Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.

Characterization of Primary Cilia in Normal Fallopian Tube Epithelium and Serous Tubal Intraepithelial Carcinoma.

OBJECTIVES: The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). METHODS: Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp-TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. RESULTS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. CONCLUSIONS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC.

Understanding Human-Animal Relations in the Context of Primate Conservation: A Multispecies Ethnographic Approach in North Morocco.

Strategies for conserving species threatened with extinction are often driven by ecological data. However, in anthropogenic landscapes, understanding and incorporating local people's perceptions may enhance species conservation. We examine the relationships shepherds, living on the periphery of the mixed oak forest of Bouhachem in northern Morocco, have with animals in the context of a conservation project for Barbary macaques (Macaca sylvanus). We analyse ethnographic data to provide insights into shepherds' conceptions of Barbary macaques and the species which bring the shepherds into the forest - goats (Capra hircus), domestic dogs (Canis lupus familiaris), and the African wolf (Canis lupus lupaster). We interpret these data within the framework of boundary theory. Our multispecies ethnographic approach illuminates the different and, in the case of the domestic dog and the Barbary macaque, complex ways shepherds perceive each species. Some shepherds show intrinsic interest in the macaques, revealing potential recruits to conservation activities. As with any ethnographic study, our interpretations of human-animal relations in Bouhachem may not extrapolate to other areas of the Barbary macaque's distribution because of the unique nature of both people and the place. We recommend that conservationists examine complex place-based relations between humans and animals to improve wildlife conservation efforts.

TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone.

Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.

A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets.

OBJECTIVE: Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. DESIGN: Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. RESULTS: 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. CONCLUSION: The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

The emerging role of MCPH1/BRIT1 in carcinogenesis.

The MCPH1 gene, also known as BRCT-repeat inhibitor of hTERT expression (BRIT1), has three BRCA1 carboxyl-terminal domains which is an important regulator of DNA repair, cell cycle checkpoints and chromosome condensation. MCPH1/BRIT1 is also known as a tumour suppressor in different types of human cancer. The expression level of the MCPH1/BRIT1 gene is decreased at the DNA, RNA or protein level in a number of types of cancers including breast cancer, lung cancer, cervical cancer, prostate cancer and ovarian cancer compared to normal tissue. This review also showed that deregulation of MCPH1/BRIT1 is significantly associated with reduced overall survival in 57% (12/21) and relapsed free survival in 33% (7/21) of cancer types especially in oesophageal squamous cell carcinoma and renal clear cell carcinoma. A common finding of this study is that the loss of MCPH1/BRIT1 gene expression plays a key role in promoting genome instability and mutations supporting its function as a tumour suppressor gene.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

The Diverse Role of CUB and Sushi Multiple Domains 1 (CSMD1) in Human Diseases.

CUB and Sushi Multiple Domains 1 (CSMD1), a tumour suppressor gene, encodes a large membrane-bound protein including a single transmembrane domain. This transmembrane region has a potential tyrosine phosphorylation site, suggesting that CSMD1 is involved in controlling cellular functions. Although the specific mechanisms of action for CSMD1 have not yet been uncovered, it has been linked to a number of processes including development, complement control, neurodevelopment, and cancer progression. In this review, we summarise CSMD1 functions in the cellular processes involved in the complement system, metastasis, and Epithelial mesenchymal transition (EMT) and also in the diseases schizophrenia, Parkinson's disease, and cancer. Clarifying the association between CSMD1 and the aforementioned diseases will contribute to the development of new diagnosis and treatment methods for these diseases. Recent studies in certain cancer types, e.g., gastric cancer, oesophageal cancer, and head and neck squamous cell carcinomas, have indicated the involvement of CSMD1 in response to immunotherapy.

Microcephalin is a new novel prognostic indicator in breast cancer associated with BRCA1 inactivation.

