Real World Data in Adaptive Biomedical Innovation: A Framework for Generating Evidence Fit for Decision-Making.

Analyses of healthcare databases (claims, electronic health records [EHRs]) are useful supplements to clinical trials for generating evidence on the effectiveness, harm, use, and value of medical products in routine care. A constant stream of data from the routine operation of modern healthcare systems, which can be analyzed in rapid cycles, enables incremental evidence development to support accelerated and appropriate access to innovative medicines. Evidentiary needs by regulators, Health Technology Assessment, payers, clinicians, and patients after marketing authorization comprise (1) monitoring of medication performance in routine care, including the materialized effectiveness, harm, and value; (2) identifying new patient strata with added value or unacceptable harms; and (3) monitoring targeted utilization. Adaptive biomedical innovation (ABI) with rapid cycle database analytics is successfully enabled if evidence is meaningful, valid, expedited, and transparent. These principles will bring rigor and credibility to current efforts to increase research efficiency while upholding evidentiary standards required for effective decision-making in healthcare.

Depolymerization of lignosulfonates by submerged cultures of the basidiomycete Irpex consors and cloning of a putative versatile peroxidase.

Lignosulfonates are abundantly available byproducts of the paper and pulping industry, and they therefore represent a promising feedstock for new sustainable processes. For industrial applications of lignosulfonates, their molecular weight distribution is a critical factor. In order to decrease the average molecular weight of lignosulfonates, Seventeen basidiomycetes were screened for their capability to depolymerize lignosulfonates from spent sulfite liquor (SSL) in surface and liquid cultures. Five basidiomycetes polymerized the lignosulfonates under the selected conditions. Only Irpex consors was found to efficiently degrade calcium lignosulfonates when SSL (0.5%, w/w) was used as the sole carbon and nitrogen source. The average molecular weight of the lignosulfonates was reduced from ∼26 to ∼4 kDa as determined by size exclusion chromatography (SEC) within two weeks. Various extracellular enzyme activities of I. consors were determined over the culture period. High peroxidase activities were correlating with a high degradation rate and the culture was harvested at the day of highest peroxidase activity. A putative versatile peroxidase was isolated by fast protein liquid chromatography (FPLC) and its encoding cDNA was cloned.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 ameliorates acute DSS colitis.

In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp)10, (GPO)10, has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO)10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO)10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO)10-treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO)10-treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO)10 application. We therefore propose (GPO)10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.

Assessing the comparative effectiveness of newly marketed medications: methodological challenges and implications for drug development.

Comparative-effectiveness research (CER) aims to produce actionable evidence regarding the effectiveness and safety of medical products and interventions as they are used outside of controlled research settings. Although CER evidence regarding medications is particularly needed shortly after market approval, key methodological challenges include (i) potential bias due to channeling of patients to the newly marketed medication because of various patient-, physician-, and system-related factors; (ii) rapid changes in the characteristics of the user population during the early phase of marketing; and (iii) lack of timely data and the often small number of users in the first few months of marketing. We propose a mix of approaches to generate comparative-effectiveness data in the early marketing period, including sequential cohort monitoring with secondary health-care data and propensity score (PS) balancing, as well as extended follow-up of phase III and phase IV trials, indirect comparisons of placebo-controlled trials, and modeling and simulation of virtual trials.

The MKK6/p38 mitogen-activated protein kinase pathway is capable of inducing SOCS3 gene expression and inhibits IL-6-induced transcription.

In this study we show that activation of p38MAPK by IL-6 acts as an inhibitory signal on IL-6-mediated activation of STAT and the alpha2-macroglobulin promoter. We analyzed the role of MKK6/p38MAPK for IL-6 signal transduction and transcriptional activation of the suppressor of cytokine signaling (SOCS) 3 promoter. Pretreatment of cells with the p38MAPK-specific inhibitor SB202190 downregulates the induction of SOCS3-mRNA expression by IL-6. Accordingly, overexpression of a constitutively active MKK6 in HepG2 cells enhanced basal activity or IL-6-induced transcriptional activation of a SOCS3 promoter reporter construct, whereas overexpression of a dominant negative mutant of MKK6 downregulated the IL-6-mediated activation of the SOCS3 promoter. These data indicate that p38MAPK-activation is crucial for IL-6-induced SOCS3 expression and downregulation of IL-6-mediated gene induction.

Region-based fractal image compression.

A fractal coder partitions an image into blocks that are coded via self-references to other parts of the image itself. We present a fractal coder that derives highly image-adaptive partitions and corresponding fractal codes in a time-efficient manner using a region-merging approach. The proposed merging strategy leads to improved rate-distortion performance compared to previously reported pure fractal coders, and it is faster than other state-of-the-art fractal coding methods.

Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax.

