It's past your bedtime, but does it matter anymore? How longitudinal changes in bedtime rules relate to adolescents' sleep.

This study investigated how changing or maintaining parent-set bedtimes over time relates to adolescents' sleep timing, latency, and duration. Adolescents (n = 2509; Mage = 12.6 [0.5] years; 47% m) self-reported their sleep patterns, and whether they had parent-set bedtimes on two separate occasions in 2019 (T1; 12.6 years) and 2020 (T2; 13.7 years). We identified four groups based on parent-set bedtimes: (1) bedtime rules at both T1 and T2 (46%, n = 1155), (2) no bedtime rules at T1 nor T2 (26%, n = 656), (3) bedtime rules at T1 but not T2 (19%, n = 472), (4) no bedtime rules at T1 but a parent-set bedtime at T2 (9%, n = 226). As expected, the entire sample showed that bedtimes generally became later and sleep duration shorter across adolescence, but the change differed among the groups. Adolescents whose parents introduced bedtime rules at T2 reported earlier bedtimes and longer sleep duration (~20 min) compared with adolescents with no bedtime rules at T2. Importantly, they no longer differed from adolescents who consistently had bedtimes across T1 and T2. There was no significant interaction for sleep latency, which declined at a similar rate for all groups. These results are the first to suggest that maintaining or re-introducing a parent-set bedtime may be possible and beneficial for adolescents' sleep.

Can regulatory T cells improve outcomes of sensitised patients after HLA-Ab incompatible renal transplantation: study protocol for the Phase IIa GAMECHANgER-1 trial.

BACKGROUND: Kidney transplantation is the gold-standard treatment for patients with kidney failure. However, one-third of patients awaiting a kidney transplant are highly sensitized to human leukocyte antigens (HLA), resulting in an increased waiting time for a suitable kidney, more acute and chronic rejection, and a shorter graft survival compared to non-highly sensitised patients. Current standard immunosuppression protocols do not adequately suppress memory responses, and so alternative strategies are needed. Autologous polyclonally expanded regulatory T cells (Tregs) have been demonstrated to be safe in transplant settings and could be a potential alternative to modulate memory immune alloresponses. METHODS: The aim of this trial is to determine whether adoptive transfer of autologous Tregs into HLA sensitised patients can suppress memory T and B cell responses against specific HLA antigens. This is a two-part, multi-centre, prospective clinical trial, comprising an observational phase (Part 1) aiming to identify patients with unregulated cellular memory responses to HLA (Pure HLA Proteins) followed by an interventional phase (Part 2). The first 9 patients identified as being eligible in Part 1 will undergo baseline immune monitoring for 2 months to inform statistical analysis of the primary endpoint. Part 2 is an adaptive, open labelled trial based on Simon's two-stage design, with 21 patients receiving Good Manufacturing Practice (GMP)-grade polyclonally expanded Tregs to a dose of 5-10 × 106 cells/kg body weight. The primary EP is suppression of in vitro memory responses for 2 months post-infusion. 12 patients will receive treatment in stage 1 of Part 2, and 9 patients will receive treatment in stage 2 of Part 2 if ≥ 50% patients pass the primary EP in stage 1. DISCUSSION: This is a prospective study aiming to identify patients with unregulated cellular memory responses to Pure HLA Proteins and determine baseline variation in these patterns of response. Part 2 will be an adaptive phase IIa clinical trial with 21 patients receiving a single infusion of GMP-grade polyclonally expanded Tregs in two stages. It remains to be demonstrated that modulating memory alloresponses clinically using Treg therapy is achievable. TRIAL REGISTRATION: EudraCT Number: 2021-001,664-23. REC Number: 21/SC/0253. Trial registration number ISRCTN14582152.

Genetic epidemiology of malignant hyperthermia in the UK.

BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

Quantified degree of poultry exposure differs for human cases of avian influenza H5N1 and H7N9.

