PNES) vs. epilepsy (ES), on measures of childhood traumatic experience, alexithymia and maturity of psychological defensive strategies. METHODS: All subjects admitted to the Epilepsy Monitoring Unit of the University of Cincinnati Medical Center were offered self-report questionnaires (Childhood Trauma Questionnaire, Toronto Alexithymia Scale-20 and Response Evaluation Measure-71) at the outset of evaluation. Diagnosis of each subject was confirmed by video-EEG and we compared subjects with PNES to those with ES on these measures. RESULTS: 82 subjects had ES AND 96 had PNES. Those with PNES were significantly more likely to have experienced childhood trauma in all domains (p=.005 to p=.05), and were significantly more likely to have alexithymia (p=.0267). There was a significant difference in the capacity to identify feelings, and a trend towards significance in capacity to describe feelings. There were no differences in defensive styles between the two groups. CONCLUSIONS: PNES diagnosis was associated with female sex, higher alexithymia scores and higher rates of childhood trauma, but not with differences in defensive styles compared to ES. These findings add empirical evidence for theories regarding the cause of conversion disorder and may aid in the design of prospective treatment trials in patients with conversion disorder.

A grounded theory of the process of adherence to oral chemotherapy in Hispanic and caucasian children and adolescents with acute lymphoblastic leukemia.

Children and adolescents with acute lymphoblastic leukemia (ALL) receive treatment that relies on daily self- or parent/caregiver-administered oral chemotherapy for approximately 2 years. Despite the fact that pediatric ALL is uniformly fatal without adequate treatment, nonadherence to oral chemotherapy has been observed in up to one third of patients. Little is known about the reasons for nonadherence in these patients. This study used Straussian grounded theory methodology to develop and validate a model to explain the process of adherence to oral chemotherapy in children and adolescents with ALL. Thirty-eight semistructured interviews (with 17 patients and 21 parents/caregivers) and 4 focused group discussions were conducted. Three stages were identified in the process of adherence: (a) Recognizing the Threat, (b) Taking Control, and (c) Managing for the Duration. Doing Our Part was identified as the core theme explaining the process of adherence and involves the parent (or patient) taking responsibility for assuring that medications are taken as prescribed. Understanding the association between taking oral chemotherapy and control/cure of leukemia (Making the Connection) appeared to mediate adherence behaviors.

MRI-guided prostate radiation therapy planning: Investigation of dosimetric accuracy of MRI-based dose planning.

BACKGROUND AND PURPOSE: Dose planning requires a CT scan which provides the electron density distribution for dose calculation. MR provides superior soft tissue contrast compared to CT and the use of MR-alone for prostate planning would provide further benefits such as lower cost to the patient. This study compares the accuracy of MR-alone based dose calculations with bulk electron density assignment to CT-based dose calculations for prostate radiotherapy. MATERIALS AND METHODS: CT and whole pelvis MR images were contoured for 39 prostate patients. Plans with uniform density and plans with bulk density values assigned to bone and tissue were compared to the patient's gold standard full density CT plan. The optimal bulk density for bone was calculated using effective depth measurements. The plans were evaluated using ICRU point doses, dose volume histograms, and Chi comparisons. Differences in spatial uniformity were investigated for the CT and MR scans. RESULTS: The calculated dose for CT bulk bone and tissue density plans was 0.1±0.6% (mean±1 SD) higher than the corresponding full density CT plan. MR bulk bone and tissue density plans were 1.3±0.8% lower than the full density CT plan. CT uniform density plans and MR uniform density plans were 1.4±0.9% and 2.6±0.9% lower, respectively. Paired t-tests performed on specific points on the DVH graphs showed that points on DVHs for all bulk electron density plans were equivalent with two exceptions. There was no significant difference between doses calculated on Pinnacle and Eclipse. The dose distributions of six patients produced Chi values outside the acceptable range of values when MR-based plans were compared to the full density plan. CONCLUSIONS: MR-alone bulk density planning is feasible provided bone is assigned a density, however, manual segmentation of bone on MR images will have to be replaced with automatic methods. The major dose differences for MR bulk density plans are due to differences in patient external contours introduced by the MR couch-top and pelvic coil.

Screening and management of anal dysplasia and anal cancer in HIV-infected patients: a guide for practice.