We previously showed that soluble, pepsin-solubilized collagen VI increases de novo DNA synthesis in serum-starved HT1080 and 3T3 fibroblasts up to 100-fold compared with soluble collagen I, reaching 80% of the stimulation caused by 10% fetal calf serum. Here we show that collagen VI also inhibits apoptotic cell death in serum-starved cells as evidenced by morphological criteria, DNA laddering, complementary apoptosis assays (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting), and quantification of apoptosis-regulating proteins. In the presence of starving medium alone or collagen I, the proapoptotic Bax was up-regulated 2-2.5-fold, compared with soluble collagen VI and fetal calf serum, whereas levels of the antiapoptotic Bcl-2 protein remained unaffected. In accordance with its potent stimulation of DNA synthesis, soluble collagen VI carries serum-starved HT1080 and Balb 3T3 fibroblasts through G(2) as shown by fluorescence-activated cell sorting analysis, whereas cells exposed to medium and collagen I where arrested at G(1)-S. This was accompanied by a 2-3-fold increase in cyclin A, B, and D1 protein expression. Collagen VI-induced inhibition of apoptotic cell death may be operative during embryogenesis, wound healing, and fibrosis when elevated tissue and blood levels of collagen VI are observed, thus initiating a feedback loop of mesenchymal cell activation and proliferation.

Soluble collagen VI induces tyrosine phosphorylation of paxillin and focal adhesion kinase and activates the MAP kinase erk2 in fibroblasts.

Signals from the extracellular matrix can modulate cellular differentiation and gene expression. We have shown previously that in contrast to other extracellular matrix molecules pepsin-solubilized collagen VI (CVI) can stimulate DNA synthesis of various mesenchymal cell types, apparently independent of integrin-mediated signal transduction. In order to further elucidate collagen VI-induced signaling events, we exposed mouse 3T3 fibroblasts and human HT1080 fibrosarcoma cells to soluble CVI. CVI induced tyrosine phosphorylation of proteins that associate with focal adhesions, such as paxillin, focal adhesion kinase (FAK), and p130CAS. Furthermore, it activated the mitogen-activated protein kinase, erk2. Kinetic analysis showed that these phosphorylations were transient, reaching a maximum after 5 min for transformed HT1080 cells and 30 min for 3T3 fibroblasts. These effects were partly inhibited by a beta1-integrin function blocking antibody and by single chains of CVI. Our results indicate that soluble fragments of native collagen VI, a ubiquitous component of the interstitial extracellular matrix, can mediate stimulation of DNA synthesis via tyrosine phosphorylation of paxillin, FAK, p130CAS, and erk2 in the absence of classical growth factors. Thus, CVI may serve as a matrix-derived sensor that allows for rapid reconstitution of a tissue defect by activating nearby mesenchymal cells.

Nitric oxide biosynthesis in an exotoxin-induced septic lung model: role of cNOS and impact on pulmonary hemodynamics.

Nitric oxide (NO) is an important vasodilator that is produced by constitutive (cNOS) as well as inducible (iNOS) isoforms of nitric oxide synthase. The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, was found to be a potent stimulator of NO liberation in isolated endothelial cells, and that it also causes thromboxane generation and related vasoconstriction in rabbit lungs. We investigated the effect of different concentrations of HlyA on pulmonary NO synthesis in buffer-perfused rabbit lungs. NO release into the alveolar as well as the intravascular compartment was monitored on-line by chemiluminescence detection of expired NO and by measurement of (peroxy-)nitrite/nitrate release into the perfusate. HlyA induced a pressor response and an immediate dose-dependent increase of exhalative and intravascular NO liberation, further enhanced by the addition of the NOS substrate L-arginine. The nonspecific NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), but not the iNOS selective inhibitors aminoguanidine and 2-(2-aminoethyl)-2-thiopseudourea-dihydrobromide, blocked the HlyA-evoked NO liberation into both the alveolar and the intravascular compartments. Enhancement of NO formation (L-arginine) slightly reduced, and inhibition of NO synthesis (L-NMMA) amplified greatly, the HlyA-elicited vasoconstrictor response. Inhibition of the pressor response by a thromboxane receptor antagonist did not interfere with the exotoxin-elicited NO formation. We conclude (1) that marked NO biosynthesis occurs in this model of the septic lung, (2) that the signal transduction in response to HlyA proceeds via activation of cNOS directly related to exotoxin activity and not to secondary changes in shear stress, and (3) that this vasodilator release mitigates the HlyA-induced pulmonary vasoconstriction. These findings may have important implications for therapeutic approaches using NOS inhibitors in sepsis.

Ability to reproduce head position after whiplash injury.

STUDY DESIGN: A two-group design with repeated measures. OBJECTIVES: To determine if there is loss of the ability to reproduce target position of the cervical spine individuals who have sustained a whiplash injury. SUMMARY OF BACKGROUND DATA: The ability to sense position is a prerequisite for functional movement. Injury may have a deleterious effect on this ability, resulting in inaccurate positioning of the head and neck with respect to the body coordinates and to the environment. METHODS: Eleven subjects with history of whiplash injury (age, 42 +/- 8.7 years) and 11 age-matched asymptomatic subjects (age, 43 +/- 3.1 years) participated in the study. Effects of whiplash injury on the ability to replicate a target position of the head were assessed. Maximum rotation of the neck and ability to reproduce the target angle were measured using a standard cervical range-of-motion device. Subjects' perception of "neutral" position was also assessed. RESULTS: Analysis of variance indicated the whiplash subjects were less accurate in reproducing the target angle than were control subjects. These whiplash subjects tended to overshoot the target. In addition, the subjects in the whiplash group were often inaccurate in their assessment of neutral position. CONCLUSIONS: Subjects who have experienced a whiplash injury demonstrate a deficit in their ability to reproduce a target position of the neck. These data are consistent with the hypothesis that these subjects possess an inaccurate perception of head position secondary to their injury. This study has implications for the rehabilitation of individuals with whiplash injury.

Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro.

Suggestions exist that, in addition to traditional growth factors, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed fibroblasts because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell growth on an individual ECM protein were not correlated. To test if ECM can stimulate cell growth, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of 20 microg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human fibroblasts and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor alpha2beta1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell growth via a pathway that is independent of the integrin alpha2beta1 and that the stimulatory region appears to be within the native helical portion of the collagen.

[Laterality and sex differences in quantitative fingerprint ridge analysis in a middle European sample (Giessen, Hessen)]. / Seiten- und Geschlechtsunterschiede quantitativer Hautleistenbefunde der Fingerbeeren bei einer mitteleuropäischen Stichprobe (Giessen, Hessen).

An examination of the quantitative characteristics of the skin of the digital pulps in a Central European population sample (Giessen, Hessen) of 625 persons (273 males and 352 females) was made. From the accumulation concerning the distribution of the ridge counts of each finger resulted for the left and for the right hand of men as well as for the right hand of females the following sequence I-IV-V-III-II, while the count of ridges diminished. On the left hand of females both first positions are exchanged. All pairs of fingers show, however, only partially significant higher values on the right homologous parts. The total amounts of the ridge values on the right and on the left hand differ significantly. With regard to males the amount of the ridges of the right hand is on the average four to five ridges higher than the left hand. Concerning females the difference is on the average four to five ridges. The following characteristics of the skin ridges have been examined with regard to sex differences: ridge counts of the single fingers, amounts of ridge counts of the right and left hand, total ridge count (TRC) and absolute ridge count (ALZ). Each of the examined characteristics show that males have higher ridge values than females. But the sexes distinguish significantly only with regard to four of ten fingers, in the amounts of ridge counts of the right and left hand and the TRC. The TRC of females amounts to 127.62, the TRC of males to 138.43. The values of TRC are distributed normally in both sexes. The rates of the correlation coefficient range in males in the field of 0.43 and 0.82, in females in the field of 0.42 and 0.82 and all are significantly positive. The homologous fingers correlate most strongly with each other. With respect to tendency concerning increasing distance of the fingers from each other, a decrease of correlation can be stated on the hand.

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

Scanning mutagenesis of the arginine-rich region of the human immunodeficiency virus type 1 Rev trans activator.

The structural proteins of human immunodeficiency virus type 1, for example, Gag and Env, are encoded by unspliced and incompletely spliced viral transcripts. The expression of these mRNAs in the cytoplasm, along with their commensurate translation, is absolutely dependent on the virally encoded Rev trans activator. Previous studies have demonstrated that Rev binds directly to its substrate mRNAs via an arginine-rich element that also serves as its nuclear localization sequence. In an attempt to define the specific amino acid residues that are important for in vivo activity, we have constructed a series of missense mutations that scan across this region. Our data demonstrate that all eight arginine residues within this element can, individually, be substituted for either leucine or lysine with no apparent loss of function. Importantly, these findings suggest that no single amino acid within the arginine-rich domain of Rev is, by itself, essential for activity and that considerable functional redundancy is therefore likely to exist within this region. Interestingly, one mutant in which a tryptophan had been substituted for a serine failed to accumulate exclusively in the nucleus but still bound RNA in a manner that was indistinguishable from that of the wild-type protein. This observation indicates that features of the arginine-rich region that are additional to those required for RNA binding are important for Rev's correct accumulation in the nucleus.

Matrix in signal transduction and growth factor modulation.

The extracellular matrix (ECM) is indispensable for the survival of multicellular organisms. It provides the adherent cells with crucial clues for migration, proliferation and differentiation. These clues are transmitted to the interior of the cell by ECM receptors like the integrins. Signaling by the ECM occurs by induction of assembly and disassembly of cytoskeletal structures or by modulation of classical signal transduction pathways such as activation of phosphatidylinositol-proteases, growth factors and cytokines that are specifically bound to its constituents and thereby stored, localized and modulated in terms of their biological activities. Finally, both the quantity and the quality of growth factor signaling appear to be dependent on the temporal and spatial activation of ECM receptors, supporting the requirement of a crosstalk between matrix and growth factor receptors.

Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.

Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.

Introducing and implementing on-line bibliographic retrieval services in a scientific research and development organization.

The National Bureau of Standards Library recently implemented on-line bibliographic retrieval services. Methods are given to orient and aid users in availing themselves of the services. Results are presented, based on appraisal of the services by users: value to users; most used data bases; problems requiring search revision; reasons for unsatisfactory results; purposes for request and use of search results; impact on subsequent library use; and future searching requirements. On-line capability impacts on library financing as a whole and on the library role in the community are described.