Sequencing by binding rivals SMOR error-corrected sequencing by synthesis technology for accurate detection and quantification of minor (< 0.1%) subpopulation variants.

BACKGROUND: Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications, including the detection of drug resistant pathogens and somatic variant detection in oncology. A recently available sequencing approach termed 'sequencing by binding (SBB)' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using SBB for the detection of ultra-rare drug resistant subpopulations in Mycobacterium tuberculosis (Mtb) using a targeted amplicon assay and compares the performance of SBB to single molecule overlapping reads (SMOR) error corrected sequencing by synthesis (SBS) data. RESULTS: SBS displayed an elevated error rate when compared to SMOR error-corrected SBS and SBB techniques. SMOR error-corrected SBS and SBB technologies performed similarly within the linear range studies and error rate studies. CONCLUSIONS: With lower sequencing error rates within SBB sequencing, this technique looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.

Propranolol in the treatment of infantile haemangiomas: lessons from the European Propranolol In the Treatment of Complicated Haemangiomas (PITCH) Taskforce survey.

BACKGROUND: Oral propranolol is widely prescribed as first-line treatment for infantile haemangiomas (IHs). Anecdotally, prescribing practice differs widely between centres. OBJECTIVES: The Propranolol In the Treatment of Complicated Haemangiomas (PITCH) Taskforce was founded to establish patterns of use of propranolol in IHs. METHODS: Participating centres entered data on all of their patients who had completed treatment with oral propranolol for IHs, using an online data capture tool. RESULTS: The study cohort comprised 1097 children from 39 centres in eight European countries. 76·1% were female and 92·8% had a focal IH, with the remainder showing a segmental, multifocal or indeterminate pattern. The main indications for treatment were periocular location (29·3%), risk of cosmetic disfigurement (21·1%) and ulceration and bleeding (20·6%). In total 69·2% of patients were titrated up to a maintenance regimen, which consisted of 2 mg kg(-1) per day (85·8%) in the majority of cases. 91·4% of patients had an excellent or good response to treatment. Rebound growth occurred in 14·1% upon stopping, of whom 53·9% were restarted and treatment response was recaptured in 91·6% of cases. While there was no significant difference in the treatment response, comparing a daily maintenance dose of < 2 mg kg(-1) vs. 2 mg kg(-1) vs. > 2 mg kg(-1) , the risk of adverse events was significantly higher: odds ratio (OR) 1 vs. adjusted OR 0·70, 95% confidence interval (CI) 0·33-1·50, P = 0·36 vs. OR 2·38, 95% CI 1·04-5·46, P = 0·04, Ptrend < 0·001. CONCLUSIONS: The PITCH survey summarizes the use of oral propranolol across 39 European centres, in a variety of IH phases, and could be used to inform treatment guidelines and the design of an interventional study.

Technical note: Isolation and characterization of porcine mammary epithelial cells.

Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), ß-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 µg/mL prolactin to culture medium for 3 d induced the production of ß-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

GD3 synthase regulates epithelial-mesenchymal transition and metastasis in breast cancer.

The epithelial-mesenchymal transition (EMT) bestows cancer cells with increased stem cell properties and metastatic potential. To date, multiple extracellular stimuli and transcription factors have been shown to regulate EMT. Many of them are not druggable and therefore it is necessary to identify targets, which can be inhibited using small molecules to prevent metastasis. Recently, we identified the ganglioside GD2 as a novel breast cancer stem cell marker. Moreover, we found that GD3 synthase (GD3S)--an enzyme involved in GD2 biosynthesis--is critical for GD2 production and could serve as a potential druggable target for inhibiting tumor initiation and metastasis. Indeed, there is a small molecule known as triptolide that has been shown to inhibit GD3S function. Accordingly, in this manuscript, we demonstrate that the inhibition of GD3S using small hairpin RNA or triptolide compromises the initiation and maintenance of EMT instigated by various signaling pathways, including Snail, Twist and transforming growth factor-ß1 as well as the mesenchymal characteristics of claudin-low breast cancer cell lines (SUM159 and MDA-MB-231). Moreover, GD3S is necessary for wound healing, migration, invasion and stem cell properties in vitro. Most importantly, inhibition of GD3S in vivo prevents metastasis in experimental as well as in spontaneous syngeneic wild-type mouse models. We also demonstrate that the transcription factor FOXC2, a central downstream effector of several EMT pathways, directly regulates GD3S expression by binding to its promoter. In clinical specimens, the expression of GD3S correlates with poor prognosis in triple-negative human breast tumors. Moreover, GD3S expression correlates with activation of the c-Met signaling pathway leading to increased stem cell properties and metastatic competence. Collectively, these findings suggest that the GD3S-c-Met axis could serve as an effective target for the treatment of metastatic breast cancers.

