Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

Human prophylactic vaccine adjuvants and their determinant role in new vaccine formulations

Adjuvants have been considered for a long time to be an accessory and empirical component of vaccine formulations. However, accumulating evidence of their crucial role in initiating and directing the immune response has increased our awareness of the importance of adjuvant research in the past decade. Nevertheless, the importance of adjuvants still is not fully realized by many researchers working in the vaccine field, who are involved mostly in the search for better target antigens. The choice of a proper adjuvant can be determinant for obtaining the best results for a given vaccine candidate, but it is restricted due to intellectual property and know-how issues. Consequently, in most cases the selected adjuvant continues to be the aluminum salt, which has a record of safety, but predominantly constitutes a delivery system (DS). Ideally, new strategies should combine immune potentiators (IP) and DS by mixing both compounds or by obtaining structures that contain both IP and DS. In addition, the term immune polarizer has been introduced as an essential concept in the vaccine design strategies. Here, we review the theme, with emphasis on the discussion of the few licensed new adjuvants, the need for safe mucosal adjuvants and the adjuvant/immunopotentiating activity of conjugation. A summary of toxicology and regulatory issues will also be discussed, and the Finlay Adjuvant Platform is briefly summarized.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70&#8197;
81;M) for 24, 48, and 72&#8197;h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48&#8197;h of treatment with 50&#8197;
81;M KA, and 31% for cells submitted to 48&#8197;h of treatment with 70&#8197;
81;M KA. Similarly, in U87 cells treated with KA for 48&#8197;h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

A phthalide derivative isolated from endophytic fungiPestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

MP [4-(3&#8242;,3&#8242;-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniaeisolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation ofp73, JunB, FKHR, andBim. The results indicate that MP may be a potential treatment for cervical cancer.

Ziyuglycoside II-induced apoptosis in human gastric carcinoma BGC-823 cells by regulating Bax/Bcl-2 expression and activating caspase-3 pathway

Ziyuglycoside II is an active compound of Sanguisorba officinalis L. that has anti-inflammation, antioxidation, antibiosis, and homeostasis properties. We report here on the anticancer effect of ziyuglycoside II on human gastric carcinoma BGC-823 cells. We investigated the effects of ziyuglycoside II on cell growth, cell cycle, and cell apoptosis of this cell line. Our results revealed that ziyuglycoside II could inhibit the proliferation of BGC-823 cells by inducing apoptosis but not cell cycle arrest, which was associated with regulation of Bax/Bcl-2 expression, and activation of the caspase-3 pathway. Our study is the first to report the antitumor potential of ziyuglycoside II in BGC-823 gastric cancer cells. Ziyuglycoside II may become a potential therapeutic agent against gastric cancer in the future.

Detection of human cytomegalovirus DNA in various blood components after liver transplantation

The quantification of human cytomegalovirus (HCMV DNA) by real-time PCR is currently a primary option for laboratory diagnosis of HCMV infection. However, the optimal sample material remains controversial due to the use of different PCR assays. To explore the best blood component for HCMV DNA surveillance after liver transplantation, whole blood (WB), serum (SE), and plasma (PL) specimens were collected simultaneously from targeted patients and examined for HCMV DNA using one commercially available assay. The HCMV DNA-positive rate with WB (16.67%) was higher than that with either SE or PL (8.33%, both P<0.01). Quantitative DNA levels in WB were of greater magnitude than those in SE (WB-SE mean log-transformed difference, 0.99; 95%CI=0.74-1.25; P<0.0001) and PL (WB-PL mean log-transformed difference, 1.37; 95%CI=1.07-1.66; P<0.0001). Dynamic monitoring revealed that HCMV DNA in WB was positive sooner and had higher values for a longer period of time during therapy. With earlier positive detection, higher sensitivity, and yield of greater viral loads, WB compared favorably to SE or PL and hence is recommended as the superior material for HCMV DNA surveillance after liver transplantation. In addition, infant recipients require more intensive monitoring and prophylactic care because of their higher susceptibility to primary HCMV infection.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10&#8722;8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10&#8722;7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Changes in cardiac aldosterone and its synthase in rats with chronic heart failure: an intervention study of long-term treatment with recombinant human brain natriuretic peptide

