[Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity].

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

Overexpression of the RNA-binding proteins Lin28B and IGF2BP3 (IMP3) is associated with chemoresistance and poor disease outcome in ovarian cancer.

BACKGROUND: RNA-binding proteins have an important role in messenger RNA (mRNA) regulation during tumour development and carcinogenesis. In the present study, we examined the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; hereafter refered to as IMPs) and Lin28 family expressions in epithelial ovarian carcinoma (EOC) patients and correlated their expression levels with the response to chemotherapy, hCTR1 expression and patient survival. METHODS: Patients clinical information, real-time RT-PCR, immunohistochemistry, western blot, Transwell migration invasion assays, and cytotoxicity assays were used. RESULTS: From 140 EOC patients, high expression of IMP3 or Lin28B was associated with poor survival, and women diagnosed at advanced stages with elevated IMP3 and Lin28B were at higher risk of developing chemoresistance. High IMP3 levels combined with high Lin28B levels significantly correlated with the poorest 5-year survival rates. Knockdown of IMP3 or Lin28B decreased cell proliferation, migration, and invasion, and increased the platinum sensitivity, but not taxol sensitivity, of ovarian cancer cells through increased expression of hCTR1, a copper transporter involved in platinum uptake. High expression of hCTR1 correlated with low expression of IMP3/Lin28B and better progression-free survival in advanced-stage EOC patients. CONCLUSION: Testing for a combination of elevated IMP3 and Lin28B levels could further facilitate the identification of a patient subgroup with the worst prognosis.

Three-dimensional power Doppler ultrasound in cervical carcinoma: monitoring treatment response to radiotherapy.

OBJECTIVES: To investigate, using three-dimensional power Doppler ultrasound (3D-PDU), alterations in cervical intratumoral vascularization during and after radiotherapy. METHODS: Between 2004 and 2009 we enrolled into the study 37 patients with FIGO Stages IB1-IIB cervical carcinoma who were undergoing radiotherapy. Serial 3D-PDU scans were performed during treatment, providing ultrasonographic measurement of tumor size, vascularization index, flow index and vascularization flow index, as well as monthly for 3 months post-treatment and tri-monthly thereafter, until vascularity was undetectable on two consecutive occasions. Physical examination, cervical cytology and serum marker evaluation were performed every 3-6 months for the first 5 years following treatment. Patients evaluated after a 2-year tumor-free interval and those with clinically assessed positive findings at follow-up underwent 3D-PDU to detect possible local disease. RESULTS: A total of 329 3D-PDU scans were performed in the 37 women. Cervical tumors and intratumoral vascularization disappeared within 3 months following radiotherapy, except in one patient with persistent disease. Nine patients had disease relapse, in four of whom the recurrence was local. In three of these four, there was recurrence of tumor and vascularization after a complete response. At follow-up, 3D-PDU detected local disease with 75.0% sensitivity and 98.5% specificity, while serum markers detected local disease among 34 patients with squamous cell carcinoma with 20.0% sensitivity and 77.3% specificity. CONCLUSIONS: Compared with serum markers in cervical squamous cell carcinoma, 3D-PDU has higher sensitivity and specificity for detecting local recurrence or persistence in cervical carcinoma. Thus, 3D-PDU combined with clinical assessment may be a new and safe method for monitoring radiotherapy treatment response and detecting local recurrence.

Molecular diversity of bacteria in Yunnan yellow cattle (Bos taurs) from Nujiang region, China.