Primary cutaneous amyloidosis of the glans penis. Two case reports and a review of the literature.

BACKGROUND: Primary cutaneous amyloidosis of the penis is a rare entity. OBJECTIVES: We describe two cases of primary penile cutaneous amyloidosis presenting as localized lesions on the penis and not associated with systemic amyloidosis. METHODS: A comprehensive literature review of all published cases. RESULTS: The rarest form of cutaneous amyloidosis, nodular amyloidosis, is the most frequent presentation of penis-limited cutaneous amyloid with a low incidence of systemic disease progression. CONCLUSIONS: Although primary nodular amyloidosis is a rare entity it seems to be the most frequent presentation of penile-limited cutaneous disease, with a low incidence of systemic involvement.

Regulation of DCIS to invasive breast cancer progression by Singleminded-2s (SIM2s).

Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of mammary epithelial cell differentiation. SIM2s is highly expressed in mammary epithelial cells and downregulated in human breast cancer. Loss of Sim2s causes aberrant mouse mammary ductal development, with features suggestive of malignant transformation, whereas overexpression of SIM2s promotes precocious alveolar differentiation in nulliparous mouse mammary glands, suggesting that SIM2s is required for establishing and enhancing mammary gland differentiation. To test the hypothesis that SIM2s regulates tumor cell differentiation, we analyzed SIM2s expression in human primary breast ductal carcinoma in situ (DCIS) samples and found that SIM2s is lost with progression from DCIS to invasive ductal cancer (IDC). Using a MCF10DCIS.COM progression model, we have shown that SIM2s expression is decreased in MCF10DCIS.COM cells compared with MCF10A cells, and reestablishment of SIM2s in MCF10DCIS.COM cells significantly inhibits growth and invasion both in vitro and in vivo. Analysis of SIM2s-MCF10DCIS.com tumors showed that SIM2s promoted a more differentiated tumor phenotype including the expression of a broad range of luminal markers (CSN2 (ß-casein), CDH1 (E-cadherin), and KER18 (keratin-18)) and suppressed genes associated with stem cell maintenance and a basal phenotype (SMO (smoothened), p63, SLUG (snail-2), KER14 (keratin-14) and VIM (vimentin)). Furthermore, loss of SIM2s expression in MCF10DCIS.COM xenografts resulted in a more invasive phenotype and increased lung metastasis likely due to an increase in Hedgehog signaling and matrix metalloproteinase expression. Together, these exciting new data support a role for SIM2s in promoting human breast tumor differentiation and maintaining epithelial integrity.

Actions of exendin-4 therapy on cognitive function and hippocampal synaptic plasticity in mice fed a high-fat diet.

High-calorie diet has been shown to impair learning ability and hippocampal synaptic plasticity in rodents. This study examined effects of daily treatment with the glucagon-like peptide-1 mimetic, exendin-4, on cognitive function and hippocampal synaptic plasticity in a model of diet-induced obesity, which exhibits compromised cognitive performance. Mice fed a high-fat diet were treated with exendin-4 (25 nmol kg(-1) bodyweight; twice daily) or saline vehicle (0.9% (w/v) NaCl) over 21 days. In addition to improving metabolic control, exendin-4-treated mice exhibited a marked increase in recognition index highlighting improved learning and memory. High-fat diet resulted in the elimination of in vivo electrophysiological long-term potentiation, which was rescued following exendin-4 treatment. This study shows that exendin-4 therapy improves cognitive function and ameliorates impaired hippocampal synaptic plasticity in dietary-induced obesity.

