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Control over supramolecular recognition between proteins and nanoparticles (NPs) is of fundamental importance in therapeutic applications and sensor development. Most NP-protein binding approaches use 'tags' such as biotin or His-tags to provide high affinity; protein surface recognition provides a versatile alternative strategy. Generating high affinity NP-protein interactions is challenging however, due to dielectric screening at physiological ionic strengths. We report here the co-engineering of nanoparticles and protein to provide high affinity binding. In this strategy, 'supercharged' proteins provide enhanced interfacial electrostatic interactions with complementarily charged nanoparticles, generating high affinity complexes. Significantly, the co-engineered protein-nanoparticle assemblies feature high binding affinity even at physiologically relevant ionic strength conditions. Computational studies identify both hydrophobic and electrostatic interactions as drivers for these high affinity NP-protein complexes.

Chronic graft-versus-host disease (cGVHD) remains a frequent cause of nonrelapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Despite recent advances, options for steroid-refractory (SR) cGVHD are limited. In previous trials of low-dose interleukin-2 (LD IL-2), the immunomodulatory properties of regulatory T cells (Tregs) have been harnessed to treat SR-cGVHD safely and effectively. In the present study, we combined a single infusion of Treg-enriched lymphocytes (Treg DLI) from the original stem cell donor with in vivo Treg expansion using LD IL-2 (1 × 106 IU/m2 per day for 8 weeks) in 25 adult patients with SR-cGVHD. Treg were not expanded ex vivo. Treg DLI was initiated at 0.1 × 106 cells per kg patient and escalated to a maximum dose of 1 × 106 cells per kg. Treg DLI plus LD IL-2 was well tolerated and led to partial responses (PR) in 5 of 25 patients (20%) after 8 weeks of therapy. Ten additional patients (40%) had stable disease with minor responses not meeting PR criteria. Patients at all dose levels had similar Treg expansion without significant changes in CD4+ conventional T cells or CD8+ T cells. High-throughput sequencing of the T-cell receptor β locus showed selective improvement of Treg diversity. A subset of DLI-derived Treg clones showed preferential expansion at week 8 and long-term persistence 1-year postinfusion. We demonstrate for the first time that infusion of polyclonal healthy donor Tregs followed by expansion with LD IL-2 is safe in patients with SR-cGVHD, thus establishing a foundation for future adoptive Treg therapies in the posttransplant setting. This trial was registered at www.clinicaltrials.gov as #NCT01937468.

Protein delivery into cells is a potentially transformative tool for treating "undruggable" targets in diseases associated with protein deficiencies or mutations. The vast majority of these targets are accessed via the cytosol, a challenging prospect for proteins with therapeutic and diagnostic relevance. In this review we will present promising non-viral approaches for intracellular and ultimately cytosolic delivery of proteins using nanocarriers. We will also discuss the mechanistic properties that govern the efficacy of nanocarrier-mediated protein delivery, applications of nanomaterials, and key challenges and opportunities in the use of nanocarriers for intracellular protein delivery.

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480μM. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5'-OH group or with the 2'-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.

The WHO was very clear that an HbA(1c) of 48 mmol/mol (6.5%) and above is diagnostic of diabetes. They were less clear regarding results that fell below 48 mmol/mol. The WHO recognize that individuals with HbA(1c) values below the cut-off point may still have diabetes, but give no guidance on how to investigate further. It is important that these individuals, who may be at increased risk of developing diabetes, are monitored correctly; a recommended scheme is given in Fig. 2; following these recommendations will ensure at-risk people are not overlooked and will be monitored closely. Even although it is not recommended to combine HbA(1c) with glucose measurement for diagnosis, the WHO did not discount the value of a fasting glucose and an oral glucose tolerance test to diagnose diabetes in selected individuals; it is the responsibility of the investigating doctor to choose how best to diagnose on an individual basis. This new method of diagnosing diabetes will identify a different cohort as having diabetes than is currently being diagnosed; but the process of investigation that does not require a fasting sample makes investigation easier, allowing more people to be investigated.

Source Citation The Blood Pressure Lowering Treatment Trialists' Collaboration, Sundstrom J, Arima H, et al. Blood pressure-lowering treatment based on cardiovascular risk: a meta-analysis of indiv...

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.