The rumen content of four Yunnan Yellow Cattle (Bos taurs) were collected to determine the bacteria diversity by using 16S rRNA gene sequence analysis. A total of 129 sequences were examined and the sequences were referred as 107 OTU (Operational Taxonomy Unit) according to the similarity level of 97% in gene sequence. Similarity analysis revealed that Yunnan Yellow Cattle had 12 sequences (10 OTU) shared 97% or greater similarity with cultured rumen bacteria Butyrivibrio fibrisolvens, Succiniclasticum ruminis, Ruminococcus bromii, Clostridium proteoclasticum, Ruminococcus flavefaciens, Pseudobutyrivibrio ruminis, Jeotgalicoccus psychrophilus, and Prevotella ruminicola, which accounting for 9.3% of the total clones (9.2% of the total OTU). The further 12 sequences (9 OTU) shared 90-97% similarity with cultured bacteria Clostridium aminobutyricum, butyrate-producing bacterium, Schwartzia succinivorans, Prevotella ruminicola, Eubacterium ruminantium, Ruminococcus albus, and Clostridium termitidis, also accounting for 9.3% of the total sequences (8.3% of the total OTU). The remaining 105 sequences (90 OTU) shared less than 90% similarity with cultured bacteria, accounting for 81.4% of the total sequences (82.5% of the total OTU). According to the phylogenetic analysis, all sequences were phylogenetically placed within phyla of low G+C subdivision (accounting for 72.1 and 72.5% of the total clones and OTU, respectively) and CFB subdivision (Cytophaga-Flexibacter-Bacteroides; accounting for 27.9 and 27.5% of the total clones and OTU, respectively). Among the examined clones, rare bacteria Jeotgalicoccus psychrophilus was detected in the rumen of cattle.

Molecular determinants of U-type inactivation in Kv2.1 channels.

Kv2.1 channels exhibit a U-shaped voltage-dependence of inactivation that is thought to represent preferential inactivation from preopen closed states. However, the molecular mechanisms underlying so-called U-type inactivation are unknown. We have performed a cysteine scan of the S3-S4 and S5-P-loop linkers and found sites that are important for U-type inactivation. In the S5-P-loop linker, U-type inactivation was preserved in all mutant channels except E352C. This mutation, but not E352Q, abolished closed-state inactivation while preserving open-state inactivation, resulting in a loss of the U-shaped voltage profile. The reducing agent DTT, as well as the C232V mutation in S2, restored U-type inactivation to the E352C mutant, which suggests that residues 352C and C232 may interact to prevent U-type inactivation. The R289C mutation, in the S3-S4 linker, also reduced U-type inactivation. In this case, DTT had little effect but application of MTSET restored wild-type-like U-type inactivation behavior, suggestive of the importance of charge at this site. Kinetic modeling suggests that the E352C and R289C inactivation phenotypes largely resulted from reductions in the rate constants for transitions from closed to inactivated states. The data indicate that specific residues within the S3-S4 and S5-P-loop linkers may play important roles in Kv2.1 U-type inactivation.

Suicidal ideation among HIV+ former blood and/or plasma donors in rural China.

Suicidal ideation is life-threatening and is prevalent among people living with HIV (PLWH). A dearth of such studies was conducted in China. This study investigated the prevalence of suicidal ideation and associated factors among PLWH who were former blood and/or plasma donors (FBPD) in a rural county in central China. Prospective respondents were randomly selected from a local registry; 176 PLWH who were FBPD joined the study. With informed consent, these PLWH and their spouse were interviewed separately and anonymously. Respectively, 34 and 8% of the sampled (index) PLWH self-reported having suicidal ideation and making a suicidal attempt in the last year. The multivariate analyses results showed that the index PLWH's Physical Function subscale score of the Medical Outcomes Study HIV Health Survey scale (Odds ratio (OR)=6.67, 95% CI=1.69-26.27, 75 percentiles), the Depression subscale score of the Depression, Anxiety, and Stress Scales (DASS; OR=9.26, 95% CI=1.32-64.77), and the spouse's Depression subscale score of the DASS (OR=7.64, 95% CI=1.37-42.77) were independently associated with the index PLWH's suicidal ideation. HIV-related variables (e.g., duration of diagnosis, treatment and side effects) and perceived discrimination of the index PLWH, and HIV status of the PLWH's spouse, were not significant factors. Depression is a risk factor for suicides. Moreover, depression may be contagious and the depression status of the spouse also matters. Treatments for depression and prevention intervention for suicides targeting PLWH in rural China are not readily available. Such services are greatly warranted and need to be provided to both the PLWH and his/her spouse.

