bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization
Authors: Vanz Ana LS , Renard Gaby , Palma Mario S , Chies Jocelei M , Dalmora Sérgio L , Basso Luiz A , Santos Diógenes S 
Published: April 2008

Abstract

Background
Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.

Results
Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.

Conclusion
The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.
Protein C: a potential biomarker in severe sepsis and a possible tool for monitoring treatment with drotrecogin alfa (activated)
Authors: Shorr Andrew F , Nelson David R , Wyncoll Duncan LA , Reinhart Konrad , Brunkhorst Frank , Vail George Matthew , Janes Jonathan 
Published: April 2008

Abstract
Introduction
Drotrecogin alfa (activated; DrotAA) treatment, a 96-hour infusion, reduces 28-day mortality in severe sepsis to approximately 25%. The question remains whether a longer infusion or higher dose could increase rate of survival. The goal of this study was to identify a dependable, sensitive measure with which to monitor disease progression and response in patients during DrotAA treatment.

Methods
Data on severe sepsis patients included in PROWESS (placebo-controlled, double-blind, randomized study of 850 DrotAA and 840 placebo individuals) and ENHANCE (single-arm, open-label study of 2,375 DrotAA patients) studies were analyzed. In these studies, DrotAA (24 μg/kg per hour) or placebo was infused for 96 hours and patients were followed for 28 days. Data on six laboratory measures and five organ dysfunctions were systematically analyzed to identify a potential surrogate end-point for monitoring DrotAA therapy and predicting 28-day mortality at the end of therapy. To allow comparison across variables, sensitivity and specificity analyses identified cut-off values for preferred outcome, and relative risks for being above or below cut-offs were calculated, as was the 'proportion of treatment effect explained' (PTEE) to identify biomarkers that contribute to benefit from DrotAA.

Results
Protein C was the only variable that correlated with outcome across all analyses. Using placebo data, a baseline protein C under 40% was established as a useful predictor of outcome (odds ratio 2.12). Similar odds ratios were associated with cut-off values of other biomarkers, but the treatment benefit associated with DrotAA was significantly greater below the cut-off than above the cut-off only for protein C (relative risk for 28-day mortality 0.66 versus 0.88; P = 0.04). Protein C was the only end-of-infusion biomarker that potentially explained at least 50% of the benefit from DrotAA (PTEE 57.2%). The PTEE was 41% for cardiovascular Sequential Organ Failure Assessment score and for d-dimer. At the end of infusion (day 4), protein C categories (≤40%, 41% to 80%, and > 80%) remained significantly related to mortality, regardless of treatment assignment.

Conclusion
Based on systematic analyses of 11 variables measured in severe sepsis clinical trials, protein C was the only variable consistently correlated with both DrotAA treatment effect and survival. Further study is needed to determine whether longer infusions or higher doses of DrotAA would achieve the goal of normalizing protein C in more patients with severe sepsis.
Antiviral NK cell responses in HIV infection: II. viral strategies for evasion and lessons for immunotherapy and vaccination
Authors: Iannello Alexandre , Debbeche Olfa , Samarani Suzanne , Ahmad Ali 
Published: April 2008

AbstractAs is the case in other viral infections, humans respond to HIV infection by activating their NK cells. However, the virus uses several strategies to neutralize and evade the host’s NK cell responses. Consequently, it is not surprising that NK cell functions become compromised in HIV-infected individuals in early stages of the infection. The compromised NK cell functions also adversely affect several aspects of the host’s antiviral adaptive immune responses. Researchers have made significant progress in understanding how HIV counters NK cell responses of the host. This knowledge has opened new avenues for immunotherapy and vaccination against this infection. In the first part of this review article, we gave an overview of our current knowledge of NK cell biology and discussed how the genes encoding NK cell receptors and their ligands determine innate genetic resistance/susceptibilty of humans against HIV infections and AIDS. In this second part, we discuss NK cell responses, viral strategies to counter these responses, and finally, their implications for anti-HIV immunotherapy and vaccination.
CD44 mobilization in allogeneic dendritic cell–T cell immunological synapse plays a key role in T cell activation
Authors: Hegde Venkatesh L , Singh Narendra P , Nagarkatti Prakash S , Nagarkatti Mitzi 
Published: April 2008

AbstractCD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)–T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44-deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC–T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C-θ enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-γ production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)-specific, syngeneic DC–T cell interaction using OVA-specific T cells derived from OT-II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.
Developmental Dependence on NurRE and EboxNeuro for Expression of Pituitary Proopiomelanocortin
Authors: Lavoie Pierre-Luc , Budry Lionel , Balsalobre Aurélio , Drouin Jacques 
Published: April 2008

