The cystatin C/creatinine ratio, a marker of glomerular filtration quality: associated factors, reference intervals, and prediction of morbidity and mortality in healthy seniors.

The ratio of cystatin C (cysC) to creatinine (crea) is regarded as a marker of glomerular filtration quality associated with cardiovascular morbidities. We sought to determine reference intervals for serum cysC-crea ratio in seniors. Furthermore, we sought to determine whether other low-molecular weight molecules exhibit a similar behavior in individuals with altered glomerular filtration quality. Finally, we investigated associations with adverse outcomes. A total of 1382 subjectively healthy Swiss volunteers aged 60 years or older were enrolled in the study. Reference intervals were calculated according to Clinical & Laboratory Standards Institute (CLSI) guideline EP28-A3c. After a baseline exam, a 4-year follow-up survey recorded information about overall morbidity and mortality. The cysC-crea ratio (mean 0.0124 ± 0.0026 mg/μmol) was significantly higher in women and increased progressively with age. Other associated factors were hemoglobin A1c, mean arterial pressure, and C-reactive protein (P < 0.05 for all). Participants exhibiting shrunken pore syndrome had significantly higher ratios of 3.5-66.5 kDa molecules (brain natriuretic peptide, parathyroid hormone, β2-microglobulin, cystatin C, retinol-binding protein, thyroid-stimulating hormone, α1-acid glycoprotein, lipase, amylase, prealbumin, and albumin) and creatinine. There was no such difference in the ratios of very low-molecular weight molecules (urea, uric acid) to creatinine or in the ratios of molecules larger than 66.5 kDa (transferrin, haptoglobin) to creatinine. The cysC-crea ratio was significantly predictive of mortality and subjective overall morbidity at follow-up in logistic regression models adjusting for several factors. The cysC-crea ratio exhibits age- and sex-specific reference intervals in seniors. In conclusion, the cysC-crea ratio may indicate the relative retention of biologically active low-molecular weight compounds and can independently predict the risk for overall mortality and morbidity in the elderly.

Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Rapid Recurrence of a Benign Meningial Perineurioma.

BACKGROUND We describe a rare case of a rapidly recurring benign meningial-based perineurioma. Clinical, radiologic, and pathologic features of a rapidly recurring falxial perineurioma are described; the perineurioma was discovered incidentally in an 86-year-old woman. CASE DESCRIPTION Due to progressive gait disturbances and radiologically proven progression after a 3-year symptom-free interval, subtotal resection of a large falxial-based meningeal tumor was performed. CONCLUSIONS The pathologic examination confirmed the diagnosis of a perineurioma (World Health Organization grade I). Follow-up magnetic resonance tomography 5 months later due to neurologic deterioration revealed an abnormally rapidly growing and extensive local tumor recurrence. Due to the mass effect, reoperation was performed and adjuvant radiation of 20 Gy to the tumor bed was implemented thereafter. Meningeal-based perineuriomas of the central nervous system are extremely rare, and literature on proper management is scarce. Although histologic classification reveals a benign lesion, follow-up may be considered for this type of tumor.

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Prime and boost vaccination against HER2/neu in breast cancer