The authors have investigated the expression of the microcephalin (MCPH1) protein to evaluate its prognostic importance in breast cancer. Microcephalin is a damage response protein involved in the regulation of BRCA1 and BRCA2. BRCA1 mutations are often associated with basal-like breast cancer, which are also often negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. MCPH1 immunohistochemistry was performed on 319 breast cancers prepared as tissue microarray and correlated with pathology, survival, ER, PR, HER2, EGFR, CK5/6, CK14 and BRCA1 expression. After performing continuous data analysis, mean microcephalin expression decreased with increasing grade (P < 0.006). Mean microcephalin expression was lower in ER/PR negative (P < 0.001) and triple negative cancers (P < 0.004). Conversely, an association with HER2-positive cancers was also identified (P < 0.034). Reduced microcephalin also correlated with reduced nuclear BRCA1 staining (P < 0.001). No association was identified with basal markers. After dichotomising the data into low and high microcephalin expression, reduced expression was identified in 29% (93/319) of breast cancers. An association with low expression was identified in invasive ductal carcinomas with breast cancer-specific survival (BCSS) (P = 0.052). Multivariate analysis of ductal carcinomas showed that microcephalin, together with lymph node involvement and tumour size were independent predictors of BCSS (P = 0.037). Microcephalin expression is reduced in 29% of breast cancers, particularly in higher grade tumours and BRCA1-negative cases. Microcephalin is an independent predictor of BCSS in invasive ductal breast cancer patients and may prove to be a useful biomarker for the identification of aggressive breast cancers.

The Role of Csmd1 during Mammary Gland Development.

The Cub Sushi Multiple Domains-1 (CSMD1) protein is a tumour suppressor which has been shown to play a role in regulating human mammary duct development in vitro. CSMD1 knockdown in vitro demonstrated increased cell proliferation, invasion and motility. However, the role of Csmd1 in vivo is poorly characterised when it comes to ductal development and is therefore an area which warrants further exploration. In this study a Csmd1 knockout (KO) mouse model was used to identify the role of Csmd1 in regulating mammary gland development during puberty. Changes in duct development and protein expression patterns were analysed by immunohistochemistry. This study identified increased ductal development during the early stages of puberty in the KO mice, characterised by increased ductal area and terminal end bud number at 6 weeks. Furthermore, increased expression of various proteins (Stat1, Fak, Akt, Slug/Snail and Progesterone receptor) was shown at 4 weeks in the KO mice, followed by lower expression levels from 6 weeks in the KO mice compared to the wild type mice. This study identifies a novel role for Csmd1 in mammary gland development, with Csmd1 KO causing significantly more rapid mammary gland development, suggesting an earlier adult mammary gland formation.

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis.

BACKGROUND: Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). RESULTS: We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. CONCLUSIONS: These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.

Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma.

CUB and SUSHI multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene that maps to chromosome 8p23, a region deleted in many tumour types including 50% of breast cancers. CSMD1 has homologies to proteins implicated in carcinogenesis. We aimed to study the expression pattern of the CSMD1 protein and evaluate its prognostic importance in invasive ductal carcinoma (IDC). An anti-CSMD1 antibody was developed and validated. The expression pattern of CSMD1 in normal breast and IDC samples was investigated by immunohistochemistry in 275 patients. Univariate and multivariate Cox regression analyses were performed. In normal breast duct epithelial cells, luminal, membranous and cytoplasmic CSMD1 staining was identified. Reduced expression of CSMD1 was detected in 79/275 (28.7%) of IDC cases. Low CSMD1 expression was significantly associated with high tumour grade (P = 0.003). CSMD1 expression was associated with overall survival (OS; HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not with disease-free survival (DFS; HR = 0.81, 95%CI: 0.46-1.43, P = 0.48). Multivariate analysis showed that CSMD1, together with Nottingham Prognostic Index, was considered an independent predictor of OS (HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not DFS (HR = 0.84, 95%CI: 0.46-1.5, P = 0.573). Reduction of CSMD1 expression was significantly associated with high tumour grade and decreased OS. Therefore, our results support the idea that CSMD1 is a tumour suppressor gene and suggest its possible use as a new prognostic biomarker. The membrane expression pattern of CSMD1 suggests that it may be a receptor or co-receptor involved in the process of signal transduction.

Genomic and Expression Analyses Define MUC17 and PCNX1 as Predictors of Chemotherapy Response in Breast Cancer.

Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy (P < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. In vitro knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively (P < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers (n = 53, n = 303). Kaplan-Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, in vitro knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.

Transitions in emerging adulthood and stress among young Australian women.