Ha-Ras transformation of MCF10A cells leads to repression of Singleminded-2s through NOTCH and C/EBPbeta.

We have previously shown that Singleminded-2s (SIM2s), a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of transcription factors, is downregulated in breast cancer samples and has tumor suppressor activity. However, the mechanism by which SIM2s is repressed in breast cancer cells has not been determined. In this study, we show that transformation of MCF10A cells by Harvey-Ras (Ha-Ras) induces CCAAT/enhance binding protein beta (C/EBPbeta) and activates the NOTCH signaling pathway to block SIM2s gene expression. NOTCH-mediated repression acts through a C-repeat binding factor 1 (CBF1)-independent mechanism, as introduction of CBF1 had no effect on SIM2s expression. Consistent with C/ebpbeta-dependent inhibition of SIM2s, C/ebpbeta(-/-) mouse mammary glands express high levels of SIM2s and reestablishment of C/ebpbeta isoforms decreased SIM2s mRNA levels in C/ebpbeta immortalized mammary epithelial cell lines. These studies illustrate a novel pathway of tumor suppressor gene silencing in Ha-Ras-transformed breast epithelial cells and identify SIM2s as a target of C/EBPbeta and NOTCH signaling.

Determinants of the variability of aflatoxin-albumin adduct levels in Ghanaians.

Hepatocellular carcinoma (HCC) is a multifactorial disease with various host and environmental factors involved in its etiology. Of these, aflatoxin exposure has been established as an important risk factor in the development of HCC; the presence of aflatoxin-albumin (AA) adducts in the blood serves as a valuable biomarker of human exposure. In this study, the relationship between a variety of different HCC host factors and the incidence of AA adduct levels was examined in a Ghanaian population at high risk for HCC. These factors included age, gender, hepatitis virus B (HVB) and hepatitis C virus (HCV) status, and genetic polymorphisms in both microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). Blood samples were analyzed for AA adducts and HBV and HCV status. GSTM1 and GSTT1 deletion polymorphisms and mEH exon 3 and exon 4 single-nucleotide polymorphisms (SNPs) were determined from urine samples. In univariate analysis, age, HBV and HVC status, and GSTT1 and mEH exon 3 genotypes were not associated with AA adduct levels. However, mean adduct levels were significantly higher in both females and individuals typed heterozygous for mEH exon 4 (vs. wild types). Stratification analysis also showed that gender along with mEH exon 4 genotype and HBV status had a significant effect on adduct levels. Both females typed HBsAg+ and males with mEH exon 4 heterozygote genotypes showed significantly higher adduct levels as compared to the HBsAg- and wild types, respectively. Understanding the relationships between these host factors and the variability in aflatoxin-adduct levels may help in identifying susceptible populations in developing countries and for targeting specific public health interventions for the prevention of aflatoxicoses in populations with HCC and chronic liver diseases.

Noninvasive identification of interindividual variation in xenobiotic-metabolizing enzymes: implications for cancer epidemiology and biomarker studies.

In this study, DNA extracted from frozen urine was used in the analysis of polymorphisms in genes coding for xenobiotic-metabolizing enzymes (XMEs). These included single-nucleotide polymorphisms (SNP) in microsomal epoxide hydrolase (mEH), that is, substitutions of tyrosine by histidine in codon 113 (Y113H) and histidine by arginine in codon 139 (H139R), and deletion polymorphisms in glutathione S-transferase (GST) M1 and T1 genes. The concentration of DNA extracted from urine of a Ghanaian population (n = 91) exposed to aflatoxins in their diet ranged from 82.5 to 573 ng/ml urine. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) procedures were used for the characterization of mEH polymorphisms, whereas a multiplex PCR method was utilized to identify GST deletion polymorphisms. In total, 91% and 94% of 91 samples were genotyped for mEH exon 3 and exon 4 polymorphisms, respectively. In the multiplex analysis of GST polymorphisms, 94% and 91% of 91 individuals were genotyped for GSTM1 and GSTT1 polymorphisms, respectively. The polymorphisms in the mEH exon 4, GSTM1 and GSTT1, were not in Hardy-Weinberg equilibrium (HWE) except for mEH exon 3. Representative genotypes identified by PCR-RFLP were cloned and sequenced, then confirmed by comparison with reference sequences of human DNA published in the GenBank BLAST database. These results demonstrate that XMEs can be genotyped from urine with reliable accuracy and may be useful in cancer and molecular epidemiology studies.

Penile intraepithelial neoplasia: clinical spectrum and treatment of 35 cases.

BACKGROUND: Penile intraepithelial neoplasia (PIN) is the term used to describe erythroplasia of Queyrat (EQ), Bowen's disease (BD) and bowenoid papulosis (BP). These conditions are distinct clinical entities and have different epidemiological and aetiological associations and prognostic implications. OBJECTIVES: To describe the presentation and treatment of patients with PIN. METHODS: Thirty-five patients presenting with PIN over a 7-year period are described. RESULTS: Our observations include: (i) patients with BP are younger than those with EQ or BD and sometimes have a history of immunosuppression; (ii) patients with BP usually have a history or clinical evidence of previous genital human papillomavirus infection; (iii) patients with EQ often have a concurrent penile dermatosis (lichen sclerosus or lichen planus); (iv) patients with PIN are usually uncircumcised; and (v) response to treatment of BP depends on the integrity of the immune system. CONCLUSIONS: We recommend vigorous treatment of all patients with PIN, including circumcision. Smoking should be actively discouraged. Patients should have life-long follow-up and partners of patients with BP should be screened for other forms of intraepithelial neoplasia (cervical and anal).

Physicochemical considerations in the preparation of amorphous ritonavir-poly(ethylene glycol) 8000 solid dispersions.

A systematic study of the properties of ritonavir and the influence of polyethylene glycol 8000 (PEG) on ritonavir revealed that amorphous ritonavir dispersions in PEG would have an improved dissolution profile and could exhibit long-term stability. Ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, is highly lipophilic [distribution coefficient (log D)= 4.3, 25 degrees C, pH 6.8], poorly water soluble (400 microg/mL in 0.1 N HCl, 1 microg/mL at pH 6.8, 37 degrees C), and exhibits an exceedingly slow dissolution rate (0.03 mg/cm(2)-min in 0.1 N HCl at 37 degrees C). These properties indicated that a solid dispersion containing ritonavir might be useful for overcoming problems associated with slow dissolution. In addition, ritonavir is a good glass former [glass-transition temperature (T(g))/melting point (T(m)) > 0.7]. Amorphous ritonavir has an apparent solubility of 4 mg/mL in 0.1 N HCl at 37 degrees C and shows reasonable stability at 25 degrees C. Amorphous ritonavir, therefore, has properties desirable for preparing a solid dispersion containing this phase. Since PEG, a commonly used polymer, improved the aqueous solubility of crystalline ritonavir, it was expected to have a positive influence on the dissolution rate of ritonavir. Moreover, PEG was found to have negligible plasticizing effect on amorphous ritonavir, which was beneficial for the stability of the dispersion. Finally, solid dispersions of amorphous ritonavir in PEG were prepared, and these dispersions had improved in vitro dissolution rate and were physically stable for > 1.5 years at 25 degrees C when protected from moisture. The performance of this solid dispersion has been attributed to the physicochemical properties of amorphous ritonavir.

The dysfunctional foreskin.

Although important, the foreskin is not absolutely essential to penile function. Dysfunction is common and a cause of considerable morbidity, and is sometimes associated with mortality from squamous cell carcinoma (SCC). Penile dermatoses herald, complicate or cause preputial dysfunction. Their effective diagnosis and management reduces morbidity and possibly mortality. Medical treatment has its limitations and circumcision is often required.

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists.

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.