Preliminary evidence suggests that direct poultry contact may play a lesser role in transmission of avian influenza A(H7N9) than A(H5N1) to humans. To better understand differences in risk factors, we quantified the degree of poultry contact reported by H5N1 and H7N9 World Health Organization-confirmed cases. We used publicly available data to classify cases by their degree of poultry contact, including direct and indirect. To account for potential data limitations, we used two methods: (1) case population method in which all cases were classified using a range of sources; and (2) case subset method in which only cases with detailed contact information from published research literature were classified. In the case population, detailed exposure information was unavailable for a large proportion of cases (H5N1, 54%; H7N9, 86%). In the case subset, direct contact proportions were higher in H5N1 cases (70·3%) than H7N9 cases (40·0%) (χ 2 = 18·5, P < 0·001), and indirect contact proportions were higher in H7N9 cases (44·6%) than H5N1 cases (19·4%) (χ 2 = 15·5, P < 0·001). Together with emerging evidence, our descriptive analysis suggests direct poultry contact is a clearer risk factor for H5N1 than for H7N9, and that other risk factors should also be considered for H7N9.

Psychosocial Evaluation in Patients Undergoing Left Ventricular Assist Device Implantation Using the Transplant Evaluation Rating Scale.

BACKGROUND: It has been recommended that all candidates for left ventricular assist device (LVAD) implantation undergo preoperative psychologic evaluation for risk assessment. OBJECTIVE: We used the transplant evaluation rating scale (TERS) that was established for pretransplant evaluation to investigate the psychosocial assessment of patients undergoing LVAD implantation. METHODS: This study retrospectively analyzed data from 125 patients with advanced heart failure who were evaluated by the TERS before LVAD implantation. Postoperative follow-up included survival, total length of hospital stay, readmissions, and post-LVAD out-of-hospital days after discharge. The cohort was stratified according to the TERS scores into low-, moderate-, and high-risk groups. The outcomes were analyzed to evaluate whether the TERS score was associated with post-LVAD adverse events. RESULTS: The TERS, when stratified into 3 risk groups showed significant difference in 8 of the 10 psychosocial domains (p < 0.001). The mean number of outpatient days after discharge was significantly different between the low-, moderate-, and high-risk groups (p < 0.001). All other outcomes were not significantly different. CONCLUSIONS: This study showed that the TERS is successful in stratifying our patients with an LVAD into 3 risk groups, indicating the internal validity of this test. The number of out-of-hospital (outpatient) days after discharge was significantly shorter in the TERS high-risk group, which may affect the quality of life and cost of post-LVAD care.

Model for end-stage liver disease predicts right ventricular failure in patients with left ventricular assist devices.

High rates of right ventricular failure continue to affect postoperative outcomes in patients implanted with left ventricular assist devices (LVADs). Development of right ventricular failure and implantation with right ventricular assist devices is known to be associated with significantly increased mortality. The model for end-stage liver disease (MELD) score is an effective means of evaluating liver dysfunction. We investigated the prognostic utility of postoperative MELD on post-LVAD implantation outcomes. MELD scores, demographic data, and outcomes including length of stay, survival, and postoperative right ventricular failure were collected for 256 patients implanted with continuous flow LVADs. Regression and Kaplan-Meier analyses were used to investigate the relationship between MELD and all outcomes. Increased MELD score was found to be an independent predictor of both right heart failure and necessity for RVAD implantation (OR 1.097, CI 1.040-1.158, p = 0.001; OR 1.121, CI 1.015, p = 0.024, respectively). Patients with RV failure and who underwent RVAD implantation had reduced postoperative survival compared to patients with RV dysfunction (no RV failure = 651.4 ± 609.8 days, RV failure = 392.6 ± 444.8 days, RVAD = 89.3 ± 72.8 days; p < 0.001). In conclusion, MELD can be used to reliably predict postoperative right heart failure and the necessity for RVAD implantation. Those patients with RV failure and RVADs experience significantly increased postoperative mortality compared to those without RV dysfunction.

Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions.

Playing for more than winning: Exploring sports participation, physical activity, and belongingness and their relationship with patterns of adolescent substance use and mental health.