When people with established type 2 diabetes first need insulin therapy there is often a delay in accessing services. Since the report of the UK Prospective Diabetes Study was published it has been recognised that people with type 2 diabetes have a progressive disease and a need for increasing treatment. The authors set up a fast-track service to enable GPs and practice nurses to refer to a nurse-led service at the diabetes centre.

This trial compares to methods used to find the insulin dose when starting insulin in people with type 2 diabetes in whom maximal tolerated oral hypoglycaemic agents have failed to control glycaemia. One method of initiating insulin (based on height, weight, and fasting blood plasma glucose and insulin resistance) was compared with a conventional method of initiating 10 units of isophane insulin twice daily.Sixty subjects were randomised to calculated (CALC) or conventional (C). Follow-up was at three, six and 12 months with diabetes specialist nurse (DSN) support. The outcomes that were compared were glycated haemoglobin levels (HbA1c), hypoglycaemia, weight gain, insulin dose, quality of life, treatment satisfaction, blood pressure, and frequency of DSN contact.HbA1c levels values were reduced significantly in the CALC group during the first three months after starting insulin (p = 0.0001) and improved by one per cent overall during 12 months (p = 0.03). No difference was found in rates of hypoglycaemia, blood pressure, quality of life, and treatment satisfaction. Weight gain was seen in both groups but was significantly higher in the CALC group. People in the CALC group needed significantly less DSN time (p = 0.01).HbA1c target values were achieved more quickly and with less DSN contact using the CALC method. The difference in weight gain with the CALC method needs further investigation.

Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs. Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme·drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide· topoisomerase II (rather than etoposide·DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

The epidemiological study of ethnic groups provides valuable information for physicians in a number of ways. First, it assists in the diagnostic approach in individual patients, especially for doctors unfamiliar with patients of a particular ethnic background. Second, it provides population data to assist in health care planning and provision for the present and future in districts where ethnic minorities are resident in significant numbers. Third, it may provide pointers for the study of pathogenic mechanisms. Fourth, changing epidemiology in migrant ethnic minorities may help to distinguish the degree of influence of genetic and environmental pathogenic factors. These studies are particularly valuable when minority populations are of a size which allows meaningful data to be collected on uncommon conditions. Furthermore the contrasts between migrant and indigenous populations are most clearly seen if migration has occurred over a restricted period. For these and other reasons the study of the migrant population from the Indian subcontinent (defined as India, Pakistan and Bangladesh) which has settled in Leicester since the mid-1960s has proved particularly rewarding. This report summarizes the distinctive epidemiological features of these people now living in Leicester.

Initial dietary advice for Asian diabetics attending a general practice diabetic clinic was given in Asian languages. Between 2 and 3 years later a questionnaire interview was conducted to assess the effectiveness of the advice given. Forty of the 59 Asian diabetic patients in the practice attended for interview. The majority continued to use sugar or 'gur' (jaggery), Asian sweets, and Asian snacks (which have a high fat content). Fat and oil consumption per person per month was also high. Failure to achieve dietary objectives seemed to result from lack of sufficient emphasis on the types of foods usually consumed and an inadequate understanding of eating and cooking habits of Asians. We conclude that for dietary advice to be successful, emphasis must be placed on specific types of food eaten together with an understanding of the sociocultural implications of eating practices in ethnic groups.

Glucose-stimulated insulin secretion was assessed in relation to the DNA polymorphism flanking the insulin gene in 16 women who had had gestational diabetes. When not pregnant they were studied by a Constant Glucose Infusion for 1 hr with Model Assessment (CIGMA). The patients with either heterozygous 1/3 alleles or homozygous 1/1 or 3/3 alleles in the 5' flanking region of the insulin gene had similar plasma insulin and C-peptide concentrations and similar estimates of beta-cell function. This suggests that the large DNA insert (Class 3 allele) near the insulin gene does not directly affect insulin secretion. The association of polymorphisms with Type 2 diabetes may be due to linkage disequilibrium with other disease-related alleles.

