[Study on the significance of intravoxel incoherent motion quantitative index combined with time-signal intensity curve in the early diagnosis of residual lesions in non-small cell lung cancer after argon-helium cryosurgery].

Objective: To study the application of intravoxel incoherent motion (IVIM) quantitative index combined with time-signal intensity curve (TIC) of dynamic contrast enhanced 3.0T magnetic resonance in the early precise diagnosis of residual lesions in non-small cell lung cancer (NSCLC) after argon-helium cryosurgery. Methods: One hundred NSCLC patients who underwent argon-helium cryosurgery were collected and divided into the residual group (21 cases) and non-residual group (79 cases) according to the result of needle biopsy and follow-up. The apparent diffusion coefficient (ADC), slow apparent diffusion coefficient (sADC), fast apparent diffusion coefficient (fADC), fraction of fast apparent diffusion coefficient (ffADC) and TIC type of IVIM quantitative index between the two groups were compared at 7 days and 1 month after argon-helium cryosurgery, respectively. The diagnosis performance of each quantitative index was analyzed by receiver operating characteristic (ROC) curve and the best cut-off value was computed. The specificity and sensitivity of TIC types were calculated as diagnostic criteria. The diagnosis performance of IVIM quantitative index combined with TIC type was evaluated and compared with the conventional MRI and DWI. Results: The differences of ADC, sADC and ffADC at 7 days and 1 month after argon-helium cryosurgery between the residual group and non-residual group were statistically significant (all P<0.05), in which the diagnosis performance of sADC and ffADC were better. The AUC of sADC and ffADC at 7 days after argon-helium cryosurgery were 0.861 and 0.895, the sensitivity were 81.0% and 90.5%, and the specificity were 77.2% and 73.4%, respectively. The AUC of sADC and ffADC at 1 month after argon-helium cryosurgery were 0.836 and 0.883, the sensitivity were 100.0% and 76.2%, and the specificity were 58.2% and 89.9%, respectively. The diagnosis performance of TIC type Ⅱ&Ⅲ was best. The sensitivity and specificity were 80.9% and 58.2% at 7 days after treatment, 85.7% and 62.0% at 1 month after treatment, respectively. At 7 days after treatment, the sensitivity and specificity of IVIM combined with TIC were 97.5% and 85.7%, while at 1 month after treatment, the sensitivity and specificity of IVIM combined with TIC were 97.5% and 90.5%, respectively. The diagnosis performance of IVIM quantitative index combined with TIC type was better than conventional MRI and DWI. Conclusion: The combination of IVIM quantitative index and TIC type can be used in the early diagnosis of residual lesions after argon-helium cryosurgery for NSCLC, whose effect is better than conventional MRI and DWI.

Learning curve for endoscopic submucosal dissection of esophageal neoplasms.

There is a significant learning curve for endoscopic submucosal dissection of esophageal neoplasms that has not been fully characterized. This retrospective study included 33 consecutive superficial esophageal neoplasms for analysis of the learning curve for esophageal endoscopic submucosal dissection based on a single, novice endoscopist's experience. The study was divided into three periods (T1, T2, and T3) of 10 endoscopic submucosal dissection procedures in chronological order, with 13 procedures in the last period. Patient factors (age, sex, coexistent esophageal varices, or submucosal fibrosis) and tumor factors (location at upper esophagus, involving >3/4 esophageal circumference) for endoscopic submucosal dissection were not statistically different between the periods. The mean procedure time was 74.6 min/cm(2) , 23.4 min/cm(2) , and 10.5 min/cm(2) for T1, T2, and T3, respectively. The procedure time decreased over time (P = 0.02) and post hoc test revealed significant difference was only between T3 and T1 (P = 0.019). The en bloc resection rate was 50%, 100%, and 92.3% for T1, T2, and T3, respectively (P for trend = 0.015). R0 resection rate was 40%, 100%, and 84.6% for T1, T2, and T3, respectively (P for trend = 0.023). Two patients had complications: each one patient in T1 and T3 period experienced major bleeding during the procedure (P for trend = 0.875). None of the patients had esophageal perforation. The results of the study concluded that at least 30 cases of endoscopic submucosal dissection of esophageal neoplasms are needed for a novice endoscopist to gain early proficiency in this technique.

