Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Yes-associated protein (YAP) functions as a tumor suppressor in breast.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

Systematic review of the impact of breast-conserving surgery on cancer outcomes of multiple ipsilateral breast cancers.

BACKGROUND: The clinical effectiveness of treating ipsilateral multifocal (MF) and multicentric (MC) breast cancers using breast-conserving surgery (BCS) compared with the standard of mastectomy is uncertain. Inconsistencies relate to definitions, incidence, staging and intertumoral heterogeneity. The primary aim of this systematic review was to compare clinical outcomes after BCS versus mastectomy for MF and MC cancers, collectively defined as multiple ipsilateral breast cancers (MIBC). METHODS: Comprehensive electronic searches were undertaken to identify complete papers published in English between May 1988 and July 2015, primarily comparing clinical outcomes of BCS and mastectomy for MIBC. All study designs were included, and studies were appraised critically using the Newcastle-Ottawa Scale. The characteristics and results of identified studies were summarized. RESULTS: Twenty-four retrospective studies were included in the review: 17 comparative studies and seven case series. They included 3537 women with MIBC undergoing BCS; breast cancers were defined as MF in 2677 women, MC in 292, and reported as MIBC in 568. Six studies evaluated MIBC treated by BCS or mastectomy, with locoregional recurrence (LRR) rates of 2-23 per cent after BCS at median follow-up of 59·5 (i.q.r. 56-81) months. BCS and mastectomy showed apparently equivalent rates of LRR (risk ratio 0·94, 95 per cent c.i. 0·65 to 1·36). Thirteen studies compared BCS in women with MIBC versus those with unifocal cancers, reporting LRR rates of 2-40 per cent after BCS at a median follow-up of 64 (i.q.r. 57-73) months. One high-quality study reported 10-year actuarial LRR rates of 5·5 per cent for BCS in 300 women versus 6·5 per cent for mastectomy among 887 women. CONCLUSION: The available studies were mainly of moderate quality, historical and underpowered, with limited follow-up and biased case selection favouring BCS rather than mastectomy for low-risk patients. The evidence was inconclusive, weakening support for the St Gallen consensus and supporting a future randomized trial.

A review of the ethnic differences in breast cancer.

Women of African descent have a lower incidence of breast cancer than their white counterparts; however, the overall age-adjusted breast cancer mortality rates are higher. They also present at a younger age, and have more advanced disease that exhibits poor prognostic features including significantly larger tumors of higher grade, higher rates of estrogen receptor and progesterone receptor negativity and a higher rate of p53 mutations and HRAS1 proto-oncogene expression, all of which confer a poor prognosis. While there are many possible contributory factors to the discrepancies in outcome in women of African descent, there is no satisfactory explanation as to why women of African origin tend to present at a younger age with hormone receptor-negative tumors and more adverse prognostic features.

Dehydroepiandrosterone Heightens Aggression and Increases Androgen Receptor and Aromatase mRNA Expression in the Brain of a Male Songbird.

Dehydroepiandrosterone (DHEA) is a testosterone/oestrogen precursor and known modulator of vertebrate aggression. Male song sparrows (Melospiza melodia morphna) show high aggression during breeding and nonbreeding life-history stages when circulating DHEA levels are high, and low aggression during molt when DHEA levels are low. We previously showed that androgen receptor and aromatase mRNA expression are higher during breeding and/or nonbreeding in brain regions associated with reproductive and aggressive behaviour, although the potential role of DHEA in mediating these seasonal changes remained unclear. In the present study, nonbreeding male song sparrows were captured and held in the laboratory under short days (8 : 16 h light/dark cycle) and implanted with s.c. DHEA-filled or empty (control) implants for 14 days. DHEA implants increased aggression in a laboratory-based simulated territorial intrusion. Brains of DHEA-implanted birds showed higher aromatase mRNA expression in the preoptic area (POA) and higher androgen receptor mRNA expression in the periventricular nucleus of the medial striatum (pvMSt) and ventromedial nucleus of the hypothalamus. The DHEA-induced increases in aromatase expression in the POA and androgen receptor expression in the pvMSt are consistent with previously reported seasonal increases in these markers associated with naturally elevated DHEA levels. This suggests that DHEA facilitates seasonal increases in aggression in nonbreeding male song sparrows by up-regulating steroid signalling/synthesis machinery in a brain region-specific fashion.

Sensitive determination of cell number using the CyQUANT cell proliferation assay.

