Better quality of life and reduced fecal incontinence in rectal cancer patients with the watch-and-wait follow-up strategy.

INTRODUCTION: The watch-and-wait (WW) strategy is an alternative to anterior resection in patients with rectal cancer (RC) that have had a complete clinical response to neoadjuvant treatment. Few reports describe the quality of life and functional anorectal disorders (FADs) in that population. AIM: To analyze and compare the FADs and quality of life in patients with locally advanced adenocarcinoma of the rectum treated with neoadjuvant therapy, divided into two different strategy groups: group 1 (G1), WW; and group 2 (G2), anterior resection. MATERIALS AND METHODS: Thirty patients (G1: n = 20 and G2: n = 10) that had finished neoadjuvant therapy at least 12 months prior were included. Mean patient age was 59.5 years (range: 41-79) and 15 of the patients were men. The FADs were evaluated through: a) clinical history, b) 21-day bowel diary, c) Jorge and Wexner fecal incontinence scale, d) anorectal manometry (ARM), and fecal incontinence quality of life scale (FIQL). RESULTS: Bowel diary: fecal incontinence (40%) and urge to defecate (45%) in G1 vs. fecal incontinence (60%) and urge to defecate (30%) in G2, with no significant differences (p = NS). Fecal incontinence scale: fecal incontinence in G1 was significantly less severe than that in G2 (median 6.5 points vs. 13 points [p = 0.0142]). ARM: no differences between the two groups. Quality of life: significantly different between the two groups (FIQL/G1: 3.7 vs. FIQL/G2: 2.8; p < 0.03). CONCLUSIONS: The WW follow-up strategy in patients with locally advanced rectal cancer was associated with better quality of life and reduced fecal incontinence.

Better quality of life and reduced fecal incontinence in rectal cancer patients with the watch-and-wait follow-up strategy. / Mejor calidad de vida y menor incontinencia fecal en pacientes con cáncer de recto con la estrategia de seguimiento watch and wait.

INTRODUCTION: The watch-and-wait (WW) strategy is an alternative to anterior resection in patients with rectal cancer (RC) that have had a complete clinical response to neoadjuvant treatment. Few reports describe the quality of life and functional anorectal disorders (FADs) in that population. AIM: To analyze and compare the FADs and quality of life in patients with locally advanced adenocarcinoma of the rectum treated with neoadjuvant therapy, divided into two different strategy groups: group 1 (G1), WW; and group 2 (G2), anterior resection. MATERIALS AND METHODS: Thirty patients (G1: n = 20 and G2: n = 10) that had finished neoadjuvant therapy at least 12 months prior were included. Mean patient age was 59.5 years (range: 41-79) and 15 of the patients were men. The FADs were evaluated through: a) clinical history, b) 21-day bowel diary, c) Jorge and Wexner fecal incontinence scale, d) anorectal manometry (ARM), and fecal incontinence quality of life scale (FIQL). RESULTS: Bowel diary: fecal incontinence (40%) and urge to defecate (45%) in G1 vs. fecal incontinence (60%) and urge to defecate (30%) in G2, with no significant differences (p = NS). Fecal incontinence scale: fecal incontinence in G1 was significantly less severe than that in G2 (median 6.5 points vs. 13 points [p = 0.0142]). ARM: no differences between the two groups. Quality of life: significantly different between the two groups (FIQL/G1: 3.7 vs. FIQL/G2: 2.8; p < 0.03). CONCLUSIONS: The WW follow-up strategy in patients with locally advanced rectal cancer was associated with better quality of life and reduced fecal incontinence.

Successful laparoscopic trans-peritoneal repair of an incisional inguinal hernia, resulting from deep lymph node dissection for melanoma: A case report.

