Association between autophagy and KRAS mutation with clinicopathological variables in colorectal cancer patients.

Autophagy is a host defensive mechanism responsible for eliminating harmful cellular components through lysosomal degradation. Autophagy has been known to either promote or suppress various cancers including colorectal cancer (CRC). KRAS mutation serves as an important predictive marker for epidermal growth factor receptor (EGFR)-targeted therapies in CRC. However, the relationship between autophagy and KRAS mutation in CRC is not well-studied. In this single-centre study, 92 formalin-fixed paraffin-embedded (FFPE) tissues of CRC patients (42 Malaysian Chinese and 50 Indonesian) were collected and KRAS mutational status was determined by quantitative PCR (qPCR) (n=92) while the expression of autophagy effector (p62, LC3A and LC3B) was examined by immunohistochemistry (IHC) (n=48). The outcomes of each were then associated with the clinicopathological variables (n=48). Our findings demonstrated that the female CRC patients have a higher tendency in developing KRAS mutation in the Malaysian Chinese population (p<0.05). Expression of autophagy effector LC3A was highly associated with the tumour grade in CRC (p<0.001) but not with other clinicopathological parameters. Lastly, the survival analysis did not yield a statistically significant outcome. Overall, this small cohort study concluded that KRAS mutation and autophagy effectors are not good prognostic markers for CRC patients.

Simultaneous Correction of a Tibia Deformity and Non-union in Achondroplasia: A Case Report.

Tibial non-union with deformity in abnormal bone is rarely reported in literature. We report a case of a 65 years old male with a history of achondroplasia. The patient presented after a mechanical fall with an undisplaced right midshaft tibia fracture associated with pre-existing varus and procurvatum tibial deformities, which was initially managed non-operatively. However, after nine months he developed a painful non-union. Because of the symptomatic non-union as well as the pre-existing deformities, osteotomy of the tibia and fibula was performed with the application of a Truelok-Hexapod (TL-Hex, Orthofix) frame. We were able to achieve compression at the fracture site, and the software guided TL-Hex frame enabled gradual three-dimensional correction of the deformity. At six months, bony union and simultaneous correction of the tibia deformity were achieved. At two years, the patient was able to ambulate well without pain and perform his activities of daily living. We present a case of tibial non-union with pre-existing deformity in an achondroplasia patient successfully treated with a circular frame application.

The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation.

Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM_006094)--an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22-11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5'-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2'-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.

Ratio of Klebsiella/Bifidobacterium in early life correlates with later development of paediatric allergy.

Several studies have reported that intestinal microbial colonisation patterns differ between non-allergic and allergic infants. However, the microbial signature underlying the pathogenesis of allergies remains unclear. We aim to gain insight into the development of the intestinal microbiota of healthy infants and infants who develop allergy in early life, and identify potential microbiota biomarkers of later allergic disease. Using a case-control design in a Chinese sub-cohort of a Singaporean birth cohort (GUSTO), we utilised 16S rRNA gene sequencing to assess intestinal microbial composition and diversity of 21 allergic and 18 healthy infants at 3 weeks, 3 months and 6 months of age, and correlated the microbiota with allergy at ages 18 and 36 months. Pronounced differences in intestinal microbiota composition between allergic and healthy infants were observed at 3 months of age. The intestine of healthy infants was colonised with higher abundance of commensal Bifidobacterium. Conversely, Klebsiella, an opportunistic pathogen, was significantly enriched in the allergic infants. Interestingly, infants with a high Klebsiella/Bifidobacterium (K/B) ratio (above the population median K/B ratio) at age 3 months had an odds ratio of developing allergy by 3 years of age of 9.00 (95% confidence interval 1.46-55.50) compared to those with low K/B ratio. This study demonstrated a relationship between the ratio of genera Klebsiella and Bifidobacterium during early infancy and development of paediatric allergy in childhood. Our study postulates that an elevated K/B ratio in early infancy could be a potential indicator of an increased risk of allergy development. This line of research might enable future intervention strategies in early life to prevent or treat allergy. Our study provides new insights into microbial signatures associated with childhood allergy, in particular, suggests that an elevated K/B ratio could be a potential early-life microbiota biomarker of allergic disease.

