[Comparison of 1-year outcomes between Roux-en-Y gastric bypass and sleeve gastrectomy with Roux-en-Y duodenojejunal bypass in the patients of obesity with type 2 diabetes mellitus].

Objective: To compare the 1-year effects of Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy with Roux-en-Y duodenal bypass (SG+RYDJB) on weight loss, remission of diabetes, and postoperative complications in patients with obesity and type 2 diabetes. Methods: A single-center retrospective cohort study was conducted at the First Affiliated Hospital of Nanjing Medical University from January 2020 to December 2020. Sixty-four patients with type 2 diabetes and body mass index (BMI) of 27.5-40.0 kg/m2 were included in this study and divided into the RYGB group (n=34) and the SG+RYDJB group (n=30). In both procedures, the biliopancreatic branch was measured 100 cm distal to the Treitz ligament, and the food branch was measured 100 cm distal to the gastric or duodenojejunal anastomosis. Patients were followed up by telephone or WeChat, a free messaging and calling app at 1, 3, 6, and 12 months postoperatively to determine their weight loss and remission of diabetes. The primary outcomes were the weight loss and reduction in blood glucose concentrations at 1 year after surgery and postoperative complications. Other postoperative changes, including body weight, BMI, percentage of total weight loss (%TWL), percentage of excess weight loss (%EWL), glycated hemoglobin A1c (HbA1c), and fasting blood glucose at 1 year after surgery were also assessed. Results: There were no significant differences in baseline data between the two groups (all P>0.05). No conversion to open surgery or death occurred in either group. Operation time was longer in the SG+RYDJB than the RYGB group (137.8±22.1 minutes vs. 80.0±24.9 minutes, t=9.779, P<0.001) and the incidence of perioperative complications was higher in the SG+RYDJB than the RYGB group (20% [6/30] vs. 2.9% [1/34], χ2=4.761, P=0.029). However, the postoperative hospital stay was similar between the two groups [3.0 (3.0, 4.3) days vs. 3.0 (4.0, 6.0) days, U=641.500, P=0.071]. Perioperative complications comprised small gastric pouch anastomotic leakage in one patient in the RYGB group and leakage (three patients) and bleeding (two patients with gastrointestinal bleeding and one with trocar site bleeding) in the SG+RYDJB group. Long-term complications were as follows. The incidence of anemia was significantly higher in the RYGB than the SG+RYDJB group (26.5% [9/34] vs. 3.3% [1/30], χ2=6.472, P=0.011). However, there were no significant differences in incidences of postoperative reflux, dumping syndrome, alopecia, diarrhea, constipation or foul-smelling flatus between the two groups (all P>0.05). Compared with 1 year before surgery, the body weights and fasting plasma glucose concentrations of patients in the SG+RYDJB and RYGB group (72.4±10.6 kg vs. 98.5±14.2 kg, respectively; 68.2±10.0 kg vs. 91.9±14.8 kg, respectively), BMI (25.2±2.9 kg/m2 vs. 34.3±4.2 kg/m2, respectively; 24.3±2.4 kg/m2 vs. 32.7±3.7 kg/m2, respectively) (5.5±1.6 vs. 10.6±3.3, respectively; 5.8±2.1 vs. 9.0±3.4, respectively); HbA1c (5.7±0.8 vs. 9.7±1.2, respectively; 9.1±1.9 vs. 5.9±0.9, respectively) were significantly lower at 1 year after surgery (all P<0.05). However, the % TWL (26.5%±6.0% vs. 25.6%±4.4%, t=0.663, P=0.510) and % EWL (109.1%±38.2% vs. 109.4%±40.3%, t=-0.026, P=0.026), rate of complete remission of diabetes at 1 year (80.0% [24/30] vs. 82.4% [28/34], χ2=0.058, P=0.810] did not differ significantly between the two groups (all P>0.05). Conclusions: Although SG+RYDJB surgery compared with RYGB is more difficult to perform, it can achieve similar weight loss and remission of diabetes and is associated with a lower incidence of anemia because of the preservation of the pylorus.