INTRODUCTION: Deep pelvic lymph node dissection for cancer may result in incisional inguinal hernias. We present a case report of successful laparoscopic trans-peritoneal repair of a large ventral inguinal hernia that developed following ileo-inguinal lymph node dissection (CLND) for melanoma. CASE PRESENTATION: A successful 3 port laparoscopic trans-peritoneal procedure was performed on a 56-year-old female for the repair of a left inguinal hernia, developed 13 months following CLND for melanoma. The large oval 18 × 14 cm inguinal defect, with superior margins bordering the conjoint tendon and inferior margins bordering the ileo-psoas muscle, femoral vessels and nerve, was not closed in order to avoid excessive tension and was repaired by fixing a 25 × 20 cm intra-peritoneal mesh to abdominal borders at superior and lateral margins with permanent fasteners and at the inferior margin by a cyanoacrylate-glued overlap to protect femoral vessels and nerves from damage. No hernia recurrence was observed 8 months following this procedure. DISCUSSION: Incisional inguinal hernias, following CLND, are rare but present a challenge to surgeons due to the difficulty in identifying both anatomical plains and safe sites for stable repair. CONCLUSIONS: We report a laparoscopic trans-peritoneal approach for the safe, reproducible and efficacious repair of incisional inguinal hernias that result from CLND. In our opinion prevention of hernia recurrence can be achieved by a intraperitoneal large mesh fixed at superior and lateral margin borders with permanent fasteners and using cyanoacrylate glue to overlap inferior margin borders in order to prevent vessels and/or nerve injury.

Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression.

BACKGROUND: Molecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic colorectal cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway. Nevertheless, even if inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance, its use is limited by the development of MEK inhibitor (MEKi) resistance. METHODS: We have generated in vitro and in vivo different CRC models in order to underline the mechanisms of MEKi resistance. RESULTS: The three different in vitro MEKi resistant models, two generated by human CRC cells quadruple wild type for KRAS, NRAS, BRAF, PI3KCA genes (SW48-MR and LIM1215-MR) and one by human CRC cells harboring KRAS mutation (HCT116-MR) showed features related to the gene signature of colorectal cancer CMS4 with up-regulation of immune pathway as confirmed by microarray and western blot analysis. In particular, the MEKi phenotype was associated with the loss of epithelial features and acquisition of mesenchymal markers and morphology. The change in morphology was accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently to RAS mutation status. To extend these in vitro findings, we have obtained mouse colon cancer MC38- and CT26-MEKi resistant syngeneic models (MC38-MR and CT26-MR). Combined treatment with MEKi, EGFR inhibitor (EGFRi) and PD-L1 inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both models. CONCLUSIONS: These results suggest a strategy to potentially improve the efficacy of MEK inhibition by co-treatment with EGFR and PD-L1 inhibitors via modulation of host immune responses.

Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

[Functional dyspepsia and the satiety test: its usefulness in clinical practice]. / Dispepsia funcional y test de saciedad: utilidad en la práctica clínica.

INTRODUCTION: According to the Rome III Criteria, functional dyspepsia (FD) is classified as postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS). On the other hand, the satiety test (ST) has been used to evaluate gastric accommodation and emptying, distinguishing healthy individuals from those with dyspepsia. AIMS: To determine whether the ST can distinguish dyspeptic individuals from healthy ones and to evaluate its usefulness in differentiating the two FD subtypes. METHODS: Adults with FD were consecutively enrolled in a cross-sectional study within the time frame of August 2011 and October 2012. Healthy subjects participated as controls. The ST consisted of the intake of a nutritional supplement (Fortisip®, Nutricia Bagó®) at a constant speed; satiety was graded at 5-minute intervals (1 to 5 points). Intake was suspended when the maximum score was reported. The total ingested volume and caloric intake was recorded and the Mann-Whitney U test was used in the statistical analysis. RESULTS: The study included 39 dyspeptic patients and 20 control individuals. The patients were predominantly women (84.6 vs. 25%; p < 0.0001) and they were similar in age (39.59 ± 13.53 vs. 34.70 ± 9.85 years) and BMI (24.32 ± 3.52 vs. 25.82 ± 3.34 kg/m2) with respect to the controls. The FD subtype percentages were PDS: 61%, EPS: 31%, and Mixed syndrome: 8%. There was a lower ingested volume and caloric intake on the part of the dyspeptic patients (185 vs. 300 ml and 277 vs. 520 Kcal, respectively. Both: P<.001). No differences in the ST were observed between the two pure dyspepsia subtypes. CONCLUSIONS: There was a difference in the ST between healthy individuals and those with dyspepsia, but the ingested volume and caloric intake in the two FD subtypes were similar.

