Emulating trial to evaluate the effectiveness of routine supportive care on mortality among cancer patients experiencing distress at the time of diagnosis.

INTRODUCTION: Few studies have evaluated the effectiveness of interventions for distress during cancer diagnosis on clinical outcomes in a real-world setting. We aimed to evaluate whether routine information and psychosocial support to patients experiencing distress at the time of diagnosis could decrease the risk of mortality within 1 and 3 years after diagnosis. MATERIAL AND METHODS: We conducted a retrospective cohort study of 4880 newly diagnosed cancer patients who reported distress scores of ≥4 using the tablet or kiosk-based screening between July 2014 and December 2017 at a university-affiliated cancer center in Seoul, South Korea. We performed an emulated target trial with two groups: those that received information and psychosocial support and those that did not. Cox proportional hazards models were used to identify the associations between information and psychosocial support and all-cause mortality. RESULTS: Of all the patients, 16.6 % had routine information and psychosocial support. The hazard ratio (HR) for one-year mortality comparing participants with information and psychosocial support to those without it were 0.73 (95 % confidence interval (CI) = 0.54, 0.99). Age < 50 and 50 - <60 group had a stronger effect of information and psychosocial support on reducing mortality within one-year than these in age ≥ 60 (p for interaction = 0.03). In terms of three-year mortality, the HR comparing participants with information and psychosocial support to those without it was 0.93 (95 % CI = 0.76, 1.14). CONCLUSION: This large-scale real-world study suggests that timely psychosocial care benefits newly diagnosed cancer patients who had distress during pre-treatment period.

Highly tumor-specific DNA nanostructures discovered by in vivo screening of a nucleic acid cage library and their applications in tumor-targeted drug delivery.

Enormous efforts have been made to harness nanoparticles showing extravasation around tumors for tumor-targeted drug carriers. Owing to the complexity of in vivo environments, however, it is very difficult to rationally design a nanoconstruct showing high tumor specificity. Here, we show an approach to develop tumor-specific drug carriers by screening a library of self-assembled nucleic acid cages in vivo. After preparation of a library of 16 nucleic acid cages by combining the sugar backbone and the shape of cages, we screened the biodistribution of the cages intravenously injected into tumor-bearing mice, to discover the cages with high tumor-specificity. This tumor specificity was found to be closely related with serum stability, cancer cell uptake efficiency, and macrophage evasion rate. We further utilized the cages showing high tumor specificity as carriers for the delivery of not only a cytotoxic small molecule drug but also a macromolecular apoptotic protein exclusively into the tumor tissue to induce tumor-specific damage. The results demonstrate that our library-based strategy to discover tumor-targeted carriers can be an efficient way to develop anti-cancer nanomedicines with tumor specificity and enhanced potency.

Multistep regulation of autophagy by WNK1.

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.

BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression.

Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4), a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment.

Negative Pressure Wound Therapy on Closed Surgical Wounds With Dead Space: Animal Study Using a Swine Model.

BACKGROUND: Closed incisional wound surgery frequently leaves dead space under the repaired skin, which results in delayed healing. The purpose of this study was to evaluate the effect of negative pressure wound therapy (NPWT) on incisional wounds with dead space after primary closure by evaluating the fluid volume through the suction drain, blood flow of the skin, tensile strength, and histology of the wounds. METHODS: Bilateral 25-cm-long incisional wounds with dead space were created on the back of 6 pigs by partially removing the back muscle and then suturing the skin with nylon sutures. NPWT (experimental group) or gauze dressing (control group) was applied over the closed incision for 7 days. Analysis of the wound included monitoring the amount of closed suction drain, blood perfusion unit, tensile strength of the repaired skin, and histology of the incision site. RESULTS: The drainage amount was significantly reduced in the experimental group (49.8 mL) compared to the control group (86.2 mL) (P = 0.046). Skin perfusion was increased in the experimental group with statistical significance compared to the control group (P = 0.0175). Collagen staining was increased in the experimental group. The tensile strength of the incision site was significantly higher in the experimental group (24.6 N at 7 days, 61.67 N at 21 days) compared to the control group (18.26 N at 7 days, 50.05 N at 21 days) (P = 0.02). CONCLUSION: This study explains some of the mechanism for using NPWT in closed incision wounds with dead space. It demonstrates that NPWT significantly reduces drainage amount, increases skin perfusion, increases tensile strength, and has the tendency to promote collagen synthesis for closed wound with dead space indicating enhanced healing.

Anti-metastatic effects of Rheum Palmatum L. extract in human MDA-MB-231 breast cancer cells.

Rheum palmatum L. (RP) has been widely used in traditional medicine for the treatment of various diseases in Asian countries. The molecular mechanism of its anti-metastasis effect remains elusive. The present study assessed the effect of RP ethanol extract (RPE) on the highly metastatic human MDA-MB-231 breast cancer cells in vitro. At a non-toxic concentration, RPE inhibited migration, motility and invasion in a concentration-dependent manner. To investigate the mechanisms involved, real-time PCR and Western blot analyses were performed. Results showed that RPE down-regulated the levels of extracellular matrix degradation-associated proteins, including MMP-2/-9, uPA and uPAR, and up-regulated PAI-1. In addition, RPE affected NF-κB by degrading IkBα, and affected the mitogen-activated protein kinase signal transduction pathway by depressing the activation of p38, ERK and Akt. These results suggest that RPE has potential anti-metastatic activity and warrants further investigation.

