Comparative evaluation of MCP gene in worldwide strains of Megalocytivirus (Iridoviridae family) for early diagnostic marker.

Members of the Iridoviridae family have been considered as aetiological agents of iridovirus diseases, causing fish mortalities and economic losses all over the world. Virus identification based on candidate gene sequencing is faster, more accurate and more reliable than other traditional phenotype methodologies. Iridoviridae viruses are covered by a protein shell (capsid) encoded by the important candidate gene, major capsid protein (MCP). In this study, we investigated the potential of the MCP gene for use in the diagnosis and identification of infections caused Megalocytivirus of the Iridoviridae family. We selected data of 66 Iridoviridae family isolates (53 strains of Megalocytivirus, eight strains of iridoviruses and five strains of Ranavirus) infecting various species of fish distributed all over the world. A total of 53 strains of Megalocytivirus were used for designing the complete primer sets for identifying the most hypervariable region of the MCP gene. Further, our in silico analysis of 102 sequences of related and unrelated viruses reconfirms that primer sets could identify strains more specifically and offers a useful and fast alternative for routine clinical laboratory testing. Our findings suggest that phenotype observation along with diagnosis using universal primer sets can help detect infection or carriers at an early stage.

Effect of the sexual abstinence period recommended by the World Health Organization on clinical outcomes of fresh embryo transfer cycles with normal ovarian response after intracytoplasmic sperm injection.

This study was to investigate whether the sexual abstinence period (SAP) recommended by the World Health Organization (WHO) affects clinical outcomes. We compared the rate of clinical outcomes between 2-7 and ≥8 days of SAP in first fresh embryo transfer after intracytoplasmic sperm injection (ICSI) in groups of young maternal age (YMA: <38 years) and old maternal age (OMA: ≥38 years). We conducted a retrospective study of 449 first ICSI cycles with a normal ovarian response. SAP was identified before collecting the semen samples. Semen analysis was performed based on the guidelines recommended by WHO (2010). Sperm preparation was made using the swim-up method. Patients' baseline characteristics in the YMA and OMA groups did not differ. The rates of fertilisation, top-quality embryos on day 3, biochemical pregnancy, clinical pregnancy, ongoing pregnancy, abortion and implantation per cycle were not significantly different between 2-7 and ≥8 days of SAP in the YMA or OMA group. In conclusion, SAP beyond the recommended period by WHO was not associated with the rates of a lower fertilisation and pregnancy in human in vitro fertilisation (IVF). We think that a new criterion of SAP for clinical application in human IVF needs to be considered by WHO.

Generation of embryonic stem-like cells from in vivo-derived porcine blastocysts at a low concentration of basic fibroblast growth factor.

Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0 × 105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5 × 105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.

Investigation of Werner protein as an early DNA damage response in actinic keratosis, Bowen disease and squamous cell carcinoma.

BACKGROUND: Werner protein (WRN) has DNA helicase activity and participates in recombination, replication and repair of DNA. Loss-of-function mutations in WRN gives rise to genetic instability and diseases such as premature ageing and cancer. Upregulation of WRN promotes proliferation and survival of cancer cells. AIM: To evaluate the expression pattern of WRN in closely related skin cancers and their correlation with age, sex and UV exposure. METHODS: Immunohistochemistry was used to investigate expression of WRN in formalin-fixed, paraffin wax-embedded tissue specimens of 9 squamous cell carcinoma (SCC), 15 actinic keratosis (AK), 11 Bowen disease (BD) and 11 normal-appearing peripheral tissue samples, obtained from patients during surgical resections. RESULTS: WRN expression was significantly increased in BD, AK and SCC compared with normal controls, with the mean WRN staining score being highest in BD, followed by AK and SCC. However, age, sex and sun exposure were not associated with WRN expression. CONCLUSIONS: To our knowledge, this is the first report to date investigating the expression of WRN in skin cancers. The overtly high expression of WRN in premalignant lesions and in in situ cancer, with relatively low WRN expression in SCC, may indicate that WRN contributes as a checkpoint for early DNA damage response in skin tumorigenesis.

In Situ Photo-Crosslinkable Protein Bioadhesive for Bone Graft Fixation.