People living with HIV infection have a significantly higher rate of anal cancer as compared with that of uninfected people. It is believed that high-grade anal dysplasia secondary to human papillomavirus infection is a precursor to anal cancer. Considering this, screening and treatment of high-grade anal dysplasia is a possible means of preventing the development of anal cancer. No national or international guidelines exist to guide practice for screening and management of anal dysplasia. On the basis of a review of research and expert recommendations, a guide to practice for screening and management of anal dysplasia and anal cancer is made for clinicians.

A sensory feedback circuit coordinates muscle activity in Drosophila.

Drosophila larval crawling is a simple behavior that allows us to dissect the functions of specific neurons in the intact animal and explore the roles of genes in the specification of those neurons. By inhibiting subsets of neurons in the PNS, we have found that two classes of multidendritic neurons play a major role in larval crawling. The bipolar dendrites and class I mds send a feedback signal to the CNS that keeps the contraction wave progressing quickly, allowing smooth forward movement. Genetic manipulation of the sensory neurons suggests that this feedback depends on proper dendritic morphology and axon pathfinding to appropriate synaptic target areas in the CNS. Our data suggest that coordination of muscle activity in larval crawling requires feedback from neurons acting as proprioceptors, sending a "mission accomplished" signal in response to segment contraction, and resulting in rapid relaxation of the segment and propagation of the wave.

Hox gene function and interaction in the milkweed bug Oncopeltus fasciatus (Hemiptera).

Studies in genetic model organisms such as Drosophila have demonstrated that the homeotic complex (Hox) genes impart segmental identity during embryogenesis. Comparative studies in a wide range of other insect taxa have shown that the Hox genes are expressed in largely conserved domains along the anterior-posterior body axis, but whether they are performing the same functions in different insects is an open question. Most of the Hox genes have been studied functionally in only a few holometabolous insects that undergo metamorphosis. Thus, it is unclear how the Hox genes are functioning in the majority of direct-developing insects and other arthropods. To address this question, we used a combination of RNAi and in situ hybridization to reveal the expression, functions, and regulatory interactions of the Hox genes in the milkweed bug Oncopeltus fasciatus. Our results reveal many similarities and some interesting differences compared to Drosophila. We find that the gene Antennapedia is required for the identity of all three thoracic segments, while Ultrabithorax, abdominal-A and Abdominal-B cooperate to pattern the abdomen. The three abdominal genes exhibit posterior prevalence like in Drosophila, but apparently via some post-transcriptional mechanism. The functions of the head genes proboscipedia, Deformed, and Sex combs reduced were shown previously, and here we find that the complex temporal expression of pb in the labium is like that of other insects, but its regulatory relationship with Scr is unique. Overall, our data reveal that the evolution of insect Hox genes has included many small changes within general conservation of expression and function, and that the milkweed bug provides a useful model for understanding the roles of Hox genes in a direct-developing insect.

Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede.

The diversity of the arthropod body plan has long been a fascinating subject of study. A flurry of recent research has analyzed Hox gene expression in various arthropod groups, with hopes of gaining insight into the mechanisms that underlie their evolution. The Hox genes have been analyzed in insects, crustaceans and chelicerates. However, the expression patterns of the Hox genes have not yet been comprehensively analyzed in a myriapod. We present the expression patterns of the ten Hox genes in a centipede, Lithobius atkinsoni, and compare our results to those from studies in other arthropods. We have three major findings. First, we find that Hox gene expression is remarkably dynamic across the arthropods. The expression patterns of the Hox genes in the centipede are in many cases intermediate between those of the chelicerates and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. Second, we found two 'extra' Hox genes in the centipede compared with those in Drosophila. Based on its pattern of expression, Hox3 appears to have a typical Hox-like role in the centipede, suggesting that the novel functions of the Hox3 homologs zen and bicoid were adopted somewhere in the crustacean-insect clade. In the centipede, the expression of the gene fushi tarazu suggests that it has both a Hox-like role (as in the mite), as well as a role in segmentation (as in insects). This suggests that this dramatic change in function was achieved via a multifunctional intermediate, a condition maintained in the centipede. Last, we found that Hox expression correlates with tagmatic boundaries, consistent with the theory that changes in Hox genes had a major role in evolution of the arthropod body plan.

Exploring myriapod segmentation: the expression patterns of even-skipped, engrailed, and wingless in a centipede.

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. Here, we present the expression patterns of the genes even-skipped (eve), engrailed, and wingless in a centipede, Lithobius atkinsoni. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods.