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Tissue engineering benefits from novel materials with precisely tunable physical, chemical and mechanical properties over a broad range. Here we report a practical approach to prepare Bombyx morisilk fibroin hydrogels using the principle of non-solvent induced phase separation (NIPS). A combination of reconstituted silk fibroin (RSF) and methanol (non-solvent), with a final concentration of 2.5% w/v and 12.5% v/v respectively, maintained at 22 °C temperature turned into a hydrogel within 10 hours. Freeze-drying of this gel gave a foam with a porosity of 88%, a water uptake capacity of 89% and a swelling index of 8.6. The gelation kinetics and the loss tangent of the gels were investigated by rheometry. The changes in the morphology of the porous foams were visualized by SEM. The changes in RSF chemical composition and the relative fraction of its secondary structural elements were analyzed by ATR-FTIR along with Fourier self-deconvolution. And, the changes in the glass transition temperature, specific heat capacity and the relative fraction of crystallinity of RSF were determined by TM-DSC. Data suggested that RSF–water–methanol behaved as a polymer–solvent–non-solvent ternary phase system, wherein the demixing of the water–methanol phases altered the thermodynamic equilibrium of RSF–water phases and resulted in the desolvation and eventual separation of the RSF phase. Systematic analysis revealed that both gelation time and the properties of hydrogels and porous foams could be controlled by the ratios of RSF and non-solvent concentration as well as by the type of non-solvent and incubation temperature. Due to the unique properties we envisage that the herein prepared NIPS induced RSF hydrogels and porous foams can possibly be used for the encapsulation of cells and/or for the controlled release of both hydrophilic and hydrophobic drugs.

In mouse models, loss of the candidate tumor suppressor gene Ubiquitin Specific Protease 44 (USP44) is associated with aneuploidy and cancer. USP44is also transcriptionally silenced in human cancers. Here we investigated the molecular mechanism of USP44silencing and whether this correlated with aneuploidy in colorectal adenomas. DNA methylation at the USP44CpG island (CGI) promoter was measured using combined bisulfite restriction analysis (COBRA) in colorectal cancer (CRC) cell lines (n = 18), and with COBRA and bisulfite sequencing in colorectal adenomas (n = 89) and matched normal colonic mucosa (n = 51). The USP44CGI was hypermethylated in all CRC cell lines, in most colorectal adenomas (79 of 89, 89%) but rarely in normal mucosa samples (3 of 51, 6%). USP44expression was also compared between normal mucosa and paired hypermethylated adenomas in six patients using qRT-PCR. Hypermethylation of the USP44CGI in adenomas was associated with a 1.8 to 5.5-fold reduction in expression compared with paired normal mucosa. Treatment of CRC cell lines with the DNA hypomethylating agent decitabine resulted in a 14 to 270-fold increase in USP44expression. Whole genome SNP array data showed that gain or loss of individual chromosomes occurred in adenomas, but hypermethylation did not correlate with more aneuploidy. In summary, our data shows that USP44is epigenetically inactivated in colorectal adenomas, but this alone is not sufficient to cause aneuploidy in colorectal neoplasia.