AbstractCell-specific expression of the pituitary proopiomelanocortin (POMC) gene depends on the combinatorial action of a large number of DNA-binding transcription factors (TFs). These include general and cell-restricted factors, as well as factors that act as effectors of signaling pathways. We have previously defined in the distal POMC promoter a composite regulatory element that contains targets for basic helix-loop-helix TFs conferring cell specificity and for NGFI-B orphan nuclear receptors that are responsive to CRH signaling and to glucocorticoid negative feedback. These factors act on neighboring regulatory elements, the EboxNeuro and NurRE, respectively. Currently, the EboxNeuro is thought to be the target of NeuroD1 during fetal development, but this factor may not account for activity in the adult pituitary; it is also unknown whether the NurRE and NGFI-B-related factors are active before establishment of the hypothalamic-pituitary portal system. In order to assess the importance of these regulatory elements and their cognate TFs throughout pituitary organogenesis and in the adult, we have assessed the activity of mutant POMC promoters in transgenic mice throughout development. These experiments indicate that the EboxNeuro and cognate basic helix-loop-helix factors are required throughout development and in the adult gland, beyond expression of NeuroD1. Similarly, the data reveal sustained importance of the NurRE and its cognate factors throughout pituitary development. These data contrast the sustained dependence throughout development on the same regulatory elements with the highly dynamic patterns of TF expression and the modulation of their activity in response to signaling pathways.
Regulation of Self-Renewal and Pluripotency by Sox2 in Human Embryonic Stem Cells
Authors: Fong Helen , Hohenstein Kristi A. , Donovan Peter J. 
Published: April 2008

Abstract
Human embryonic stem (hES) cells, derived from blastocysts, are capable of unlimited self-renewal and differentiation into all cell lineages of the body. Because of their pluripotent nature, hES cells are valuable tools for understanding human development and advancing the field of regenerative medicine. However, one key to harnessing the therapeutic power of hES cells for biomedical applications begins with determining how these cells maintain their pluripotent and undifferentiated state. Studies in mice have implicated three factors in regulating pluripotency in embryonic stem cells, Oct4, Nanog, and Sox2. However, significant differences in growth regulation between mouse embryonic stem and hES cells have been identified, suggesting a need to determine when and how factors work in hES cells. To date, the transcription factors Oct4 and Nanog have been identified as critical regulators of stem cell fate by functional studies in hES cells. To determine the role of Sox2 in maintaining hES cell pluripotency and self-renewal, we used RNA interference to specifically knock down Sox2 gene expression. Reduction of Sox2 expression in hES cells results in loss of the undifferentiated stem cell state, as indicated by a change in cell morphology, altered stem cell marker expression, and increased expression of trophectoderm markers. In addition, knockdown of Sox2 results in reduced expression of several key stem cell factors, including Oct4 and Nanog, linking these three factors together in a pluripotent regulatory network.
Disclosure of potential conflicts of interest is found at the end of this article.
Concise Review: Adult Multipotent Stromal Cells and Cancer: Risk or Benefit?
Authors: Lazennec Gwendal , Jorgensen Christian 
Published: April 2008

Abstract
This review focuses on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anticancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages, including chondrocytes, osteoblasts, and adipocytes. Recent evidence also suggests that they could play a role in the progression of carcinogenesis and that MSCs could migrate toward primary tumors and metastatic sites. It is possible that MSCs could also be involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immunosuppressive properties and proangiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express antitumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSC use in cell-mediated gene strategies.
Disclosure of potential conflicts of interest is found at the end of this article.
Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
Authors: Vazin Tandis , Chen Jia , Lee Chun-Ting , Amable Rose , Freed William J. 
Published: April 2008

Abstract
Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by β-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect.
Disclosure of potential conflicts of interest is found at the end of this article.
Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone
Authors: Agasse Fabienne , Bernardino Liliana , Kristiansen Heidi , Christiansen Søren H. , Ferreira Raquel , Silva Bruno , Grade Sofia , Woldbye David P.D. , Malva João O. 
Published: April 2008

Abstract
Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 μM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal-regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c-Jun-NH2-terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell-based brain therapy.
Disclosure of potential conflicts of interest is found at the end of this article.
The influence of traumatic acid on the growth and metabolite content of the green alga Chlorella vulgaris Beijerinck
Authors: Pietryczuk Anna , Piotrowska Alicja , Czerpak Romuald 
Published: April 2008

Abstract
The effect of exogenous traumatic acid, (2E)-dodecene-1,12-dioic acid (TA), at concentrations of 10-6 - 10-3 M on the growth and development of the unicellular green alga Chlorella vulgaris was examined over 7 days. TA plays an important role in algal growth and development. Treatment with 10-5 M TA resulted in the greatest increase in cell numbers, by 77%, chlorophyll a content, by 126%, chlorophyll b, by 45%, total carotenoids, by 65%, monosaccharides, by 111%, and water-soluble proteins, by 37%, in relation to the control. After three days of treatment with 10-5 M TA cultures were analysed by SDS-PAGE, which showed the presence of new polypeptides with molecular weights 13-103 kDa.
Resuscitation of Seventeen-year Stressed Salmonella typhimurium
Authors: Amel Dhiaf , Amina Bakhrouf 
Published: April 2008