Risk and protective factors in association with mental health problems among people living with HIV who were former plasma/blood donors in rural China.

A random sample of 271 people living with HIV (PLWH) who were former plasma/blood donors and a convenience sample of 67 HIV negative villagers were anonymously interviewed. Compared with the non-PLWH, PLWH reported higher prevalence of symptoms of depression (adjusted OR = 2.53, p=0.001), anxiety (adjusted OR = 1.85, p=0.04), and stress (adjusted OR = 1.77, p=0.06). Of the PLWH respondents, 81.7% received Highly Active Antiretroviral Therapy (HAART); 32.1% of whom reported some side effects. Respectively 13.7%, 37.4%, and 38.4% PLWH perceived discrimination from their family members, relatives/friends, and neighbors. Absence of HAART, poor physical function, perceived discrimination from relatives and friends, and low level of resilience were associated with depression (stepwise regression; beta = - 0.28-0.17, R-square = 0.22), anxiety and stress (R-square = 0.32 and 0.16, respectively). The majority of respondents (70.1%) desired group intervention as a means for providing psychological support services. Relevant programs should both remove risk factors (e.g., absence of medical treatment, HIV-related discrimination) and promote protective factors (e.g., resilience). Support group is one of the potentially useful approaches to provide psychological support services.

Clinical implications of insulin-like growth factor 1 system in early-stage cervical cancer.

This study was aimed to identify the expression and the correlation of insulin-like growth factor-1 (IGF-1) system and their prognostic impacts in cervical cancer. Seventy-two patients with early-stage cervical cancer were eligible. We obtained the serum levels of total IGF-1 and IGF binding protein-3 (IGFBP-3) by enzyme-linked immunosorbent assay and the expression of IGF-1 receptor (IGF-1R) in cancerous tissue by immuno-fluorescent (IF) stains. The 5-year recurrence-free and overall survival rates were significantly lower (P=0.003 and P=0.01, respectively) among patients with high-grade expression of tissue IGF-1R, compared with those with low-grade expression. After adjustment for other factors, preoperative serum total IGF-1 or IGFBP-3 levels failed to predict cancer death and recurrence. High-grade expression of IGF-1R and elevated preoperative squamous cell carcinoma antigen level were independent predictors of both death and recurrence, and combination of both factors could further help identify the subgroup of patients at higher death risk. The IF staining indicates the colocalisation of IGF-1 and IGF-1R in the cancerous tissues, whereas the IGF-1R expression is not correlated with circulating levels of IGF-1 or IGFBP-3. In early-stage cervical cancer, IGF-1 system may have a paracrine or autocrine function and the adverse impacts on prognosis by IGF-1R overexpression are implicated.

Potential contribution of a voltage-activated proton conductance to acid extrusion from rat hippocampal neurons.

We examined the potential contribution of a voltage-gated proton conductance (gH+) to acid extrusion from cultured postnatal rat hippocampal neurons. In neurons loaded with Ca2+- and/or pH-sensitive fluorophores, transient exposures to 25-139.5 mM external K+ (K+o) or 20 microM veratridine in the presence of 2 mM Ca2+o (extracellular pH (pHo) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi), respectively. In contrast, under external Ca2+-free conditions, the same stimuli failed to affect [Ca2+]i but caused an increase in pHi, the magnitude of which was related to the [K+]o applied and the change in membrane potential. Consistent with the properties of gH+s in other cell types, the magnitude of the rise in pHi observed in the absence of external Ca2+ was not affected by the removal of external Na+ but was sensitive to external Zn2+ and temperature and was dependent on the measured transmembrane pH gradient (DeltapHmemb). Increasing DeltapH(memb) by pretreatment with carbonylcyanide-p-trifluoromethoxyphenylhydrazone augmented both the high-[K+]o-evoked rise in pHi and the Zn2+-sensitive component of the rise in pHi, suggestive of increased acid extrusion via a gH+. The inhibitory effect of Zn2+ at a given DeltapHmemb was further enhanced by increasing pHo from 7.35-7.8, consistent with a pHo-dependent inhibition of the putative gH+ by Zn2+. Under conditions designed to isolate H+ currents, a voltage-dependent outward current was recorded from whole-cell patch-clamped neurons. Although the outward current appeared to show some selectivity for protons, it was not sensitive to Zn2+ or temperature and the H+-selective component could not be separated from a larger conductance of unknown selectivity. Nonetheless, taken together, the results suggest that a Zn2+-sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H+ efflux under depolarizing conditions.

