SpCas9 activity prediction by DeepSpCas9, a deep learning-based model with high generalization performance.

We evaluated SpCas9 activities at 12,832 target sequences using a high-throughput approach based on a human cell library containing single-guide RNA-encoding and target sequence pairs. Deep learning-based training on this large dataset of SpCas9-induced indel frequencies led to the development of a SpCas9 activity-predicting model named DeepSpCas9. When tested against independently generated datasets (our own and those published by other groups), DeepSpCas9 showed high generalization performance. DeepSpCas9 is available at http://deepcrispr.info/DeepSpCas9.

Paip1 Indicated Poor Prognosis in Cervical Cancer and Promoted Cervical Carcinogenesis.

PURPOSE: This study was aimed to investigate the role of poly(A)-binding protein-interacting protein 1 (Paip1) in cervical carcinogenesis. MATERIALS AND METHODS: The expression of Paip1 in normal cervical epithelial tissues and cervical cancer (CC) tissues were detected by immunohistochemistry. In vivo and in vitro assays were performed to validate effect of Paip1 on CC progression. RESULTS: Paip1 was found to be up-regulated in CC, which was linked with shorter survival. Knockdown of Paip1 inhibited cell growth, induced apoptosis and cell cycle arrest in CC cells, whereas its overexpression reversed these effects. The in vivo tumor model confirmed the pro-tumor role of Paip1 in CC growth. CONCLUSION: Altogether, the investigation demonstrated the clinical significance of Paip1 expression, which prompted that the up-regulated of Paip1 can presumably be a potential prognostic and progression marker for CC.

Overexpression and Implications of Melanoma-associated Antigen A12 in Pathogenesis of Human Cutaneous Squamous Cell Carcinoma.

BACKGROUND/AIM: Melanoma-associated antigen A12 (MAGEA12) has recently been reported as a repressor of tumor-suppressor genes. This study aimed to investigate the implications of MAGEA12 expression in the pathogenesis of cutaneous squamous cell carcinoma (cSCC). MATERIALS AND METHODS: MAGEA12 and p21 expression were investigated in 15 samples of normal skin and 111 of cSCC tissues by immunohistochemistry. The biological functions of MAGEA12 in cSCC were also investigated both in vitro and in vivo. RESULTS: Expression of both MAGEA12 and p21 was significantly increased in cSCC. MAGEA12 expression showed a positive correlation, while p21 expression showed negative correlation with the recurrence-free survival of patients with cSCC. In addition, MAGEA12 knockdown significantly attenuated proliferative, migratory, invasive, and tumorigenic activities of cSCC cells and was negatively correlated with p21 expression both in vitro and in vivo. CONCLUSION: MAGEA12-mediated down-regulation of p21 may be involved in cSCC pathogenesis and MAGEA12 may serve as a molecular biomarker in cSCC.

General rules for functional microRNA targeting.

The functional rules for microRNA (miRNA) targeting remain controversial despite their biological importance because only a small fraction of distinct interactions, called site types, have been examined among an astronomical number of site types that can occur between miRNAs and their target mRNAs. To systematically discover functional site types and to evaluate the contradicting rules reported previously, we used large-scale transcriptome data and statistically examined whether each of approximately 2 billion site types is enriched in differentially downregulated mRNAs responding to overexpressed miRNAs. Accordingly, we identified seven non-canonical functional site types, most of which are novel, in addition to four canonical site types, while also removing numerous false positives reported by previous studies. Extensive experimental validation and significantly elevated 3' UTR sequence conservation indicate that these non-canonical site types may have biologically relevant roles. Our expanded catalog of functional site types suggests that the gene regulatory network controlled by miRNAs may be far more complex than currently understood.

Human telomerase reverse transcriptase (hTERT) promotes cancer invasion by modulating cathepsin D via early growth response (EGR)-1.

