Hydroxyl-radical activated water for inactivation of Escherichia coli O157:H7, Salmonella and Listeria monocytogenes on germinating mung beans.

The following reports on the generation of hydroxyl-radical activated water prepared by passing a hydrogen peroxide solution containing Fe(III) catalyst through a UV-C reactor. The activated water was subsequently evaluated for antimicrobial activity against Escherichia coli O157:H7 in suspension or when inoculated onto mung beans. Hydroxyl-radical generation was assessed through the oxidation of methylene blue when reacted with activated water prepared from solutions of different pH (4-10), UV-C dose (32-128 mJ/cm2), hydrogen peroxide (0-1000 mg/L) and Fe(III) concentration (0-100 mg/L). Methylene blue oxidation was associated with high concentrations of each reactant with a positive correlation with Fe(III) concentration. Inactivation curves of E. coli O157:H7 in activated water were diphasic with an initial slow rate that increased after 15 min contact time. In contrast to the methylene blue assay, the antimicrobial action of activated water was associated with high hydrogen peroxide (500 mg/mL) and low Fe(III) catalyst (1 mg/L) with no significant interaction with UV-C dose. Evidence would suggest that the mode-of-inactivation was through a radical propagation reaction that is rate-limited by the reduction of Fe (III) to Fe (II). Here, the initial activation process via UV-C illumination results in photo-reduction of Fe(III) and propagates the formation of hydroxyl-radicals. Fe(III) to Fe(II) cycling continues with oxidation of cell structures that ultimately leads to loss of viability due to accumulation of cellular damage. When activated water was used to soak mung beans inoculated with E. coli O157:H7 a 1 log reduction was obtained with a 19% increase in germinated beans and 8.5% higher sprout yield relative to controls. The oxidation reduction potential decreased from 477 mV to 288 mV and pH increased from 3.97 to 5.47, over the 24 h mung bean soak period. The reduction of Salmonella and Listeria monocytogenes on mung beans soaked in activated water was <1 log CFU/g with all three pathogens growing back over the sprouting period. From the results it can be concluded that activated water can enhance the germination of mung beans along with sprout yield but has limited capacity when applied alone as a seed disinfection method.

Pre-oxidation of spent lettuce wash water by continuous Advanced Oxidation Process to reduce chlorine demand and cross-contamination of pathogens during post-harvest washing.

A continuous Photo-Fenton Advanced-Oxidation-Process (AOP) for reducing the chlorine-demand of spent lettuce wash water was developed based on the generation of hydroxyl-radicals from the UV-C degradation of hydrogen peroxide in the presence of ferric-catalyst. It was found that an interaction between UV-C and hydrogen peroxide or ferric-catalyst concentration was associated with high hydroxyl-radical generation as determined from the oxidation of methylene blue. The optimal AOP treatment was identified as 320 mJ/cm2 UV-C dose, 9.6 mg/L H2O2, and 9 mg/L ferric-catalyst. When the treatment was applied to simulated lettuce spent wash water (6.6 g romaine lettuce per liter of distilled water containing 100 mg bentonite; pH 6.9) the chlorine demand was reduced from 150 ppm to 130 ppm. The chlorination of AOP treated water did not result in a greater log reduction of pathogens (Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella) on lettuce but did reduce cross-contamination between batches during washing. The chlorinated byproducts formed in AOP treated water exhibited higher antimicrobial activity compared to untreated controls. Although the treatment was successful in reducing cross-contamination of lettuce batches the cytotoxicity of disinfection byproducts requires to be assessed.

C3 Assignment: Camera Cubemap Color Assignment for Creative Interior Design.

Color design for 3D indoor scenes is a challenging problem due to many factors that need to be balanced. Although learning from images is a commonly adopted strategy, this strategy may be more suitable for natural scenes in which objects tend to have relatively fixed colors. For interior scenes consisting mostly of man-made objects, creative yet reasonable color assignments are expected. We propose C3 Assignment, a system providing diverse suggestions for interior color design while satisfying general global and local rules including color compatibility, color mood, contrast, and user preference. We extend these constraints from the image domain to [Formula: see text], and formulate 3D interior color design as an optimization problem. The design is accomplished in an omnidirectional manner to ensure a comfortable experience when the inhabitant observes the interior scene from possible positions and directions. We design a surrogate-assisted evolutionary algorithm to efficiently solve the highly nonlinear optimization problem for interactive applications, and investigate the system performance concerning problem complexity, solver convergence, and suggestion diversity. Preliminary user studies have been conducted to validate the rule extension from 2D to 3D and to verify system usability.

