Monitoring gastric cancer progression with circulating tumour DNA.

BACKGROUND: Circulating tumour DNA (ctDNA) is an emerging candidate biomarker for malignancies and may be useful for monitoring the disease status of gastric cancer. METHODS: We performed targeted deep sequencing of plasma cell-free DNA (cfDNA) by massively parallel sequencing in patients with tumours harbouring TP53 mutations. The quantitative values of TP53-ctDNA during the clinical course were compared with the tumour status. RESULTS: Three out of ten patients with TP53 mutations in primary tumours showed detectable TP53 mutation levels in preoperative cfDNA. Although the cfDNA concentrations were not always reflective of the disease course, the ctDNA fraction correlated with the disease status. CONCLUSIONS: ctDNA may serve as a useful biomarker to monitor gastric cancer progression and residual disease.

Effect of obesity on sperm retrieval outcome and reproductive hormone levels in Japanese azoospermic men with and without Klinefelter syndrome.

Obesity is reported to have adverse effects on semen quality and the endocrine system. In this study, we evaluated the effect of obesity on sperm retrieval outcome and reproductive hormone levels in Japanese men with non-obstructive azoospermia (NOA). This study is based on the clinical records of 217 men [172 with a 46,XY karyotype, 45 with Klinefelter syndrome (KS)] with NOA who underwent microdissection testicular sperm extraction at Nagoya City University Hospital between January 2004 and December 2014. Body mass index (BMI) and serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and total testosterone (TT) were measured in all patients. In a subset of patients, bioavailable testosterone (cBAT) also was calculated. Values were evaluated separately in patients with and without KS. Sperm retrieval rates (SRRs) in 46,XY men with a BMI <25 kg/m2 and ≥25 kg/m2 were 29.3% and 18.4%, respectively (p = 0.142), while SRRs in KS men with a BMI <25 kg/m2 and ≥25 kg/m2 were 25.0% and 35.3%, respectively (p = 0.460). TT level in men with a BMI ≥25 kg/m2 was lower than that in men with a BMI <25 kg/m2 , regardless of KS status. According to Pearson product-moment correlation coefficients, TT and cBAT levels tended to have negative correlations with BMI; however, statistical significance was observed only for TT level in 46,XY men (r = 0.340, p < 0.001). LH and FSH levels were negatively correlated with BMI in KS men (r = -0.466, p = 0.001 and r = -0.647, p < 0.001, respectively), but not in 46,XY men. These results suggest that obesity may be irrelevant to sperm retrieval outcome in patients with NOA. The negative correlations between gonadotropins and BMI in patients with KS suggest an underlying suppressive effect on gonadotropin excretion, which is distinctive in obese patients with KS.

Chromosomal anomalies and sperm retrieval outcomes of patients with non-obstructive azoospermia: a case series.

Some preoperative factors affecting the outcome of microdissection testicular sperm extraction (micro-TESE) have been previously evaluated. However, other than Klinefelter syndrome (KS), no other chromosomal anomalies have been discussed in the context of sperm retrieval outcomes. The objective of this study was to describe chromosomal anomalies and their relationship with sperm retrieval outcomes in patients with non-obstructive azoospermia (NOA). Of the 197 NOA patients whose clinical records were retrospectively reviewed, 144 (73.1%) had normal 46,XY karyotype, 40 (20.3%) had KS (47,XXY), and 13 (6.6%) had other chromosomal anomalies (autosomal in seven cases and sex-chromosomal anomalies in six). Of the seven patients with autosomal anomalies, two had the reportedly normal variant 46,XY,inv(9)(p12;q13). Testicular volume and serum hormone levels (luteinizing hormone, follicle-stimulating hormone, and total testosterone) of the patients with chromosomal anomalies other than KS were comparable to those of the patients with normal karyotype. The sperm retrieval rate of the patients with 46,XY karyotype, KS, or other chromosomal anomalies were 27.1%, 22.5%, and 15.4%, respectively, with no statistically significant difference. However, among the samples collected from the 13 patients with chromosomal anomalies other than KS, only those from the two patients with the normal variant 46,XY,inv(9)(p12;q13) contained spermatozoa. Among our series of NOA patients, the incidence of autosomal anomalies was higher than that generally noted among neonates, which suggests that not only sex-chromosomal anomalies but also autosomal anomalies may affect the development of NOA. Furthermore, our findings suggest that sperm retrieval outcome is more unfavorable in NOA patients with chromosomal anomalies than in NOA patients with 46,XY karyotype or KS, despite the use of micro-TESE.

Excitatory synaptogenesis between identified Lymnaea neurons requires extrinsic trophic factors and is mediated by receptor tyrosine kinases.