[Analysis of early severe postoperative complications and risk factors in 4255 patients who underwent bariatric and metabolic surgery in a single cente].

Objective: To analyze the incidence of early severe complications following bariatric and metabolic surgery and the experience of their diagnosis, treatment, and risk factors. Methods: In this retrospective observational study, the clinical data of 4255 patients who underwent bariatric and metabolic surgery between May 2010 and May 2022 in the Department of Bariatric and Metabolic Surgery of the First Affiliated Hospital of Nanjing Medical University were retrospectively collected. Among these patients, 1125 were male and 3130 were female. The mean age and body mass index (BMI) of the patients at the time of operation were 31.3±4.5 years and 36.5±6.4 kg/m2, respectively. Regarding surgical type, 2397 patients underwent sleeve gastrectomy (SG), 489 underwent Roux-en-Y gastric bypass (RYGB), 1028 underwent sleeve gastrectomy plus jejunojejunal bypass (SG+JJB), and 341 underwent single anastomosis duodenal switch (SADS). The inclusion criteria were patients (1) with a Clavien-Dindo grade of ≥III; (2) who were undergoing SG, RYGB, SG-JJB, or SADS; and (3) who had complete clinical data. The exclusion criteria were patients (1) undergoing revisional surgery and (2) other operations during the bariatric and metabolic surgery. The Clavien-Dindo classification was used to analyze the incidence of early severe postoperative complications and their prognosis. Early severe postoperative complications were defined as Clavien-Dindo ≥ III complications within 30 days after surgery. Meanwhile, multivariate logistic regression model was used to identify risk factors of the complications. Results: Summary of early severe complications following bariatric and metabolic surgery: (1) of the 4255 patients, 22 (12 male and 10 female) exhibited early severe complications (0.52%). The mean age and BMI of these patients were 41.1±9.9 years and 36.9±8.2 kg/m2, respectively. Preoperatively, 7 patients had hypertension, 10 had type 2 diabetes mellitus, 1 had respiratory failure, and 1 had heart failure. The severe complications included 9 patients (0.21%) with grade IIIa, 11 (0.26%) with grade IIIb, 1 (0.02%) with grade IVa, and 1 (0.02%) with grade V complications. The incidences of severe postoperative complications in the different surgical procedures were 0.17% for SG (4/2397), 0.61% for RYGB (3/489), 0.58% for SG+JJB (6/1028), and 2.64% for SADS (9/341). The common severe complications were leakage (0.28%, 12 patients), bleeding (0.14%, 6 patients), and obstruction (0.05%, 2 patients). (2) Management of complications: Grade IIIa complications (including eight patients with leakage and one with severe inflammation) were treated with antibiotics, nasogastric and nutritional tube placements, and CT-guided drainage. For grade IIIa complications, five patients with bleeding were treated with reoperation, and all the patients recovered; four patients with leakage were treated with reoperation, wherein three were converted to RYGB and one patient underwent resuturing of the leakage site; two patients with obstruction were treated with adhesiolysis. The patient with grade IVa complication (including respiratory failure complicated with acid aspiration) was treated in the ICU. For the grade V complication, bleeding in a patient with SG+JJB was treated with reoperation, which confirmed the bleeding of short gastric vessels. Unfortunately, the patient died. (3) Risk factor analysis of early severe complications: univariate analysis detected that sex, age, type 2 diabetes mellitus, operation time, and surgical type were associated with postoperative complications (P<0.05). However, multivariate analysis indicated that an age of ≥31.3 years (odds ratio [OR] = 5.423, 95% confidence interval [CI]: 1.004-29.278, P=0.049) and surgical type (SADS: OR = 19.758, 95%CI: 5.803-67.282, P<0.001; RYGB: OR = 9.752, 95%CI: 2.456-38.723, P=0.001; SG+JJB: OR = 5.706, 95%CI: 1.966- 16.559, P=0.001) were independent risk factors of early severe complications following bariatric and metabolic surgery. Conclusion: Bariatric and metabolic surgery is safe. Its common postoperative complications include leakage, bleeding, and obstruction, which require early detection, diagnosis, and treatment to improve treatment outcomes. Age and surgical type are independent risk factors of early severe complications following bariatric and metabolic surgery.