Osteoprotegerin, inflammation and dyslipidemia are associated with abdominal aortic calcification in non-diabetic patients on peritoneal dialysis.

BACKGROUND AND AIMS: Abdominal aortic calcification (AC) has been reported to be associated with cardiovascular disease (CVD) in hemodialysis patients but is rarely discussed in peritoneal dialysis (PD) patients. We examined the independent predictors and predictive power for survival of AC in prevalent PD patients. METHODS AND RESULTS: AC was detected by computed tomography (CT) and represented as the percentage of the total aortic cross-section area affected by AC (%AC). The predictors of %AC ≥ 15 were examined by multiple logistic regression analysis. Cox proportional hazard analysis was used to determine the hazard ratios associated with high %AC. A total of 183 PD patients were recruited to receive CT scans and divided into group 1 (%AC < 15, n = 97), group 2 (%AC ≥ 15, n = 41), and group 3 (diabetic patients, n = 45). Group 1 patients had lower osteoprotegerin (OPG) levels than group 2 patients (798 ± 378 vs. 1308 ± 1350 pg/mL, p < 0.05). The independent predictors for %AC ≥ 15 included the atherogenic index, OPG, and C-reactive protein (CRP). The age-adjusted hazard ratios associated with %AC ≥ 15 were 3.46 (p = 0.043) for mortality and 1.90 (p = 0.007) for hospitalization. CONCLUSIONS: %AC can predict mortality and morbidity in non-diabetic PD patients, and 15% is a good cut-off value for such predictions. There are complex associations among mineral metabolism, inflammation, and dyslipidemia in the pathogenesis of AC.

Innovative technique to reconstruct two branches of the right hepatic artery in living donor liver transplantation.

Hepatic arterial thrombosis is a critical complication in living donor liver transplantation (LDLT). Two separate branches of the right hepatic artery (RHA) are sometimes observed and addressed by anastomosis of the larger branch first, then checking backflow from the smaller branch. If not good, the smaller branch must be reconstructed. We used the cystic artery as a conduit for the reconstruction. Meticulous dissection was performed to identify all branches of the hepatic artery in the donor operation. The length of cystic artery preserved was as long as possible. The cystic arterial stump was anastomosed to the stump of the posterior branch the of RHA under microscopic guidance on the back table. Patency was checked through the stump of the anterior branch of the RHA. With this technique, only one orifice, the stump of right anterior hepatic artery, was used for hepatic artery reconstruction. We have performed this technique in two patients. Both had good arterial flow after living donor liver transplantation. This innovative technique is easy and safe, and requires only one anastomosis, which, in theory, decreases the adds of developing hepatic arterial thrombosis.

Indication for splenectomy in the era of living-donor liver transplantation.

Although end-stage liver disease (ESLD) is often associated with splenomegaly and thrombocytopenia, splenectomy is not necessary in liver transplantation (OLT) except in special situations. In this paper, we examined the indications for splenectomy in the era of living-donor living transplantation. Six of 46 patients underwent splenectomies. Among them, one received a cadaveric graft. Three splenectomies were performed at 6, 7, and 44 days after OLT because of a huge spleen, massive ascites, or impaired liver function. The other two patients received simultaneous splenectomy during OLT to prevent rejection of ABO-incompatible grafts with a positive anti-T-cell test; and one, for postoperative therapy of hepatitis C. All six patients had a good response to splenectomy. We concluded that splenectomy may be indicated for recipients with severe thrombocytopenia, small-for-size syndrome, ABO incompatibility with positive anti-T/B-cell tests and post-OLT therapy for hepatitis C.

Inflammatory pain-induced signaling events following a conditional deletion of the N-methyl-D-aspartate receptor in spinal cord dorsal horn.