BACKGROUND: Promoting adolescent sports participation and physical activity may be effective low-barrier prevention strategies for co-occurring adolescent substance use (SU) and mental health symptoms (MH). The objectives of this study were to: 1) explore associations between profiles of SU/MH and sports participation; and 2) determine whether physical activity and belongingness account for these associations. METHODS: Data came from a representative sample of 11,994 grade 9-12 Ontarian students (ages ~14-18) previously grouped into five SU/MH profiles based on patterns of use and symptoms. A series of multinomial logistic regressions, adjusted for socio-demographics and school clustering, were used to predict the risks of students belonging to SU/MH profiles based on: 1) school sports participation (>=weekly), 2) sports and physical activity (>=60minutes; 0-7 days), and 3) sports, physical activity, and school belongingness. RESULTS: Greater school sports participation, physical activity, and belongingness were each associated with reduced risks of belonging to most profiles with elevations in SU and/or MH symptoms relative to the low SU/MH profile (Relative Risk Ratios: sports=0.62-0.87, physical activity=0.78-0.98, belonging=0.75-0.83). Frequency of physical activity accounted for ~32-60% of the associations between sports and SU/MH profiles, while school belongingness accounted for the remaining associations. Physical activity and belongingness remained independently associated with SU/MH profiles. CONCLUSIONS: Findings suggest possible indirect associations between school sports participation and SU/MH profiles through physical activity and school belongingness, which may be promising prevention targets that have independent associations over and above sports. School sports participation may be one of a number of ways to achieve these goals.

Spontaneous closure of a traumatic acquired Gerbode defect in a dog.

A one-year-old female English Springer Spaniel with no previous history of cardiac disease presented to the Queen's Veterinary School Hospital following a blunt traumatic incident with an acquired, direct Gerbode defect and associated third-degree atrioventricular block. Two months after the initial incident, follow-up echocardiography showed a nearly closed Gerbode defect with just trivial residual flow. A 24-h Holter monitor indicated second-degree atrioventricular block with occasional junctional tachycardia. A reassessment 22 months later confirmed spontaneous closure of the previously acquired traumatic Gerbode defect, with 2:1 second-degree atrioventricular block. The dog remained clinically asymptomatic, and free of treatment. To our knowledge, this is the first reported case of spontaneous closure of a previously acquired traumatic Gerbode defect in a dog.

Unilateral external jugular vein aneurysm in a dog.

An 11-month-old Staffordshire Bull Terrier was referred with a two-month history of fluctuating unilateral jugular groove swelling, which appeared to enlarge after exercise. There was no history of trauma. Multimodal imaging findings (using transdermal and transesophageal ultrasound and dual phase computed tomography angiography) were consistent with large, saccular, left jugular vein aneurysm, running parallel to the left carotid artery. There did not appear to be any arteriovenous communication present. There were no cardiac abnormalities found on echocardiography. Following surgical excision, histopathological analysis supported the clinical suspicion of a congenital external jugular venous aneurysm.

A data-centric approach to generative modelling for 3D-printed steel.

The emergence of additive manufacture (AM) for metallic material enables components of near arbitrary complexity to be produced. This has potential to disrupt traditional engineering approaches. However, metallic AM components exhibit greater levels of variation in their geometric and mechanical properties compared to standard components, which is not yet well understood. This uncertainty poses a fundamental barrier to potential users of the material, since extensive post-manufacture testing is currently required to ensure safety standards are met. Taking an interdisciplinary approach that combines probabilistic mechanics and uncertainty quantification, we demonstrate that intrinsic variation in AM steel can be well described by a generative statistical model that enables the quality of a design to be predicted before manufacture. Specifically, the geometric variation in the material can be described by an anisotropic spatial random field with oscillatory covariance structure, and the mechanical behaviour by a stochastic anisotropic elasto-plastic material model. The fitted generative model is validated on a held-out experimental dataset and our results underscore the need to combine both statistical and physics-based modelling in the characterization of new AM steel products.

The sup-pf-2 mutations of Chlamydomonas alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