Nucleus z in the rat: spinal afferents from collaterals of dorsal spinocerebellar tract neurons.

Proprioceptive information from the hindlimb of the cat is now known to be relayed to the somatosensory thalamus and cortex via axons in the dorsolateral fasciculus and a medullary relay in nucleus z. The aim of this study was to identify nucleus z in the rat, to locate the cells of origin of spinal afferents to nucleus z, and to determine whether they are collaterals of the dorsal spinocerebellar tract. The location and extent of nucleus z were studied by filling the axon terminals of collaterals of the dorsal spinocerebellar tract (dsc) with horseradish peroxidase (HRP), which was injected into the inferior cerebellar peduncle. Nucleus z in the rat was found to be similar in location to nucleus z in other mammals. It was located just below the dorsal surface of the medulla, bounded laterally by the rostral pole of the cuneate nucleus and medially by the nucleus of the solitary tract. The cells of origin of the spinal afferents to nucleus z were studied by using the retrograde transport of HRP. They were located in Clarke's column (dorsal nucleus) and in lamina 10 of the dorsal horn. They were similar in location and morphology to neurons giving rise to the dorsal spinocerebellar tract, but were smaller in average diameter. A double retrograde labeling technique was used to determine whether the spinal afferents to nucleus z are collaterals of neurons giving rise to the dsc. It was estimated that up to 92% of the spinal afferents to nucleus z were collaterals of dsc neurons, while approximately 3% of all dsc neurons gave rise to collaterals terminating in nucleus z.

Effect of food-simulating liquids on surface characteristics of composite and polyacid-modified composite restoratives.

The chemical environment is one aspect of the oral environment that could have an appreciable influence on the in vivo degradation of composite resins. The effects of food-simulating liquids on the surface roughness and hardness of composite (Silux Plus, Z100, Spectrum TPH, and P50) and polyacid-modified composite resins (F2000 and Dyract AP) were thus investigated and compared. Sixty disks of each material were made. Half were used for microhardness testing and the remaining half for studying surface roughness using profilometry. Each group of 30 disks was subdivided into six groups of five and conditioned for one week as follows--Group 1 (control): air at 37 degrees C; Group 2: distilled water at 37 degrees C; Group 3: 0.02 N citric acid at 37 degrees C; Group 4: 0.02 N lactic acid at 37 degrees C Group 5: heptane at 37 degrees C; Group 6: 50% ethanol-water solution at 37 degrees C. Data were analyzed using one-way ANOVA and Scheffé's test at a significance level of 0.05. Results showed that the surface roughness of all restoratives evaluated was not significantly affected by food-simulating liquids. No significant change in surface hardness was noted with conditioning of Spectrum TPH, Dyract AP, and F2000 in the various food-simulating liquids. The BIS-GMA-based composites Silux Plus, Z100, and P50 appeared to be more susceptible to the softening effects of some food-simulating liquids.

A novel isoform of the 8p22 tumor suppressor gene DLC1 suppresses tumor growth and is frequently silenced in multiple common tumors.

