Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC.

BACKGROUND:

The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry.

AIMS:

To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease.

METHODS:

GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms.

RESULTS:

Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied.

CONCLUSIONS:

The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1e) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M-r of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M-r 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to -9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 mug (.) kg(-1) (.) min(-1), followed by 2 h at 83.0 mug (.) kg(-1) (.) min(-1); corresponding to 0.4 and 2.0 mU (.) kg(-1) (.) min(-1)). At the lower dose, the exogenons glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P

Survival of pathogenic bacteria during mesophilic anaerobic-digestion of animal waste

The survival of pathogenic bacteria was investigated during the operation of a full-scale anaerobic digester which was fed daily and operated at 28-degrees-C. The digester had a mean hydraulic retention time of 24 d. The viable numbers of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter jejuni were reduced during mesophilic anaerobic digestion. Escherichia coli had the smallest mean viable numbers at each stage of the digestion process. Its mean T90 value was 76-9 d. Yersinia enterocolitica was the least resistant to the anaerobic digester environment; its mean T90 value was 18.2 d. Campylobacter jejuni was the most resistant bacterium; its mean T90 value was 438.6 d. Regression analysis showed that there were no direct relationships between the slurry input and performance of the digester and the decline of pathogen numbers during the 140 d experimental period.

Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.

How much gas can we get from grass?

Grass biomethane has been shown to be a sustainable gaseous transport biofuel, with a good energy balance, and significant potential for economic viability. Of issue for the designer is the variation in characteristics of the grass depending on location of source, time of cut and species. Further confusion arises from the biomethane potential tests (BMP) which have a tendency to give varying results. This paper has dual ambitions. One of these is to highlight the various results for biomethane potential that may be obtained from the same grass silage. The results indicated that methane potential from the same grass silage varied from 350 to 493 L CH4 kg−1 VS added for three different BMP procedures. The second ambition is to attempt to compare two distinct digestion systems again using the same grass: a two stage continuously stirred tank reactor (CSTR); and a sequentially fed leach bed reactor connected to an upflow anaerobic sludge blanket (SLBR–UASB). The two engineered systems were designed, fabricated, commissioned and operated at small pilot scale until stable optimal operating conditions were reached. The CSTR system achieved 451 L CH4 kg−1 VS added over a 50 day retention period. The SLBR–UASB achieved 341 L CH4 kg−1 VS added at a 30 day retention time.

A microbiological survey of a constructed wetland system supplied by dairy 'dirty water' effluent

A constructed wetland at Greenmount College, Co. Antrim, N. Ireland was built in 2004 to study the treatment of ‘dirty water’ effluent from the Greenmount dairy unit. The effluent has a mean BOD5 of c.1000 mg/L and contains milking parlour wash-water and runoff from silage clamps and yard areas lightly contaminated with cattle manure. The nominal water retention time of this wetland is 100 days. The primary purposes of the wetland are to eliminate organic pollution and eutrophication risk from nitrogen and phosphorus compounds. However the wetland should also effectively remove any zoonotic pathogens present in manure and milk. Accordingly, a 12-month microbiological survey of water in the five ponds of the wetland commenced in August 2007. The aims of the survey are to determine changes, as effluent passes through the wetland system, in a broad range of indicator organisms (faecal coliforms, Escherichia coli, Enterococcus faecalis and Clostridium perfringens) and the occurrence of several pathogens - Salmonella, Campylobacter, Cryptosporidium and Mycobacterium avium subsp. paratuberculosis (Map). The highest indicator organism counts - E. coli and faecal coliforms, 103-104 CFU/ml - are observed in pond 1, and a significant reduction (1-3 log10) in all indicator organisms occurs as water passes through the wetland from pond 1 to pond 5. Hence the wetland is efficient at reducing levels of indicator organisms in the dairy effluent. Salmonella and Campylobacter spp. are being detected intermittently in all the ponds, whilst Cryptosporidium and Map have yet to be detected, and so the ability of the wetland to reduce/eliminate specific pathogens is less clear at present.

The use of water soluble mucoadhesive gels for the intravesical delivery of epirubicin to the bladder for the treatment of non-muscle invasive bladder cancer

Objectives: To develop an epirubicin-loaded, water-soluble mucoadhesive gels that have the correct rheological properties to facilitate their delivery into the bladder via a catheter, while allowing for their spread across the bladder wall with limited expansion of the bladder and increasing the retention of epirubicin in the bladder and flushing with urine.

Methods: Epirubicin-loaded hydroxyl ethyl cellulose (HEC) and hydroxy propyl methyl cellulose (HPMC) gels were manufactured and tested for their rheological properties. Their ability to be pushed through a catheter was also assessed as was their in-vitro drug release, spreading in a bladder and retention of epirubicin after flushing with simulated urine.