Bone grafting is a fundamental dental surgery procedure widely used for implant placement and periodontal disease management treatments. Despite its broad applications, vertical bone augmentation presents unique challenges, including the risk of graft displacement due to gravitational and masticatory forces. Traditional physical stabilization methods introduce additional complexities and risks, underscoring the need for innovative fixation technologies. This study aimed to develop an in situ photo-crosslinkable bioadhesive hydrogel (iPBAH) as a multifunctional bone graft binder to enhance the process of bone reconstruction. The bioadhesive is composed of mussel-derived adhesive protein (MAP) fused with the cell-adhesive peptide RGD. The numerous tyrosine residues in MAP facilitate rapid photo-crosslinking, enabling efficient hydrogel formation using visible blue light. Subsequently, iPBAH underwent comprehensive characterization to evaluate its suitability as a multifunctional bone graft binder. iPBAH efficiently underwent in situ crosslinking through harmless exposure to visible light within minutes and displayed several exceptional properties, including a microporous structure, underwater adhesion, extended durability, high compressive strength, and biocompatibility. In vivo assessments, using male Sprague-Dawley rats, demonstrated that iPBAH binder significantly enhanced bone regeneration in a rat calvarial bone defect model. The in situ crosslinking of the iPBAH binder during bone graft transplantation can effectively fill irregular and complex defect shapes while simultaneously preventing graft material leakage. The improved physical attributes of the bound graft material can enhance its resistance to external forces, thereby ensuring sustained retention over time. Moreover, the interaction between iPBAH and surrounding tissues promotes adhesion and integration of the graft material with host tissues in the defect area. In addition, the included RGD peptide in iPBAH can augment inherent cell recruitment, adhesion, and growth, consequently expediting osteogenesis.

Isolated acute cellular rejection of the liver after simultaneous liver and kidney transplantation: a case report.

Simultaneous liver and kidney transplantation (SLKT) is now considered the treatment of choice for patients with concurrent end-stage liver and kidney diseases. Even though the early postoperative mortality rate following SLKT is reported to be high compared to that of liver transplantation alone, the liver graft from the same donor has been argued to induce better kidney graft acceptance as evidenced by a low rate of acute renal rejection episodes. There have been many reports of a low incidence of acute renal rejection following SLKT; however, only a few cases were proven by simultaneous biopsies. The authors experienced a case of biopsy-proven isolated acute cellular rejection of the liver graft following SLKT.

Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells.

We examined the anti-invasive activity of ursolic acid (UA) on the highly metastatic HT1080 human fibrosarcoma cell line. UA reduced tumor cell invasion through a reconstituted basement membrane in a transwell chamber. A significant down-regulation of matrix metalloproteinase-9 [MMP-9; Mr 92,000 gelatinase/type IV collagenase (gelatinase B)] by UA was detected by Northern blot analysis. However, MMP-2 [Mr 72,000 gelatinase/type IV collagenase (gelatinase A)] and membrane-type MMP were constantly expressed, and the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 also was not changed after 3 and 6 days of treatment with UA. Quantitative gelatin-based zymography confirmed a markedly reduced expression of MMP-9 but not MMP-2 after treatment with UA. To confirm the UA-induced down-regulation of MMP-9 expression, we constructed a secreted alkaline phosphatase (SEAP) reporter vector including MMP-9 promoter. After transfection of MMP-9/SEAP reporter vector into HT1080 cells, reduced SEAP activity was detected after treatment with UA. These results suggest that down-regulation of MMP-9 contributes to the anti-invasive activity of UA in HT1080 cells.

Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells.

We have previously reported that ursolic acid, a pentacyclic triterpene acid, inhibited the invasion of HT1080 human fibrosarcoma cells by reducing the expression of matrix metalloproteinase-9. Since the chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether ursolic acid acts through the glucocorticoid receptor. The expression of matrix metalloproteinase-9 is thought to be regulated similarly with matrix metalloproteinase-1 and matrix metalloproteinase-3 as containing common 2-O-tetradecanoylphorbol-acetate responsible region, where AP-1 proteins can bind. Dexamethasone has been studied to repress the 2-O-tetradecanoylphorbol-acetate-induced expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 through a glucocorticoid receptor-mediated manner. In Northern blot analysis, we found that ursolic acid reduced the expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 induced by 2-O-tetradecanoylphorbol-acetate. Similarly, ursolic acid down-regulated 2-O-tetradecanoylphorbol-acetate-induction of matrix metalloproteinase-9 gene in the same manner of dexamethasone. RU486, a potent glucocorticoid receptor antagonist, was used for identifying that ursolic acid-induced down-regulation of matrix metalloproteinase-9 expression is mediated by its binding to glucocorticoid receptor. The effect of ursolic acid on the matrix metalloproteinase-9 expression was blocked by RU486, suggesting that ursolic acid acts via a glucocorticoid receptor in the regulation of matrix metalloproteinase-9. Western blot analysis and immunocytochemistry showed that ursolic acid increased glucocorticoid receptor fraction in the nucleus, although it decreased the synthesis of glucocorticoid receptor mRNA. In addition, ursolic acid did not decrease the expression of c-jun and DNA-binding activity of AP-1 to its cognate sequences. Taken together, we suggest that ursolic acid may induce the repression of matrix metalloproteinase-9 by stimulating the nuclear translocation of glucocorticoid receptor, and the translocated glucocorticoid receptor probably down-modulating the trans-activating function of AP-1 to 2-O-tetradecanoylphorbol-acetate responsible element of matrix metalloproteinase-9 promoter region.