DNAH6 is a novel candidate gene associated with sperm head anomaly.

Globozoospermia and acephalic spermatozoa are two rare sperm head anomalies associated with male infertility. Combination of the two phenotypes in the same patient is extremely rare, so the underlying pathogenesis of this disorder remains unclear. Here, we report a 35-year-old infertile male, who presented with 30% of sperm-lacked heads and 69% of sperm round-headed or small-headed with neck thickening in his ejaculate. Subsequent whole-exome sequencing (WES) analysis identified compound heterozygous variants within the DNAH6 gene. DNAH6 is a testis-specific-expressed protein that was localised to the neck region in the spermatozoa of normal control; however, immunofluorescent staining failed to detect DNAH6 protein in the patient's spermatozoa. Quantitative real-time PCR analysis also showed the complete absence of DNAH6 mRNA in the patient's spermatozoa. Moreover, two cycles of in vitro fertilisation (IVF)-assisted reproduction were carried out, but pregnancy was not achieved after embryo transfer. Therefore, rare sequence variants in DNAH6 might be susceptibility risks for human sperm head anomaly.

TSGA10 is a novel candidate gene associated with acephalic spermatozoa.

Acephalic spermatozoa is a rare teratozoospermia associated with male infertility. However, the pathogenesis of this disorder remains unclear. Here, we report a 27 years old infertile male from a consanguineous family, who presented with 99% headless sperm in his ejaculate. Electron microscopic and immunofluorescence analysis suggested breakage at the midpiece of the patient's sperm cells. Subsequent whole-exome sequencing analysis identified a homozygous deletion within TSGA10 (c.211delG; p.A71Hfs*12), which resulted in the production of truncated TSGA10 protein. TSGA10 is a testis-specific protein that localized to the midpiece in the spermatozoa of a normal control; however, immunostaining failed to detect TSGA10 protein in the patient's sperm. Western blot analysis also showed complete absence of TSGA10 protein in the patient. One cycle of in vitro fertilization-assisted reproduction was conducted, but pregnancy was not achieved after embryo transfer, possibly due to poor embryo quality. Therefore, we speculate that the presence of rare sequence variants within TSGA10 may be associated with acephalic spermatozoa in humans.

Headless spermatozoa in infertile men.

Spermatozoa morphology, an important parameter in a semen specimen's potential fertility evaluation, is a significant factor for in vitro fertilisation in assisted reproductive technology. Eleven sterile men with headless spermatozoa, a type of human teratozoospermia, are presented. Their ejaculates' headless spermatozoa percentages were high with rare normal spermatozoa forms. Additionally, abnormal morphology (e.g. round-headed or microcephalic spermatozoa) was also found. Spermatozoa motility was somewhat affected, potentially because of the missing mitochondrial sheath at the sperm tail base. Patients who underwent assisted reproductive technology treatment experienced adverse pregnancy outcomes. Work types and corresponding environments seemed irrelevant, but specific family history may have prompted its genetic origin. Computer-assisted semen analysis systems easily mistake headless spermatozoa as oligozoospermia because of nonrecognition of the loose head. However, morphological testing, especially with an electronic microscope, clearly identifies abnormal spermatozoa. Future exploration requires more methods investigating the frequency and percentage of this morphological abnormality in different populations with varied fertility levels. Such research would estimate the probable correlation of the abnormality with other semen parameters and examine the potential developmental or genetic origins. During clinical work, medical staff should detect these cases, avoid misdiagnosis and provide proper consultation about diagnosis and assisted reproductive technology treatment.