We describe here the development and characterization of the CyQUANT cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range using a wide variety of cell types. Under the recommended assay conditions, standard curves were linear (r(2)>0.995), detecting as few as 10-50 cells and as many as 25,000-50,000 cells with a single dye concentration, depending on cell type. Increasing the dye concentration extended the linear range of the assay to 100,000-250,000 cells. Results of cell proliferation and growth inhibition studies with the assay were similar to those obtained in published studies using other standard assays. CyQUANT assay measurements of serum-stimulated cell proliferation correlated well with measurements made using [3H]-thymidine. Also, the assay was used to analyze cellular DNA or RNA content, with the addition of a nuclease digestion step to the protocol. The assay procedure is simple and convenient, with no wash steps, and is readily amenable to automation.

Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Gait abnormalities and inhibitive casts in cerebral palsy. Literature review.

Although cerebral palsy is primarily a central nervous system disorder, its major manifestations are musculoskeletal. The authors focus on normal tonic reflexes of the foot and the developmental consequences of failure to inhibit these reflexes. Various gait abnormalities seen in cerebral palsy are reviewed. Finally, the use of inhibitive casts as a conservative modality for treating hyperactive reflexes in the spastic cerebral palsy child is discussed. The reduction of abnormal tone facilitates the development of a normal gait pattern in these children.

New techniques to establish the subtalar joint's functional axis.

A new technique to establish the functional subtalar joint axis in live models is presented. Previous studies had a poor reference to the body planes because they relied on cadaver bone models and were not referenced to actual live function. The lower value for the function axis is due to the response of the midtarsal joint and forefoot to loading and ground reactive forces. The techniques were applied to the clinical setting by establishing a reference line on lateral RCSP radiographs and calculating the actual axis to this reference line. The axis deviated from the reference line by a small range (4 degrees) and can be corrected by reference to the calcaneal inclination angle for high or low angular differences. This simple technique can be used clinically to determine axial changes that may be required for surgical procedures and for evaluating the effect of orthosis control.

Abnormal biomechanics of flatfoot deformities and related theories of biomechanical development.

The flatfoot deformity has been investigated over this century by many leading authorities with regard to its etiologies and pathogenesis. A large number of articles in the early orthopedic literature concerning the flatfoot deformity defined the primary abnormalities of the structure without strong emphasis on its relationship to abnormal functional etiologies leading to the pathogenesis of the deformity. This article emphasizes abnormal biomechanics and theories of development.

Network meta-analysis of indomethacin versus ibuprofen versus placebo for PDA in preterm infants.

OBJECTIVES: To evaluate the effects of indomethacin or ibuprofen compared with placebo on closure, morbidity and mortality in preterm infants <37 weeks' gestation with echocardiographically and/or clinically important patent ductus arteriosus (PDA) at >24 h of life. DATA SOURCES: MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, Cochrane Library, clinicaltrials.gov, controlled-trials.com, American Pediatric and European Paediatric Research Societies and Effective Care of the Newborn Infant. REVIEW METHODS: Systematic review with network meta-analysis of randomised studies comparing intravenous indomethacin, ibuprofen or placebo for PDA in preterm infants at >24 h of life. RESULTS: Ten trials compared intravenous indomethacin versus intravenous ibuprofen, nine intravenous indomethacin versus placebo and one intravenous ibuprofen versus placebo. Both intravenous indomethacin (pooled RR 2.39 (95% CI 2.05 to 2.78)) and intravenous ibuprofen (RR 2.40 (95% CI 2.03 to 2.84)) closed a PDA more effectively than placebo. Intravenous ibuprofen was associated with approximately 30% greater risk of chronic lung disease than intravenous indomethacin (RR 1.28 (95% CI 1.03 to 1.60)) or placebo (RR 1.29 (95% CI 0.99 to 1.70)). Differences in risk or benefit were not significant between any combination of intravenous indomethacin, intravenous ibuprofen or placebo groups for intraventricular haemorrhage, necrotising enterocolitis and death. Reporting on neurological outcomes was insufficient for pooling. CONCLUSIONS: Intravenous indomethacin or ibuprofen administered to preterm infants for PDA at >24 h of life promoted ductal closure, but other short-term benefits were not seen. Treatment with intravenous ibuprofen may increase the risk of chronic lung disease. Good-quality evidence of treatment effect on morbidity, mortality and improved neurodevelopment is urgently needed.

Cadherin switching dictates the biology of transitional cell carcinoma of the bladder: ex vivo and in vitro studies.