Human telomerase reverse transcriptase (hTERT) contributes to tumor progression as well as maintaining telomere length, however, the mechanism by which hTERT promotes invasiveness is not yet completely understood. This study aims to unravel the precise mechanism through which hTERT promotes cancer invasion. We established an hTERT-overexpressed immortalized cell line (IHOK/hTERT). In orthotopic xenograft models, IHOK/hTERT harbors higher tumorigenicity than IHOK/Control. IHOK/hTERT showed much higher migration and invasion activities compared to IHOK/Control. IHOK/hTERT co-cultured with fibroblasts displayed increased invasion compared to IHOK/hTERT without fibroblasts. We screened for genes that play an important role in intermodulation between cancer cells and fibroblasts using a microarray and identified fibroblast activation protein (FAP). hTERT knockdown showed decreased expression of FAP and early growth response (EGR)-1, one of the transcriptional regulators of FAP in IHOK/hTERT and oral cancer cell line YD10B. Furthermore, EGR-1 knockdown in IHOK/hTERT and YD10B showed reduced invasion and reduced cathepsin D expression compared to Control-siRNA cells. Taken together, this study provides evidence that hTERT overexpression is responsible for the upregulation of the cysteine protease cathepsin D by regulating EGR-1 to activate invasiveness in cancer progression.

A distinct role for interleukin-6 as a major mediator of cellular adjustment to an altered culture condition.

Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial-mesenchymal transition (EMT)/ mesenchymal-epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7-transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL-6) from IHOK. We found IL-6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL-6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions.

RUNX3 confers sensitivity to pheophorbide a-photodynamic therapy in human oral squamous cell carcinoma cell lines.

Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, screening process should be preceded to enhance sensitivity to PDT. Thus, we investigated a molecular biomarker to determine the sensitivity to pheophorbide a (Pa)-PDT in immortalized human oral keratinocytes (IHOK) and oral squamous cell carcinoma (OSCC) cell lines. Two IHOK and several OSCC cell lines were used. After Pa-PDT, cell viability was reduced by more than 50%, and reactive oxygen species were generated in IHOK and OSCC cell lines. Additionally, apoptosis occurred in PDT-treated cells. IHOK(S) and IHOK(P), the two IHOK cell lines derived from the same source, showed a difference in cytotoxicity after Pa-PDT. To explain this difference in cytotoxicity, we looked at the expression of Wnt signaling-related genes in these two cell lines, for the morphology of IHOK(S) which was spindle like and elongated and distinct from IHOK(P) and the parent cell. Among the relevant genes, runt-related transcription factor 3 (RUNX3), an apoptosis-related gene, was selected as a potential marker that confers sensitivity to PDT. We found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Additionally, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT, suggesting that RUNX3 might be a biomarker to determine sensitivity to Pa-PDT. This was the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. Hence, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT on oral precancerous and cancerous lesions.

Human telomerase reverse transcriptase is a promising target for cancer inhibition in squamous cell carcinomas.

BACKGROUND/AIM: The present study aimed to investigate whether the down-regulation of human telomerase reverse transcriptase (hTERT) may induce an anti-invasive effect in oral squamous cell cancer cell lines. MATERIALS AND METHODS: A genetically-engineered squamous carcinoma cell line overexpressing hTERT in immortalized oral keratinocytes transfected by human papilloma virus (HPV)-16 E6/E7 (IHOK) was used. In vivo tumorigenicity was examined using an orthotopic xenograft model of nude mice. For evaluating anti-invasive activity by knockdown of hTERT expression, transwell invasion assay and real-time polymerase chain reaction (PCR) for matrix metalloproteinases (MMP) were employed. RESULTS: The down-regulation of hTERT expression reduced the invasive activity and MMP expression. This result was re-confirmed in the HSC3 oral squamous carcinoma cell line. CONCLUSION: Targeting hTERT may lead to novel therapeutic approaches.

Reciprocal interaction between carcinoma-associated fibroblasts and squamous carcinoma cells through interleukin-1α induces cancer progression.

Crosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) has earned recognition as an interaction that plays a pivotal role in carcinogenesis. Thus, we attempted to clarify whether increase in the level of CAFs promotes cancer progression by proportionally enhancing the interaction between cancer cells and CAFs. We first analyzed clinical correlation between the levels of fibroblasts and cancer progression and found that the level of CAFs made a noticeable difference on the prognosis of patients with oral squamous cell carcinoma (OSCC). In vivo animal study also demonstrated that tumor volume depended on the dose of CAFs that was co-injected with OSCC cells. The same tendency was observed in an in vitro study. We also found that interleukin-1α (IL-1α) secreted from OSCC cells had dual effects on CAFs: IL-1α not only promoted the proliferation of CAFs but also upregulated the secretion of cytokines in CAFs such as CCL7, CXCL1, and IL-8. The induction activity of cytokine secretion by IL-1α surpassed that of proliferation in OSCC cells. In summary, we unraveled an important interactive mechanism of carcinogenesis: IL-1α released from carcinoma stimulates the proliferation of CAFs and the simultaneous increase in cytokine secretion from CAFs promotes cancer progression in human OSCC. On the basis of these findings, we propose that the level of CAFs is eligible for being selected as a prognostic factor that will be useful in routine diagnosis. We also propose that blockage of reciprocal interaction between cancer cells and CAFs will provide an insight for developing novel chemotherapeutic strategy.