Abnormal expression of PTEN and PIK3CA in pemetrexed-resistant human pancreatic cancer cell line Patu8988.

The aim of this study was to investigate the expression of PTEN and PIK3CA in the pemetrexed-resistant human pancreatic cancer cell line Patu8988, and to evaluate their effects on the biological behavior of pancreatic cancer cells. PTEN and PIK3CA gene and protein expressions were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot, respectively, in a pemetrexed-resistant pancreatic cancer cell line and in the parent strain of the pancreatic cancer cells. The discrepancies between the two types of cell lines were detected by a transwell test. RT-PCR and western blot analyses revealed that PTEN and PIK3CA were overexpressed in the pemetrexed-resistant pancreatic cancer cell line. PTEN and PIK3CA were shown to be upregulated by 89 and 76% (western blot), respectively, in the pemetrexed-resistant cell line, compared to the normal pancreatic cancer cell line. The migratory and invasive abilities of the pemetrexed-resistant pancreatic cancer cell were significantly reduced compared to those of the parent strain (P < 0.05; transwell assay). Both PTEN and PIK3CA expression was abnormally enhanced in the pemetrexed-resistant cell line Patu8988; the co-existence of high levels of PTEN and PIK3CA in the pemetrexed-resistant pancreatic cancer line cells induced a significant decrease in their migratory and invasive capacities. This suggested that the mechanism of pemetrexed resistant may be affected by PTEN and PIK3CA, and that these may alter the biological behavior of cancer cells.

Genetic variations in MOV10 and CACNB2 are associated with hypertension in a Chinese Han population.

Human hypertension is a complex, multifactorial disease. Multiple variants associated with hypertension have been identified in the large numbers of genome-wide association studies, meta-analysis, and case-control studies. The present study investigated the association between the single nucleotide polymorphisms (SNPs) of five candidate genes and the susceptibility and prognosis of hypertension in a Chinese Han population. A hospital-based case-control study in a Chinese Han population was carried out, including 500 hypertension patients and 506 healthy controls. The five SNP markers were detected using the Sequenom MassArray(®) iPLEX System. The association of genotypes with susceptibility to hypertension was analyzed using odds ratio, with 95% confidence interval and logistic regression. All five variants conformed to Hardy-Weinberg proportions in the controls. No significant differences were noted in the genotype distributions for AGTR1, PRRC2A, and CALCA polymorphisms in patients with hypertension (N = 500) and healthy controls (N = 506). SNP rs2932538, a variant in MOV10, was found to be significantly associated with an increased risk of hypertension. However, SNP rs4373814, a variant in CACNB2, showed a relevant association with a decreased risk of hypertension. In conclusion, the results of our case-control study confirmed the significant association of the SNP rs2932538 in MOV10 and SNP rs4373814 in CACNB2 with an increased risk of hypertension in a Chinese Han population, suggesting that the SNP rs2932538 may be a poor prognostic indicator for hypertension, while SNP rs4373814 may be a good prognostic indicator for hypertension in the same region. However, our findings need to be replicated in larger epidemiological and functional studies.

Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis.

AIMS: Vibrio anguillarum is a universal marine pathogen causing vibriosis. Vibrio anguillarum encounters different osmolarity conditions between seawater and hosts, and its outer membrane proteins (OMPs) play a crucial role in the adaptation to changes of the surroundings. In the present study, proteomic approaches were applied to investigate the salt-responsive OMPs of V. anguillarum. METHODS AND RESULTS: Lower salinity (0.85% NaCl) is more suitable for growth, survival and swimming motility of the bacterium. Comparative two-dimensional electrophoresis (2-DE) analysis reveals six differentially expressed protein spots among three different salinities, which were successfully identified as OmpU, maltoporin, flagellin B, Omp26La, Omp26La and OmpW respectively. CONCLUSIONS: OmpW and OmpU were highly expressed at 3.5% salinity, suggesting their role in the efficient efflux of NaCl. Maltoporin was downregulated in higher salinity, indicating that higher osmolarity inhibits carbohydrate transport and bacterial growth. Omp26La, the homologue of OmpV, functions as a salt-responsive protein in lower salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing salt stress-responsive proteins of V. anguillarum using proteomic approaches. Our results provide a useful strategy for delineating the osmoregulatory mechanism of the marine pathogens.

Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy.

Nasopharyngeal carcinoma (NPC) is an epithelial cancer that metastasizes predictably to cervical lymph nodes or distant organs. To assess whether the chemokine receptors of NPC cells play important roles in metastasis and are associated with radiotherapy history, the significance of various chemokine receptors (CCR1-10, CXCR1-6, XCR1, and CX3CR1) in NPC cell lines (TW01, TW04, HONE1, BM1, and AS1) and 52 NPC tumour biopsies from 48 patients with NPC was evaluated by mRNA and cytometric analyses, chemotaxis and actin polymerization assays, and immunohistochemical staining. Quantitative real-time reverse transcription-polymerase chain reaction revealed substantial expression of CCR7, CCR9, CXCR4, and CXCR6 mRNA in all the NPC cell lines. Of these, however, only CCR7, CXCR4, and CXCR6 were functional in NPC cells. Negative immunoreactivity for CCR7, CXCR4, and CXCR6 was demonstrated in almost all nasopharyngeal (NP) specimens from patients with primary NPC (n = 12) and in those with regional metastatic NPC (n = 15). However, expression of two or three of these chemokine receptors was demonstrated in NP specimens from patients with liver metastasis. Strong positivity was demonstrated for all three of these chemokine receptors in almost all of the regional and distant metastasis specimens. Significant differences in the expression of CCR7, CXCR4, and CXCR6 were found between primary tumours and metastases (p < 0.001, p < 0.001, and p < 0.002, respectively). This observation was further confirmed by laser capture microdissection of freshly frozen tumours from primary (n = 5) and metastatic (n = 8) NPC sites (p = 0.04, 0.03, and 0.03 for CCR7, CXCR4, and CXCR6, respectively). Finally, significant differences in CXCR4 expression were demonstrated between de novo and post-radiotherapy groups (1/22 vs. 5/8; p < 0.003). It appears reasonable to conclude, therefore, that CCR7, CXCR4, and CXCR6 are expressed and active in human NPC metastases, while CXCR4 expression is associated with radiotherapy history.

Correction/mutation of acid alpha-D-glucosidase gene by modified single-stranded oligonucleotides: in vitro and in vivo studies.

Deficiency in acid alpha-D-glucosidase results in Pompe's disease. Modified single-stranded oligonucleotide (ODN) was designed to correct the acid alpha-D-glucosidase gene with a C1935 --> A (Asp --> Glu) point mutation which causes a complete loss of enzymatic activity for glycogen digestion in the lysosome. The ODN vectors contained a stretch of normal oligonucleotide flanked by phosphorothioated sequences. The 25mer and 35mer ODNs were homologous to the target sequence, except for a mismatched base in the middle. The ODNs caused permanent and inheritable restoration of acid alpha-D-glucosidase activity in skin fibroblast cells carrying this mutation derived from a Pompe's disease patient. Gene correction was confirmed by amplification refractory mutation system-PCR (ARMS-PCR), restriction fragment length polymorphism (RFLP) and direct DNA cloning and sequencing. The increased acid alpha-D-glucosidase activity was detected using 4-MUG as the artificial substrate. The correction efficiency, ranging from 0.5 to 4%, was dependent on the length and polarity of the MSSOV used, the optimal design being a sense-strand 35mer ODNs. Repeated treatment of the mutant fibroblast cells with the ODNs substantially increased correction. We also constructed ODN vectors to trigger specific and in vivo nonsense mutation in the mouse acid alpha-D-glucosidase gene. The ODNs were in complex with YEEE-K(18), an asialoglycoprotein-receptor ligand tagged with polylysine and targeted to hepatocytes and renal cells in vivo through intravenous injection. The mutated genotype was detected in the liver and the kidney by ARMS-PCR and glycogen accumulation in the lysosome of the liver cells. The studies demonstrate the utility of single-stranded ODN to direct targeted gene correction or mutation in a human hereditary disease and in an animal model. Our data open the possibility of developing ODN vector as a therapeutic approach for treatment of human hereditary diseases caused by point mutation.

