Different Mechanisms for Visual Attention at the Hand-movement Goal and Endogenous Visual Attention.

Visual perception is closely related to body movements and action, and it is known that processing visual stimuli is facilitated at the hand or at the hand-movement goal. Such facilitation suggests that there may be an attentional process associated with the hands or hand movements. To investigate the underlying mechanisms of visual attention at a hand-movement goal, we conducted two experiments to examine whether attention at the hand-movement goal is a process independent from endogenous attention. Endogenous attention is attention that is intentionally focused on a location, feature, or object. We controlled the hand-movement goal and endogenous attention separately to investigate the spatial profiles of the two types of attention. A visual target was presented either at the goal of hand movement (same condition) or at its opposite side (opposite condition) while steady-state visual-evoked potential (SSVEP) was used to estimate the spatial distributions of the facilitation effect from the 2 types of attention around the hand-movement goal and around the visual target through EEG. We estimated the spatial profile of attentional modulation for the hand-movement goal by taking the difference in SSVEP amplitude between conditions with and without hand movement, thereby obtaining the effect of visual endogenous attention alone. The results showed a peak at the hand-movement goal, independent of the location of the visual target where participants intentionally focused their attention (endogenous attention). We also found differences in the spatial extent of attentional modulation. Spatial tuning was narrow around the hand-movement goal (i.e., attentional facilitation only at the goal location) but was broadly tuned around the focus of endogenous attention (i.e., attentional facilitation spreading over adjacent stimulus locations), which was obtained from the condition without hand movement. These results suggest the existence of two separate mechanisms, one underlying the attention at the hand-movement goal and another underlying endogenous attention.

Tumor-Specificity, Neurotoxicity, and Possible Involvement of the Nuclear Receptor Response Pathway of 4,6,8-Trimethyl Azulene Amide Derivatives.

BACKGROUND: Very few papers covering the anticancer activity of azulenes have been reported, as compared with those of antibacterial and anti-inflammatory activity. This led us to investigate the antitumor potential of fifteen 4,6,8-trimethyl azulene amide derivatives against oral malignant cells. METHODS: 4,6,8-Trimethyl azulene amide derivatives were newly synthesized. Anticancer activity was evaluated by tumor-specificity against four human oral squamous cell carcinoma (OSCC) cell lines over three normal oral cells. Neurotoxicity was evaluated by cytotoxicity against three neuronal cell lines over normal oral cells. Apoptosis induction was evaluated by Western blot and cell cycle analyses. RESULTS: Among fifteen derivatives, compounds 7, 9, and 15 showed the highest anticancer activity, and relatively lower neurotoxicity than doxorubicin, 5-fluorouracil (5-FU), and melphalan. They induced the accumulation of a comparable amount of a subG1 population, but slightly lower extent of caspase activation, as compared with actinomycin D, used as an apoptosis inducer. The quantitative structure-activity relationship analysis suggests the significant correlation of tumor-specificity with a 3D shape of molecules, and possible involvement of inflammation and hormone receptor response pathways. CONCLUSIONS: Compounds 7 and 15 can be potential candidates of a lead compound for developing novel anticancer drugs.

A case of intussusceptions at two parts of the ileum caused by an ileus tube.

An 87-year-old woman was admitted to our hospital for paralytic ileus, and she was treated using an ileus tube. Although her symptoms improved, abdominal fullness developed again on day 3 after ileus tube insertion. Abdominal computed tomography indicated intussusceptions at the ileum and the terminal part of the ileum;therefore, an emergency surgery was performed. During the surgery, antegrade intussusceptions were found in the ileum 60cm from the ileocecal valve and the terminal part of the ileum into the ascending colon. The intussusception of the anal side was resolved by manual reduction, but the oral side needed a partial resection of small bowel because of the presence of necrosis. There were no lesions, such as tumors, at the intussusceptions sites. Therefore, the two intussusceptions were thought to be caused by the ileus tube. We diagnosed a rare case of intussusceptions in the two parts of the ileum as a complication of the placement of an ileus tube.

ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations.

Age-associated telomere shortening in mouse oocytes.

