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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.

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. DOI: http://dx.doi.org/10.7554/eLife.24903.001, eLife digest Many of the molecules that are essential for life are too small to be visible inside cells. So, scientists use large complex proteins called antibodies that bind to these molecules to detect whether they are present and show where they are in a cell. As well as being useful tools in experiments, these antibodies can be used to help identify and treat diseases. The body produces antibodies in response to an infection. The antibodies used in experiments are purified from animal blood, but this method of producing antibodies has flaws. For example, it can be difficult to make identical batches of antibody that always behave in the same way. So scientists have developed “alternative binding proteins” that can be made in the laboratory. These proteins are much less complicated and can be developed more quickly than antibodies, and can easily be adapted for a variety of uses. An alternative binding protein called an Affimer behaves in a similar way to an antibody by binding tightly to its target molecule, but is much more stable to acidity and high temperature. Tiede et al. have now tested how well the Affimer works in a wide range of different experiments that normally use antibodies to analyse the amount of a particular molecule inside a cell. The results of the tests show that the Affimer behaves in the same way as antibodies, and sometimes works more effectively. Tiede et al. show that an Affimer can help to reveal how a particular molecule works within a cell, to create detailed pictures of molecules in cells and tissues, and to identify a tumour. It can also be used alongside a new technique called ‘super-resolution microscopy’ that allows researchers to watch the activity of individual molecules. Future challenges are to test the Affimer in even more applications and to encourage its wider use by researchers, alongside other alternative binding proteins, as as replacements for some antibodies. This could ultimately lead to the development of faster and more efficient diagnostic, imaging and therapeutic tests. DOI: http://dx.doi.org/10.7554/eLife.24903.002

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.

Purpose/Objective(s) Randomized controlled trials for patients with squamous cell carcinoma of the anus have been successfully performed, despite the rarity of the disease. The RTOG 9811 and ACT2 trials demonstrated no improvement in cancer outcomes when neoadjuvant or adjuvant cisplatin 5FU chemotherapy was used. To capture as many anal cancer patients as possible in future clinical trials, we developed an umbrella of trials testing efficacy and acceptable morbidity across different loco-regional risk strata. Materials/Methods We formed a network of UK and international multi-disciplinary trialists and identified the following priorities:- i] the need for national consensus guidance for the introduction of intensity modulated radiation therapy (IMRT) based on the successful principles and fractionation used in the ACT2 protocol prior to future trials; ii] to evaluate a strategy using selective chemoradiation therapy (CRT) after local excision of anal margin tumors; iii] to evaluate reduced dose CRT to the primary tumor in order to limit late effects whilst maintaining elective nodal irradiation in early stage disease; iv] to evaluate dose escalated CRT in locally advanced T3/4 and N+ stage disease. Results The PLATO (personalizing radiotherapy dose in anal cancer) umbrella trial comprising of the ACT3, 4 and 5 trials is funded by Cancer Research UK and is due to commence recruitment in Q3 2016.The ACT 3 trial (n = 90) is a non-randomized phase II study that will evaluate a strategy of local excision for T1N0 anal margin tumors with selective postoperative involved field CRT using 41.4 Gy in 23 fractions (F) and concurrent capecitabine reserved for patients with margins

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon. NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis. Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008). NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.

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.

One hundred and nine samples comprising carcinomas, adenomas, dysplastic, inflamed and normal mucosa from patients with sporadic colon cancer and ulcerative colitis (UC) were analysed for c-Ki-ras mutations. DNA was extracted from archival paraffin-embedded material, amplified using the polymerase chain reaction (PCR) and the PCR products analysed using restriction enzyme digestion. Forty-two per cent (14/33) of the sporadic carcinoma controls contained Ki-ras codon 12 mutations in contrast to 24% (8/33) of ulcerative colitis carcinomas. A significantly higher c-Ki-ras mutation rate was observed in rectal carcinomas (72%) in comparison to colonic carcinomas (28%) in control patients (P less than 0.04), while the opposite was observed in UC patients. The difference between the incidence of c-Ki-ras mutations in rectal carcinomas in UC (9%) and in sporadic rectal carcinomas (72%) was also significant (P less than 0.01). This lower prevalence rate and different site distribution of c-Ki-ras mutations in UC carcinomas compared to sporadic carcinomas suggests that specific genetic differences may underlie the causation of carcinomas arising in these situations. Images Figure 2 Figure 3 Figure 4 Figure 5

Hereditary pancreatitis is an autosomal dominant disorder with incomplete penetrance. It is characterised by recurring episodes of severe abdominal pain and often presents in childhood. Recently, a mutation in the cationic trypsinogen gene was identified in this disease. Previously, only one mutation at residue 117 of the trypsinogen gene has been found in the five separate hereditary pancreatitis families, four from the USA and one from Italy. Alteration of the Arg117 site is believed to disrupt a fail-safe mechanism for the inactivation of trypsin, leading to autodigestion of the pancreas under certain conditions. Molecular analysis of the trypsinogen gene was carried out on a hereditary pancreatitis family from the UK. The same G to A mutation at residue 117 was identified in this family, suggesting that this is a common mutation in hereditary pancreatitis.

The frequency of p53 overexpression and K-ras codon 12 mutation was investigated in a series of colorectal adenomas. p53 was detected by immunohistochemistry in only 5% of tumours, whereas K-ras mutation was found in eight of 30 adenomas examined. In vitro, mutant p53 and ras genes cooperate to transform primary rat cells into a tumourigenic cell line. The presence of both p53 overexpression and K-ras mutation in a benign tubulovillous polyp in the present series suggests that in vivo this combination of events is insufficient to cause malignant transformation of a large bowel adenoma.

Previously we identified MCPH1, a DNA damage response protein involved in the regulation of BRCA1 and BRCA2, as the defective protein in one form of microcephaly. BRCA1 mutations are associated with basal-like breast cancer, which are often also negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. Our data indicate that MCPH1 plays a role in response to chemotherapeutic agents used in the treatment of breast cancer due to its role in DNA repair and the spindle checkpoint.

Background Mutations in the A bnormal Sp indle M icrocephaly 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.