For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.

Objective: To clarify the value of contrast-enhanced harmonic ultrasonography (US) with Sonazoid, a second-generation US contrast agent, in the differential diagnosis of liver tumors compared to dynamic CT. Methods: A total of 249 hepatic nodules in 214 patients were studied; these included 177 hepatocellular carcinomas (HCCs), 42 liver metastases, 20 liver hemangiomas, 6 dysplastic nodules and 4 focal nodular hyperplasias (FNHs). After the injection of Sonazoid, nodules were scanned using real-time contrast-enhanced harmonic US in the vascular phases, i.e. the early and late vascular phases, and the Kupffer phase. Results: Six enhancement patterns were identified to be significant for the differential diagnosis of hepatic tumors. In HCCs, the presence of intratumoral vessels supplied from the periphery and fast washout (sensitivity, 96.6%; specificity, 94.4%) were the most typical characteristics. In metastases, the presence of rim-like enhancement with peripheral tumor vessels (sensitivity, 88.1%; specificity, 100%) was the typical pattern. In hemangiomas, the presence of intratumoral hypoperfusion images with globular or cotton wool-like pooling, which continue to the late vascular phase (sensitivity, 90.0%; specificity, 99.6%), was typical. In dysplastic nodules, the presence of portal enhancement without arterial supply in the early vascular phase and the presence of intratumoral uptake in the Kupffer phase (sensitivity, 83.3%; specificity, 100%) were the most typical patterns. In FNHs, the presence of a spoke-wheel pattern in the early vascular phase with dense staining in the late vascular phase, and positive uptake within the nodule in the Kupffer phase (sensitivity, 100%; specificity, 100%) were the most typical patterns. Conclusion: Contrast-enhanced harmonic US with Sonazoid allowed intimate vascular and Kupffer imaging and, therefore, is useful for the differential diagnosis of hepatic tumors.

To further clarify the molecular features of colorectal neuroendocrine carcinomas (NECs), we immunohistochemically examined tumor samples from 25 NECs, including 9 small cell NECs (SCNECs) and 16 large cell NECs (LCNECs), 20 neuroendocrine tumors (NETs), and 21 poorly differentiated adenocarcinomas (PDCs) for the expression of several biomarkers (p53, β-catenin, Bcl-2, Rb, p16, p21, cyclin D1, and cyclin E) and used sequencing analysis to identify gene alterations of TP53, APC, CTNNB1, KRAS, and BRAF. The frequencies of aberrant p53 expression (88%), β-catenin nuclear expression (48%), and high expression of cyclin E (84%) were significantly higher in NECs than in NETs (0%, 5%, and 5%, P < .01, respectively). The immunohistochemical results of NECs and PDCs were similar. TP53, APC, KRAS, and BRAF gene mutations were variously detected in NECs and PDCs but not in any NETs. The frequencies of decreased expression of Rb (56%) and high expression of p16 (56%) and Bcl-2 (64%) were significantly higher in NECs than in PDCs (5%, 19%, and 5%, P < .05, respectively) or NETs (10%, 5%, and 5%, P < .01, respectively). Such immunohistochemical characteristics of NECs were more evident in SCNECs than in large cell NECs (P < .01). In conclusion, the molecular features of colorectal NECs are similar to those of adenocarcinomas and not to those of NETs. Decreased expression of Rb and high expression of p16 and Bcl-2 are characteristics of NECs, suggesting that Rb-p16 pathway disruption may contribute to the promotion of proliferative activity in colorectal NECs. SCNECs may be a prototype of NECs.