The N-methyl-d-aspartate (NMDA) receptor in the spinal cord dorsal horn (SCDH) is one of the mechanisms involved in central sensitization during chronic pain. Previously, this laboratory created a spatio-temporal knockout (KO) of the N-methyl-d-aspartate receptor I (NR1) subunit in the mouse SCDH. The NR1 KO completely blocks NR1 gene and subsequent NMDA receptor expression and function in SCDH neurons. In the NR1 KO mice, the mechanical and cold allodynia induced at 24 h after complete Freund's adjuvant (CFA) was reduced. However, the protective effects of KO were transient and were not seen at 48 h after CFA. These observations suggest the presence of NMDA-independent pathways that contribute to CFA-induced pain. CFA induces the activation of several signaling cascades in the SCDH, including protein kinase C (PKC)gamma and extracellular signal-regulated kinases (ERK1/2). The phosphorylation of PKCgamma and ERK1/2 was inhibited in the SCDH of NR1 KO mice up to 48 h after CFA treatment, suggesting that these pathways are NMDA receptor-dependent. Interestingly, neuronal cyclooxygenase (COX) -2 expression and microglial p38 phosphorylation were induced in the SCDH of the NR1 KO at 48 h after CFA. Our findings provide evidence that inflammatory reactions are responsible for the recurrence of pain after NR1 KO in the SCDH.

prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme.

The paired-related homeobox gene, prx-1, is expressed in the postmigratory cranial mesenchyme of all facial prominences and is required for the formation of proximal first arch derivatives. We introduced lacZ into the prx-1 locus to study the developmental fate of cells destined to express prx-1 in the prx-1 mutant background. lacZ was normally expressed in prx-1(neo); prx-1(lacZ )mutant craniofacial mesenchyme up until 11.5 d.p.c. At later time points, lacZ expression was lost from structures that are defective in the prx-1(neo) mutant mice. A related gene, prx-2, demonstrated overlapping expression with prx-1. To test the idea that prx-1 and prx-2 perform redundant functions, we generated prx-1(neo;)prx-2 compound mutant mice. Double mutant mice had novel phenotypes in which the rostral aspect of the mandible was defective, the mandibular incisor arrested as a single, bud-stage tooth germ and Meckel's cartilage was absent. Expression of two markers for tooth development, pax9 and patched, were downregulated. Using a transgene that marks a subset of prx-1-expressing cells in the craniofacial mesenchyme, we showed that cells within the hyoid arch take on the properties of the first branchial arch. These data suggest that prx-1 and prx-2 coordinately regulate gene expression in cells that contribute to the distal aspects of the mandibular arch mesenchyme and that prx-1 and prx-2 play a role in the maintenance of cell fate within the craniofacial mesenchyme.

Paired-related homeobox genes cooperate in handplate and hindlimb zeugopod morphogenesis.

The closely related homeobox genes prx-1 and prx-2 are expressed in lateral plate and limb bud mesoderm, but targeted inactivation of these genes failed to demonstrate a limb phenotype. Here we report that mice carrying compound mutations in prx-1 and prx-2 have severe limb deformities. In the forelimb autopod, pre- and postaxial polydactyly were found most commonly, but also syndactyly, oligodactyly, and abnormal digit placement affecting posterior elements were observed. In the hindlimb, preaxial polydactyly with variable expressivity was seen in all cases. Extreme distal digit duplications were seen in both the fore- and hindlimbs. prx-1; prx-2 double-mutant mice also displayed extreme shortening and impaired ossification of the hindlimb zeugopods. Integrity of the forelimb apical ectodermal ridge was abnormal as determined by expression of FGF8 and BMP4. Expression of msx-1 and msx-2, markers for BMP signaling pathways, was absent in regions of the posterior handplates, while expression of Shh and patched was unaffected. The mutant phenotypes were dosage dependent, since prx-1 -/-; prx-2 +/- mice also displayed severe limb abnormalities. These data suggest that prx-1 and prx-2 cooperatively regulate handplate and hindlimb zeugopod morphogenesis through BMP-mediated signaling pathways.

Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase.

We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2'-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2'-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA-protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), as a dramatic elevation of KM at pH values above 7.5. KM values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt from the 5' terminus to a previously unknown site on the VP39 surface.

Unpredictability of triphenyltetrazolium chloride in staining irreversible ischaemia-reperfusion injury in the skeletal muscle of rats.

