CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Improved recovery time and sensitivity to H2 and NH3 at room temperature with SnOx vertical nanopillars on ITO.

Nanostructured SnO2 is a promising material for the scalable production of portable gas sensors. To fully exploit their potential, these gas sensors need a faster recovery rate and higher sensitivity at room temperature than the current state of the art. Here we demonstrate a chemiresistive gas sensor based on vertical SnOx nanopillars, capable of sensing < 5 ppm of H2 at room temperature and 10 ppt at 230 °C. We test the sample both in vacuum and in air and observe an exceptional improvement in the performance compared to commercially available gas sensors. In particular, the recovery time for sensing NH3 at room temperature is more than one order of magnitude faster than a commercial SnO2 sensor. The sensor shows an unique combination of high sensitivity and fast recovery time, matching the requirements on materials expected to foster widespread use of portable and affordable gas sensors.

Humidity-enhanced sub-ppm sensitivity to ammonia of covalently functionalized single-wall carbon nanotube bundle layers.

A low-cost method for carbon nanotubes (CNTs) network production from solutions on flexible polyethylene naphthalate substrates has been adopted to prepare high quality and well characterized SWCNT bundle layers to be used as the active layer in chemiresistor gas sensors. Two types of SWCNTs have been tested: pristine SWCNTs, deposited from a surfactant solution, and covalently functionalized SWCNTs, deposited from a dimethyl-acetamide solution. The humidity effects on the sensitivity of the SWCNTs network to NH3 have been investigated. The results show that relative humidity favors the response to NH3, confirming recent theoretical predictions. The COOH-functionalized sample displays the largest response owing to both its hydrophilic nature, favoring the interaction with H2O molecules, and its largest surface area. Compared to data available in the literature, the present sensors display a remarkable sensitivity well below the ppm range, which makes them quite promising for environmental and medical applications, where NH3 concentrations (mostly of the order of tens of ppb) have to be detected.

Synthesis of branched Au nanoparticles with tunable near-infrared LSPR using a zwitterionic surfactant.

Asymmetric branched gold nanoparticles are obtained using for the first time in the seed-growth approach a zwitterionic surfactant, laurylsulfobetaine, whose concentration in the growth solution allows to control both the length to base-width ratio of the branches and the LSPR position, that can be tuned in the 700-1100 nm near infrared range.

Cyclical Cushing's syndrome in a patient with a bronchial neuroendocrine tumor (typical carcinoid) expressing ghrelin and growth hormone secretagogue receptors.

A 56-yr-old woman was referred with a diagnosis of Cushing's disease. Hypertension and severe hypokalemia were present and high urinary free cortisol/cortisone ratio was detected, raising a suspicion of an ectopic ACTH syndrome. Inferior petrosal sinus sampling, thoracic computed tomography, and octreotide scans were negative. Remission and relapse periods lasting 3-4 months were observed during the 3.5 yr of follow-up. Finally a thoracic computed tomography scan showed a basal paracardic nodule in the left lung. After surgery, a well-differentiated neuroendocrine tumor (typical bronchial carcinoid) was diagnosed, staining positively for ACTH. RT-PCR revealed expression of proopiomelanocortin, CRH receptor, and V3 vasopressin receptor. Somatostatin receptor type 1, 2, 3, and 5 mRNA was detected only in tumoral tissue. Interestingly, we observed the simultaneous presence of ghrelin and both GH secretagogue (GHS) receptors (1a and 1b) mRNA in tumoral tissue but not in the normal lung. This finding correlates with the in vivo ACTH hyperresponsiveness to hexarelin (a GHS). This is the first report of a cyclical ectopic ACTH-secreting tumor with an in vivo ACTH response to hexarelin coupled with the tumoral expression of ghrelin and GHS receptors. This finding might imply an autocrine/paracrine modulatory effect of ghrelin in bronchial ACTH-secreting tumors.

Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box.

The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.

Molecular diagnosis of Stickler syndrome: a COL2A1 stop codon mutation screening strategy that is not compromised by mutant mRNA instability.

We have developed a novel strategy for screening families with type 1 Stickler syndrome due to COL2A1 nonsense mutations, using a modified RNA-based protein truncation test. To overcome the problem of the unavailability of collagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessible cells (lymphoblasts and fibroblasts), which were pre-incubated with cycloheximide to prevent nonsense-mutation-induced mRNA decay. The five overlapping RT-PCR fragments covering the COL2A1 coding region were then transcribed and translated in vitro to identify smaller truncated protein products which result from a premature stop codon. This method was used to screen a 4-generation Stickler family and a protein truncating mutation was identified, which was present in all affected individuals. Targeted sequencing identified the mutation as a G(+1) to A substitution at the 5' splice donor site of intron 25, which led to the activation of a cryptic splice site 8-bp upstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon. Mutant mRNA was undetectable without cycloheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay. As well as providing further evidence that type 1 Stickler syndrome results from COL2A1 premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency may also be an important, but previously unrecognized, molecular basis of Stickler syndrome. This rapid new test for COL2A1 nonsense mutations is of particular clinical importance to Stickler syndrome families, where the identification of individuals who are at risk of this potentially preventable form of blindness will allow them to undergo regular ophthalmological surveillance and preventative or early ameliorative treatment.

Transforming growth factor beta1: implications in adrenocortical tumorigenesis.

