KRAS inhibition activates an actionable CD24 'don't eat me' signal in pancreas cancer.

KRAS G12C inhibitor (G12Ci) has produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the tumor biology of the KRAS G12C allele and possible bypass mechanisms, we developed a novel autochthonous KRAS G12C -driven PDAC model. Compared to the classical KRAS G12D PDAC model, the G12C model exhibit slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRAS G12C tumors produced modest impact despite stimulating a 'hot' tumor immune microenvironment. Immunoprofiling revealed that CD24, a 'do-not-eat-me' signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRAS G12D -driven PDAC. Our study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC. Significance: Lack of faithful preclinical models limits the exploration of resistance mechanisms to KRAS G12C inhibitor in PDAC. We generated an autochthonous KRAS G12C -driven PDAC model, which revealed allele-specific biology of the KRAS G12C during PDAC development. We identified CD24 as an actionable adaptive mechanisms in cancer cells induced upon KRAS G12C inhibition and blocking CD24 sensitizes PDAC to KRAS inhibitors in preclinical models.

Production of a 135-residue long N-truncated human keratinocyte growth factor 1 in Escherichia coli.

BACKGROUND: Palifermin (trade name Kepivance®) is an amino-terminally truncated recombinant human keratinocyte growth factor 1 (KGF-1) with 140 residues that has been produced using Escherichia coli to prevent and treat oral mucositis following radiation or chemotherapy. In this study, an amino-terminally shortened KGF-1 variant with 135 residues was produced and purified in E. coli, and its cell proliferation activity was evaluated. RESULTS: We expressed soluble KGF-1 fused to thioredoxin (TRX) in the cytoplasmic fraction of E. coli to improve its production yield. However, three N-truncated forms (KGF-1 with 140, 138, and 135 residues) were observed after the removal of the TRX protein from the fusion form by cleavage of the human enterokinase light chain C112S (hEKL C112S). The shortest KGF-1 variant, with 135 residues, was expressed by fusion with TRX via the hEKL cleavage site in E. coli and purified at high purity (> 99%). Circular dichroism spectroscopy shows that purified KGF-1135 had a structure similar to that of the KGF-1140 as a random coiled form, and MCF-7 cell proliferation assays demonstrate its biological activity. CONCLUSIONS: We identified variations in N-terminus-truncated KGF-1 and selected the most stable form. Furthermore, by a simple two-step purification, highly purified KGF-1135 was obtained that showed biological activity. These results demonstrate that KGF-1135 may be considered an alternative protein to KGF-1.

Inoculation of ACC deaminase-producing endophytic bacteria down-regulates ethylene-induced pathogenesis-related signaling in red pepper (Capsicum annuum L.) under salt stress.

Pathogenesis-related (PR) signaling plays multiple roles in plant development under abiotic and biotic stress conditions and is regulated by a plethora of plant physiological as well as external factors. Here, our study was conducted to evaluate the role of an ACC deaminase-producing endophytic bacteria in regulating ethylene-induced PR signaling in red pepper plants under salt stress. We also evaluated the efficiency of the bacteria in down-regulating the PR signaling for efficient colonization and persistence in the plant endosphere. We used a characteristic endophyte, Methylobacterium oryzae CBMB20 and its ACC deaminase knockdown mutant (acdS- ). The wild-type M. oryzae CBMB20 was able to decrease ethylene emission by 23% compared to the noninoculated and acdS- M. oryzae CBMB20 inoculated plants under salt stress. The increase in ethylene emission resulted in enhanced hydrogen peroxide concentration, phenylalanine ammonia-lyase activity, ß-1,3 glucanase activity, and expression profiles of WRKY, CaPR1, and CaPTI1 genes that are typical salt stress and PR signaling factors. Furthermore, the inoculation of both the bacterial strains had shown induction of PR signaling under normal conditions during the initial inoculation period. However, wild-type M. oryzae CBMB20 was able to down-regulate the ethylene-induced PR signaling under salt stress and enhance plant growth and stress tolerance. Collectively, ACC deaminase-producing endophytic bacteria down-regulate the salt stress-mediated PR signaling in plants by regulating the stress ethylene emission levels and this suggests a new paradigm in efficient colonization and persistence of ACC deaminase-producing endophytic bacteria for better plant growth and productivity.

High-level production of keratinocyte growth factor 2 in Escherichia coli.

Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.

Effect of Polydeoxyribonucleotide (PDRN) Treatment on Corneal Wound Healing in Zebrafish (Danio rerio).