The physiological mechanisms involved in isoproterenol (ISO)-induced chronic heart failure (CHF) are not fully understood. In this study, we investigated local changes in cardiac aldosterone and its synthase in rats with ISO-induced CHF, and evaluated the effects of treatment with recombinant human brain natriuretic peptide (rhBNP). Sprague-Dawley rats were divided into 4 different groups. Fifty rats received subcutaneous ISO injections to induce CHF and the control group (n=10) received equal volumes of saline. After establishing the rat model, 9 CHF rats received no further treatment, rats in the low-dose group (n=8) received 22.5 μg/kg rhBNP and those in the high-dose group (n=8) received 45 μg/kg rhBNP daily for 1 month. Cardiac function was assessed by echocardiographic and hemodynamic analysis. Collagen volume fraction (CVF) was determined. Plasma and myocardial aldosterone concentrations were determined using radioimmunoassay. Myocardial aldosterone synthase (CYP11B2) was detected by quantitative real-time PCR. Cardiac function was significantly lower in the CHF group than in the control group (P<0.01), whereas CVF, plasma and myocardial aldosterone, and CYP11B2 transcription were significantly higher than in the control group (P<0.05). Low and high doses of rhBNP significantly improved hemodynamics (P<0.01) and cardiac function (P<0.05) and reduced CVF, plasma and myocardial aldosterone, and CYP11B2 transcription (P<0.05). There were no significant differences between the rhBNP dose groups (P>0.05). Elevated cardiac aldosterone and upregulation of aldosterone synthase expression were detected in rats with ISO-induced CHF. Administration of rhBNP improved hemodynamics and ventricular remodeling and reduced myocardial fibrosis, possibly by downregulating CYP11B2 transcription and reducing myocardial aldosterone synthesis.

Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Securin, Cdc20 and Cdc27 in predicting the outcome of human breast cancer

Deregulated proliferation has been recognized among the most important factors promoting breast cancer development and progression. The aim of the project is to gain understanding of the role of specific cell cycle regulators of metaphase-anaphase transition and evaluate their potential in breast cancer prognostication and treatment decisions. Metaphase-anaphase transition is triggered by activation of anaphase promoting complex (APC) which is activated by a cascade of regulatory proteins, among them securin, Cdc20 and Cdc27. These proteins promote the metaphase–anaphase transition and participate in the timely separation of the chromatids. This study is based on a patient material of approximately 600 breast cancer patients and up to 22 years of follow-up. As the main observation, based on DNA cytometric and immunohistochemical methods, securin, Cdc20 and Cdc27 protein expressions were associated with abnormal DNA content and outcome of breast cancer. In the studied patient material, high securin expression alone and in combination with Cdc20 and Cdc27 predicted up to 9.8-fold odds for aneuploid DNA content in human breast cancer. In Kaplan–Meier analyses, high expression of securin systematically indicated decrease in breast cancer survival as compared to low expression cases. The adverse effect of high securin expression was further strengthened by combining it with Cdc20 or Cdc27 expressions, resulting in up to 6.8-fold risk of breast cancer death. High securin and Cdc20 expression was also associated with triple-negative breast cancer type with high statistical significance. Securin, Cdc20 or Cdc27 have not previously been investigated in a clinically relevant large breast cancer patient material or in association with DNA ploidy. The present findings suggest that the studied proteins may serve as potential biomarkers for identification of aggressive course of disease and unfavourable outcome of human breast cancer, and that they may provide a future research aim for understanding abnormal proliferation in malignant disease.