BACKGROUND: Emerging adulthood involves major transitions in social roles and high levels of stress, which may affect later health. PURPOSE: To examine cross-sectionally and longitudinally the relationships of stress to roles in four life domains-residential independence from family of origin, employment, relationships, and motherhood-among young adult women. METHOD: 8,749 young women participating in the Australian Longitudinal Study on Women's Health provided data at Survey 1, ages 18-23, and Survey 2, ages 22-27. RESULTS: Contrary to expectation, major life transitions were associated with low and reducing levels of stress. Cross-sectionally, living independently, not being a student, being married, and being a mother were associated with the lowest stress. Normative transitions such as moving out of home, finding work, or motherhood were associated with no change in stress, while marrying was associated with a decrease in stress. Three types of transition were associated with increases in stress: non-normative transitions to more "adolescent" statuses, no transition, and transitions occurring earlier than normative. CONCLUSION: High levels of stress at this age are associated with unusual changes, delays in changing, or changing earlier than one's peers. The normative transitions of young adulthood are not associated with high levels of stress.

A MAPK/c-Jun-mediated switch regulates the initial adaptive and cell death responses to mitochondrial damage in a neuronal cell model.

Parkinson's disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor c-Jun can act in either a pro- or anti-apoptotic manner, depending on the level of stress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategies.

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both p = 0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (p = 0.003 and p = 0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).

Biosocial Conservation: Integrating Biological and Ethnographic Methods to Study Human-Primate Interactions.

Biodiversity conservation is one of the grand challenges facing society. Many people interested in biodiversity conservation have a background in wildlife biology. However, the diverse social, cultural, political, and historical factors that influence the lives of people and wildlife can be investigated fully only by incorporating social science methods, ideally within an interdisciplinary framework. Cultural hierarchies of knowledge and the hegemony of the natural sciences create a barrier to interdisciplinary understandings. Here, we review three different projects that confront this difficulty, integrating biological and ethnographic methods to study conservation problems. The first project involved wildlife foraging on crops around a newly established national park in Gabon. Biological methods revealed the extent of crop loss, the species responsible, and an effect of field isolation, while ethnography revealed institutional and social vulnerability to foraging wildlife. The second project concerned great ape tourism in the Central African Republic. Biological methods revealed that gorilla tourism poses risks to gorillas, while ethnography revealed why people seek close proximity to gorillas. The third project focused on humans and other primates living alongside one another in Morocco. Incorporating shepherds in the coproduction of ecological knowledge about primates built trust and altered attitudes to the primates. These three case studies demonstrate how the integration of biological and social methods can help us to understand the sustainability of human-wildlife interactions, and thus promote coexistence. In each case, an integrated biosocial approach incorporating ethnographic data produced results that would not otherwise have come to light. Research that transcends conventional academic boundaries requires the openness and flexibility to move beyond one's comfort zone to understand and acknowledge the legitimacy of "other" kinds of knowledge. It is challenging but crucial if we are to address conservation problems effectively.

Loss of CSMD1 expression disrupts mammary duct formation while enhancing proliferation, migration and invasion.

The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the 'normal' MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Pattern of expression of genes linked to epigenetic silencing in human breast cancer.

Epigenetic mechanisms such as DNA methylation are now recognized to play an important role in neoplasia. The aim of this study is to relate the pattern of expression of multiple cancer genes known to undergo epigenetic inactivation by promoter hypermethylation in breast cancer with histologic and outcome data. We used immunohistochemistry to study expression of the tumor suppressor gene p16, estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), and the DNA repair gene MGMT (O6 -methylguanine-DNA methyltransferase) in a panel of 200 breast cancers. Methylation-specific polymerase chain reaction was used to confirm MGMT promoter methylation. Loss of expression of MGMT, ERalpha, ERbeta, PR, and p16 was observed in 19%, 24%, 13%, 40%, and 50% of cases, respectively. A significant correlation was seen between grade III tumor and loss of expression of ERalpha, ERbeta, PR (all P < .0001), and MGMT (P = .04), whereas loss of expression of p16 was associated with grades I and II tumors (P < .001). Cases that expressed 3 or less of the 5 proteins studied had significantly reduced survival (P = .0016). Methylation-specific polymerase chain reaction in a subset of 20 cancers showed DNA methylation associated with the loss of MGMT expression (P < .001). In conclusion, there is silencing of several key genes in breast cancer affecting molecular pathways involved in cell immortalization, DNA repair, and hormonal regulation, and this correlates significantly with risk of cancer-specific death. This expression profile could be linked to epigenetic events, and if so, these pathways have potential as targets for therapeutic strategies based on reversal of epigenetic silencing.