The prevalence of diabetic nephropathy was studied in a population of 847 juvenile-onset (insulin-dependent) diabetics diagnosed under the age of 17 years who attended diabetic clinics in Leicestershire between 1930 and 1985. Seven hundred and eighty-nine patients (93.2 per cent) were traced. Eight patients with non-diabetic proteinuria and four classified as maturity-onset diabetics of the young were excluded from further analysis. The mean age at onset was 9.3 +/- 4.2 years (mean +/- SD) and the mean duration of diabetes 17.0 +/- 10.6 years (range 3-63 years). Overall, 28 patients (3.6 per cent) developed persistent proteinuria and 39 patients (five per cent) intermittent proteinuria. End-stage renal failure developed in eight patients. In patients with diabetes of 20 years duration (n = 254) the prevalence of persistent proteinuria was 9.1 per cent and of intermittent proteinuria, 7.9 per cent. Systolic and diastolic blood pressures were elevated in diabetics with persistent proteinuria compared to patients without proteinuria (154/89 +/- 27/15 vs 123/76 +/- 15/9 mm Hg mean +/- SD; p less than 0.001). Systolic blood pressure was also raised in patients with intermittent proteinuria (133/77 +/- 17/12, p less than 0.005). In addition, proteinuria (intermittent and persistent) was significantly associated with the use of once-daily insulin therapy in childhood and poor clinic attendance. This study suggests that twice-daily insulin therapy from diagnosis in childhood and regular contact with the diabetic clinic decreased the prevalence of diabetic nephropathy in this population.

20 patients with non insulin dependent diabetes mellitus (NIDDM), and within 20% of their ideal body weight were studied. They had failed to achieve adequate diabetic control following 3 months of dietary therapy. They were randomly allocated to insulin or sulphonylurea therapy for 3 months and then "crossed over" for the same period of time. Patients were maintained at euglycaemia (plasma glucose 4-7 mmol/l) for 24 hr using an open-loop intravenous insulin infusion, and then underwent a standard 75 gm oral glucose tolerance test (OGTT) following each mode of therapy. Mean glycosylated haemoglobin and preprandial blood glucose were 8.7% and 7.7 mmol/l respectively after sulphonylurea, and 7.8% (p less than 0.05) and 6.6 mmol/l (p less than 0.05) after insulin therapy. There was no significant difference in change in body weight. Following a 75 gm OGTT mean plasma insulin at 1/2 hr and 1 hr was 14.0 mu/l and 16.5 mu/l after sulphonylurea, and 23.4 mu/l (p less than 0.05) and 22.1 mu/l (p less than 0.05) after insulin therapy. Plasma C-peptide responses were also improved at 1/2, 1 and 1 1/2 hr after a period of insulin therapy being 0.61 nmol/l, 0.65 nmol/l and 0.59 nmol/l respectively. After sulphonylurea therapy comparable plasma C-peptide responses were 0.31, 0.41 and 0.37 nmol/l respectively (p less than 0.05). There was no significant difference in the total amount of intravenous insulin required for 24 hr euglycaemia. Our study shows that short term insulin therapy in patients with NIDDM who had failed on diet alone has advantages over sulphonylurea therapy in that it achieves better diabetic control and an improved B-cell response to glucose stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the effect of short term intensive insulin therapy in non-insulin-dependent diabetes mellitus (NIDDM) we studied 10 patients who had been on maximal doses of sulphonylurea therapy, with a glycosylated haemoglobin value persistently above the normal range. All patients were non-ketonuric, had negative islet cell antibodies, and had been on sulphonylureas for a mean duration of 5.6 yr. Patients were maintained at euglycaemia (plasma glucose 4-7 mmol/l) for 24 hr using an open-loop intravenous insulin regimen, and then underwent a standard 75 g oral glucose tolerance test (OGTT). This was repeated after 3 months of treatment with insulin. Mean fasting plasma glucose and glycosylated haemoglobin were 10.1 mmol/l and 12.2% respectively before, and 7.1 mmol/l (p less than 0.001) and 8.4% (p less than 0.001) after treatment. There was no significant change in body weight. Plasma insulin and C-peptide responses to 75 g OGTT did not change significantly, but the total amount of intravenously infused insulin required for 24-hr euglycaemia fell from a mean value of 138 u before treatment to 87 u (p less than 0.001) at the end of insulin therapy. Remission, with glycosylated haemoglobin in the normal range for more than 3 months after stopping insulin, was observed in 5 out of the 10 patients. All 5 who failed to achieve remission had markedly blunted maximal insulin responses of less than 10 mu/l on both OGTT's. Our study shows that insulin treatment in NIDDM appears to exercise a beneficial effect by lowering insulin resistance. We suggest that this may be of advantage early on in patients with NIDDM in preserving B-cell reserve.