OBJECTIVE: To find out whether pale staining with triphenyltetrazolium chloride (TTC) in skeletal muscle of rat hindlimbs which had been subjected to ischaemia-reperfusion definitely indicated irreversible tissue damage. DESIGN: Laboratory study. SETTING: University hospital, Taiwan. MATERIAL: 77 Female Wistar rats. INTERVENTIONS: Ischaemia of one hindlimb was caused by wrapping of a tourniquet above knee joint for 1.5 (n = 14). 2 (n = 15), 2.5 (n = 17), 3 (n = 17) or 4 (n = 14) hours. Each ischaemic group was divided into three subgroups to receive nil, 1 or 1.5 hours reperfusion, respectively. Gastrocnemius and soleus muscles were excised bilaterally. MAIN OUTCOME MEASURES: TTC reduction in the ischaemic limbs presented as a percentage of the opposite control limb measured by a spectrophotometric assay. Density of red formazan deposition in the ischaemic limbs assessed by microscopic examination of the TTC histochemical stain in the 3 hour ischaemic group. RESULTS: In the 2, 2.5, and 3 hour ischaemic groups the TTC reduction in the ischaemic limbs after 1.5 hours reperfusion increased significantly as compared with that with one hour reperfusion (p < 0.01 in each case). The density of red formazan deposition in the muscle after 3 hours ischaemia and 1.5 hours reperfusion was significantly higher than that of only 1 hour of reperfusion. CONCLUSION: Lack of TTC staining does not necessarily represent irreversible ischaemia-reperfusion injury of the skeletal muscle in rats.

p53 gene mutational spectra in hepatocellular carcinomas induced by 2-acetylaminofluorene and N-nitroso-2-acetylaminofluorene in rats.

In this study, the mutation frequencies of the p53 gene in rat hepatocellular carcinomas (HCCs) induced by N-nitroso-2-acetylaminofluorene (NO-AAF) and 2-acetylaminofluorene (AAF) were 19.23% (20 of 104) and 31.1% (33 of 106), respectively. Four noteworthy features of the mutation spectrums of the p53 gene in HCCs induced by both NO-AAF and AAF were observed: (i) There was preferential clustering of mutations at exons 5-8 in both the NO-AAF or AAF groups. (ii) Nearly all the mutations (98%) induced by NO-AAF and AAF were point mutations. (iii) A high frequency of the p53 mutations were transition mutations, and the ratios of transition to transversion in the NO-AAF and the AAF group were 13:6 and 21:12, respectively. Almost all the mutations were G-->A transitions and guanosine was the major target base. (iv) The frequency and base location of p53 mutations were significantly associated with cancer cell differentiation. In poorly differentiated HCCs (58 individual tumor samples), mutations were detected in 24 of 58 samples (41.1%) and clustered mostly in exons 7 and 8 (19 of 24 samples), whereas in well-differentiated HCCs (105 individual tumor samples), the incidence of mutations was low (one of 10 in the AAF group, 17 of 95 in the NO-AAF group), and the mutations were located in exon 5 (11 of 18). The biological significance of these different mutational spectra among p53 genes deserves further investigation.

Reduction of reperfusion injury in rat skeletal muscle following administration of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor.

We used cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 (TXA2) receptor, and superoxide dismutase (SOD) with catalase to examine their protective effect against reperfusion injury in rat skeletal muscle. In 5 groups of 6 wistar rats three hours of ischaemia were induced in one hind limb by application of a tourniquet to the proximal thigh; the contralateral limb served as an internal, nonischaemic control. The first group did not receive any drug nor was it reperfused. In the other four groups, normal saline (reperfusion control), dimethylsulphoxide (DMSO), cinnamophilin, or SOD with catalase was given before removal of the tourniquet and one hour of reperfusion followed. Skeletal muscle injury was measured by a quantitative spectrophotometric assay of triphenyltetrazolium chloride (TTC) reduction and by muscle weight gain. One hour of reperfusion significantly (p<0.05) lowered TTC reduction in ischaemic limbs in the reperfusion control group in comparison with the rats in 3h ischaemia alone. Among the four reperfusion groups, only the cinnamophilin group had significantly lower decrease of TTC reduction and significantly lower muscle weight gain. These results demonstrate the protective effect of cinnamophilin against reperfusion injury of the ischaemic skeletal muscle in rats.