ObjectiveThe objective of this study was to comparatively analyse the gastric fluid (GF) microbiota between patients with functional dyspepsia (FD) and healthy controls (HC), and to assess the effect of probiotics on the microbiota.DesignTwenty-four Japanese patients with FD who met the Rome III definition and 21 age-matched and gender-matched HC volunteers were enrolled. The patients with FD had been treated with LG21, a probiotic strain. The GF was sampled after an overnight fast using a nasogastric tube. The bile acids concentration was determined by ELISA. The V3-V4 region of 16S rRNA gene was amplified using bacterial DNA from the GF, and then about 30 000 high-quality amplicons per sample were grouped into operational taxonomic units for analyses.ResultsThe ratio of GF samples in which the bile acids were detectable was significantly greater in the FD than in the HC groups. In the bacterial composition analysis at the phylum level, the GF microbiota had a Bacteroidetes > Proteobacteria abundance and an absence of Acidobacteria in the FD group, in contrast, the GF microbiota had a Bacteroidetes < Proteobacteria abundance and the presence of Acidobacteria in the HC group. Probiotic therapy in patients with FD shifted the composition of the GF microbiota to that observed in the HC volunteers.ConclusionsAlteration in the GF microbiota was found in patients with FD compared with HC volunteers. Reflux of the small intestinal contents, including bile acid and intestinal bacteria, to the stomach was suggested to induce a bacterial composition change and be involved in the pathophysiology underlying FD. Probiotics appear effective in the treatment of FD through the normalisation of gastric microbiota.Trial registration numberUMINCTR 000022026; Results.

AGVD is the main cause of pseudolesions in the posterior edge of segment IV of the liver.

The current data show that propofol, mixed with clinical reagents (propofol MCT/LCT), resulted in the down-regulation of high oxidative stress due to scavenging hydroxyl radical, as demonstrated by in vitro or in vivo electron spin resonance analysis. These results led to reduced levels of hydroxyl radical, formed by brain injury such as stroke, and may therefore provide advantages for neuroprotection during anesthesia for craniotomy, e.g., in cases of brain disease.

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy-and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRL-RRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.Leukemias and lymphomas are hematological malignant diseases characterized by impaired differentiation, increased clonal cell proliferation, and hematopoiesis suppression; the standard treatment for these tumors today is still predominantly based on nonspecific cytotoxics that disrupt nucleic acid and protein synthesis, often with severe side effects and relatively poor outcomes (1-3). However, selective anti-leukemia drugs have recently been developed (4), thus conceptually validating the scientific hope for a revolutionary targeted pharmacology against this group of diseases.Over the past decade, we have selected phage-display random peptide libraries in vitro and in vivo to isolate and exploit tumor-specific and angiogenesis-related ligand-receptor systems toward targeted drug design and translation (5-7). Because cell trafficking and homing from the blood and/or lymphatic vessels to lymphoid and myeloid tissues to virtually all organs are essential leukocyte functions, we reasoned that targeting membranes would be a suitable approach to discover leukemia-specific ligands. Cell surface-binding peptide motifs have been reported in lymphoma and leukemia lines (8 -11). Unfortunately, so far their corresponding receptors are either unknown (9, 10) or relatively nonspecific adhesion molecules such as certain integrins (8, 11) to which ligand binding does not enable clear enough differentiation between normal leukocytes and tumor cells; ...

Objective: To assess whether low-dose, long-term maintenance interferon (IFN) therapy inhibits recurrence after complete ablation of hepatocellular carcinoma (HCC) and improves patient survival. Methods and Patients: From June 1999 through May 2006, a total of 127 HCC cases that met the requirements of both tumor diameter 3 cm or less, and number of tumors three or fewer, were curatively treated by radiofrequency ablation therapy (RFA). Among them, 43 patients received three million IU of IFN-α2b twice per week or pegylated IFN-α2a 90 µg once per week or once per 2 weeks without discontinuation (IFN maintenance group). The remaining 84 patients, whose sex, age, and platelet counts were randomly matched to those of the IFN maintenance group, did not receive IFN treatment (control group). Results: Cumulative first, second, and third recurrence rates were significantly reduced in the IFN maintenance group compared with the control group by Kaplan-Meier estimate. The 5-year survival rate was 66% for the control group and 83% for the IFN maintenance group (p = 0.004). Multivariate analysis using the Cox proportional hazards model identified IFN maintenance therapy as an independent risk factor for survival, and the risk ratio was 0.21 (95% CI: 0.05–0.73). In conclusion, low-dose, long-term maintenance IFN therapy after curative RFA therapy of HCC significantly inhibits recurrence, and consequently improves patient survival.