The sup-pf-2 mutation is a member of a group of dynein regulatory mutations that are capable of restoring motility to paralyzed central pair or radial spoke defective strains. Previous work has shown that the flagellar beat frequency is reduced in sup-pf-2, but little else was known about the sup-pf-2 phenotype (Huang, B., Z. Ramanis, and D.J.L. Luck. 1982. Cell. 28:115-125; Brokaw, C.J., and D.J.L. Luck. 1985. Cell Motil. 5:195-208). We have reexamined sup-pf-2 using improved biochemical and structural techniques and by the analysis of additional sup-pf-2 alleles. We have found that the sup-pf-2 mutations are associated with defects in the outer dynein arms. Biochemical analysis of sup-pf-2-1 axonemes indicates that both axonemal ATPase activity and outer arm polypeptides are reduced by 40-50% when compared with wild type. By thin-section EM, these defects correlate with an approximately 45% loss of outer dynein arm structures. Interestingly, this loss is biased toward a subset of outer doublets, resulting in a radial asymmetry that may reflect some aspect of outer arm assembly. The defects in outer arm assembly do not appear to result from defects in either the outer doublet microtubules or the outer arm docking structures, but rather appear to result from defects in outer dynein arm components. Analysis of new sup-pf-2 mutations indicates that the severity of the outer arm assembly defects varies with different alleles. Complementation tests and linkage analysis reveal that the sup-pf-2 mutations are alleles of the PF28/ODA2 locus, which is thought to encode the gamma-dynein heavy chain subunit of the outer arm. The sup-pf-2 mutations therefore appear to alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella.

Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is though to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown (Huang et al., 1982; Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) to analyze the relationship between the DRC and the dynein arms. Our results suggest that some of the DRC components are located at the base of the second radial spoke in close association with the inner dynein arms. (a) Averages of axoneme cross-sections indicate that inner arm structures are significantly reduced in three DRC mutants (pf3 < pf2 < sup-pf-3 < wt). (b) These defects are more pronounced in distal/medial regions of the axoneme than in proximal regions. (c) Analysis of flagellar extracts by fast protein liquid chromatography and SDS-PAGE indicates that a specific dynein I2 isoform is missing in pf3 and reduced in pf2 and sup-pf-3. Comparison with ida4 and pf3ida4 extracts reveals that this isoform differs from those missing in ida4. (d) When viewed in longitudinal section, all three DRC mutants lack a crescent-shaped density above the second radial spoke, and pf3 axonemes lack additional structures adjacent to the crescent. We propose that the crescent corresponds in part to the location of the DRC, and that this structure is also directly associated with a subset of the inner dynein arms. This position is appropriate for a complex that is thought to mediate signals between the radial spokes and the dynein arms.

Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain.

We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors of the paralyzed central pair mutant pf6 (Luck, D.J.L., and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the beta-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an approximately 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by approximately 5 kb of genomic DNA (Mitchell, D.R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region that is predicted to encode a small alpha-helical coiled-coil domain of the beta-DHC. This domain may play a role in protein-protein interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.

Validity, reliability and acceptability of the Imperial Nutritional Screening System (INSYST): a tool that does not require the body mass index.

BACKGROUND: Nutritional screening tools are central to identifying malnourished patients, but their efficacy is often reduced as a result of difficulties in obtaining height for body mass index (BMI) calculations. The present study aimed to evaluate the validity, reliability and acceptability of the Imperial Nutritional Screening System (INSYST); a tool that does not require the BMI. METHODS: Patients were screened by the researcher within 72 h of admission using INSYST I & II, Malnutrition Universal Screening Tool (MUST) and Mini Nutritional Assessment (MNA), including taking height and weight. Routine INSYST data, completed by nursing staff, were subsequently collected. At risk and malnourished patients were combined for statistical analysis. Inter-tool and inter-rater agreement (kappa, kappa) was evaluated. Sensitivity and specificity were calculated. Nurses were timed using INSYST. Acceptability, including ease and speed of use, was evaluated. RESULTS: Kappa (agreement) scores (all P < 0.001) were substantial for INSYST I versus MUST and MNA (kappa = 0.73 and kappa = 0.76, respectively) and moderate for INSYST II (both kappa = 0.53). The sensitivity of INSYST I and II was high (95-100%), whereas specificity was lower (65-83%). The agreement between dietitian and nurse for INSYST I was substantial kappa = 0.77 and that for INSYST II was fair kappa = 0.39 (both P

The short-term effects of prescribed burning on biomass removal and the release of nitrogen and phosphorus in a treatment wetland.