Breast cancer is the most common cancer among women with approximately 180,000 new cases being diagnosed yearly in the United States (1). HER2/neu gene amplification and subsequent protein overexpression is found in 20–30% of breast cancer patients and can lead to the promotion of various metastasis-related properties (2–4) and/or resistance to cancer therapies such as chemotherapy and radiation (5). ^ The protein product of the HER2/neu gene, p185, is a proven target for immunological therapy. Recently, passive immunotherapy with the monoclonal antibody Trastuzumab® has validated an immunological approach to HER2/neu+ breast cancer. Immunity to HER2/ neu, when found in breast cancer patients, is of low magnitude. Vaccination-induced HER2/neu-specific antibodies and HER2/neu-specific cytotoxic T cells could result in long-lived immunity with therapeutic benefit. Many features of DNA vaccines and attenuated viral vectors may contribute to the efficacy of prime-boost vaccination. In particular, vaccines capable of eliciting strong cell-mediated immunity are thought to hold the greatest promise for control of cancer (6–9). ^ To optimize cellular immunization to HER2/neu in my study, the HER2/neu gene was presented to the immune system using a priming vector followed by a second vector used as the boost. In both animals and humans, priming with DNA and boosting with a poxviruses, vaccinia or canarypox appears to be particularly promising for induction of a broad immune responses (10). ^ I tested three gene vaccines encoding the HER2/neu gene: (1) a plasmid, SINCP, that contains part of the genome of Sindbis virus; (2) Viral Replicon Particles (VRP) of Venezuela Equine Encephalitis virus (VEE) and (3) E1/E2a-deleted human Type 5 Adenovirus. In SINCP and the VRP, the caspid and envelope genes of the virus were deleted and replaced with the gene for HER2/neu. SINCP-neu, VRP- neu and Adeno-neu when used alone were effective vaccines protecting healthy mice from challenge with a breast cancer cell line injected in the mammary fat pad or injected i.v. to induce experimental lung metastasis. However, SINCP-neu, VRP-neu or Adeno-neu when used alone were not able to prolong survival of mice in therapeutic models in which vaccination occurred after injection of a breast cancer cell line. ^ When the vaccines were combined in a mixed regimen of a SINCP- neu prime VRP-neu or Adeno-neu boost, there was a significant difference in tumor growth and survival in the therapeutic vaccine models. In vitro assays demonstrated that vaccination with each of the three vaccines induced IgG specific for p185, the gene product of HER2/neu, induced p185-specific T lymphocytes, as measured by tetramer analysis. Vaccination also induced intracellular INF-γ and a positive ELISPOT assay. These findings indicate that SINCP-neu, VRP-neu and Adeno-neu, used alone or in combination, may have clinical potential as adjuvant immunotherapy for the treatment of HER2/neu-expressing tumors. ^

Miscellany of Hebrew Literature. Vol. 1

[ed. by Israel Davis]

Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

PRETERM BIRTH AND OCCUPATIONAL PHYSICAL ACTIVITY: A CASE-CONTROL STUDY OF THE RELATIONSHIP BETWEEN LENGTH OF GESTATION AND THE PHYSICAL DEMAND OF U.S. ARMY MILITARY OCCUPATIONAL SPECIALTIES (UNITED STATES)

A case-control study has been conducted examining the relationship between preterm birth and occupational physical activity among U.S. Army enlisted gravidas from 1981 to 1984. The study includes 604 cases (37 or less weeks gestation) and 6,070 controls (greater than 37 weeks gestation) treated at U.S. Army medical treatment facilities worldwide. Occupational physical activity was measured using existing physical demand ratings of military occupational specialties.^ A statistically significant trend of preterm birth with increasing physical demand level was found (p = 0.0056). The relative risk point estimates for the two highest physical demand categories were statistically significant, RR's = 1.69 (p = 0.02) and 1.75 (p = 0.01), respectively. Six of eleven additional variables were also statistically significant predictors of preterm birth: age (less than 20), race (non-white), marital status (single, never married), paygrade (E1 - E3), length of military service (less than 2 years), and aptitude score (less than 100).^ Multivariate analyses using the logistic model resulted in three statistically significant risk factors for preterm birth: occupational physical demand; lower paygrade; and non-white race. Controlling for race and paygrade, the two highest physical demand categories were again statistically significant with relative risk point estimates of 1.56 and 1.70, respectively. The population attributable risk for military occupational physical demand was 26%, adjusted for paygrade and race; 17.5% of the preterm births were attributable to the two highest physical demand categories. ^

Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.

Lower Cretaceous nannofossils of the northwestern Australian margin

Moderately to sparsely nannofossiliferous Neocomian siliciclastics and rich Aptian-Albian nannofossil chalks were cored at two Leg 123 sites on the abyssal plains off northwestern Australia. At Site 765, the basal 70 m of cored section yields questionable Tithonian and Berriasian to early Hauterivian assemblages of moderate diversity containing Cruelellipsis cuvillieri, Tegumentum striatum, Speetonia colligata, and Crucibiscutum salebrosum. The overlying Hauterivianlower Aptian is represented by 140 m of sediments barren of nannofossils. Above this, the remaining 80 m of the Lower Cretaceous section has been assigned to the Rhagodiscus angustus Zone (late Aptian-early Albian in age) and the Prediscosphaera columnata Zone (middle-late Albian in age). Common species include Rhagodiscus angustus, Prediscosphaera columnata, Eprolithus floralis, Eprolithus sp., Chiastozygus litterarius, Rucinolithus irregularis, and Flabellites biforaminis. At Site 766, the Neocomian, represented by 200 m of sediment, yields C. cuvillieri, T. striatum, S. colligata, and C. salebrosum. Within the overlying Aptian-Albian sequence of 80 m, the Rhagodiscus angustus, and P. columnata zones were recognized. The paleobiogeographic patterns and implications are discussed, with special emphasis paid to the bipolar high-latitude distribution pattern of C. salebrosum in the Valanginian-Hauterivian. Biostratigraphically important species are discussed and their occurrence in the Indian Ocean is compared with one from the Tethys and Boreal realms. Two new species, Serbiscutum gaultensis and Eprolithus bettenstaedtii, are described.