Interaction between 3-(p-tolylamino)-1,5-azulenequinone and the deoxyguanosine residue in various oligonucleotides upon photolysis.

Eight single-stranded oligodeoxyribonucleotides 32P-labeled at the 5'-end were synthesized; they were annealed with the complementary oligodeoxyribonucleotides to form the corresponding double-stranded helices. These duplexes possessed standard Watson-Crick base pairs, locally perturbed sites of a base mismatch, or a bulge. Further, 5'-32P-labeled oligodeoxyribonucleotides with a hairpin loop were also synthesized. Cleavage of these single- and double-stranded oligodexyribonucleotides selectively at the deoxyguanosine residue was accomplished by use of 3-(p-tolylamino)-1,5-azulenequinone 1 upon irradiation with 350 nm UV light. The single strands were cleaved more efficiently than the double-helices. For the helices containing a deoxyguanosine residue at a bulge, at a hairpin loop or toward the end, the cleaving efficiency was increased. Computation results indicate that two possibilities exist for agent 1 to form two "Watson-Crick type" hydrogen bonds with guanine in single-stranded oligodeoxyribonucleotides; yet, only one possibility exists in duplexes.

Inhibition of thymidylate synthase activity by antisense oligodeoxynucleotide and possible role in thymineless treatment.

Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer. However, efficacy of TS-targeted anticancer drugs is limited by the development of drug resistance as a result of TS gene amplification. In this work, a phosphorothioated antisense oligonucleotide (ODN), designated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 microM concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine shortage, suggesting that ATS-2 is specifically effective for dividing cells. When used in combination with the anticancer drug FdUrd, ATS-2 exerted a additive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a second cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell division and ODN uptake. In contrast to HEK, which was shown to have very low levels of TdR kinase activity in [(3)H]thymidine incorporation experiments, [(3)H]thymidine incorporation in HeLa was 15-fold greater than that of HEK. We found that HeLa cells were sensitive to FdUrd but were rather resistant to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensitive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.

Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure.

The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris-HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.

Formation of DNA triple helix containing N(4)-(6-aminopyridin-2-yl)-2'-deoxycytidine.

A cytidinyl derivative, N(4)-(6-aminopyridin-2-yl)- 2'-deoxycytidine ((p)C), could interact with a CG base pair to support the triple-helix (triplex) formation of oligodeoxyribonucleotides. Characteristics of (p)C in the formation of both intramolecular triplex, i.e., a "paper clip type" triplex ((P)CT) and intermolecular triplex, i.e., a "linear type" triplex (LT) was monitored by optical methods and isothermal titration calorimetric measurements. Experimental results revealed that the LT with (p)C*CG internally was independent of the solution pH. Only single substitution of (p)C, situated internally but not terminally, facilitated the (P)CT formation by the UV thermal melting study at the neutral pH. However, the best stabilization of the PCT in acidic conditions occurred when (p)C at the end of the triplex rather than internally. In addition, an LT, but not a (P)CT, containing an alternating (p)CT(p)CT(p)C sequence, could be formed in the conditions of 20 mM MgCl(2) and/or 5 mM spermine. Thus, the presence of several nucleotides of (p)C in proximity along the Hoogsteen strand may lead to structural distortion such that the more flexible LT with multiple substitutions is formed in favor of the more rigid PCT.