This study aimed to develop a corneal epithelial injury model in zebrafish (Danio rerio) and investigate the effectiveness of polydeoxyribonucleotide (PDRN) treatment on in vivo corneal epithelial regeneration and wound healing. Chemical injury to zebrafish cornea was produced by placing a small cotton swab containing 3% acetic acid solution. PDRN treatment was performed by immersing corneal-injured zebrafish in water containing PDRN (2 mg/mL) for 10 min at 0, 24, 48, and 72 h post-injury (hpi). The level of corneal healing was evaluated by fluorescein staining, histological examination, transcriptional profiling, and immunoblotting techniques. Fluorescein staining results demonstrate that PDRN treatment significantly (p < 0.05) reduced the wounded area of the zebrafish eye at 48 and 72 hpi, suggesting that PDRN may accelerate the corneal re-epithelialization. Histopathological evaluation revealed that injured corneal epithelial cells were re-organized at 72 hpi upon PDRN treatment with increased goblet cell density and size. Moreover, transcriptional analysis results demonstrate that PDRN treatment induced the mRNA expression of adora2ab (6.3-fold), pax6a (7.8-fold), pax6b (29.3-fold), klf4 (7.3-fold), and muc2.1 (5.0-fold) after the first treatment. Besides, tnf-α (2.0-fold) and heat-shock proteins (hsp70; 2.8-fold and hsp90ab1; 1.6-fold) have modulated the gene expression following the PDRN treatment. Immunoblotting results convincingly confirmed the modulation of Mmp-9, Hsp70, and Tnf-α expression levels upon PDRN treatment. Overall, our corneal injury model in zebrafish allows for understanding the morphological and molecular events of corneal epithelial healing, and ophthalmic responses for PDRN treatment following acid injury in zebrafish.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Association of ST2 Elevation in the Early Third Trimester with Heart Failure and Pre-Eclampsia in the Peripartum Period.

Background: Although high-risk pregnancies are common in clinical practice, there are limited data on the association of soluble suppression of tumorigenicity 2 (ST2) with pregnancy-related complications. The rates of maternal complications, including heart failure (HF) during the peripartum period, were evaluated according to the ST2 level. Materials and Methods: A single-center retrospective cohort study included and stratified 259 women with high-risk pregnancies in their early third trimester according to the ST2 levels. The primary endpoint was the occurrence of peripartum HF based on symptoms, N-terminal pro-brain natriuretic peptide, or echocardiography associated with fluid retention. The secondary endpoints consisted of pre-eclampsia, silent pleural effusion, and pericardial effusion during the peripartum period. We performed a logistic model for the association between ST2 and maternal complications. Results: Of the 259 patients (mean age: 36.4 years, mean gestational duration: 31.6 weeks), advanced age ≥35 years and twin gestation were the most prevalent risk factors. Patients with ST2 ≥ 35 ng/mL showed enlarged cardiac chambers. Peripartum HF occurred in 2 (1.6%) out of 121 patients with ST2 < 35 ng/mL and in 47 (34%) out of 138 patients with ST2 ≥ 35 ng/mL. Those with ST2 ≥ 35 ng/mL were more likely to have the secondary endpoints (40.6% vs. 5.8%, p < 0.001). After adjustment, ST2 ≥ 35 ng/mL was associated with a six-fold occurrence of peripartum HF and a four-fold increase in the secondary endpoints. Conclusions: In women with high-risk pregnancies, peripartum HF and pre-eclampsia were not uncommon, and ST2 ≥ 35 ng/mL in the third trimester was independently related to maternal complications.

Protein Kinase B2 (PKB2/AKT2) Is Essential for Host Protection in CVB3-Induced Acute Viral Myocarditis.

Protein kinase B2 (AKT2) is involved in various cardiomyocyte signaling processes, including those important for survival and metabolism. Coxsackievirus B3 (CVB3) is one of the most common pathogens that cause myocarditis in humans. The role of AKT2 in CVB3 infection is not yet well understood. We used a cardiac-specific AKT2 knockout (KO) mouse to determine the role of AKT2 in CVB3-mediated myocarditis. CVB3 was injected intraperitoneally into wild-type (WT) and KO mice. The mice's survival rate was recorded: survival in KO mice was significantly decreased compared with WT mice (WT vs. KO: 73.3 vs. 27.1%). Myocardial damage and inflammation were significantly increased in the hearts of KO mice compared with those of WT mice. Moreover, from surface ECG, AKT2 KO mice showed a prolonged atria and ventricle conduction time (PR interval, WT vs. KO: 47.27 ± 1.17 vs. 64.79 ± 7.17 ms). AKT2 deletion induced severe myocarditis and cardiac dysfunction due to CVB3 infection. According to real-time PCR, the mRNA level of IL-1, IL-6, and TNF-α decreased significantly in KO mice compared with WT mice on Days 5 after infection. In addition, innate immune response antiviral effectors, Type I interferon (interferon-α and ß), and p62, were dramatically suppressed in the heart of KO mice. In particular, the adult cardiac myocytes isolated from the heart showed high induction of TLR4 protein in KO mice in comparison with WT. AKT2 deletion suppressed the activation of Type I interferon and p62 transcription in CVB3 infection. In cardiac myocytes, AKT2 is a key signaling molecule for the heart from damage through the activation of innate immunity during acute myocarditis.