A computational search for box C/D snoRNA genes in the Drosophila melanogaster genome.

MOTIVATION: In eukaryotes, the family of non-coding RNA genes includes a number of genes encoding small nucleolar RNAs (mainly C/D and H/ACA snoRNAs), which act as guides in the maturation or post-transcriptional modifications of target RNA molecules. Since in Drosophila melanogaster (Dm) only few examples of snoRNAs have been identified so far by cDNA libraries screening, integration of the molecular data with in silico identification of these types of genes could throw light on their organization in the Dm genome. RESULTS: We have performed a computational screening of the Dm genome for C/D snoRNA genes, followed by experimental validation of the putative candidates. Few of the 26 confirmed snoRNAs had been recognized by cDNA library analysis. Organization of the Dm genome was also found to be more variegated than previously suspected, with snoRNA genes nested in both the introns and exons of protein-coding genes. This finding suggests that the presence of additional mechanisms of snoRNA biogenesis based on the alternative production of overlapping mRNA/snoRNA molecules. AVAILABILITY: Additional information is available at http://www.bioinformatica.unito.it/bioinformatics/snoRNAs.

The clot gene of Drosophila melanogaster encodes a conserved member of the thioredoxin-like protein superfamily.

The conversion of pyruvoyl-H(4)-pterin to pyrimidodiazepine (PDA), which is an essential step in the biosynthesis of the red components of Drosophila eye pigments known as drosopterins, requires the products of the genes sepia and clot. While the product of sepia has been shown to correspond to the enzyme PDA-synthase, the role of clot remains unknown, although the clot(1) allele was one of the first eye-color mutants to be isolated in Drosophila melanogaster,and much genetic and biochemical data has become available since. Here we report the cloning of the clot gene, describe its molecular organization and characterize the sequence alterations associated with the alleles cl(1) and cl(2). The coding properties of the gene show that it encodes a protein related to the Glutaredoxin class of the Thioredoxin-like enzyme superfamily, conserved members of which are found in human, mouse and plants. We suggest that the Clot protein is an essential component of a glutathione redox system required for the final step in the biosynthetic pathway for drosopterins.

Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated Drosophila ng-1 gene.

The steroid hormone ecdysone controls multiple aspects of insect development, including larval moults and metamorphosis, and can induce specific genetic responses in different tissues. The definition of the molecular mechanisms able to mediate this tissue-specific responsiveness may greatly contribute to understanding how such an accurate genetic response is achieved. In this work we have identified, by transgenic analysis, the regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile. Our results show that an ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the FKH regulatory protein, which can potentially modulate the ecdysone genetic regulated response.

minifly, a Drosophila gene required for ribosome biogenesis.

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes.

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences, the ecdysone response elements (EcREs), whose repertoire still remains to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), we have analysed the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. We report here that the ng-EcRE contacts the ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and that this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive nuclear receptors cross-talking along the development, a mechanism that might play a relevant role in determining the temporal and spatial specificity of the ecdysone response. Finally, we show that the ng-EcRE can promote functional interactions in vitro as well as in vivo, acting as a transcriptional enhancer able to confer a specific developmental expression profile to a minimal promoter in transgenic flies.

Paclitaxel (Taxol®): A new natural product with major anticancer activity.