Estimating residue evolutionary conservation by introducing von Neumann entropy and a novel gap-treating approach.

Evolutionary conservation derived from a multiple sequence alignment is a powerful indicator of the functional significance of a residue, and it can help to predict active sites, ligand-binding sites, and protein interaction interfaces. The results of the existing algorithms in identifying the residue's conservation strongly depend on the sequence alignment, making the results highly variable. Here, by introducing the amino acid similarity matrix, we propose a novel gap-treating approach by combining the evolutionary information and von Neumann entropies to compute the residue conservation scores. It is indicated through a series of tested results that the new approach is quite encouraging and promising and may become a useful tool in complementing the existing methods.

Identification and characterization of a new type of asymmetrical dicentric chromosome derived from a single maternal chromosome 18.

Molecular cytogenetic analysis identified a new type of dicentric chromosome involving different breakpoints at 18q in a female fetus. The chromosome anomaly was designated as an asymmetrical pseudoisodicentric chromosome 18, 46,XX,psu dic(18)(pter-->q11.2::q21.3-->pter)mat. A series of BAC clones for 18q11.2 and q21.3 regions were used to identify one breakpoint within the region q11.2 between 19.8 and 21.6 Mb from the telomere of 18p and another breakpoint within q21.3 between 55.4 and 56.9 Mb from the telomere of 18p by FISH analysis. Real-time quantitative PCR and microsatellite analysis further verified that the dicentric chromosome was maternal in origin and resulted from a break-reunion between sister chromatids of a single maternal chromosome. We propose that a loop-type configuration of sister chromatids took place and that the break-reunion occurred at cross sites of the loop to form an asymmetrical isodicentric chromosome during either mitosis or meiosis. In this case, the asymmetrical pseudoisodicentric resulted in an 18pter--> q11.2 duplication and an 18q21.3-->qter deletion, which could have led to certain dysmorphic features of 18q- syndrome in this fetus.

Prediction of protein subcellular localization by support vector machines using multi-scale energy and pseudo amino acid composition.

As more and more genomes have been discovered in recent years, there is an urgent need to develop a reliable method to predict the subcellular localization for the explosion of newly found proteins. However, many well-known prediction methods based on amino acid composition have problems utilizing the sequence-order information. Here, based on the concept of Chou's pseudo amino acid composition (PseAA), a new feature extraction method, the multi-scale energy (MSE) approach, is introduced to incorporate the sequence-order information. First, a protein sequence was mapped to a digital signal using the amino acid index. Then, by wavelet transform, the mapped signal was broken down into several scales in which the energy factors were calculated and further formed into an MSE feature vector. Following this, combining this MSE feature vector with amino acid composition (AA), we constructed a series of MSEPseAA feature vectors to represent the protein subcellular localization sequences. Finally, according to a new kind of normalization approach, the MSEPseAA feature vectors were normalized to form the improved MSEPseAA vectors, named as IEPseAA. Using the technique of IEPseAA, C-support vector machine (C-SVM) and three multi-class SVMs strategies, quite promising results were obtained, indicating that MSE is quite effective in reflecting the sequence-order effects and might become a useful tool for predicting the other attributes of proteins as well.

Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patients.

The purpose of this study was to investigate the clinical usefulness of the color Doppler vascularity index (CDVI) in patients with colon cancer before surgery. Forty-four patients with sonographically visible tumor mass of colon cancer were investigated. The CDVI of each tumor was determined using transabdominal color Doppler ultrasound. The CDVI was defined as the ratio of the number of the colored pixels within a tumor section to the number of total pixels in that specific tumor section and was calculated by using Encomate software (Electronic Business Machine Co. Ltd., Taipei, Taiwan). The correlation between the CDVI and clinicopathological factors, mode of recurrence, and patient survival was studied. For comparison, microvessel density (the mean number of microvessels in three areas of highest vascular density at x200 magnification) of the tumors of these 44 patients was also evaluated by using immunohistochemical staining of surgical specimens with anti-CD34. The microvessel density was not correlated with Dukes' classification, clinicopathological factors, and survival. The CDVI was significantly higher in the patients with lymph node metastases and vascular invasion than in those without such metastases and invasion (P = 0.006 and P = 0.0098, respectively). Moreover, in patients with a high CDVI (> 15%) and positive vascular invasion, survival was significantly poorer than in those with low CDVI (< or = 15%) and negative invasion (P = 0.0037 and 0.0039, respectively). Multivariate analysis indicated that liver metastasis, vascular invasion, and CDVI are independent prognostic factors in the patients with colon cancer. According to the mode of recurrence in 36 patients who underwent curative resection, the frequency of the distant organ recurrence was significantly higher in the high CDVI group (40%) than in the low CDVI group (0%). The CDVI is a good preoperative indicator of recurrence and patient survival in colon cancer. Thus, the CDVI may be helpful in stratifying patients for adjuvant therapy.

Large-scale mitochondrial DNA deletions in skeletal muscle of patients with end-stage renal disease.

End-stage renal disease (ESRD) is associated with enhanced oxidative stress. This disease state provides a unique system for investigating the deleterious effect of exogenous sources of free radicals and reactive oxygen species (ROS) on mitochondrial DNA (mtDNA). To test the hypothesis that uremic milieu might cause more severe damage to mtDNA, we investigated the prevalence and abundance of mtDNA deletions in the skeletal muscles of ESRD patients. The results showed that the frequencies of occurrence of the 4977 bp and 7436 bp deletions of mtDNA in the muscle tissues of the older ESRD patients were higher than those of the younger patients. The frequency of occurrence of the 4977 bp-deleted mtDNA in the muscle was 33.3% for the patients in the age group of < 40 years, 66.6% in the 41-60-year-old group, 100% in the 61-80-year-old group, and 100% in patients >80 years of age, respectively. Only 22% of the normal aged controls carried the 4977 bp mtDNA deletion, whereas 77% (17/22) of the ESRD patients exhibited the mtDNA deletion. Using a semiquantitative PCR method, we determined the proportion of the 4977 bp-deleted mtDNA from the muscles that had been confirmed to harbor the deletion. We found that the proportions of the 4977 bp-deleted mtDNA in the muscle were significantly higher than those of the aged matched controls. Using long-range PCR techniques, a distinctive array of mtDNA deletions was demonstrated in the muscle of uremic patients. In summary, we found diverse and multiple mtDNA deletions in the skeletal muscles of ESRD patients. These deletions are more prevalent and abundant in ESRD patients than those found in normal populations. Accumulation of uremic toxins and impaired free radical scavenging systems may be responsible for the increased oxidative stress in ESRD patients. Such stress may result in oxidative damage and aging-associated mutation of the mitochondrial genome.

Allelic loss at chromosome band 6q14 correlates with favorable prognosis in hepatocellular carcinoma.