In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha.

BACKGROUND/AIMS: It has been hypothesized that liver stem cells may be activated and proliferate upon liver injury and may participate in the development of liver cancer. GP7TB, a rat liver epithelial tumor cell line, possesses characteristics of liver stem-like cells and can develop into a tumor in syngeneic Fischer 344 rat. We found that protein kinase C-alpha (PKC-alpha) is overexpressed in GP7TB cells. The importance of PKC-alpha for this liver tumor cell was elucidated. METHODS: Antisense oligonucleotide (ODN) was applied to suppress the production of PKC-alpha in GP7TB cells in vitro and in vivo. Cell viability was measured by acid phosphatase assay. The cellular levels of PKC-alpha and Bcl-2 were monitored by Western-blot analysis. Activation of nuclear factor NF-kappaB was analyzed by electrophoretic mobility shift assay. Cell cycle phase distribution was monitored by FACScan. Cell apoptosis was detected by TUNEL assay and histochemical staining of tumor tissue sections. The in vivo experiment was conducted by implanting tumor mass of GP7TB in the liver of F-344 rat and continuous delivery of the ODN by a mini-osmotic pump. RESULTS: Antisense ODN effectively suppressed the level of PKC-alpha that resulted in the decrease of Bcl-2 and nuclear NF-kappaB. The cumulative viable cells also decreased dramatically for the antisense-treated group. FACScan showed that the cells were arrested at early S-phase. These cells in turn went into apoptosis without completing a cell cycle. It was found that growth of the tumor was suppressed efficiently by antisense ODN. Cell apoptosis was found in the orthotopic tumor. The normal liver cells were not affected. CONCLUSIONS: A lethal effect of depressing the level of PKC-alpha in GP7TB cells and success in suppressing orthotopic tumor growth in vivo suggests that PKC-alpha antisense ODN would be a promising therapeutic agent for some liver cancers.

"Paper-clip" type triple helix formation by 5'-d-(TC)3Ta(CT)3Cb(AG)3 (a and b = 0-4) as a function of loop size with and without the pseudoisocytosine base in the Hoogsteen strand.

The formation of a DNA "paper-clip" type triple helix (triplex) with a common sequence 5'-d-(TC)(3)T(a)()(CT)(3)C(b)()(AG)(3) (a and b = 0-4) was studied by UV thermal melting experiments and CD spectra. These DNA oligomers form triplexes and duplexes under slightly acidic and neutral conditions, respectively. The stability of the formed triplexes (at pH 4.5) or duplexes (at pH 7.0 or 8.0) does not vary significantly with the size of the loops (a and b = 1-4). At pH 6.0, the triplex stability is, however, a function of a and b. It is also interesting to note that the oligomer 5'-d-(TC)(3)(CT)(3)(AG)(3) (a and b = 0) forms a stable triplex at pH 4.5 with a slightly lower T(m) value, due to dissociation of a base triad at one end and a distorted base triad at the other, observed by (1)H NMR. Thus, we have here a model system, 5'-d-(TC)(3)T(a)(CT)(3)C(b)(AG)(3), that could form a triplex effectively with (a and b = 1-4) and without (a and b = 0) loops under acidic conditions. In addition, the triplex formation of oligomers with replacement of one, two, or three 2'-deoxycytidine in the Hoogsteen strand by either 2'-deoxypseudoisocytidine (D) or 2'-O-methylpseudoisocytidine (M) was also studied in the sequence 5'-d-(TX)(3)T(2)(CT)(3)C(2)(AG)(3) (where X is C, D, or M). Both CD spectra and UV melting results showed that only D3 [(TX)(3) = (TD)(3)] and M3 [(TX)(3) = (TM)(3)] were able to form the paper-clip structure under both neutral and acidic conditions. This is because the N(3)H of a pseudoisocytosine base can serve as a proton donor without protonation. We hereby proved that the 2'-deoxypseudoisocytidine, similar to 2'-O-methylpseudoisocytidine, could replace 2'-deoxycytidine in the Hoogsteen strand to provide triplex formation at neutral pH.