TGFbeta1, a multifunctional growth modulator, inhibits the proliferation of epithelial cells. TGFbeta1 signaling is dependent on the heterodimerization of the TGFbeta1 receptor II (TGFbeta1RII) with the TGFbeta1 receptor I (TGFbeta1RI). The cytoplasmic proteins Smads are the mediators of the TGFbeta1 signal. TGFbeta1 regulates adult and fetal adrenal growth and function. Previously we have shown by Northern analysis that TGFbeta1mRNA was well expressed in normal adrenal and in adrenocortical adenomas but reduced in carcinomas. To investigate whether TGFbeta1 receptors may act as tumor suppressors of adrenal tumorigenesis, 16 adenomas and 12 carcinomas were studied. We have used SSCP analysis to scan for inactivating mutations in carcinomas. All tumor samples were negative for somatic alterations of both genes. A competitive RT-PCR system was developed to compare the levels of expression of TGFbeta1, TGFbeta1R-I and TGFbeta1R-II, Smad-2 and Smad-4 genes in all tumors. In our study, we confirmed the presence of reduced levels of TGFbeta1 in carcinomas. On the contrary, Smad-4 gene levels were elevated in carcinomas when compared to that of adenomas. No significant differences were observed in gene expression of TGFbeta1RI and Smad-2. Our results suggest that mutations of TGFbeta1 receptors appear not to be involved in adrenal tumorigenesis. Adrenal carcinomas showed a significant reduction of the TGFbeta1 mRNA levels but on the contrary Smad 4 mRNA levels were significantly increased.

Interaction of collagen alpha1(X) containing engineered NC1 mutations with normal alpha1(X) in vitro. Implications for the molecular basis of schmid metaphyseal chondrodysplasia.

Collagen X is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal nonhelical NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggests a critical role for NC1 in collagen X structure and function. In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that although alpha1(X) containing normal NC1 domains can form electrophoretically stable trimers, engineered SMCD NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal alpha1(X). To allow the detection of more subtle interactions that may interfere with assembly but not produce SDS-stable final products, we have developed a competition-based in vitro co-expression and assembly approach. Our studies show that alpha1(X) chains containing SMCD mutations reduce the efficiency of normal alpha1(X) trimer assembly, indicating that interactions do occur between mutant and normal NC1 domains, which can impact on the formation of normal trimers. This finding has important implications for the molecular pathology of collagen X mutations in SMCD. Although we have previously demonstrated haploinsufficiency as one in vivo mechanism (Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1998) J. Clin. Invest. 101, 1490-1499), the current study suggests dominant interference is also possible if the mutant protein is expressed in vivo. Furthermore, we establish that a conserved 13-amino acid aromatic motif (amino acids 589-601) is critical for the interaction between the NC1 domains, suggesting that this region may initiate assembly and the other NC1 mutations interfered with secondary interactions important in folding or in stabilizing the assembly process.

Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.

The protein truncation test (PTT) is a mutation-detection method used to scan for premature termination (nonsense) mutations. PCR amplification of the DNA or mRNA source material is performed using forward primers containing a T7-promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source material, but success of the PTT and other RNA-based mutation detection methods can be severely compromised by nonsense mutation-induced mRNA decay, a well-documented process that is often overlooked in mutation detection strategies. In this study, we develop an RNA-based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the mutant mRNA. The effectiveness of this method for mutation detection in abundant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the protection of type I collagen (COL1A1) mRNA containing nonsense mutations that normally resulted in mutant mRNA degradation. Stabilisation of mutant mismatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Importantly, our strategy also stabilised very low-level (or illegitimate) nonsense-containing transcripts in lymphoblasts from patients with Bethlem myopathy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRCA1). The greatly increased sensitivity and reliability of this RT-PCR/PTT protocol has broad applicability to the many genetic diseases in which only blood-derived cells may be readily available for analysis.

Blood IgA, IgM and IgE levels are influenced by sex and HLA phenotype.

In the present study we have investigated the HLA-B8,DR3-associated control of serum IgA levels in 110 randomly selected HLA-typed healthy subjects. Serum IgG, IgA, IgM and IgE levels were measured in all individuals. The values were analyzed according to age, sex and HLA-DR phenotype. No correlation between age and serum IgA, IgM and IgE levels was found in our homogeneous sample, since all subjects studied were in a narrow age range, i.e. 20-49 years. In contrast, a significant positive correlation was found for IgG levels. The mean IgM level was significantly higher in females than in males, whereas the mean IgA and IgE levels were significantly lower in females. HLA-DR2-positive subjects displayed the significantly highest levels of serum IgE, whereas HLA-B8,DR3-positive subjects showed the significantly lowest levels of serum IgA. Thus, our results show that in our random sample sex and DR phenotype are involved in the control of serum IgA, IgM and IgE levels. The genetic control of sex and HLA-B8,DR3 of serum IgA levels could be related to the high incidence of autoimmune disorders among females and HLA-B8,DR3-positive individuals. The results showing higher levels of IgE in HLA-DR2-positive subjects might be consistent with the observation of a strong association between HLA-DR2 and IgE immune hyperresponsiveness to some allergen(s).

Derivatives of 2H,3H-benzimidazo(1,2-b)oxazole.

The Authors report the synthesis and chemical properties of a number of derivatives of 2H,3H-benzimidazo(1,2-b)oxazole. These substances were prepared in order to investigate their pharmacological activity.