Vapor-Phase Hydroxyl or Chlorine Radical Treatment for Inactivating Listeria monocytogenes on Mushrooms (Agaricus bisporus) without Negatively Affecting Quality or Shelf Life.

ABSTRACT: Processes based on generating vapor-phase hydroxyl radicals or chlorine radicals were developed for inactivating Listeria monocytogenes on mushrooms without negatively affecting quality. Antimicrobial radicals were generated from the UV-C degradation of hydrogen peroxide or hypochlorite and ozone gas. Response surface modeling was used to identify the interaction among the operating parameters for the hydroxyl radical process: UV-C254nm intensity, hydrogen peroxide concentration, and ozone delivered. There was an inverse relationship between hydrogen peroxide concentration and UV-C intensity in terms of the log reduction of L. monocytogenes. The independent parameters for the chlorine radical process were hypochlorite concentration, pH, and UV-C intensity. From predictive models, the optimal hydroxyl radical treatment was found to be 5% (v/v) H2O2, 2.86 mW/cm2 UV-C intensity (total UV-C dose 144 mJ/cm2), and 16.5 mg of ozone. The optimal parameters for the chlorine radical process were 10 ppm of hypochlorite (pH 3.0), 11.0 mg of ozone, and 4.60 mW/cm2 UV-C intensity. When inoculated mushrooms were treated with the optimal hydroxyl radical and chlorine radical processes, the reduction of L. monocytogenes was found to be 2.42 ± 0.42 and 2.61 ± 0.30 log CFU, respectively, without any negative effects on mushroom quality (weight loss and Browning index during 14 days of storage at 4°C). These reductions were significantly greater than those from application of the individual elements of the radical processes and those in the control process, which used a 90-s dip in 1% (v/v) hydrogen peroxide. The study has demonstrated that hydroxyl radical and chlorine radical vapor-phase treatments are equally effective at inactivating L. monocytogenes on mushrooms and can be considered as a preventative control step.

Development and validation of a novel 29-plex Y-STR typing system for forensic application.

Short tandem repeat within the male-specific part of the human Y chromosome (Y-STR) is an effective forensic tool in mixture identification, patrilineal relationship evaluation, and familial searches. Despite their usefulness, current Y-STR-based genotyping systems often lack the discriminatory power to resolve genetic relationships between distant relatives or within patrilocal populations. In this study, we developed a novel Y-STR 29-plex typing system, which combined the 17 Y-STR loci used in the AmpFLSTR® Yfiler® PCR Amplification Kit (Yfiler), eight Y-STR loci with a low-medium mutation rate, and four rapidly mutating Y-STR loci. The system was generated to achieve greater discriminatory power between male subjects and improved ability to infer haplogroup classifications. The system was extensively tested on data from 752 individuals for its sensitivity, male specificity, species specificity, mixture resolution, reproducibility, concordance, stutter and size accuracy, precision, and population genetics, following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The results demonstrated that the Y-STR 29-plex typing system was time-efficient, reproducible, accurate, sensitive, and robust to familial searching and paternal biogeographic ancestry inference.

Droplet-Transmitted Infection Risk Ranking Based on Close Proximity Interaction.

We propose an automatic method to identify people who are potentially-infected by droplet-transmitted diseases. This high-risk group of infection was previously identified by conducting large-scale visits/interviews, or manually screening among tons of recorded surveillance videos. Both are time-intensive and most likely to delay the control of communicable diseases like influenza. In this paper, we address this challenge by solving a multi-tasking problem from the captured surveillance videos. This multi-tasking framework aims to model the principle of Close Proximity Interaction and thus infer the infection risk of individuals. The complete workflow includes three essential sub-tasks: (1) person re-identification (REID), to identify the diagnosed patient and infected individuals across different cameras, (2) depth estimation, to provide a spatial knowledge of the captured environment, (3) pose estimation, to evaluate the distance between the diagnosed and potentially-infected subjects. Our method significantly reduces the time and labor costs. We demonstrate the advantages of high accuracy and efficiency of our method. Our method is expected to be effective in accelerating the process of identifying the potentially infected group and ultimately contribute to the well-being of public health.

Generation of integration-free induced pluripotent stem cells from a patient with spina bifida.

