Aberrant promoter methylation and reduced expression of p16 gene in esophageal squamous cell carcinoma from Kashmir valley: a high-risk area

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in Jammu and Kashmir region of India and has multi-factorial etiology involving dietary habits, genetic factors, and gene environmental interactions. Inactivation of the p16 gene expression by aberrant promoter methylation plays an important role in the progression of esophageal carcinoma. In the present investigation, we have studied the role of p16 promoter methylation in 69 histopathologically confirmed ESCC tissues and compared it with corresponding normal adjacent tissues for DNA methylation in the CpG island in the p16 promoter region by methylation-specific polymerase chain reaction (MSP) and p16 protein expression by immunoblotting. The results showed loss of p16 expression in 67% (46/69) of tumor tissues compared to only 3% in control tissues (2/69). Promoter methylation was observed in 52% (36/69) of tumor tissues and it gradually increased with the increasing severity of histological grades of the cancer (P = 0.0001). Loss of p16 expression with promoter methylation was observed in 26 of 36 cases (72%). Analysis of patients dietary habits revealed a strong association between promoter methylation and high consumption of hot salted tea (P < 0.05) which is a most favourite drink commonly consumed by Kashmiri people.     

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.     

Sequence analysis of the internal transcribed spacer 2 (ITS2) from Philornis seguyi (García, 1952) and Philornis torquans (Nielsen, 1913) (Diptera: Muscidae)

Philornis Meinert, 1890 (Diptera: Muscidae) is a genus of Neotropical dipterans that parasitise birds. The currently used external morphological characters to distinguish between species within this genus present some limitations. We used the second internal transcribed spacer region (ITS2) of the rRNA gene as a molecular marker to differentiate adult specimens of Philornis identified morphologically as Philornis torquans and Philornis seguyi from different localities. Specimens identified as P. seguyi from Magdalena (Buenos Aires Province) showed an ITS2 sequence different from that for P. torquans, whereas all other specimens of P. seguyi had sequences identical to those for P. torquans. These findings do not necessarily confirm that specimens from Magdalena indeed belong to P. seguyi, nor that P. seguyi is a valid species. Instead, they alert us about the potential for species misidentification when using morphological characters alone. The use of molecular approaches to aid the identification of Philornis spp. will shed light on the systematics of this group. P. torquans is reported for the first time in Mendoza Province and Uruguay.     

Formin leaky cap allows elongation in the presence of tight capping proteins

Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.     

Detection of X chromosome aneuploidy using Southern blot analysis during routine population-based screening for fragile X syndrome

We report herein two cases where detection of X chromosome aneuploidy (cytogenetically proved 45,X/46XX and 47,XXX) was made possible by molecular diagnosis during population-based carrier screening for Fragile X syndrome, using Southern blot analysis. This study emphasizes the value of molecular analysis for gene dosage to suggest chromosomal aneuploidy.     

Improving the Effectiveness of Artificial MicroRNA (amiR)-Mediated Resistance against Turnip Mosaic Virus by Combining Two amiRs or by Targeting Highly Conserved Viral Genomic Regions

A drawback of recent antiviral therapies based on the transgenic expression of artificial microRNAs (amiRs) is the ease with which viruses generate escape mutations. Here, we show two alternative strategies for improving the effectiveness of resistance in plants. First, we expressed two amiRs complementary to independent targets in the viral genome, and second, we designed amiRs complementary to highly conserved RNA motifs in the viral genome.     

Positive and negative unintended human-induced effects on Iberian mole abundance at the edge of its distribution area

Humans can unintentionally induce both positive and negative effects on wildlife presence and abundance, with organisms living in or associated with agricultural areas being good examples. Our study focused on a 1500 ha area (75 sampled 100 m × 50 m plots) at the driest edge of the endemic Iberian mole Talpa occidentalis distribution range, where the species is listed as “Vulnerable”. Here, poplar cultivations dominate wasteland and other irrigated and non-irrigated crops. The poplar irrigation system was traditionally based on a network of straight ridges, although it is rapidly being replaced with a sophisticated procedure which permits water to spread with ridges no longer being needed. In these habitats, ridges are relevant for moles because they provide dry shelters for nests. In this paper we explore (a) mole local habitat preferences and (b) the impact of changes in poplar irrigation systems on mole abundance. Iberian mole abundance positively related to earthworm biomass and numbers; however, multivariate analyses highlighted the effect of herbaceous cover (positive relationship), rocks cover and soil hardness (negative relationship), and habitat type (poplar being the preferred one). Furthermore, mole abundance was substantially higher in poplar groves where ridges were still present than where they were not. We conclude that Iberian moles in semi-arid environments are favoured by poplar plantations but, at the same time, they are highly vulnerable to recent changes in traditional agriculture practices. Therefore, this study shows how agricultural habitats can benefit some species of conservation concern, especially some temperate species at the edge of their range or in extreme ecological conditions. Changes in agricultural practices that negatively affect the suitability of such habitats can compromise these species's conservation, as we found for the Iberian mole.     

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.     

A novel p16(INK4A) mutation associated with esophageal squamous cell carcinoma in a high risk population

This study describes identification of p16(INK4A) sequence variants and their potential association with esophageal squamous cell carcinoma (ESCC) in a high risk population from Kashmir, India. We report a novel 7 base pair exon 2 deletion in 22 out of 106 (~20%) surgically resected tumor samples. The deletion beginning at the second base of codon 103, results in a frame shift causing premature termination of the protein at codon 142, with structural and functional consequences predicted by insilico analysis. The described mutation is a previously unreported variant of p16(INK4A), perhaps representing a founder mutation unique to the population.     

Effectiveness of Antegrade Access in Bladder Tumors With Inaccessible Urethra

Inaccessible urethra with no retrograde endoscopic access due to multiple/diffuse strictures or multiple urethrocutaneous fistulas with acute urinary retention due to posturethral instrumentation (transurethral resection of bladder tumor [TURBT], or TURBT with transurethral resection of the prostate [TURP]), is a rare entity. Management of such a case with a bladder tumor for TURBT/surveillance cystoscopy poses a great challenge. The authors present 12 cases of bladder tumor with inaccessible urethra, 10 cases due to multiple strictures (post-TURBT and/or TURP), and 2 cases due to urethrocutaneous fistulas (post-TURBT), who presented to our emergency department with acute urinary retention. Emergent suprapubic catheterization was used as a temporary treatment method.Key words: Suprapubic cystostomy, Inaccessible urethra, Bladder tumors, Tract seedlingBladder tumors are the most common neoplasm of the lower urinary tract, comprising 6% of all malignancies in men and 2% of those in women.¹ A majority of patients present with gross painless hematuria, usually as the sole presenting symptom.² Bladder carcinoma is unique among human neoplasms in that many of its etiologic factors are known; the urologist should be aware of the possible occupational exposures to urothelial carcinogens.³ Initial symptoms of urothelial carcinoma of the bladder (UCB) include microhematuria, painless macrohematuria, and/or irritative voiding symptoms, and require further investigation. Carcinoma in situ of the bladder causes irritative lower urinary tract symptoms (LUTS) more often than does papillary UCB. Histopathologic evaluation is necessary to assess stage and grade with sufficient certainty after the appearance of bladder tumors.⁴ Bladder tumors spread by implantation in abdominal wounds, denuded epithelium, resected prostatic fossa, or traumatized urethra⁵; implantation occurs most often with high-grade tumors.