Allograft reconstruction for large parosteal osteoma of the clavicle: a case report.

A large parosteal osteoma arising on the surface of the right clavicle of a 39-year-old male patient was suspected preoperatively as a parosteal osteosarcoma. The lesion was treated with wide resection and allograft reconstruction. In this case report, we discuss the accurate diagnosis and appropriate surgical treatment for unusual clavicular tumors.

Speech-aid prosthesis in velopharyngeal incompetency patient with cleft palate: can speech aids be applicable for adult patient?

BACKGROUND: Velopharyngeal incompetence (VPI) therapy for cleft palate (speech therapy alone, speech therapy using speech aids, or combined therapy such as speech therapy using a pharyngeal flap), is more effective in younger patients than in adult patients. Speech therapy is known as very difficult for patients who still have VPI as an adult. Because of the possibility of subsequent speech disorders, the timing of surgery for cleft palate is accelerating. Herein, we present a case of an adult with articulation disorder due to VPI who was treated by speech therapy and a speech-aid prosthesis. CASE PRESENTATION: A woman who underwent cleft palate surgery at 8 years of age still had difficulty with articulation due to VPI as a 24-year-old adult because of a lack of continuous speech therapy. We decided to use a speech-aid application using palatal lift, and a reduction program was conducted four times, along with simultaneous speech therapy, over a period of 1 year and 7 months. During the therapy period, she was able to speak normally within a relatively short period of time, and after implementation of the reduction program, the therapy was completed by completely removing the device. Long-term observations have shown normal speech function without recurrence, even after the device was removed. CONCLUSION: As seen in this case, speech therapy using speech aids can show a good result for adult patients with cleft palate who missed the usual timing for the treatment of articulation disorders, depending on the situation. Therefore, it is hereby reported as a therapy option worthy of consideration.

Specific elimination of coxsackievirus B3 infected cells with a protein engineered toxin-antitoxin system.

BACKGROUNDS: Coxsackievirus B3 (CVB3) is a member of the family Picornaviridae, and along with polio-viruses, belongs to the Enterovirus genus. The CVB3 genome is composed single-stranded RNA encoding polyproteins, which are cleaved to individual functional proteins by 2A and 3C proteases proteins which have been targeted for drug development. Here, we showed that protease activity required to activate a toxic protein may be used to prevent viral infection. METHODS: We modified the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal fragment of MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for CVB3. This fusion protein formed a stable dimer and was capable of inactivating the mRNA interferase activity of MazF which cleaves the ACA sequence in mRNA substrates. RESULTS: The incubation of 2A proteases with the fusion proteins induced cleavage between the MazE and MazF fragments from the fusion proteins; the subsequent release of MazF significantly inhibited virus replication. Additionally, we note that, CVB3 infected HeLa cells quickly died through a MazF toxin mediated effect before virus protein expression. CONCLUSION: These findings suggest that the MazEF fusion protein has a strong potential to be developed as an anti-virus therapy following CVB3 infection.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Efficacy of cortico/cancellous composite allograft in treatment of cervical spondylosis.

This was a prospective comparative study.The aim of this study was to compare the clinical and radiologic outcomes of patients treated with cortico/cancellous composite allograft or autoiliac bone graft in anterior cervical discectomy and fusion.Several methods have been developed to fuse the cervical spine for treatment of cervical spondylosis. Cortico/cancellous composite allograft might be another alternative.A total of 46 patients who underwent surgery for treatment of cervical spondylosis were evaluated between September 2010 and January 2015. The duration of operation, blood loss, perioperative complications, neck disability index (NDI), visual analogue scale (VAS), and fusion rates were compared between the 2 groups.There were no significant differences in clinical or radiologic outcomes between the patients treated with cortico/cancellous composite allograft and those treated with autoiliac bone graft. The 2 groups showed similar improvements in clinical symptoms and fusion rates. Although not statistically significant, the subsidence rate was lower in the cortico/cancellous composite group.Cortico/cancellous composite allograft is an effective alternative to conventional allograft or autograft in anterior cervical discectomy and fusion.