The critical 8p22 tumor suppressor deleted in liver cancer 1 (DLC1) is frequently inactivated by aberrant CpG methylation and/or genetic deletion and implicated in tumorigeneses of multiple tumor types. Here, we report the identification and characterization of its new isoform, DLC1 isoform 4 (DLC1-i4). This novel isoform encodes an 1125-aa (amino acid) protein with distinct N-terminus as compared with other known DLC1 isoforms. Similar to other isoforms, DLC1-i4 is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. However, differential expression of the four DLC1 isoforms is found in tumor cell lines: Isoform 1 (longest) and 3 (short thus probably nonfunctional) share a promoter and are silenced in almost all cancer and immortalized cell lines, whereas isoform 2 and 4 utilize different promoters and are frequently downregulated. DLC1-i4 is significantly downregulated in multiple carcinoma cell lines, including 2/4 nasopharyngeal, 8/16 (50%) esophageal, 4/16 (25%) gastric, 6/9 (67%) breast, 3/4 colorectal, 4/4 cervical and 2/8(25%) lung carcinoma cell lines. The functional DLC1-i4 promoter is within a CpG island and is activated by wild-type p53. CpG methylation of the DLC1-i4 promoter is associated with its silencing in tumor cells and was detected in 38-100% of multiple primary tumors. Treatment with 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B led to demethylation of the promoter and reactivation of its expression, indicating a predominantly epigenetic mechanism of silencing. Ectopic expression of DLC1-i4 in silenced tumor cells strongly inhibited their growth and colony formation. Thus, we identified a new isoform of DLC1 with tumor suppressive function. The differential expression of various DLC1 isoforms suggests interplay in modulating the complex activities of DLC1 during carcinogenesis.

Combined adjuvant cisplatin and ifosfamide chemotherapy and radiotherapy for malignant mixed müllerian tumors of the uterus.

The role of adjuvant therapy for malignant mixed müllerian tumors of the uterus has not been established. Our aim was to review our experience with sequential adjuvant therapy using cisplatin and ifosfamide chemotherapy and radiotherapy after surgical staging. A retrospective study of 43 patients from 1995 to 2004 was undertaken. Survival was calculated using the Kaplan-Meier method and compared by the log-rank test. The Cox proportional hazard regression model was used to assess the effect of treatment on survival after adjustment for age and stage. Twenty-eight patients received adjuvant chemotherapy and 28 patients had adjuvant radiotherapy. Twenty-one patients underwent sequential adjuvant chemotherapy and radiotherapy. Tumor recurrence occurred in 14 patients at a median duration of 10 months. The overall 2- and 5-year survival was 64% and 60%, respectively. The 2- and 5-year survival for stage I and II diseases was both 95%, while the 2-year survival for stage III and IV diseases was 25%. Patients who underwent sequential adjuvant therapy had an improved survival compared with patients who did not follow the protocol (P= 0.024). Our results with sequential adjuvant therapy are encouraging and justify future randomized trials.

Celecoxib reduces microvessel density in patients treated with nasopharyngeal carcinoma and induces changes in gene expression.

BACKGROUND: Celecoxib is a selective cyclooxygenase-2 inhibitor with antitumor and antiangiogenic activity. We sought to determine pharmacodynamic change in tumors of patients with nasopharyngeal carcinoma (NPC) treated with celecoxib. METHODS: Tumor biopsies were obtained before and after treatment with celecoxib 400 mg b.i.d. for 14 days in patients with newly diagnosed, untreated NPC. Tumor angiogenesis and cell proliferation were assessed by immunohistochemistry and gene expression by microarray analysis. Plasma celecoxib concentrations were obtained on days 8 and 14. RESULTS: Paired samples were analyzed in 15 patients. Microvessel density was reduced in post-treatment samples and mean celecoxib levels reached therapeutic levels. Thirty-five genes (27 down-regulated, eight up-regulated) were differentially expressed on microarray analysis (p < 0.001). Down-regulated genes included cell cycle regulation-related (cyclin-dependent kinase 2, YES1), transcription factor (TRIP-Br2), whereas the antigen processing and presentation-related gene HLA-DM B was up-regulated. CONCLUSION: Celecoxib reduced angiogenesis and induced tumor transcriptional changes. Further characterization of these transcriptional changes in vivo is needed to provide further insights into the effects of celecoxib in neoplastic tissue. Our findings provide a rationale for clinical studies aimed at assessing the efficacy of celecoxib in the treatment of NPC.