Seaweed - epiphyte - mesograzer communities were tested for their responses to elevated seawater temperature and [CO2] in benthic mesocosms experiments across four consecutive seasons of one year in Kiel, Germany

Rising seawater temperature and CO2 concentrations (ocean acidification) represent two of the most influential factors impacting marine ecosystems in the face of global climate change. In ecological climate change research full-factorial experiments across seasons in multi-species, cross-trophic level set-ups are essential as they allow making realistic estimations about direct and indirect effects and the relative importance of both major environmental stressors on ecosystems. In benthic mesocosm experiments we tested the responses of coastal Baltic Sea Fucus vesiculosus communities to elevated seawater temperature and CO2 concentrations across four seasons of one year. While increasing [CO2] levels only had minor effects, warming had strong and persistent effects on grazers which affected the Fucus community differently depending on season. In late summer a temperature-driven collapse of grazers caused a cascading effect from the consumers to the foundation species resulting in overgrowth of Fucus thalli by epiphytes. In fall/ winter, outside the growing season of epiphytes, intensified grazing under warming resulted in a significant reduction of Fucus biomass. Thus, we confirm the prediction that future increasing water temperatures influence marine food-web processes by altering top-down control, but we also show that specific consequences for food-web structure depend on season. Since Fucus vesiculosus is the dominant habitat-forming brown algal system in the Baltic Sea, its potential decline under global warming implicates the loss of key functions and services such as provision of nutrient storage, substrate, food, shelter and nursery grounds for a diverse community of marine invertebrates and fish in Baltic Sea coastal waters.

Abundance of phytoplankton, heterotrophic nanoflagellates and bacteria through the water column at time series station L4 in the Western English Channel. 2007-2011

The marine laboratories in Plymouth have sampled at two principle sites in the Western English Channel for over a century in open-shelf (station E1; 50° 02'N, 4° 22'W) and coastal (station L4; 50° 15'N, 4° 13'W) waters. These stations are seasonally stratified from late-April until September, and the variable biological response is regulated by subtle variations in temperature, light, nutrients and meteorology. Station L4 is characterized by summer nutrient depletion, although intense summer precipitation, increasing riverine input to the system, results in pulses of increased nitrate concentration and surface freshening. The winter nutrient concentrations at E1 are consistent with an open-shelf site. Both stations have a spring and autumn phytoplankton bloom; at station E1, the autumn bloom tends to dominate in terms of chlorophyll concentration. The last two decades have seen a warming of around 0.6°C per decade, and this is superimposed on several periods of warming and cooling over the past century. In general, over the Western English Channel domain, the end of the 20th century was around 0.5°C warmer than the first half of the century. The warming magnitude and trend is consistent with other stations across the north-west European Shelf and occurred during a period of reduced wind stress and increased levels of insolation (+20%); these are both correlated with the larger scale climatic forcing of the North Atlantic Oscillation.

Calcareous nannofossil species richness across the Paleocene-Eocene Thermal Maximum

The Paleocene-Eocene Thermal Maximum (PETM, ~5 million years ago) was an interval of global warming and ocean acidification attributed to rapid release and oxidation of buried carbon. We show that the onset of the PETM coincided with a prominent increase in the origination and extinction of calcareous phytoplankton. Yet major perturbation of the surface-water saturation state across the PETM was not detrimental to the survival of most calcareous nannoplankton taxa and did not impart a calcification or ecological bias to the pattern of evolutionary turnover. Instead, the rate of environmental change appears to have driven turnover, preferentially affecting rare taxa living close to their viable limits.