Paclitaxel, the first of a new class of microtubule-stabilizing anti-tumor agents, has been hailed as the most significant chemotherapy advance in 15 years. Early clinical results showed response rates of 24%-30% in platinum refractory ovarian cancer at doses ranging from 110-250 mg/m(2) repeated every three to four weeks. The dose limiting toxicity is neutropenia which is characteristically profound, but brief. Severe hypersensitivity reactions which occurred in the phase I studies can be prevented by the use of anti-allergic pre-medication. From a slow start constrained by drug availability, the clinical development of paclitaxel has accelerated dramatically as the issue of product supply has been solved. Substantial activity has been reported in advanced breast cancer (56% RR in pre-treated patients; 62% RR in patients untreated for advanced disease) and non-small cell lung cancer (NSCLC) (20%-30% RR in advanced stage IIIb/IV patients). Significant activity has been reported also in melanoma, small cell lung cancer (SCLC), bladder cancer, squamous cell carcinoma of the head and neck, and of the esophagus, cervical and endometrial cancer, as well as lymphomas. Combination chemotherapy studies have shown high activity in a variety of tumor types. Superiority over standard therapy has been demonstrated in ovarian and lung cancer in phase III randomized studies. Paclitaxel's novel mechanism of action and relative lack of clinical cross-resistance with other agents suggest a role in the combination therapy of early stage disease.

Tosca: a Drosophila gene encoding a nuclease specifically expressed in the female germline.

We describe here a Drosophila gene, tosca (tos), that is specifically expressed in the female germline. tos mRNA accumulates selectively within the pro-oocyte in germarial region 2 and persists throughout oogenesis. In the early embryo, the maternally supplied tos mRNA is evenly distributed at the syncytial blastoderm stage, but is excluded from the forming cells when cellularization begins. tos product is the first Drosophila member of the RAD2 protein family, a group of related DNA repair nucleases conserved from yeast to humans. Within the family, Tos is more closely related to ExoI, a Schizosaccharomyces pombe 5'-->3' double-stranded DNA exonuclease specifically induced in meiotic prophase I. The definite oocyte localization of tos transcript during meiosis and its ubiquitous distribution in early embryos suggest that tos may play a role in mismatch repair during genetic recombination and early cleavage divisions.

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats.

In Drosophila melanogaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which the originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (EcREs). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone functional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA.

The role of the BR-C locus on the expression of genes located at the ecdysone-regulated 3C puff of Drosophila melanogaster.

During the third larval instar, the steroid moulting hormone ecdysone activates three temporally distinct puff sets on the D. melanogaster salivary gland polytene chromosome: the so-called intermoult, early and late puffs. Hormonal regulation of intermoult puffs is quite complex and, so far, largely not understood. In order to further investigate this aspect, we have analysed the effects of mutations in a key regulator of the ecdysone response at the onset of metamorphosis, the Broad-Complex (BR-C) locus, on the expression of genes mapping at the 3C intermoult puff. On the basis of an accurate examination of 3C intermoult gene activity in single, carefully staged, third instar larvae of wild-type and BR-C mutant strains, we were able to subdivide these genes into two groups. Each group is characterised by a different temporal expression profile, so that at the beginning of the wandering stage the transcription of the first group declines as group II transcription is induced. Interestingly, the BR-C locus appears to play a regulatory role in establishing this transcriptional switch. By using mutants of each of the three lethal complementation groups, we precisely defined the role of BR-C functions in this developmental transition and we show that this locus also plays an essential role in the early pre-metamorphic hormonal response.

Dense cluster of genes is located at the ecdysone-regulated 3C puff of Drosophila melanogaster.