Trust Embedded Open Innovation: Human-Centricity in the Financial

Globalization and interconnectedness in the worldwide sphere have changed the existing and prevailing modus operandi of organizations around the globe and have challenged existing practices along with the business as usual mindset. There are no rules in terms of creating a competitive advantage and positioning within an unstable, constantly changing and volatile globalized business environment. The financial industry, the locomotive or the flagship industry of global economy, especially, within the aftermath of the financial crisis, has reached a certain point trying to recover and redefine its strategic orientation and positioning within the global business arena. Innovation has always been a trend and a buzzword and by many has been considered as the ultimate answer to any kind of problem. The mantra Innovate or Die has been prevailing in any organizational entity in a, sometimes, ruthless endeavour to develop cutting-edge products and services and capture a landmark position in the market. The emerging shift from a closed to an open innovation paradigm has been considered as new operational mechanism within the management and leadership of the company of the future. To that respect, open innovation has been experiencing a tremendous growth research trajectory by putting forward a new way of exchanging and using surplus knowledge in order to sustain innovation within organizations and in the level of industry. In the abovementioned reality, there seems to be something missing: the human element. This research, by going beyond the traditional narratives for open innovation, aims at making an innovative theoretical and managerial contribution developed and grounded on the on-going discussion regarding the individual and organizational barriers to open innovation within the financial industry. By functioning across disciplines and researching out to primary data, it debunks the myth that open innovation is solely a knowledge inflow and outflow mechanism and sheds light to the understanding on the why and the how organizational open innovation works by enlightening the broader dynamics and underlying principles of this fascinating paradigm. Little attention has been given to the role of the human element, the foundational pre-requisite of trust encapsulated within the precise and fundamental nature of organizing for open innovation, the organizational capabilities, the individual profiles of open innovation leaders, the definition of open innovation in the realms of the financial industry, the strategic intent of the financial industry and the need for nurturing a societal impact for human development. To that respect, this research introduces the trust-embedded approach to open innovation as a new insightful way of organizing for open innovation. It unveils the peculiarities of the corporate and individual spheres that act as a catalyst towards the creation of productive open innovation activities. The incentive of this research captures the fundamental question revolving around the need for financial institutions to recognise the importance for organizing for open innovation. The overarching question is why and how to create a corporate culture of openness in the financial industry, an organizational environment that can help open innovation excel. This research shares novel and cutting edge outcomes and propositions both under the prism of theory and practice. The trust-embedded open innovation paradigm captures the norms and narratives around the way of leading open innovation within the 21st century by cultivating a human-centricity mindset that leads to the creation of human organizations, leaving behind the dehumanization mindset currently prevailing within the financial industry.

Prevalence of Chronic Kidney Disease in newly diagnosed patients with Human immunodeficiency virus in Ilorin, Nigeria

AbstractIntroduction:Human immunodeficiency virus (HIV) the causative agent of Acquired immunodeficiency syndrome (AIDS) is an important cause of renal diseases in sub-Saharan Africa. There is paucity of studies on the burden of chronic kidney disease (CKD) among patients with HIV/AIDS in the North-Central zone of Nigeria.Methods:This is a cross-sectional study of 227 newly-diagnosed, antiretroviral naïve patients with HIV/AIDS seen at the HIV clinic of the Medical Out-patient Department (MOPD) of University of Ilorin Teaching Hospital (UITH). They were matched with 108 control group. Laboratory investigations were performed for the participants. CKD was defined as estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 m2 and/or albumin creatinine ratio (ACR) > 30 mg/g.Results:There were 100 (44%) males among the patients and 47 (43.5%) among the control group. The mean ages of the patients and controls were 40.3 ± 10.3 years and 41.8 ± 9.5 years respectively. CKD was observed in 108 (47.6%) among the patients and 18 (16.7%) of the controls (p = 0.01). The median CD4 T-cell count was significantly lower in patients with CKD. Ninety-three (41.0%) of the patients had dipstick proteinuria of > 2 +. The median albumin creatinine ratio (ACR) was significantly higher among the HIV-positive patients (272.3 mg/g) compared with the HIV-negative controls (27.22 mg/g) p = 0.01. The CD4 T-cell count correlates positively with eGFR (r = 0.463, p = 0.001) and negatively with ACR (r = -0.806, p = 0.001).Conclusions:CKD is very common among patients with HIV/AIDS in Ilorin. Screening and early intervention for CKD should be part of the protocols in the management of these patients.