Neurotrophic factors have well established roles in neuronal development and adult synaptic plasticity, but their precise role in synapse formation has yet to be determined. This paper provides the first direct evidence that neurotrophic factors in brain conditioned medium (CM) differentially regulate excitatory and inhibitory synapse formation. Somata of identified presynaptic and postsynaptic neurons were isolated from the CNS of Lymnaea and were cultured in a soma-soma configuration in the presence (CM) or absence [defined medium (DM)] of trophic factors. In DM, excitatory synapses did not form. When they were paired in CM or in DM containing Lymnaea epidermal growth factor (EGF); however, all presynaptic neurons reestablished their specific excitatory synapses, which had electrical properties similar to those seen in vivo. CM-induced formation of excitatory synapses required transcription and de novo protein synthesis, as indicated by the observations that synapse formation was blocked by the protein synthesis inhibitor anisomycin and the protein transcription blocker actinomycin D; the CM factor was inactivated by boiling. They were also blocked by receptor tyrosine kinase inhibitors (lavendustin A, genistein, K252a, and KT5926) but not by inactive analogs (genistin and lavendustin B), suggesting that the effect was mediated by receptor tyrosine kinases. These results, together with our previously published data, demonstrate that trophic factors are required for excitatory, but not inhibitory, synapse formation and extends the role of EGF from cell proliferation, neurite outgrowth, and survival to excitatory synapse formation.

Distribution of involved abdominal lymph nodes is correlated with the distance from the esophagogastric junction to the distal end of the tumor in Siewert type II tumors.

BACKGROUND: The optimal surgical approach for Siewert type II adenocarcinoma of the esophagogastric junction (AEG) has not yet been agreed. Here we investigated whether the distance from the esophagogastric junction (EGJ) to the distal end of the tumor was related to the distribution of involved abdominal lymph nodes in Siewert type II tumors. METHODS: A total of 288 patients with pT2-4 AEG Siewert II, treated by R0 surgical resection at 7 institutions in Japan, were retrospectively investigated. The distribution of involved abdominal nodes was correlated with the distance from the EGJ to the distal end of the tumor. RESULTS: In patients where the distance from the EGJ to the distal end of the tumor was ≤30 mm, the frequency of nodal involvement along the greater curvature or antrum was low (2.2%). In contrast, in patients where the distance was >50 mm, the incidence of this nodal involvement was 20.0%. In patients where the distance was 30-50 mm incidence was intermediate (8.0%). Multivariate analyses showed that the distance from the EGJ to the distal end of the tumor was significantly related to lymph node involvement along the greater curvature or antrum (odds ratio 3.7, 95% confidence interval 1.3-11, p = 0.006). CONCLUSIONS: When the distance from the EGJ to the distal end of the tumor is ≤ 30 mm for Siewert II AEG, esophagectomy or proximal gastrectomy is sufficient from the point of view of abdominal lymphadenectomy. However, a total gastrectomy should be considered for abdominal lymphadenectomy when this distance is > 50 mm.

Double sentinel lymph node mapping with indocyanine green and 99m-technetium-tin colloid in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

[Preemptive analgesia produced with epidural analgesia administered prior to surgery].

The effect of epidural analgesia administered before or during surgery on postoperative pain relief using continuous epidural infusion of the mixture of local anesthetics and narcotics was studied. Ninety patients undergoing abdominal hysterectomy were randomly allocated to three groups; thirty patients of group 1 who received general anesthesia alone, thirty patients of group 2 with epidural analgesia 20 min before the end of surgery under general anesthesia and thirty patients of group 3 with epidural analgesia plus general anesthesia before surgery. Epidural analgesia was induced with 2% mepivacaine solution 15 ml without epinephrine in group 2 and 3, and in group 3 followed with 5 ml of the same solution at one-hour intervals. General anesthesia was induced with thiamylal and maintained with nitrous oxide, oxygen and sevoflurane. Immediately after surgery, 5 ml of the mixture of 0.225% bupivacaine and 0.0005% fentanyl was injected epidurally and followed with continuous infusion of the same mixture at the rate of 2.1 ml.h-1 over 24 h. Visual analogue score and Prince-Henry score were significantly less in group 3 than in group 1 and group 2 at 4 hours and 24 hours after surgery (P < 0.01, P < 0.05 respectively). These results suggest that postoperative continuous epidural analgesia is more effective if the entrance of noxious stimuli into the central neural system is prevented by preincisional epidural block.

[Pharmacokinetics of epidurally administered bupivacaine during prostaglandin E1- or trimetaphan-induced hypotension].