Enhanced osteoinductivity of recombinant human bone morphogenetic protein-2 in combination with epidermal growth factor in a rabbit tibial defect model.

This study aims to explore the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone formation when treated with epidermal growth factor (EGF) using human mesenchymal stem cells (hMSCs) and a rabbit tibial defect model. The rhBMP-2 (250 ng/ml)+EGF (10 ng/ml) group showed higher alkaline phosphatase (ALP) activity, ALP expression, increased calcium amount than rhBMP-2 group. In micro-CT and histology results of animal experiments, the rhBMP-2+EGF group showed more amount of bone bridging compared to the rhBMP-2 group. Among the 8-week groups, the rhBMP-2+EGF group showed significantly higher percent bone volume and trabecular number compared to the rhBMP-2 group. The combined treatment with EGF and rhBMP-2 induced significantly higher bone formation compared to that of rhBMP-2 only in both hMSCs and a rabbit tibial defect model. Therefore, EGF is expected to facilitate bone formation effect of rhBMP-2 when both factors are treated in combination.

A8, an anti-uPA agonistic antibody, promotes metastasis of cancer cells via ERK pathway.

Urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) are expressed in many tumors and have been reported to be correlated to protein expression and poor prognosis in malignant tumors. In a previous study, we reported on the selection of human single-chain variable fragment (scFv) A8 specific to uPA from phage-displayed human naïve scFv library. In this study, scFv A8 was converted to minibody form and evaluated for its functional ability on the uPA system involved in cellular signaling and cancer cell metastasis. A8 minibody increased enzyme activity of uPA and enhanced the migration and invasion of HT1080 colon cancer cells in a dose-dependent manner. A8 increased ERK phosphorylation, and enhanced migration was blocked by U0126, but not by LY0294002, SB2203580, and SP600125. A8 minibody also enhanced migration of MDA-MB231 by mediated expressing surface uPA, but not that of MCF-7 non-expressing surface uPA. Taken together, the A8 anti-uPA antibody is a uPA agonistic antibody, enhancing migration and invasion of cancer cells that express uPA via activation of ERK pathway.

The effects of recombinant human bone morphogenetic protein-2-loaded tricalcium phosphate microsphere-hydrogel composite on the osseointegration of dental implants in minipigs.

Bone formation in tooth defect areas and the osseointegration of dental implants are very important for successful dental implant surgery. The aim of the present study was to assess the strengthening effect of a ß-TCP microsphere-hydrogel composite containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone healing and implant osseointegration. The molars and premolars on the left and right sides of the maxilla were extracted from six male minipigs, and dental implants were placed using either the ß-TCP microsphere-hydrogel carrier alone or the carrier loaded with rhBMP-2 (500 µg). The animals were kept alive for a further 8 weeks. The molars and premolars from the left and the right sides of the mandibles of another six minipigs were extracted, and the animals were kept alive for 4 weeks. Two 5-mm-diameter bone defects were then made on both sides of the mandible. The defects were filled with saline, ß-TCP microsphere-hydrogel carrier, or the carrier loaded with rhBMP-2 (300 µg), and dental implant fixtures were inserted. The animals were kept alive for a further 4 weeks. Bone formation was examined using plane radiographs, micro-CT, and the histology of undecalcified specimens. The group treated with the rhBMP-2-loaded carrier composite showed a significantly higher percentage bone volume and a greater trabecular thickness for the newly formed bone in the tooth defect areas when compared to the group treated with the carrier alone. The rhBMP-2 group had a significantly higher osseointegration, a larger percentage bone volume, greater trabecular thickness in the newly formed bone in tooth defect areas, a larger newly formed bone fraction in the fixture pitch, and a greater number of newly formed trabecular bones when compared to the other groups. We confirmed that the rhBMP-2-loaded carrier composite promotes new bone formation after tooth extraction and strengthens osseointegration of dental fixtures by improving the degree of osseointegration around the dental implant fixture.

Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting.

Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

Binding site specificity and factor redundancy in activator protein-1-driven human papillomavirus chromatin-dependent transcription.