Cytogenetic and molecular studies have frequently shown chromosome 6q deletions in non-Hodgkin lymphoma and several human cancers. There have been few studies concerning chromosome 6q deletion in hepatocellular carcinoma (HCC), and most of these studies have focused on region 6q26-27. We previously described frequent allelic loss at 6q14 in HCC. As a step toward narrowing the scope of search for tumor suppressor genes, we used a series of yeast artificial chromosome clones that map to the long arm of chromosome 6 (6q14-6q22) by fluorescence in situ hybridization (FISH) to define the minimal common region of allelic loss in 25 cases of HCC. Altogether, 12 tumors had allelic loss on 6q (48%). Eleven of the 12 tumors had polysomy of chromosome 6 with evident intratumor cytogenetic heterogeneity. The minimal common region of allelic loss lies within a 2-cM region at 6q14, flanked by D6S458 (849_d_8) and D6S275 (911_a_3). Clinicopathologic correlation between the 12 patients with allelic loss at 6q and the 13 patients without allelic loss showed no significant differences in any basic characteristics except survival. Patients with allelic loss at 6q had a much longer median survival time than those without allelic loss (50 months vs. 11 months, P = 0.0019). Only 5 of the 25 HCC patients were still alive at the time of this study, and all of them had allelic loss at 6q, which is also statistically significant (P = 0.037, alive vs. dead). The association of allelic loss at 6q with polysomy implies that this may be a progression-associated event in HCC. The correlation of allelic loss at 6q with long survival suggests a complex mechanism of tumorigenesis in HCC and is worthy of further investigation.

Recovery of allografted small intestine function.

AIM: To investigate recovery of the allografted small intestine function after clinical small bowel transplantation (SBT). METHODS: The structure of the graft was evaluated by endoscopic biopsy and histopathologic examination. Graft functions were assessed by D-xylose absorption, barium studies, nitrogen balance calculation, and blood and stool cultures. Nutritional status of the recipients was judged by measurement of body weight and serum protein concentrations. RESULTS: The recipient discontinued total parenteral nutrition (TPN) and resumed oral nutrition 100 d after SBT. On oral diet, the patient maintained a normal nutritional status, gained weight by 3 kg, and had a normal serum albumin concentration (40.2 g/L ± 0.2 g/L). Satisfactory D-xylose absorption was achieved 8 wk after the operation. Nitrogen balance of the gut was maintained well and increased gradually. Serial mucosal biopsy showed normal structures 2 wk after grafting, without evidence of rejection and graft versus host diseases (GVHD). Barium studies conducted on the 10(th) day and 38(th) day by barium studies revealed that the grafted small bowel motility showed normal patterns of peristalsis and transit. No bacterial translocations were noted. CONCLUSION: Function of the grafted small intestine recovered satisfactorily 100 d after transplantation, indicating good clinical outcome of SBT.

Gait analysis after ankle arthrodesis.

The purpose of this study was to employ a computerized motion analysis system to identify the effect of ankle arthrodesis on the three-dimensional kinematic behavior of the rear and fore foot during level walking. A three-segment rigid body model was used to describe the motion of the foot and ankle. The results demonstrated that sagittal plane motion of the hindfoot was significantly decreased in the foot of patients having had ankle arthrodesis compared to normal subjects. The kinematic data indicated a generalized stiffness of the hindfoot on the involved foot in the sagittal plane. Sagittal plane movement in the forefoot and transverse plane movements in the hindfoot and forefoot increased in patients compared to controls.

Use of a cross-leg free muscle flap to reconstruct an extensive burn wound involving a lower extremity.

A young patient sustained a high-voltage burn with extensive destruction of the soft tissue in his left lower extremity. Occlusion of the anterior and posterior tibial arteries, loss of toe extensors and the superficial and deep peroneal nerves were noted, besides the exposure of the lower end of the tibia and metatarsal bones. In the absence of proper recipient vessels, a cross-leg free latissimus dorsi muscle flap with overlying skin and depending on the vessels of the contralateral foot was used successfully for reconstruction of the defect. The pedicle was divided 3 weeks after microvascular anastomosis and the flap survived completely. This technique permits transfer of free flaps to compromised wounds without available recipient vessels, and the latissimus dorsi muscle flap, with its characteristics of large size and copious vascularity, could be split to cover exposed bones in different areas simultaneously.