Bladder cancer is the fifth most common malignancy in the UK. Clinically, the most important process in determining prognosis is the development of invasion, initially of the lamina propria and then beyond as these transitional cell carcinomas (TCCs) progress from stage pT1 to stages T2+. Cadherins and catenins are the main mediators of cell-cell interactions in epithelial tissues, and loss of membranous E-cadherin immunoreactivity is strongly correlated with high grade, advanced stage and poor prognosis in bladder cancer and other malignancies. However, the role of P-cadherin is yet to be fully elucidated in bladder TCC. The objectives of this study were to establish how the expression of cadherins and catenins determines clinical and in vitro behaviour in bladder TCC. Utilizing immunohistochemistry, immunofluorescence and western blotting, we demonstrated a significant reduction in the expression of E-cadherin and beta-catenin as grade and stage of bladder TCC progress, accompanied by a significant increase in P-cadherin expression (all p < 0.05, Pearson's chi2 test). Increased P-cadherin expression was also associated with a significantly worse bladder cancer-specific survival (log rank p = 0.008), with Cox regression showing P-cadherin to be an independent prognostic factor. Utilizing a variety of tissue culture models in a range of functional studies, we demonstrated that P-cadherin mediates defective cell-cell adhesion and enhances anchorage-independent growth. The results provide evidence that increased P-cadherin expression promotes a more malignant and invasive phenotype of bladder cancer, and appears to have a novel role late in the disease.

Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain.

We have developed a simple, sensitive, fluorescence microplate-based assay for tumor necrosis factor (TNF) biological activity. The assay employs SYTOX Green nucleic acid stain to detect TNF-induced cell necrosis in actinomycin D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymmetrical cyanine dye that is excluded from live cells but can readily penetrate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is monitored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25-500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, using mouse fibroblast-derived WEHI 164, WEHI 13var, and L929 cell lines. The procedure can also be used to detect agents that modulate TNF activity. We demonstrated complete inhibition of rhTNF-alpha using monoclonal anti-human TNF-alpha antibody and determined that approximately 20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-alpha in these cell lines. Reagents are added in a single step, followed by a 6- to 8-h incubation period, during which the cytokine exhibits its effects. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures.

Comparative effects of gentle teaching and visual screening on self-injurious behaviour.

Gentle teaching and visual screening procedures have been used to control severe behaviour problems in persons with mental retardation. An alternating treatments design was used to compare gentle teaching, visual screening and a task-training condition in the reduction of high levels of self-injury of an adult with profound mental retardation. Following baseline, a task-training condition using standard behavioural techniques was implemented to establish the effects of training the subject on age-appropriate tasks. Results showed a modest reduction in self-injury. This was followed by an alternating treatments phase in which visual screening, gentle teaching and no-treatment control conditions were compared. Both procedures were superior to the control condition in reducing self-injury, with visual screening being more effective than gentle teaching. When visual screening was implemented across two and then all three daily conditions, self-injury was further reduced to near-zero levels. Bonding occurred at the same low levels under both treatments, contrary to the predictions of gentle teaching's proponents.

Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis.

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

Effect of tamoxifen on Ki67 labelling index in human breast tumours and its relationship to oestrogen and progesterone receptor status.

This study aimed to investigate the effect of tamoxifen on breast tumour levels of oestrogen and progesterone receptor (ER and PR) and proliferation as defined by the Ki67 antibody. A group of primary breast cancer patients was randomised to receive either tamoxifen (n = 59) or placebo (n = 44) treatment in the interval between clinic and surgery (median 21 days). Frozen sections of breast tumour biopsies obtained before and after treatment were stained immunocytochemically to obtain the percentage of nuclei containing ER and PR, and a Ki67 labelling index (LI). Tamoxifen-treated patients had a median Ki67 LI of 5.6% in the first biopsy falling to 3.0% in the second biopsy (P < 0.001 by Wilcoxon's matched pairs test), whereas placebo-treated patients had a median Ki67 LI of 5.4% in the first biopsy and 5.75% in the second (no significant difference). No significant differences were observed when the median %ER or %PR staining before and after treatment were compared. The Ki67 LI tended to increase with increasing histological grade and was greater in tumours that were ER - ve compared to those that were ER + ve (> 5% nuclei stained), median 7.8% and 4.3% respectively (P = 0.011 by Mann-Whitney U-test). However, the decline in tumour Ki67 LI following anti-oestrogen treatment failed to correlate with ER and PR status or to predict recurrence over a short follow-up period. To our knowledge, this is the first time that tamoxifen treatment has been shown to reduce the Ki67 LI in human breast tumours in vivo. These data indicate that staining with the Ki67 antibody may be useful in monitoring response to anti-oestrogen therapy.