Activator protein-1 (AP-1) regulates diverse gene responses triggered by environmental cues and virus-induced cellular stress. Although many signaling events leading to AP-1 activation have been described, the fundamental features underlying binding site selection and factor recruitment of dimeric AP-1 complexes to their target genes remain mostly uncharacterized. Using recombinant full-length human AP-1 dimers formed between c-Jun and Fos family members (c-Fos, FosB, Fra-1, Fra-2) for DNA binding and transcriptional analysis, we found that each of these AP-1 complex exhibits differential activity for distinct non-consensus AP-1 sites present in human papillomavirus (HPV), and each AP-1 complex is capable of activating transcription from in vitro-reconstituted HPV chromatin in a p300- and acetyl-CoA-dependent manner. Transcription from HPV chromatin requires AP-1-dependent and contact-driven recruitment of p300. Acetylation of dimeric AP-1 complexes by p300 enhances AP-1 binding to DNA. Using a human C-33A cervical cancer-derived cell line harboring the episomal HPV type 11 genome, we illustrate binding site selectivity recognized by c-Jun, JunB, JunD, and various Fos family members in a combinatorial and unique pattern, highlighting the diversity and importance of non-canonical binding site recognition by various AP-1 family proteins.

Chromatin adaptor Brd4 modulates E2 transcription activity and protein stability.

Brd4 is a chromatin adaptor containing tandem bromodomains binding to acetylated histone H3 and H4. Although Brd4 has been implicated in the transcriptional control of papillomavirus-encoded E2 protein, it is unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Using DNase I footprinting performed with in vitro reconstituted human papillomavirus (HPV) chromatin and nucleosome-free DNA templates, we found that Brd4 facilitates E2 binding to its cognate sequences in chromatin depending on bromodomains and the E2-interacting region of Brd4. Moreover, the coactivator and corepressor function of Brd4 requires at least one intact bromodomain and is mediated by its direct association with E2 proteins encoded by cancer-inducing high risk HPV-16 and HPV-18, wart-causing low risk HPV-11, and bovine papillomavirus type 1, in part through enhancing the protein stability of E2 that is normally degraded via the ubiquitin-dependent proteasome pathway. Our findings indicate that a chromatin adaptor can bridge and enhance the binding of a sequence-specific transcription factor to chromatin and further promote the stability of a labile transcription factor via direct protein-protein interaction.

Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis.

Two laccase cDNAs, pblac1 and pblac2, were cloned from a white-rot fungus strain, Polyporus brumalis (KFRI 20912). The cloned cDNAs consisted of 1,829 bp and 1,804 bp, and their open reading frames encoded proteins of 520 and 524 amino acids, with calculated molecular masses of approximately 55.9 kDa and 56 kDa, respectively. The deduced amino acid sequences of each protein showed 70% similarity. The copper binding regions were conserved in both proteins, as in other fungal laccases. RT-PCR analysis revealed that the transcript levels of the two laccases increased progressively in shallow stationary culture liquid medium. The transcript level of each laccase was induced when the fungus was exposed to di-butyl phthalate (DBP), suggesting that the two laccases are involved in DBP degradation. The overexpression of the pblac1 gene was derived by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase, using a homologous system. The activity of laccase in the transformants was significantly higher than that of the wild type. The identification of these laccase cDNAs was a first step to characterize the molecular events related to the lignin degradation ability of this basidiomycetous fungus, as well as the degradation of many recalcitrant xenobiotics.

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

Brd4 links chromatin targeting to HPV transcriptional silencing.

The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site-specific manner as illustrated by in vitro reconstituted chromatin transcription experiments. Knockdown of Brd4 in human cells alleviates E2-mediated repression of HPV transcription. The E2-interacting domain at the extreme C terminus and the chromatin targeting activity of a bromodomain-containing region are both essential for the corepressor activity of Brd4. Interestingly, E2-Brd4 blocks the recruitment of TFIID and RNA polymerase II to the HPV E6 promoter region without inhibiting acetylation of nucleosomal histones H3 and H4, indicating an acetylation-dependent role of Brd4 in the recruitment of E2 for transcriptional silencing of HPV gene activity. Our finding that Brd4 is a component of the virus-assembled transcriptional silencing complex uncovers a novel function of Brd4 as a cellular cofactor modulating viral gene expression.

Mouse LGI3 gene: expression in brain and promoter analysis.

Leucine-rich glioma inactivated 3 (LGI3) is a member of LGI/epitempin family of which the first member, LGI1/epitempin, was shown to be mutated in glioma and autosomal dominant lateral temporal epilepsy. Similar to LGI1, LGI3 is expressed predominantly in brain and its function is unknown. In this study, we examined the expression of mouse LGI3 (mLGI3) in adult and developing brain and analyzed the 5'-upstream transcriptional regulatory regions of mLGI3 gene. In situ hybridization showed that mLGI3 was expressed in widespread areas with selective regional variation in adult brain. In developing brain, mLGI3 mRNA was expressed at low level during embryo stages and markedly increased in broad areas after birth. Analysis of the 5'- and 3'-ends of mLGI3 mRNA identified a single transcription start site and two alternative 3'-ends. Luciferase reporter analysis using Neuro-2a cells and electrophoretic mobility shift assays identified a neuronal restrictive silencer element (NRSE; -2573 approximately -2553) and a phorbol ester-sensitive AP-2 element with repressor activity (-44 approximately -33) among multiple positive and negative regulatory regions. Since NRSE and AP-2 are implicated in neuron-specific gene expression and developmental regulation of many genes in brain, respectively, these results suggested that NRSE and AP-2 might play important roles in regulation of mLGI3 expression in brain.