BACKGROUND: Oocytes may undergo two types of aging. The first is induced by exposure to an aged ovarian microenvironment before being ovulated, known as 'reproductive or maternal aging', and the second by either a prolonged stay in the oviduct before fertilization or in vitro aging prior to insemination, known as 'postovulatory aging'. However, the molecular mechanisms underlying these aging processes remain to be elucidated. As telomere shortening in cultured somatic cells triggers replicative senescence, telomere shortening in oocytes during reproductive and postovulatory aging may predict developmental competence. This study aimed to ascertain the mechanisms underlying altered telomere biology in mouse oocytes during reproductive and postovulatory aging. METHODS: We studied Tert expression patterns, telomerase activity, cytosolic reactive oxygen species (ROS) production, and telomere length in fresh oocytes from young versus reproductively-aged female mice retrieved from oviducts at 14 h post-human chorionic gonadotropin (hCG), in vivo or in vitro postovulatory-aged mouse oocytes at 23 h post-hCG. Oocytes were collected from super-ovulated C57BL/6 J mice of 6-8 weeks or 42-48 weeks of age. mRNA and protein expressions of the Tert gene were quantified using real-time quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and immunochemistry. Telomerase activity was measured by a telomeric repeat amplification protocol assay, while telomere length was measured by Q-PCR and quantitative fluorescence in situ hybridization analyses. RESULTS: The abundance of Tert expression in oocytes significantly decreased during reproductive and postovulatory aging. Immunofluorescent staining clearly demonstrated an altered pattern and intensity of TERT protein expression in oocytes during reproductive aging. Furthermore, relative telomerase activity (RTA) in oocytes from reproductively-aged females was significantly lower than that in oocytes from young females. In contrast, RTA in postovulatory-aged oocytes was similar to that in fresh oocytes. Oocytes from reproductively-aged females and postovulatory-aged oocytes showed higher ROS levels than oocytes from young females. Relative telomere length (RTL) was remarkably shorter in oocytes from reproductively-aged females compared to oocytes from young females. However, postovulatory aging had no significant effect on RTL of oocytes. CONCLUSIONS: Long-term adverse effects of low telomerase activity and increased ROS exposure are likely associated with telomere shortening in oocytes from reproductively-aged female mice.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

Six new secoiridoids from the dried fruits of Ligustrum lucidum.

Six new secoiridoid constituents, named isoligustrosidic acid (1), 6'-O-trans-cinnamoyl 8-epikingisidic acid (2), 6'-O-cis-cinnamoyl 8-epikingisidic acid (3), oleopolynuzhenide A (4), nuzhenals A (5) and B (6) were isolated from the dried fruits of Ligustrum lucidum AIT. Their structures were established on the basis of spectral and chemical data.

[Indications for and postoperative problems of distal pancreatectomy with en bloc celiac axis resection for locally advanced pancreatic body cancer].

To treat locally advanced cancer of the pancreatic body involving the common hepatic artery and/or celiac axis with perineural invasion in the nerve plexus surrounding these arteries, we have employed distal pancreatectomy with en bloc celiac axis resection (DP-CAR) without arterial reconstruction. DP-CAR has been performed in patients in whom the gastroduodenal artery and superior mesenteric artery could be preserved. Between April 1998 and December 2007, 37 patients underwent DP-CAR in our institution. The surgical margins were histologically clear (R0) in 35 (95%) patients. The postoperative morbidity rate was 59%. The primary complications were pancreatic fistula occurring in 19 patients and ischemic gastropathy in 5. Estimated overall 1- and 5-year survival rates were 72% and 17%, respectively, and the median survival was 21 months. The most common site of recurrence was the liver, where recurrence appeared significantly earlier than in other metastatic sites. DP-CAR, with its potential to achieve complete local control, has been confirmed to be advantageous only in cases that are unlikely to develop hepatic metastasis. In principle, since 2006 patients who have undergone DP-CAR also receive postoperative adjuvant chemotherapy. Patients must achieve feasible general status within 3 months after DP-CAR to be able to start adjuvant chemotherapy.

A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. RESULTS: Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. CONCLUSIONS: For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Maintenance of pluripotency and self-renewal ability of mouse embryonic stem cells in the absence of tetraspanin CD9.

We have previously demonstrated that the tetraspanin CD9 is necessary for membrane fusion between sperm and oocyte during fertilization. While knockout mice for CD9 are viable, CD9(-/-) females are sterile due to the inability of their oocytes to fuse with sperm. While CD9 is not essential for subsequent development, a role in embryonic stem (ES) cell self-renewal was hypothesised on the basis of two observations: CD9 is highly expressed in murine and human ES cells and the CD9-blocking antibody inhibits mouse ES cell colony formation and survival. To investigate whether CD9 has a direct effect on ES cells, we generated and characterised several CD9 knockout murine ES cell lines. These CD9(-/-) ES cell lines exhibited equivalent morphology and growth properties to wild-type ES cells. Furthermore, the CD9(-/-) ES cell lines also displayed similar expression of pluripotency factors Oct3/4, Sox2 and Nanog. CD9(-/-) ES cells were found to be pluripotent in vivo, as their cells injected into immunocompromised mice gave rise to teratomas consisting of tissues representative of all three germ layers. Additionally several high contribution mouse chimeras were generated by blastocyst injection with several CD9(-/-) ES cell lines. Taken together, our results reveal that CD9 is dispensable for mouse ES cell self-renewal and pluripotency. The generation of CD9(-/-) ES cells should prove to be a useful tool with which to study the function of this protein and a range of other associated cellular processes.