Nutrient removal by constructed wetlands can decline over time due to the accumulation of organic matter. A prescribed burn is one of many management strategies used to remove detritus in macrophyte-dominated systems. We quantified the short-term effects on effluent water quality and the amount of aboveground detritus removed from a prescribed burn event. Surface water outflow concentrations were approximately three times higher for P and 1.5 times higher for total Kjeldhal nitrogen (TKN) following the burn event when compared to the control. The length of time over which the fire effect was significant (P < 0.05), 3 d for TKN and up to 23 d for P fractions. Over time, the concentration of soluble reactive phosphorus (SRP) in the effluent decreased, but was compensated with increases in dissolved organic phosphorus (DOP) and particulate phosphorus (PP), such that net total P remained the same. Total aboveground biomass decreased by 68.5% as a result of the burn, however, much of the live vegetation was converted to standing dead material. These results demonstrate that a prescribed burn can significantly decrease the amount of senescent organic matter in a constructed wetland. However, short-term nutrient releases following the burn could increase effluent nutrient concentrations. Therefore, management strategies should include hydraulically isolating the burned area immediately following the burn event to prevent nutrient export.

Risk stratification with longitudinal neutrophil to lymphocyte ratio assessment after left ventricular assist device implantation.

INTRODUCTION: Inflammatory processes are well-characterized risk factors in cardiovascular disease including advanced heart failure. Previous studies have utilized individual white cell subtypes in risk analysis, and a recent study has focused on the efficacy of the neutrophil-to-lymphocyte ratio in evaluating negative outcomes following left ventricular assist device implantation. To investigate the interaction between the left ventricular assist device and white cell counts, we assessed longitudinal changes in neutrophil-to-lymphocyte ratio following left ventricular assist device implantation. METHODS: This retrospective study included 100 patients who underwent left ventricular assist device implantation between 2012 and 2013. The neutrophil-to-lymphocyte ratio was calculated prior to left ventricular assist device implantation, daily for the first 30 postoperative days, and at the first two postoperative outpatient visits. Preoperative demographic and clinical data were collected for all patients. RESULTS: The mean neutrophil-to-lymphocyte ratio immediately before left ventricular assist device implantation was 5.2 ± 4.9. After surgery, the neutrophil-to-lymphocyte ratio decreased asymptotically, from a peak of 29.2 on postoperative day 1 to 4.1 at the second outpatient visit ( p < 0.001). Lack of improvement in the neutrophil-to-lymphocyte ratio at postoperative day 10 was associated with increased length of stay, right heart failure, and a trend toward worsened survival. CONCLUSION: Our results indicate a significant inflammatory response to implantation of the left ventricular assist device, a known effect. The magnitude of this response may be effectively and easily monitored over time using the neutrophil-to-lymphocyte ratio. In general, approximately 30 days is required for the neutrophil-to-lymphocyte ratio to return to preoperative levels. After several months, the neutrophil-to-lymphocyte ratio improves to below preoperative levels. It is possible that this reduction reflects the reversal of various heart failure-mediated inflammatory processes following left ventricular assist device implantation.

Otic lesions and congenital hypothyroidism in the developing chick.

In an effort to elucidate the relation, if any, between thyroid abnormality and congenital deafness in Pendred's syndrome, an experiment was designed to study the effects of hypothyroidism on middle and inner ear hearing structures, including the auditory nerve and its central projection, in developing chick embryos. Propylthiouracil (PTU), 2 mg, was injected into the albumin of fertile chick eggs on the 10th incubation day. Single doses of L-thyroxine (range 1-100mug) were inoculated in a similar manner, either alone or with PTU. Control inocula included sterile saline or water. After hatching, each chick was examined for obvious malformations. The thyroid glands, middle and inner ear mechanisms, auditory nerve, and brainstem were studied grossly and with different histologic staining techniques. When compared to controls, chicks exposed to PTU on their 10th incubation day exhibited: increased mortality, delayed hatching, reduced size, incomplete yolk sac absorption, and death within 5 days unless exogenous thyroid hormone was provided in the first 24-48 hr after hatching. Specific, consistent, morphologic alterations were observed in their thyroid glands as well as in the sensory hair cells of the acoustic papilla and cells of the spiral ganglion of the cochlea. Our data also indicate that if 50-75 mug of L-thyroxine is given simultaneously with (or as long as 120 hr after) the PTU injection on the 10th incubation day, one cannot detect the gross defects, marked thyroid lesions, or abnormal histology in cells of the cochlea and its ganglion. A relationship between embryonic thyroid gland function and the hearing mechanism of the chick embryo is suggested.

Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.