A skin biopsy was obtained from a 14-year-old female patient with a history of Myelomeningocele. Dermal fibroblasts were isolated and reprogrammed with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. The generated induced Pluripotent Stem Cell (iPSC) clones NTDi4_09A were free of genomically integrated reprogramming genes, had a stable normal karyotype and expressed pluripotency markers. The iPSCs formed teratomas in mice, which were differentiated towards derivatives of the three germ layers in vivo. This iPSC line offers a useful resource to study a genetic profile of a patient with spina bifida.

Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes.

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/-), and p53 homozygous mutant (p53-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/- and p53-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/- and p53-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES) cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs) transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc), without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs), iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin), cardiac alpha myosin heavy chain (α-MHC), cardiac troponin T (cTnT), and connexin 43 (CX43), as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5) and gata binding protein 4 (gata4). The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs) was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID) mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI) in the future.

Germ Cell Nuclear Factor (GCNF) Represses Oct4 Expression and Globally Modulates Gene Expression in Human Embryonic Stem (hES) Cells.

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.

Analysis of indoleamine 2-3 dioxygenase (IDO) and EGFR co-expression in breast cancer tissue by immunohistochemistry.

BACKGROUND: To determine the amount of co-expression of IDO and EGFR in breast cancer patients. MATERIALS AND METHODS: In order to obtain the distribution of co-expression of IDO and EGFR in breast cancer, we tested 110 breast cancer paraffin tissue blocks with immunohistochemical methods. Then we investigated the relationship between the diagnostic and pathologic characteristics (tumor size, lymph node status, histologic grade, the gene expression of ER, PR, HER2, p53, Ki67 and PCNA) with the situation of co-expression of IDO and EGFR by reviewing the medical records of 32 breast cancer patients. RESULTS: Among 110 breast cancers, 32 cases demonstrated IDO and EGFR co-expression (29.1%), IDO and EGFR synchronous co-expression being found in 19.1% and asynchronous in 10.0%. CONCLUSIONS: IDO and EGFR were co-expressed in breast cancer, including synchronous and asynchronous co-expression. The results suggest that considering IDO and EGFR as two indicators for breast cancer treatment or prognosis analysis provides a potential option of individual treatment for the portion of breast cancer patients with co-expression of IDO and EGFR.

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF(-) (/) (-) ) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3'UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF(-) (/) (-) ESCs restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF(-) (/) (-) ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a.

Induced multipotency in adult keratinocytes through down-regulation of ΔNp63 or DGCR8.

The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.

Chromatin changes in dicer-deficient mouse embryonic stem cells in response to retinoic acid induced differentiation.

Loss of Dicer, an enzyme critical for microRNA biogenesis, results in lethality due to a block in mouse embryonic stem cell (mES) differentiation. Using ChIP-Seq we found increased H3K9me2 at over 900 CpG islands in the Dicer(-/-)ES epigenome. Gene ontology analysis revealed that promoters of chromatin regulators to be among the most impacted by increased CpG island H3K9me2 in ES (Dicer(-/-)). We therefore, extended the study to include H3K4me3 and H3K27me3 marks for selected genes. We found that the ES (Dicer(-/-)) mutant epigenome was characterized by a shift in the overall balance between transcriptionally favorable (H3K4me3) and unfavorable (H3K27me3) marks at key genes regulating ES cell differentiation. Pluripotency genes Oct4, Sox2 and Nanog were not impacted in relation to patterns of H3K27me3 and H3K4me3 and showed no changes in the rates of transcript down-regulation in response to RA. The most striking changes were observed in regards to genes regulating differentiation and the transition from self-renewal to differentiation. An increase in H3K4me3 at the promoter of Lin28b was associated with the down-regulation of this gene at a lower rate in Dicer(-/-)ES as compared to wild type ES. An increase in H3K27me3 in the promoters of differentiation genes Hoxa1 and Cdx2 in Dicer(-/-)ES cells was coincident with an inability to up-regulate these genes at the same rate as ES upon retinoic acid (RA)-induced differentiation. We found that siRNAs Ezh2 and post-transcriptional silencing of Ezh2 by let-7 g rescued this effect suggesting that Ezh2 up-regulation is in part responsible for increased H3K27me3 and decreased rates of up-regulation of differentiation genes in Dicer(-/-)ES.

Epigenetic reprogramming of the germ cell nuclear factor gene is required for proper differentiation of induced pluripotent cells.

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that germ cell nuclear factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells recapitulated the phenotype of Gcnf knockout (Gcnf(-/-)) ES cells. Reintroduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf(-/-) ES cells rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells.

Generation of viable male and female mice from two fathers.

In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These male-derived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.