Epigenetic silencing of cancer-related genes by promoter methylation is a frequent event in sporadic colorectal cancer. The CpG island methylator phenotype (CIMP+), in which discrete genes throughout the genome are simultaneously methylated, and long-range epigenetic silencing, whereby multiple genes within contiguous chromosomal regions are methylated, have been described in subsets of colorectal cancer. We previously reported the concurrent methylation of the mismatch repair gene MLH1with a cluster of flanking genes in chromosome region 3p22 in sporadic colorectal carcinoma exhibiting microsatellite instability and the BRAF V600E mutation. Herein, we aimed to determine whether methylation of MLH1and neighbouring 3p22 genes, singly or concomitantly, correlate with the germline c.-93G>A SNP within the MLH1promoter, CIMP+ and other clinicopathological and molecular features of the tumours. By studying a cohort of 946 sporadic colorectal cancer cases, we show a strong association between concordant methylation of ≥3 of five 3p22 genes with CIMP+ and the BRAF V600E mutation (P<0.001). These associations were independent of microsatellite instability, as concomitant methylation of 3p22 genes other than MLH1was found in microsatellite stable cancers. These findings show that long-range epigenetic silencing across 3p22 occurs in the context of CIMP+ and the BRAF V600E mutation, and only gives rise to microsatellite instability when this process encompasses MLH1. Furthermore, the strong relationship between long-range epigenetic silencing of 3p22 and CIMP+ provides further evidence that these two purportedly distinct epigenetic phenotypes represent a single entity with a common aetiology. Low-level methylation of MLH1and flanking 3p22 genes, as well as the BRAF V600E mutation, were detected in the apparently normal colonic mucosa of a small number of cases whose tumours showed a similar molecular profile, suggesting that these concurring genetic and epigenetic events can occur as a field defect in neoplastic development.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that restores mutagenic O6-methylguanine to guanine. MGMTmethylation is frequently observed in sporadic colorectal cancer and was recently correlated with the C>T allele at SNP rs16906252, within the transcriptional enhancer element of the promoter. MGMTmethylation has also been associated with KRASmutations, particularly G>A transitions. We studied 1123 colorectal carcinoma to define the molecular and clinicopathological profiles associated with MGMTmethylation. Furthermore, we assessed factors contributing to MGMTmethylation in the development of colorectal cancer by studying the allelic pattern of MGMTmethylation using SNP rs16906252, and the methylation status of neighbouring genes within 10q26 in selected tumours and matched normal colonic mucosa. MGMTmethylation was detected by combined bisulphite restriction analysis in 28% of tumours and was associated with a number of characteristics, including CDKN2Amethylation, absent lymphovascular space invasion and KRASmutations (but not specifically with KRASG>A transitions). In a multivariate analysis adjusted for age and sex, MGMTmethylation was associated with the T allele of SNP rs16906252 (P<0.0001, OR 5.5, 95% CI 3.8–7.9). Low-level methylation was detected by quantitative methylation-specific PCR in the normal colonic mucosa of cases, particularly those with a correspondingly methylated tumour, as well as controls without neoplasia, and this was also associated with the C>T SNP. We show that the T allele at SNP rs16906252 is a key determinant in the onset of MGMTmethylation in colorectal cancer, whereas the association of methylation at MGMTand CDKN2Asuggests that these loci may be targets of a common mechanism of epigenetic dysregulation.

Activating mutations of the BRAFand KRASgenes cause constitutive stimulation of an important cell-signaling pathway promoting tumorigenesis, and are increasingly recognized as determinants of response to targeted cancer therapies. The V600E mutation accounts for most of the BRAF mutations in cancer, and KRAS mutations are predominantly encoded by nucleotide substitutions within codons 12 and 13. We designed novel pyrosequencing assays for the detection of the common “hotspot” mutations in these genes, which demonstrated analytical sensitivities of ≤10 in titrations of mutant cell lines. The KRASpyrosequencing assay has the ability to simultaneously identify all potential nucleotide changes within the mutation cluster at codons 12 and 13, with a sequence output in the sense direction to facilitate results interpretation. These assays were used to determine the mutation status in a prospective series of 1198 sporadic colorectal cancers. The BRAF V600E mutation was detected in 13.2 of the colorectal cancers. The frequency of KRASmutations in our cohort was 32.4, with G>A transitions at position 2 of codons 12 and 13 being most prevalent. Both assays proved highly sensitive and specific when applied to clinical specimens, and were applicable to both fresh-frozen and formalin-fixed paraffin-embedded archival tissues. These assays would serve as a suitable platform for large-scale mutation detection in cancer specimens where the facility for pyrosequencing is available.