The 3C11-12 polytene bands of the Drosophila melanogaster X chromosome give rise to a prominent puff, whose regression is triggered by the increase in the titre of the steroid hormone 20-hydroxyecdysone occurring before the metamorphosis. Here, we report the molecular characterization of three genes, named ng-2, ng-3 and ng-4, which we found to be closely linked to each other and to Sgs-4, Pig-1 and ng-1, three other genes previously mapped at this polytene region. All six genes are, in fact, arranged in a tightly linked cluster spanning a DNA segment of only 11 kb. With the exception of ng-4, all the clustered genes are highly expressed only during the larval life and share the same tissue-specificity, being mainly transcribed within the salivary glands. In addition, two members of the cluster, ng-1 and ng-2, show a very high degree of sequence homology, clearly indicating that they are related to each other by means of a duplication event. Interestingly to note, the entire cluster shows a peculiar genomic location, extending across two introns of the memory gene dunce, a large gene of Drosophila whose organization has proved to be remarkably complex.

Opa-like repeats in the genome of the Medfly Ceratitis capitata.

The sequence determination of several genomic clones isolated from the Mediterranean fruitfly Ceratitis capitata identified the existence of opa-like repeats, often more than one being clustered in small chromosomal segments. These repeats have previously been shown to consist of stretches of tandemly reiterated glutamine-encoding residues, and they are found in multiple genes of several organisms. Most of the repeats described here are flanked or interrupted by stop codons in all reading frames and, thus, could not possibly be part of protein-coding sequences. Furthermore, these repeats, of which there are several hundred in the genome of the Medfly, can be used effectively for the determination of sequence polymorphisms, providing a convenient approach to obtain additional landmarks for the construction of genomic maps of this economically important insect.

Molecular characterization of a Drosophila melanogaster variant strain defective in the Sgs-4 gene dosage compensation.

The X-linked Sgs-4 gene of Drosophila melanogaster encodes a salivary glue protein. Here we report the molecular characterization of a non-dosage compensated variant strain, named Karsnas, in which males accumulate only about half of the Sgs-4 polypeptide amount as do females. The results obtained show that significant nucleotide sequence alterations are accumulated within the Sgs-4 coding and 3' untranslated region of the variant strain, thus suggesting a possible role of these sequences in the Sgs-4 dosage compensation.

Effect of ecd1 mutation on the expression of genes mapped at the Drosophila melanogaster 3C11-12 intermoult puff.

The Drosophila melanogaster ecd1 mutation causes a severe temperature-sensitive deficiency in the titre of the steroid hormone ecdysone. This mutation was used to investigate the role of ecdysone in both the transcription of the genes mapped at the 3C11-12 intermoult puff region and the puff formation. Thoroughly synchronized ecd1 larvae were shifted to the non-permissive temperature at various times of the development; after 24 or 48 h, the levels of the transcripts derived from Sgs-4, Pig-1 and ng-1, the three genes located at the 3C11-12 polytene bands, were determined. The results showed that the levels of the transcripts encoded by Pig-1 and ng-1 are unaffected by the drop in the ecdysone titre occurring in non-permissive conditions whereas the amount of Sgs-4 mRNA is greatly reduced. These data clearly indicate that transcription of the three genes mapped within the puff region is affected differently by the hormone. Furthermore, ecd1 larvae cultured at the non-permissive temperature show a prominent puff at the 3C11-12 polytene bands, indicating that ecdysone is not essential for puff induction and that puff size is not simply correlated with high-level Sgs-4 transcription.

A cDNA clone representative of a novel splicing pattern of the D. melanogaster dunce gene.

The D. melanogaster dunce gene is involved in both the learning and memory processes of the fly. The gene encodes for a cAMP-specific phosphodiesterase, a function playing a central role in the regulation of the intracellular cAMP level. Molecular cloning of dunce has so far not been completely achieved, although it is known that the gene encodes a large set of RNAs and has a complex organization, extending for more than 140 kilobases and containing several genes within its introns. Here we report the isolation and the characterization of 21/7, a cDNA clone representative of a novel dunce splicing pattern. The nucleotide sequence of this clone led to the identification of a dunce exon included in at least one transcript so far uncharacterized.