Scleral outfolding for macular translocation.

PURPOSE: To report experimental study and clinical observation of scleral outfolding for macular translocation. METHODS: In six human cadaver eyes, circumferential mattress sutures to create sclera infoldings were compared with radial-interrupted mattress sutures to create scleral outfoldings. In a 75-year-old man with macular degeneration and choroidal neovascular membrane, radial-interrupted mattress sutures were used for macular translocation. RESULTS: In the human cadaver eyes, circumferential mattress sutures for scleral infolding created an average decrease in corresponding internal anteroposterior retinal contour of 0.36 mm. Radial-interrupted mattress sutures for scleral outfoldings created an average decrease in the corresponding internal anteroposterior retinal contour of 4. 61 mm. The statistical significance of the difference between infoldings of the sclera versus outfoldings of the sclera had a P value of.0001. CONCLUSIONS: Initial experimental and clinical study suggests that radial-interrupted mattress sutures may generate more shortening of the internal scleral surface and greater macular translocation than circumferential mattress sutures. Additional studies are needed to evaluate the long-term effects of radial-interrupted mattress sutures and macular translocation for treatment of subfoveal choroidal neovascularization secondary to age-related macular degeneration.

Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells.

Regulation of arachidonate metabolism in human epidermoid carcinoma A431 cells by phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cytosolic glutathione peroxidase (GPx1) was studied. In order to study the effect of reduced glutathione (GSH) on the catalysis regulation of these oxygenation enzymes, diethyl maleate was used to deplete the intracellular GSH. In the presence of 13-hydroperoxyoctadecadienoic acid, the enzymatic catalysis of cyclooxygenase and 12-lipoxygenase was significantly increased in the GSH-depleted cells. In terms of the inhibitory effect on 12-lipoxygenase, PHGPx was more sensitive to GSH concentrations than GPx1. Inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both cyclooxygenase and 12-lipoxygenase. In conclusion, the results indicate that catalysis of cyclooxygenase and 12-lipoxygenase in A431 cells was regulated by redox-reaction, and PHGPx seems to play an important role in the controlling of these reactions.

Computer system for four-dimensional transesophageal echocardiographic image reconstruction.

This paper presents a system for reconstructing a four-dimensional (4D) heart-beating image from transesophageal echocardiographic (TEE) data acquired with a rotational approach. The system consists of the necessary processing modules for two-dimensional (2D) echocardiogram reformation and 3D/4D-image reconstruction. These include the modules of image decoding, image re-coordinating, and three-dimensional (3D) volume rendering. The system is implemented under PC platform with Windows 95 operating system (with Intel Pentium-166 CPU, 64 MB RAM on board, and 2.0 GB hard disk capacity). It takes 6 min to reconstruct a 4D echocardiographic data set. The resultant 2D/3D/4D echocardiographic image provide the tools for investigating the phenomenon of heart beating, exploring the heart structure, and reformatting the 2D echocardiograms in an arbitrary plane. The functions provided by the system can be applied for further studies, such as 3D cardiac shape analysis, cardiac function measurement, and so forth.

Assessment of left ventricular cardiac shape by the use of volumetric curvature analysis from 3D echocardiography.

A method for three-dimensional shape analysis of left ventricle (LV) is presented in this article. The method uses three-dimensional transesophageal echocardiography (TEE) as the source to derive the 3D wire-frame model and the related shape descriptors. The shape descriptors developed in this article include regional surface changing (RSC), global surface curvature (GSC), surface distance (SD), normalized surface distance (ND), and effective radius (ER) of the endocardial surface. Based on these shape descriptors, the shape of LV could be sketched in both static and dynamic manner. The results show that the new approach provides a robust but easy method to quantify regional and global LV shape from 2D and 3D echocardiograms.