Effective vitrification of human induced pluripotent stem cells using carboxylated ε-poly-l-lysine.

Derivation of human induced pluripotent stem (iPS) cells could enable their widespread application in future. Establishment of highly efficient and reliable methods for their preservation is a prerequisite for these applications. In this study, we developed a vitrification solution comprising ethylene glycol (EG) and sucrose as well as carboxylated ε-poly-l-lysine (PLL); this solution inhibited devitrification. Human iPS cells were vitrified in 200-µL vitrification solutions comprised 6.5M EG, 0.75 M sucrose and 0 or 10%w/v carboxylated PLL with 65 mol% of the amino groups converted to carboxyl groups [PLL (0.65)] in a cryovial by directly immersing in liquid nitrogen. After warming, attached colony and recovery rates of human iPS cells vitrified by adding PLL (0.65) were significantly higher than those for cells without PLL (0.65) and vitrification solution (DAP213: 2M dimethyl sulfoxide, 1M acetamide and 3M propylene glycol). Furthermore, even after warming at room temperature, attached colony and recovery rates of iPS cells vitrified with PLL (0.65) were reduced to a lesser extent than those vitrified with either DAP213 or EG and sucrose without PLL (0.65). This could be attributed to inhibition of devitrification by PLL (0.65), as differential scanning calorimetry indicated less damage after vitrification with PLL (0.65). In addition, human iPS cells vitrified in the solution with PLL (0.65) had normal karyotypes and maintained undifferentiated states and pluripotency as determined by immunohistochemistry and teratoma formation. Addition of PLL (0.65) successfully vitrified human iPS cells with high efficiency. We believe that this method could aid future applications and increase utility of human iPS cells.

Biological and biomechanical evaluations of osteochondral allografts preserved in cold storage solution containing epigallocatechin gallate.

The beneficial effects of (-)-epigallocatechin-3-O-gallate (EGCG) on the nonfrozen preservation of mammalian cells and tissues are generally not well understood. A storage solution containing EGCG was employed to test the hypothesis that EGCG is capable of extending the storage duration for the cold preservation of articular cartilages. Human articular cartilages were preserved in a storage solution composed of serum-free RPMI-1640 medium with 1% antibiotic-antimycotic solution and 1 mM EGCG at 4°C for 1, 2, and 4 weeks. The chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The chondrocyte viability of the cartilages preserved with EGCG was significantly well maintained for at least 2 weeks with high content of GAG and total collagen. These beneficial effects of EGCG were confirmed by the immunohistochemical observations of well-preserved cartilaginous structures and delayed denaturation of the extracellular matrix in preserved cartilages. There was no significant difference in the compressive elastic modulus (MPa) between the cartilages preserved with and without EGCG. These results suggest that EGCG may play an effective role in preserving osteochondral allografts, which can be exploited in devising strategies for the long-term preservation of other tissues under cold storage conditions.

Polyampholytes as cryoprotective agents for mammalian cell cryopreservation.

Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. ɛ-poly-L-lysine (PLL) derivatives show higher cryopreservation efficiency than the conventional CPAs. Culture medium solutions with 7.5 w/w% of PLL whose amino groups of more than 50 mol% were converted to carboxyl groups by succinic anhydride showed higher postthaw survival efficiency of L929 cells than those of current CPAs without the addition of any proteins. In addition, rat mesenchymal stem cells were cryopreserved more effectively than with DMSO and fully retained the potential for proliferation and differentiation. Furthermore, many kinds of cells could be cryopreserved with PLL having the appropriate ratio of COOH groups, regardless of the cell types, including adhesive and floating cells, human- and mouse-derived cells, primary cells, and established cell lines. The properties might be associated with the antifreeze protein properties. These results indicate that these polymeric extracellular CPAs may replace current CPAs and the high viability after thawing and nonnecessity of serum ensure that these CPAs may be used in various preservation fields.