The effects of prostaglandin E1 (PGE1) and trimetaphan (TMP) on the plasma concentrations and derived pharmacokinetic parameters of bupivacaine were studied in 14 women after its epidural administration. Patients, whose ages ranging from 35 to 60 years for mastectomy, received 50 mg of bupivacaine without epinephrine injected into the cervical epidural space during PGE1- or TMP-induced hypotension (80-90 mmHg of the systolic arterial pressure) under general anesthesia with nitrous oxide, oxygen and isoflurane 0.3-0.5%. No significant differences in pharmacokinetic parameters for the absorption and distribution of bupivacaine were found between the two groups. However, the mean elimination half-life of bupivacaine was significantly longer in patients with TMP [5.0 +/- 1.7 (SD) hr] compared with those with PGE1 (3.1 +/- 1.4 hr). The total clearance of bupivacaine was greater in patients with PGE1 (345 +/- 150 ml.min-1) compared with those with TMP (248 +/- 66 ml.min-1). The results of this pharmacokinetic study indicate that the plasma bupivacaine concentration decreases more rapidly during PGE1-induced hypotension than during TMP-induced hypotension.

[Depressive effect of dibucaine and bupivacaine on the growth of axons from cultured neuron of Lymnaea stagnalis].

The depressive effect of dibucaine (n = 8) was compared with that of bupivacaine (n = 9) using identified cultured neurons (A cluster) of Lymnaea stagnalis. Cultured interneurons exhibit extensive neurite outgrowth within 14-20 hours when placed in brain conditioned media. The changes of cultured neuron were recorded using a color video camera directly connected to an inverted microscope and the images were stored on digital video tape. Local anesthetics were added to the culture dish, with final concentrations of 1 x 10(-6) M-8 x 10(-4) M of dibucaine and 1 x 10(-5) M-8 X 10(-3) M of bupivacaine. We examined the damage of growth cone before and 30 minute after local anesthetics administration. Histologic damage were scored from moderate to severe compared to the control before dibucaine or bupivacaine administration. Dibucaine or bupivacaine damaged the growth cone moderately in the concentration of 1 x 10(-5) M or 4 x 10(-4) M, respectively. While dibucaine or bupivacaine damaged it severely in the concentration of 8 x 10(-5) M or 2 x 10(-3) M, respectively. These results suggest that bupivacaine is safer than dibucaine with the concentration we use in clinical practice.

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA.

The effect of dopamine on hepatic blood flow in patients undergoing epidural anesthesia.

We studied the effect of dopamine on hepatic blood flow during epidural anesthesia with the infusion of hydroxyethyl starch (HES). Hepatic blood flow was measured noninvasively via indocyanine green (ICG) clearance (indices: K [ICG disappearance rate] and R15 [15-min ICG retention rate]). Group C (n = 7) received no epidural anesthesia, Group E (n = 14) received epidural anesthesia, and Group E + D (n = 7) received a dopamine infusion (5 microg x kg(-1) x min(-1)) during epidural anesthesia. Epidural blockade extended from a median of T5 (T4-7) to L4 (L3-5) in Group E and from T5 (T4-7) to L4 (L3-S1) in Group E + D. Mean arterial pressure was maintained at preanesthetic levels in Groups E and E + D. K decreased and R15 increased in Group E (P < 0.05). In Groups C and E + D, K decreased and R15 increased slightly, but not significantly. K was smaller and R15 greater in Group E than in Group C (P < 0.05). We conclude that hepatic blood flow is decreased by epidural anesthesia, despite normotension maintained by continuous infusion of HES, but that this decrease in flow is reversed by the addition of a dopamine infusion.

[Effects of propiverine hydrochloride (P-4) and its metabolites on urinary bladder function in anesthetized rats].

The effects of P-4 and its active metabolites, 1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide[P-4(N----O)], 1-methyl-4-piperidyl benzilate N-oxide [DPr-P-4 (N----O)] and 1-methyl-4-piperidyl benzilate hydrochloride (DPr-P-4), on urinary bladder function were investigated in urethane anesthetized rats. By cystometrography, P-4 (2, 4 mg/kg, i.v.) and P-4 (N----O) (4 mg/kg, i.v.), which have direct action on smooth muscles, significantly increased the maximum vesical volume. As for rhythmic bladder contractions, P-4 (1,2,4 mg/kg, i.v.) and P-4 (N----O) (2, 4 mg/kg, i.v.) significantly decreased the frequency with a slight decrease in the amplitude. On the other hand, DPr-P-4 (N----O) (0.1, 0.5 mg/kg, i.v.) and DPr-P-4 (0.01, 0.05 mg/kg, i.v.), which have anticholinergic effects, significantly inhibited the maximum vesical pressure on the cystometrograms, and DPr-P-4 (N----O) (0.1, 0.5 mg/kg, i.v.) and DPr-P-4 (0.005, 0.05 mg/kg, i.v.) significantly inhibited the amplitude of the rhythmic bladder contractions. The effects of flavoxate and papaverine were similar to those of P-4 and P-4 (N----O), but the effects of propantheline and atropine were similar to those of DPr-P-4 (N----O) and DPr-P-4 in these two experimental methods. These results suggest that the clinical effects of P-4 are based not only on the actions of P-4 itself but also on those of its active metabolites.