Reappraisal of K-ras and p53 gene mutations in the recurrence of Dukes' B2 rectal cancer after curative resection.

BACKGROUND/AIMS: Recurrence of rectal cancer remains a major clinical problem. This study was conducted to evaluate the impact of K-ras and p53 mutations on the recurrence of rectal cancer. METHODOLOGY: A total of 166 resected Dukes' B2 stage rectal carcinomas were collected between January 1990 and April 1994. The stored frozen tissues were retrieved for immunocytochemistry of p53 and genomic analysis of K-ras and p53 genes. The data of K-ras and p53 gene mutations were correlated with clinicopathological variables. The concordance of immunocytochemistry with genomic analysis in the survey of p53-mutations was examined. The follow-up data were analyzed by Kaplan-Meier estimator. RESULTS: Sixty-nine patients (41.6%) developed recurrent tumor. A significantly higher recurrence rate (p = 0.0013) and shorter median recurrence time were noted in p53 mutated than non-mutated cancers. Mutations in K-ras gene do not significantly increase the risk of tumor recurrence (p = 0.1702). K-ras and p53 mutations are not associated with clinicopathological parameters (p > 0.05). Kappa statistic indicates highly significant reproducibility between immunocytochemistry and genomic analysis for p53 mutations (p < 0.0001). CONCLUSIONS: Presence of p53 mutation significantly increases the recurrence rate and shortens the recurrence time of the resected rectal cancers. Pre-operative routine check for p53 mutations by immunocytochemistry may be beneficial in choosing the optimal surgical strategy for rectal cancer.

Clinicopathologic and carcinogenetic appraisal of DNA replication error in sporadic T3N0M0 stage colorectal cancer after curative resection.

BACKGROUND/AIMS: DNA replication error (RER) was found to play a role in the carcinogenesis of a subset of sporadic colorectal cancers. This study was conducted to evaluate the clinicopathologic implications of RER in T3N0M0 stage colorectal cancers. To better understand the carcinogenesis of sporadic colorectal cancer, the RER status was further correlated with the alterations of K-ras, p53 and deleted in colorectal cancer (DCC) genes which were frequently involved in the adenoma-carcinoma sequence. METHODOLOGY: Seventy-eight patients with curatively resected T3N0M0 stage sporadic colorectal cancer were accumulated. The stored frozen tissues were retrieved for analyses of 1) microsatellite instability at 7 distinct chromosomal loci, 2) loss of heterozygosity at DCC gene, 3) K-ras gene mutation, 4) p53 expression, and 5) DNA content. The RER status was correlated with various clinicopathologic and molecular genetic factors. The survival of patients stratified by RER status was analyzed by Kaplan-Meier estimator. RESULTS: The RER-positive tumor was detected in 32.1% (25/78) of patients. The RER-positive cancer patients presented with distinct clinicopathologic features including young age of tumor onset, proximal tumor location, mucin production in histology, a higher rate of synchronous and metachronous colorectal cancers, and an increased incidence of extracolonic primary cancer. Patients with RER-positive tumor were found to have an improved prognosis with the 5-year survival probability of 76% and 45% in RER-positive and RER-negative groups, respectively (p < 0.05). The RER-positive tumors tended to have normal p53 expression, DNA diploidy, and a lower DNA index. The rate for the loss of heterozygosity of DCC gene was significantly lower in RER-positive tumors. RER status was not associated with K-ras mutation. CONCLUSIONS: The clinicopathologic features and carcinogenesis of RER-positive sporadic colorectal cancers were considered different from those of RER-negative tumors. The presence of RER may identify a subset of less aggressive tumors with good prognosis in T3N0M0 stage colorectal cancers.