Key findings: Epirubicin drug release was viscosity-dependent. The 1 and 1.5% HEC gels and the 1, 1.5 and 2% HPMC gels had the correct viscosity to be administered through a model catheter and spread evenly across the bladder wall under the pressure of the detrusor muscle. The epirubicin-loaded gels had an increased retention time in the bladder when compared with a standard intravesical solution of epirubicin, even after successive flushes with simulated urine.

Conclusion: The increased retention of epirubicin in the bladder by the HEC and HPMC gels warrant further investigation, using an in-vivo model, to assess their potential for use as treatment for non-muscle-invasive bladder cancer.

Use of an earthworm lysosomal biomarker for the ecological assessment of pollution from an industrial plastics fire

A low-cost field technique employing retention of the dye neutral-red by lysosomes in coelomocyte cells taken from earthworms (Lumbricus castaneus), was used as a means of assessing the ecological effects (if any) of an industrial accident. Earthworms and soil samples were collected at the site of a large industrial plastics fire in Thetford, UK along a 200 m transect leading from the factory perimeter fence, over a layer of molten plastic impregnated soil and into the surrounding forest. Coelomic fluid extracted from the earthworms was dye-loaded with neutral-red and lysosomal leaking observed. Metal residues in soil and earthworms were found to be highly elevated close to the factory perimeter and to rapidly drop to background levels within the first 50 m of the transect. Coelomocyte cells taken from earthworms adjacent to the factory perimeter showed the shortest period of neutral-red retention (2 min); cells taken from worms further into the surrounding forest had a longer retention time (12 min), whilst cells taken from worms from a control site showed even greater retention times (25 min). Thus, the neutral-red retention times correlated negatively with measured residues of heavy metals in the earthworms, the higher the body metal concentration the shorter the retention time. This field trial has demonstrated the validity of using an in vitro cellular biomarker technique for use in biological impact assessment along gradients of contamination.

Studying N-linked glycosylation of receptor tyrosine kinases

Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs.

Energy versus data integrity trade-offs in embedded high-density logic compatible dynamic memories

Current variation aware design methodologies, tuned for worst-case scenarios, are becoming increasingly pessimistic from the perspective of power and performance. A good example of such pessimism is setting the refresh rate of DRAMs according to the worst-case access statistics, thereby resulting in very frequent refresh cycles, which are responsible for the majority of the standby power consumption of these memories. However, such a high refresh rate may not be required, either due to extremely low probability of the actual occurrence of such a worst-case, or due to the inherent error resilient nature of many applications that can tolerate a certain number of potential failures. In this paper, we exploit and quantify the possibilities that exist in dynamic memory design by shifting to the so-called approximate computing paradigm in order to save power and enhance yield at no cost. The statistical characteristics of the retention time in dynamic memories were revealed by studying a fabricated 2kb CMOS compatible embedded DRAM (eDRAM) memory array based on gain-cells. Measurements show that up to 73% of the retention power can be saved by altering the refresh time and setting it such that a small number of failures is allowed. We show that these savings can be further increased by utilizing known circuit techniques, such as body biasing, which can help, not only in extending, but also in preferably shaping the retention time distribution. Our approach is one of the first attempts to access the data integrity and energy tradeoffs achieved in eDRAMs for utilizing them in error resilient applications and can prove helpful in the anticipated shift to approximate computing.

Identifying strategies to maximise recruitment and retention of practices and patients in a multicentre randomised controlled trial of an intervention to optimise secondary prevention for coronary heart disease in primary care.

Background Recruitment and retention of patients and healthcare providers in randomised controlled trials (RCTs) is important in order to determine the effectiveness of interventions. However, failure to achieve recruitment targets is common and reasons why a particular recruitment strategy works for one study and not another remain unclear. We sought to describe a strategy used in a multicentre RCT in primary care, to report researchers’ and participants’ experiences of its implementation and to inform future strategies to maximise recruitment and retention. Methods In total 48 general practices and 903 patients were recruited from three different areas of Ireland to a RCT of an intervention designed to optimise secondary prevention of coronary heart disease. The recruitment process involved telephoning practices, posting information, visiting practices, identifying potential participants, posting invitations and obtaining consent. Retention involved patients attending reviews and responding to questionnaires and practices facilitating data collection. Results We achieved high retention rates for practices (100%) and for patients (85%) over an 18-month intervention period. Pilot work, knowledge of the setting, awareness of change in staff and organisation amongst participant sites, rapid responses to queries and acknowledgement of practitioners’ contributions were identified as being important. Minor variations in protocol and research support helped to meet varied, complex and changing individual needs of practitioners and patients and encouraged retention in the trial. A collaborative relationship between researcher and practice staff which required time to develop was perceived as vital for both recruitment and retention. Conclusions Recruiting and retaining the numbers of practices and patients estimated as required to provide findings with adequate power contributes to increased confidence in the validity and generalisability of RCT results. A continuous dynamic process of monitoring progress within trials and tailoring strategies to particular circumstances, whilst not compromising trial protocols, should allow maximal recruitment and retention.