Synthesis of multicomponent systems composed of one phthalocyanine and four terpyridine ligands.

Two phthalocyanine-based multiple ligands were synthesized and characterized. Photochemical and electrochemical properties were measured for zinc(II) phthalocyanines covalently linked with four ruthenium(II) bisterpyridyl complexes. The absorption and electrochemical results are indicative of electronic interaction between two photoactive and redox-active components. Fluorescence spectroscopy of the five nuclear complexes provides evidence of an efficient photoinduced intramolecular energy transfer between the ruthenium-based metal-to-ligand charge-transfer (MLCT) chromophores and the zinc(II) phthalocyanine core. The absorption and fluorescence spectra of the phthalocyanine-based multiple ligands change dramatically as a result of the coordination of metal ions with peripheral terpyridine ligands. This change of fluorescence intensity upon addition of metal ions can apply to an output signal for metal ion sensing. The direct attachment of metal ion receptors with a zinc phthalocyanine core enhanced efficiency of the energy- and electron-transfer reaction from the core to the metal complexes.

Trophic factor-induced plasticity of synaptic connections between identified Lymnaea neurons.

Neurotrophic factors participate in both developmental and adult synaptic plasticity; however, the underlying mechanisms remain unknown. Using soma-soma synapses between the identified Lymnaea neurons, we demonstrate that the brain conditioned medium (CM)-derived trophic factors are required for the formation of excitatory but not the inhibitory synapse. Specifically, identified presynaptic [right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD4)] and postsynaptic [visceral dorsal 2/3 (VD2/3) and left pedal dorsal 1 (LPeD1)] neurons were soma-soma paired either in the absence or presence of CM. We show that in defined medium (DM-does not contain extrinsic trophic factors), appropriate excitatory synapses failed to develop between RPeD1 and VD2/3. Instead, inappropriate inhibitory synapses formed between VD2/3 and RPeD1. Similarly, mutual inhibitory synapses developed between VD4 and LPeD1 in DM. These inhibitory synapses were termed novel because they do not exist in the intact brain. To test whether DM-induced, inappropriate inhibitory synapses could be corrected by the addition of CM, cells were first paired in DM for an initial period of 12 hr. DM was then replaced with CM, and simultaneous intracellular recordings were made from paired cells after 6-12 hr of CM substitution. Not only did CM induce the formation of appropriate excitatory synapses between both cell pairs, but it also reduced the incidence of inappropriate inhibitory synapse formation. The CM-induced plasticity of synaptic connections involved new protein synthesis and transcription and was mediated via receptor tyrosine kinases. Taken together, our data provide the first direct insight into the cellular mechanism underlying trophic factor-induced specificity and plasticity of synaptic connections between soma-soma paired Lymnaea neurons.

Sevoflurane induced suppression of inhibitory synaptic transmission between soma-soma paired Lymnaea neurons.

The cellular and synaptic mechanisms by which general anesthetics affect cell-cell communications in the nervous system remain poorly defined. In this study, we sought to determine how clinically relevant concentrations of sevoflurane affected inhibitory synaptic transmission between identified Lymnaea neurons in vitro. Inhibitory synapses were reconstructed in cell culture, between the somata of two functionally well-characterized neurons, right pedal dorsal 1 (RPeD1, the giant dopaminergic neuron) and visceral dorsal 4 (VD4). Clinically relevant concentrations of sevoflurane (1-4%) were tested for their effects on synaptic transmission and the intrinsic membrane properties of soma-soma paired cells. RPeD1- induced inhibitory postsynaptic potentials (IPSPs) in VD4 were completely and reversibly blocked by sevoflurane (4%). Sevoflurane also suppressed action potentials in both RPeD1 and VD4 cells. To determine whether the anesthetic-induced synaptic depression involved postsynaptic transmitter receptors, dopamine was pressure applied to VD4, either in the presence or absence of sevoflurane. Dopamine (10(-]5) M) activated a voltage-insensitive K(+) current in VD4. The same K(+) current was also altered by sevoflurane; however, the effects of two compounds were nonadditive. Because transmitter release from RPeD1 requires Ca(2+) influx through voltage-gated Ca(2+) channels, we next tested whether the anesthetic-induced synaptic depression involved these channels. Individually isolated RPeD1 somata were whole cell voltage clamped, and Ca(2+) currents were analyzed in control and various anesthetic conditions. Clinically relevant concentrations of sevoflurane did not significantly affect voltage-activated Ca(2+) channels in RPeD1. Taken together, this study provides the first direct evidence that sevoflurane-induced synaptic depression involves both pre- and postsynaptic ion channels.