The hsa-miR-302 cluster controls ectodermal differentiation of human pluripotent stem cell via repression of DAZAP2.

INTRODUCTION: Recent studies have revealed that microRNAs (miRNAs, miRs) are important for self-renewal, differentiation, and cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC); however, their functional roles and target genes that are regulated by human PSC-specific miRs including hsa-miR-302 clusters remain largely unknown. Analysis of their target gene will give us the opportunity to understand the functional roles of such miRs. METHODS: We analyzed the expression profiles of miRs in 4 somatic cell lines, 8 human iPSC lines derived from 4 different cell types, 3 human ESC lines, and embryoid bodies differentiated from the human ESCs to identify human PSC-specific miRs. We also analyzed the simultaneous expression profiles of miRs and mRNAs to identify candidate targets of human PSC-specific miRs. Then, we constructed a vector for overexpressing one of the target gene to dissect the functions of human PSC-specific miR in maintenance of self-renew and differentiation. RESULTS: We focused on hsa-miR-302 cluster as a human PSC-specific miR and identified 22 candidate targets of hsa-miR-302 cluster that were moderately expressed in undifferentiated human PSCs and up-regulated in differentiated cells. Deleted in azoospermia-associated protein 2 (DAZAP2), one such target, was directly repressed by hsa-miR-302a, -302b, -302c and -302d, but not by hsa-miR-367. Overexpression of DAZAP2 caused a decrease in cell proliferation of undifferentiated human iPSCs, although morphology and undifferentiated marker gene expression was not affected. In addition, neural differentiation was suppressed in DAZAP2-overexpressing human iPSCs. CONCLUSION: Our study revealed that hsa-miR-302 cluster controls the cell proliferation of human PSCs and the neural differentiation of human PSCs by repression of DAZAP2, thereby highlighting an additional function of human PSC-specific miRs in maintaining pluripotency.

Cobalamin deficiency results in an abnormal increase in L-methylmalonyl-co-enzyme-A mutase expression in rat liver and COS-7 cells.

The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.

SALL3 expression balance underlies lineage biases in human induced pluripotent stem cell differentiation.

Clinical applications of human induced pluripotent stem cells (hiPSCs) are expected, but hiPSC lines vary in their differentiation propensity. For efficient selection of hiPSC lines suitable for differentiation into desired cell lineages, here we identify SALL3 as a marker to predict differentiation propensity. SALL3 expression in hiPSCs correlates positively with ectoderm differentiation capacity and negatively with mesoderm/endoderm differentiation capacity. Without affecting self-renewal of hiPSCs, SALL3 knockdown inhibits ectoderm differentiation and conversely enhances mesodermal/endodermal differentiation. Similarly, loss- and gain-of-function studies reveal that SALL3 inversely regulates the differentiation of hiPSCs into cardiomyocytes and neural cells. Mechanistically, SALL3 modulates DNMT3B function and DNA methyltransferase activity, and influences gene body methylation of Wnt signaling-related genes in hiPSCs. These findings suggest that SALL3 switches the differentiation propensity of hiPSCs toward distinct cell lineages by changing the epigenetic profile and serves as a marker for evaluating the hiPSC differentiation propensity.

CXCL4/PF4 is a predictive biomarker of cardiac differentiation potential of human induced pluripotent stem cells.

Selection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening. We aimed to identify biomarkers for predicting cardiac differentiation potential of hiPSC lines by comparing the gene expression profiles of six undifferentiated hiPSC lines with different cardiac differentiation capabilities. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA array, and microRNA array to efficiently screen biomarkers related to cardiac differentiation of hiPSCs. Statistical analysis revealed candidate biomarker genes with significant correlation between the gene expression levels in the undifferentiated hiPSCs and their cardiac differentiation potential. Of the candidate genes, PF4 was validated as a biomarker expressed in undifferentiated hiPSCs with high potential for cardiac differentiation in 13 additional hiPSC lines. Our observations suggest that PF4 may be a useful biomarker for selecting hiPSC lines appropriate for the generation of cardiomyocytes.

Telomere protection mechanisms change during neurogenesis and neuronal maturation: newly generated neurons are hypersensitive to telomere and DNA damage.

Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes that play an important role in maintaining the integrity of the genome. In proliferative stem cells and cancer cells, telomere length is maintained by telomerase, and telomere structure and functions are regulated by telomere-associated proteins. We find that telomerase levels are high in embryonic cortical neural progenitor cells (NPCs) and low in newly generated neurons (NGNs) and mature neurons (MNs). In contrast, telomere repeat-binding factor 2 (TRF2) expression is undetectable in early brain development in vivo and in cultured NPCs and is expressed at progressively higher levels as NPCs cease proliferation and differentiate into postmitotic neurons. The telomere-disrupting agent telomestatin induces a DNA damage response and apoptosis in NGNs (which have low levels of TRF2 and telomerase), whereas NPCs (which have high levels of telomerase) and MNs (which have high levels of TRF2) are resistant to telomere damage. Overexpression of TRF2 in NGNs protects them against death induced by telomestatin and other DNA-damaging agents. Knockdown of TRF2 expression in MNs and knock-out of telomerase reverse transcriptase in NPCs increased their sensitivity to telomere- and DNA-damaging agents but did not affect the vulnerability of NGNs. These findings suggest that TRF2 and telomerase function as distinct telomere protection mechanisms during the processes of neurogenesis and neuronal maturation and that hypersensitivity of NGNs to telomere damage results from relative deficiencies of both telomerase and TRF2.

Tumorigenicity-associated characteristics of human iPS cell lines.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61-77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.

Resveratrol down-regulates the androgen receptor at the post-translational level in prostate cancer cells.

Androgen receptor (AR) functions as a transcriptional factor for the development and progression of prostate cancer. Resveratrol is known to inhibit the function of AR and to repress AR expression at the transcriptional level. This study focuses on the effects of resveratrol on the AR function and the post-translational AR level. Resveratrol repressed the transcriptional activities of a mutant AR lacking the ligand-binding domain, a constitutive active form of AR, and wild-type AR in a concentration-dependent manner in human prostate cancer PC-3 cells, indicating that resveratrol does not inhibit the transcriptional activity of AR through binding to the ligand-binding domain of AR. Furthermore, the half-life of AR protein was approximately 4 h in resveratrol-treated AR-positive prostate cancer LNCaP cells, compared to approximately 13 h in control cells, as determined by cycloheximide chase. These results indicate that resveratrol down-regulates AR protein through a post-translational mechanism and suggest that the inhibitory effect of resveratrol on AR function is partly attributable to a decrease in the post-translational AR level.

Randomized controlled trial of perioperative antimicrobial therapy based on the results of preoperative bile cultures in patients undergoing biliary reconstruction.

BACKGROUND: The high frequency of surgical site infections (SSIs) after hepato-pancreato-biliary (HPB) surgery is a problem that needs to be addressed. This prospective, randomized, controlled study examined whether perioperative prophylactic use of antibiotics based on preoperative bile culture results in HPB surgery could decrease SSI. METHODS: Participants comprised 126 patients who underwent HPB (bile duct, gallbladder, ampullary, or pancreatic) cancer surgery with biliary reconstruction at Hokkaido University Hospital between August 2008 and March 2013 (UMIN Clinical Trial Registry #00001278). Before surgery, subjects were randomly allocated to a targeted group administered antibiotics based on bile culture results or a standard group administered cefmetazole. The primary endpoint was SSI rates within 30 days after surgery. Secondary endpoint was SSI rates for each operative procedure. RESULTS: Of the 126 patients, 124 were randomly allocated (targeted group, n = 62; standard group, n = 62). Frequency of SSI after surgery was significantly lower in the targeted group (27 patients, 43.5%) than in the standard group (44 patients, 71.0%; P = 0.002). Among patients who underwent pancreaticoduodenectomy and hepatectomy, SSI occurred significantly less frequently in the targeted group (P = 0.001 and P = 0.025, respectively). CONCLUSIONS: This study demonstrated that preoperative bile culture-targeted administration of prophylactic antibiotics decreased SSIs following HBP surgery with biliary reconstruction.

A xenogeneic-free system generating functional human gut organoids from pluripotent stem cells.

Functional intestines are composed of cell types from all 3 primary germ layers and are generated through a highly orchestrated and serial developmental process. Directed differentiation of human pluripotent stem cells (hPSCs) has been shown to yield gut-specific cell types; however, these structures do not reproduce critical functional interactions between cell types of different germ layers. Here, we developed a simple protocol for the generation of mature functional intestinal organoids from hPSCs under xenogeneic-free conditions. The stem cell-derived gut organoids produced here were found to contain distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, that were derived from all 3 germ layers; moreover, they demonstrated intestinal functions, including peptide absorption, and showed innervated bowel movements in response to stimulation with histamine and anticholinergic drugs. Importantly, the gut organoids obtained using this xenogeneic-free system could be stably maintained in culture for prolonged periods and were successfully engrafted in vivo. Our xenogeneic-free approach for generating gut organoids from hPSCs provides a platform for studying human intestinal diseases and for pharmacological testing.

Sox2 expression defines a heterogeneous population of neurosphere-forming cells in the adult murine brain.

The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population, we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice, we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore, all neurospheres derived from these neurogenic regions express Sox2, suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain, with differing capacities for cell production. In vitro, all neurospheres express Sox2, but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example, both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres, but their expression is mutually exclusive; likewise, these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus, we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain, but that not all NSCs maintain the ability to form all neural cell types in vivo.