Nonfrozen preservation of articular cartilage by epigallocatechin-3-gallate reversibly regulating cell cycle and NF-kappaB expression.

Epigallocatechin-3-O-gallate (EGCG) is known to have beneficial effects on the nonfrozen preservation of mammalian cells and tissues. In this study, we aimed at testifying the hypothesis that the deleterious effects of cold preservation of articular cartilages can be ameliorated by the addition of EGCG to the storage media. Articular cartilages were preserved in a storage solution composed of serum-free RPMI 1640 media with 1 mM EGCG at 4 degrees C for 1-4 weeks. The regulatory effects of EGCG on cell cycle progression as well as expression levels of CyCliNS (CCNs) and nuclear factor-kappaB (NF-kappaB) were investigated in articular chondrocytes. Chondrocyte viability of cartilages preserved with EGCG was significantly well maintained for 2 weeks with high contents of glycosaminoglycan and total collagen. These beneficial effects of EGCG were confirmed by histological and immunohistochemical observations showing well-preserved cartilaginous structures and delayed denaturation of extracellular matrices. The compressive elastic modulus of cartilages preserved with EGCG was close to that of fresh specimens. Increased cell population at the G(0)/G(1) phase by EGCG returned to the normal level after EGCG removal, whereas decrease at the G(2)/M phase did not. Negatively regulated expression of CCND1, CCNE2, or NF-kappaB in EGCG-treated cells was restored by removing EGCG, but not CCNA2 and CCNB1. After 8 weeks of in vivo implantation into full-thickness cartilage defects in rabbits, the cartilages preserved with EGCG were found to be integrated with the host environment and support tissue regeneration. It is suggested that EGCG plays effective roles in preserving and repairing articular cartilages by reversibly regulating cell cycle at G(0)/G(1) phase and NF-kappaB expression.

Beneficial storage effects of epigallocatechin-3-o-gallate on the articular cartilage of rabbit osteochondral allografts.

A fresh osteochondral allograft is one of the most effective treatments for cartilage defects of the knee. Despite the clinical success, fresh osteochondral allografts have great limitations in relation to the short storage time that cartilage tissues can be well-preserved. Fresh osteochondral grafts are generally stored in culture medium at 4 degrees C. While the viability of articular cartilage stored in culture medium is significantly diminished within 1 week, appropriate serology testing to minimize the chances for the disease transmission requires a minimum of 2 weeks. (-)-Epigallocatechin-3-O-gallate (EGCG) has differential effects on the proliferation of cancer and normal cells, thus a cytotoxic effect on various cancer cells, but a cytopreservative effect on normal cells. Therefore, a storage solution containing EGCG might extend the storage duration of articular cartilages. Rabbit osteochondral allografts were performed with osteochondral grafts stored at 4 degrees C in culture medium containing EGCG for 2 weeks and then the clinical effects were examined with macroscopic and histological assessment after 4 weeks. The cartilaginous structure of an osteochondral graft stored with EGCG was well-preserved with high cell viability and glycosaminoglycan (GAG) content of the extracellular matrix (ECM). After an osteochondral allograft, the implanted osteochondral grafts stored with EGCG also provided a significantly better retention of the articular cartilage with viability and metabolic activity. These data suggest that EGCG can be an effective storage agent that allows long-term preservation of articular cartilage under cold storage conditions.

Preservation of platelets by adding epigallocatechin-3-o-gallate to platelet concentrates.

The effect of epigallocatechin-3-O-gallate (EGCG), a major component of green tea, on platelet preservation was evaluated. Single donor platelets (N = 10) were collected and preserved by the standard method. EGCG was added to the platelet concentrates before preservation and then the functional and biochemical parameters were monitored throughout the storage period. After 6 days of preservation, the aggregability of the platelets was significantly maintained by addition of 50 and 100 microg/ml of EGCG. Platelet prothrombinase activity was also significantly retained by the addition of EGCG. The accumulation of P-selectin and RANTES in the plasma preserved with EGCG was less than those preserved without EGCG, which indicated that EGCG might inhibit platelet activation. Furthermore, EGCG reduced the increase of LDH in plasma during preservation and inhibited the activation of caspase-3 and cleavage of gelsolin, thereby showing that EGCG could inhibit the apoptosis of platelets. These results suggest that EGCG may play an effective role in preserving platelets by inhibiting the activation and apoptosis of platelets.

Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts.

We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G(2)/M phases of cell cycle with concomitant increase in that of the G(0)/G(1) phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.

A Population-Based Case-Control Study on the Risk Factors of Congenital Heart Malformations / 한국역학회지

The multifactorial hypothesis is proposed as a working hypothesis which encompass both the genetic and environmental factors known to participate in the etiology of congenital heart malformations. So, it is believed that avoidance of suspected environmental factors in early pregnancy is the most certain preventive measure of congenital heart malformations. This study has been undertaken in order to find the possible environmental risk factors for congenital heart malformations in Korea. A total of 114 mothers of first graders of the elementary schools with congenital heart malformations confirmed through the screening program in Kyonggi Province from 1992 to 1995 were included as cases. And 206 mothers of healthy students matched by sex and elementary schools comprised the control group. Environmental risk factors including drug use during the first trimester of pregnancy, and other confounders were collected by telephone interview using standardized questionnaires by well trained interviewers. The result of multivariate logistic regression analysis showed that congenital heart malformation were associated with family history of congenital heart malformations(OR=2.94, 95% CI: 1.08, 7.96), the order of birth(OR=0.49, 95% CI: 0.31, 0.79). And the coffee consumption over 14 cups/week during early pregnancy showed marginal significance(OR=3.52, 95%CI: 0.98, 12.62). The mother's age at the subject birth and father's smoking at home were significant in linear trend test(p<0.05). It is recommended that the genetic counselling and the avoidance of known environmental risk factors in early pregnancy were needed to prevent congenital heart malformations.

A Day Care Model for Rehabilitation of Chronic Psychotic Patients / 신경정신의학

OBJECTIVES: The purpost of this study was to present general system,operation,and program of a day care model managed by a psychiatrist,to evaluate the results of performing this model,and thus to developa day care model applied to Korean situation appropriately for promoting maintenance and rehabilitation of chronic psychotic patients. METHODS: We performed this day care model(hereinafter called as this model) from March 1997 to February 1998. The subjects were composed of 23 psychotic patients. PANSS, Quality of Life Scale,Life Satisfaction Self-Rating Scale were used before beginning this model-and after three months of performing this model. Program Helpfulness Scale,13 Therapeutic Factors Scale were used at one month of day care and after three months of day care. RESULTS: 1) This model helped maintenance and rehabilitation of chronic psychotic patients through reducing their symptoms and increasing quality of life. 2) This model used the various group therapy, especially creative art therapy and activeity therapy, played an important fole,for recovering Physical,psychological,and social functions. 3) Important therapeutic factors at early and late period of this model were identification with therapists,guidance of therapists,and the corrective recatitulation of the primary family group. Important therapeutic factors at early period were group cohesiveness and altruism, and at late period instillation of hope. 4) This model was useful for integration the multidisciplinary therapeutic team and the various therapeutic methods. CONCLUSION: This model would be applied to day care in this country effectively and efficiently for maintenance and rehabilitation of chronic psychotic patients.

The Community Mental Health Project of Kyonggi Province

No abstract available.

Indigenous Malaria Surveillance in Korea / 한국역학회지

Malaria, one of the compulsory notifiable diseases, has been diappeared from Korea based on that fact no notification on malaria case was received from local health agencies during the last decade or so. Recently, Indigenous malaria has been re-emerged since 1993 and 549 cases was notificated till 1996. We conducted a surveillance system on the resurgent malaria outbreaks in the northern area of Kyonggi Province around the Imjin River. Malaria Surveillance Networks(MSNs) were established in Paju and Yoncheon between August 1996 and December 1996. When a febrile patient visits a clinic or a hospital, clinician takes a blood sample and refer to district malaria laboratory for the sample. The blood sample is examined in the malaria laboratory(public health center), and if malaria parasites are found, a radical or curative treatment is offered to patients. MSNs took 94 febrile cases and identified 23 malaria cases(24.5%). All malaria cases were infected by the indigenous vivax malaria. In Paju, 14 of 62 febrile cases(22.6%) were malaria outbreaks and 9 of 32 febrile cases(28.1%) in Yoncheon. In Korea resurgent malaria, malaria surveillance system should be operated for a program based on the district public health center with the coupled laboratory and dispensary.