Identification of cis-acting promoter elements that support expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) in v-src transformed Madin-Darby canine kidney cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) expressed in tumor cells is believed to be important for the pericellular degradation of extracellular matrices during invasion and metastasis. To analyze the mechanism by which MT1-MMP becomes expressed in cancer cells, we assessed the MT1-MMP promoter region for the presence of cis-acting promoter elements that support transcription in transformed cells. Our tumor model consisted of Madin-Darby canine kidney (MDCK) cells transformed by v-src (src4 cells). MT1-MMP mRNA was only faintly detected in parental cells but was strongly expressed in the src4 cells. In parallel, src4 cells invaded into collagen gels, whereas MDCK cells did not. When MDCK and src4 cells were transiently transfected with a plasmid containing of -3000 to -99 nt from the upstream region of the MT1-MMP gene, the promoter activity was 2.6-fold higher in src4 cells than in MDCK cells. Furthermore, the region between -399 and -356 nt was found to contain the src4-specific enhancer element(s). Tandem Sp1 binding sites were also found to be essential in promoting transcription. An Egr-1 site that partially overlaps with the Sp1 sites was found to cooperate with the src4-specific enhancer and to also contribute weakly to the basal promoter activity. The presence of transcription factors that bind to the src4-specific enhancer site was detected by mobility-shift assays in src4 cell nuclear extracts but only weakly in MDCK extracts. Thus, we have identified a novel enhancer element that acts specifically in the transformed cells to enhance MT1-MMP expression.

Glucocorticoid receptor-induced down-regulation of MMP-9 by ginseng components, PD and PT contributes to inhibition of the invasive capacity of HT1080 human fibrosarcoma cells.

We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HT1080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP-9 by PD and PT affects the invasive capacity of HT1080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HT1080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT. These results suggest that GR-induced down-regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HT1080 cells.

Enhancement of recombinant glucoamylase expression by introducing yeast GAL7 mRNA termination sequence.

Glucoamylase gene (STA1) of Saccharomyces diastaticus was expressed in recombinant Saccharomyces cerevisiae systems. The yeast, GAL7 mRNA termination sequence, was introduced in the 3' noncoding region of the STA1 structural gene which was under the control of the SUC2 promoter and STA1 secretion signal sequence. This plasmid was named YEpSSG7 and was introduced into yeast S. cerevisiae MMY2 to construct recombinant S. cerevisiae MMY2SSG7. The GAL7 mRNA termination sequence enhanced the glucoamylase expression level by 3-5 times depending on the culture conditions compared to the result from the strain S. cerevisiae MMY2SUCSTA which did not contain the GAL7 mRNA termination sequence. Such an enhancement was not due to plasmid stability or plasmid copy number effects. Such an enhancement was primarily due to the fact that GAL7 mRNA termination sequence stabilized the STA1 mRNA 3' end.

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells.

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand.

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro-D-mannitol.

Described herein is an efficient method for the synthesis of the sixteen positional isomers of methylated and acetylated or benzoylated 1,5-anhydro-D-mannitol. The compounds are generated simultaneously by partial methylation of 1,5-anhydro-D-mannitol and subsequent benzoylation, and the individual isomers are obtained in pure form by high-performance liquid chromatography. Debenzoylation of the latter and acetylation yielded the desired acetates. The the benzoates and the electron-ionization mass spectra of the acetates and the tetra-O-methyl derivative are reported herein as are the linear temperature-programmed gas-liquid chromatography retention indices of the acetates and the tetra-O-methyl derivative on three different capillary columns.

Production and secretion patterns of cloned glucoamylase in plasmid-harboring and chromosome-integrated recombinant yeasts employing an SUC2 promoter.

To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8- to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosome-integrated recombinant yeast culture was lower than that in the plasmid-harboring recombinant yeast culture owing to gene dosage effect.

Insulin concentration is critical in culturing human neural stem cells and neurons.

Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling.

Absolute energy calibration for relativistic electron beams with pointing instability from a laser-plasma accelerator.

The pointing instability of energetic electron beams generated from a laser-driven accelerator can cause a serious error in measuring the electron spectrum with a magnetic spectrometer. In order to determine a correct electron spectrum, the pointing angle of an electron beam incident on the spectrometer should be exactly defined. Here, we present a method for absolutely calibrating the electron spectrum by monitoring the pointing angle using a scintillating screen installed in front of a permanent dipole magnet. The ambiguous electron energy due to the pointing instability is corrected by the numerical and analytical calculations based on the relativistic equation of electron motion. It is also possible to estimate the energy spread of the electron beam and determine the energy resolution of the spectrometer using the beam divergence angle that is simultaneously measured on the screen. The calibration method with direct measurement of the spatial profile of an incident electron beam has a simple experimental layout and presents the full range of spatial and spectral information of the electron beams with energies of multi-hundred MeV level, despite the limited energy resolution of the simple electron spectrometer.

E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53.

Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53.