What Is the Role of Epidural Steroid Injections in Lumbar Spinal Disease with Moderate Disability?

UNLABELLED: Epidural steroid injections have been gaining popularity as an alternative to surgical treatment of radicular pain with associated spinal derangement. To determine the effectiveness and indications of lumbar epidural steroid injections in patients with or without surgery, we performed a prospective observational study. We gathered data from 262 degenerative short-segment spinal disease patients (affected at one or 2 levels) with greater than 12 weeks of medication-resistant radicular pain without neurological deficits but with moderate disability (visual analog scale < 6.5; Oswestry Disability Index < 35). All patients received initial fluoroscopically guided transforaminal epidural steroid injections of the affected vertebral level(s) corresponding to their symptoms. Those with inadequate responses or who wanted subsequently surgery underwent decompression surgery. Clinical and demographic characteristics were assessed to compare the differences between the groups. RESULTS: Of the 262 patients who received epidural steroid injections, 204 did not have operations for up to one year. However, 58 patients experienced inadequate relief of pain or wanted operations and therefore underwent surgery. At baseline, the 2 groups had similar mean disability indices and pain scores, as well as gender ratios, ages, and durations of symptoms (P > 0.05). In the patients who underwent surgery, the mean disability and pain scores were not significantly decreased after injection compared to those in the injection-alone group, although the scores for the injection plus surgery patients decreased significantly after surgery (P < 0.05). In contrast, patients who underwent epidural steroid injection alone experienced a significant decrease in disability and pain after injection, and that persisted up to one year of follow-up (P < 0.05). Epidural steroid injection can decrease the pain and disability in the majority of a moderate disability group for up to one year, although a significant number of patients underwent surgery regardless of injection. We recommend epidural steroid injection as a first-line treatment in patients with moderate disability that can be converted to surgery without significant delay. KEY WORDS: Epidural steroid injection, spinal surgery, lumbar spinal disease, lumbar radiculopathy, lumbar radicular pain.

Effect of Leukocyte-Rich and Platelet-Rich Plasma on Healing of a Horizontal Medial Meniscus Tear in a Rabbit Model.

There are limited reports on the effect of platelet-rich plasma (PRP) on meniscus healing. The purpose of this study was to investigate the effect of leukocyte-rich PRP (L-PRP) on potential healing of the horizontal medial meniscus tears in a rabbit model. A horizontal medial meniscus tear was created in both knees of nine skeletally mature adult rabbits. Left or right knees were randomly assigned to a L-PRP group, or a control group. 0.5 mL of L-PRP from 10 mL of each rabbit's whole blood was prepared and injected into the horizontal tears in a L-PRP group. None was applied to the horizontal tears in a control group. The histological assessment of meniscus healing was performed at two, four, and six weeks after surgery. We found that there were no significant differences of quantitative histologic scoring between two groups at 2, 4, and 6 weeks after surgery (p > 0.05). This study failed to show the positive effect of single injection of L-PRP on enhancing healing of the horizontal medial meniscus tears in a rabbit model. Single injection of L-PRP into horizontal meniscus tears may not effectively enhance healing of horizontal medial meniscus tears.

Nonvolatile memory thin-film transistors using biodegradable chicken albumen gate insulator and oxide semiconductor channel on eco-friendly paper substrate.

Nonvolatile memory thin-film transistors (TFTs) fabricated on paper substrates were proposed as one of the eco-friendly electronic devices. The gate stack was composed of chicken albumen gate insulator and In-Ga-Zn-O semiconducting channel layers. All the fabrication processes were performed below 120 °C. To improve the process compatibility of the synthethic paper substrate, an Al2O3 thin film was introduced as adhesion and barrier layers by atomic layer deposition. The dielectric properties of biomaterial albumen gate insulator were also enhanced by the preparation of Al2O3 capping layer. The nonvolatile bistabilities were realized by the switching phenomena of residual polarization within the albumen thin film. The fabricated device exhibited a counterclockwise hysteresis with a memory window of 11.8 V, high on/off ratio of approximately 1.1 × 10(6), and high saturation mobility (µsat) of 11.5 cm(2)/(V s). Furthermore, these device characteristics were not markedly degraded even after the delamination and under the bending situration. When the curvature radius was set as 5.3 cm, the ION/IOFF ratio and µsat were obtained to be 5.9 × 10(6) and 7.9 cm(2)/(V s), respectively.

ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase.

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases.

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.