Abstract

Salmonella enterica serovar typhimurium was stressed by incubation in seawater microcosms for seventeen years. The microcosms were prepared in such a way as to allow progressive evaporation of the water. Despite being introduced into the sterile seawater at very high concentrations, the Salmonella rapidly declined to levels undetectable by plate counts on nutrient agar. After two years of starvation, about half of the seawater volume had evaporated from each microcosm, and salt crystals appeared. Inoculation of the salty suspension did not result in any culturable strains in selective and non-selective media. However, incubation of samples in nutrient-rich broth, without supplemental growth factors, allowed resuscitation of stressed cells, yielding colonies that remained viable for extended periods of time. After three years the total volume of water had evaporated from each microcosm, and only salt crystals remained. These microcosms were then incubated at room temperature for seventeen years. Resuscitation of VBNC Salmonella from the salt crystals was conducted in vitro and in vivo; recovery occurring after incubation in both nutrient broth and in mice. Recovery occurred in the Salmonella administered orally into the mice, but not in those administered by intra-peritoneal injection. The identity of initial, stressed and revived strains was confirmed by DGGE analysis and sequencing of 16S RNA gene fragments.
Decapod crustaceans of soft-sediments on the Aegean Sea coast of Turkey (the eastern Aegean Sea)
Authors: Ateş A. , Katağan Tuncer 
Published: April 2008

Abstract
58 species of decapod crustacean were identified (1 Dendobranchiata, 14 Caridea, 5 Thalassinidea, 14 Anomura, 24 Brachyura) on soft substrates of the Aegean Sea coast of Turkey. Samples were collected from 60 sampling sites along the Turkish Aegean shore (eastern Aegean Sea) between July 2000 and September 2000 with dredge and a van Veen grab at bottom depths of 7 to 150 m. Different species dominated in each ecological zone with the anomuran squat lobster, Galathea intermedia Lilljeborg, 1851, being the most dominant species across the whole sampling area, comprising 11.71% of the individuals collected. This report represents the first published investigations into soft-sediment decapod communities in the coastal waters of the Turkish Aegean Sea.
Diversity and vertical distribution of planktonic ciliates in a stratified mesotrophic lake: relationship to environmental conditions
Authors: Mieczan Tomasz 
Published: April 2008

Abstract
The density and biomass of planktonic ciliates significantly differed between sample sites in a deep, mesotrophic lake, with the lowest numbers in the hypolimnion and the highest in the epilimnion. The community composition varied greatly with depth. Oligotrichs dominated in the epilimnion, where prostomatids and peritrichs were also important components of the community. The density of oligotrichs and peritrichs decreased in the metalimnion, and they were absent from the hypolimnion. In the deep metalimnion the most abundant groups were prostomatids, haptorids and scuticociliates. In the hypolimnion, the ciliate community was dominated by scuticociliates and heterotrichs. In the epilimnion and metalimnion a significant positive correlation was observed between ciliate numbers, chlorophyll a and heterotrophic flagellate density. In all three zones the number of ciliates was most strongly correlated with water temperature, concentrations of total organic carbon (TOC), total phosphorus (Ptot) and bacterial density, whilst in the hypolimnion there was a significant correlation between ciliate numbers and the dissolved oxygen concentration.
Nephrocalcinosis and disordered calcium metabolism in two children with SHORT syndrome
Authors: Reardon William , Temple I. Karen 
Published: April 2008

AbstractReports of SHORT syndrome have, to date, focused on the clinical features which lie at the core of the diagnosis but there has been little by way of report of long‐term outcome, either in terms of medical complications or of intellectual development. We now report two children in whom nephrocalcinosis has developed and a third, adult, with similar findings. It may be that clinicians should be looking among cases of unexplained hypercalcaemia for an alternative phenotypic presentation of this short stature syndrome. © 2008 Wiley‐Liss, Inc.
Spatiotemporal Expression of SSeCKS in Injured Rat Sciatic Nerve
Authors: Chen Li , Qin Jing , Cheng Chun , Niu Shuqiong , Liu Yonghua , Shi Shuxian , Liu Hai'ou , Shen Aiguo 
Published: April 2008

AbstractSSeCKS (src suppressed C kinase substrate) functions in the control of cell signaling and cytoskeletal arrangement. It is expressed in brain and spinal cord, but little is known about its expression in peripheral nerves. In this study, in rats, real‐time polymerase chain reaction and Western blot analysis showed that expression of SSeCKS in crushed sciatic nerve reached its highest level 6 hr after crushing, whereas in a transection model, SSeCKS peaked at 2 days in the proximal stump and 12 hr in the distal stump. Immunohistochemical analysis demonstrated up‐regulation of SSeCKS protein surrounding the crush site and in the two stumps of the transected nerve. In addition, SSeCKS colocalized with growth‐associated protein 43 and with S100, which also changed with time after injury. These findings support the idea that SSeCKS participates in the adaptive response to peripheral nerve injury and may be associated with regeneration. Anat Rec, 291:527–537, 2008. © 2008 Wiley‐Liss, Inc.