Structure-based design of a T-cell receptor leads to nearly 100-fold improvement in binding affinity for pepMHC

T-cell receptors (TCRs) are proteins that recognize peptides from foreign proteins bound to the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell. This interaction enables the T cells to initiate a cell-mediated immune response to terminate cells displaying the foreign peptide on their MHC. Naturally occurring TCRs have high specificity but low affinity toward the peptide-MHC (pepMHC) complex. This prevents the usage of solubilized TCRs for diagnosis and treatment of viral infections or cancers. Efforts to enhance the binding affinity of several TCRs have been reported in recent years, through randomized libraries and in vitro selection. However, there have been no reported efforts to enhance the affinity via structure-based design, which allows more control and understanding of the mechanism of improvement. Here, we have applied structure-based design to a human TCR to improve its pepMHC binding. Our design method evolved based on iterative steps of prediction, testing, and generating more predictions based on the new data. The final design function, named ZAFFI, has a correlation of 0.77 and average error of 0.35 kcal/mol with the binding free energies of 26 point mutations for this system that we measured by surface plasmon resonance (SPR). Applying the filter that we developed to remove nonbinding predictions, this correlation increases to 0.85, and the average error decreases to 0.3 kcal/mol. Using this algorithm, we predicted and tested several point mutations that improved binding, with one giving over sixfold binding improvement. Four of the point mutations that improved binding were then combined to give a mutant TCR that binds the pepMHC 99 times more strongly than the wild-type TCR.     

Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering

Localized and release-controlled delivery systems for the sustained expression of the biologic potency of rhBMPs are essential. A substantial number of biomaterials have been investigated thus far. Most fail after implantation or administration mainly due to either being too soft, difficult to control and/or stabilize mechanically. In the second part of this review, we review a representative selection of rhBMP-2 and rhBMP-7 carrier materials and delivery systems ranging from simple nano/microparticles to complex 3-D scaffolds in sites of orthopaedic and craniofacial bone regeneration and repair.     

Electron Microscopic Localization of LacZ Expression in the Proximal Convoluted Tubular Cells of the Kidney in Transgenic Mice Carrying Chimeric Erythropoietin/lacZ Gene Constructs

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5′-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5′-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5′-lacZ construct and two additional constructs carrying the 6.5-kb 5′-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3′-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5′-flanking sequence with or without the body of the gene and the 1.2-kb 3′-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.     

The molecular biology of human renin and its gene

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.     

Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1)

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) = 1.4 +/- 0.66 h; n = 3) but almost completely inhibited at either 15 or 4 degrees C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited alpha-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated alpha-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates alpha-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.     

Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts

Familial HDL deficiency (FHD) is the heterozygous form of Tangier disease (TD). Mutations of the ABCA1 gene cause FHD and TD. FHD/TD cells are unable to normally efflux cholesterol onto nascent HDL particles, which are rapidly catabolized. TD fibroblasts have an abnormal pattern of PLC and PLD activation following cell stimulation with HDL(3) or apolipoprotein A-I (apoA-I). We examined cellular cholesterol efflux in FHD and TD fibroblasts by phospholipid-derived-molecules, compared with that of normal cells. We used the PKC agonist 1,2-dioctanoylglycerol (DOG) and phorbol myristate acetate (PMA) to activate PKC, calphostin C, and GO 6976, as inhibitors of PKC; phosphatidic acid (PA), which is the product of PLD, and lysophosphatidic acid (LPA), phosphatidylcholine, sphingomyelin, and beta-cyclodextrin to investigate their potential effect in modulating cellular cholesterol efflux in [(3)H]cholesterol-labeled and cholesterol-loaded fibroblasts. Phosphatidylcholine, sphingomyelin, and beta-cyclodextrin promoted cholesterol efflux in an identical fashion in control, FHD, or TD fibroblasts. In a dose-dependent fashion, DOG (0-200 microM) increased apoA-I-mediated cellular cholesterol efflux by 40% in controls, 71% in FHD, and 242% in TD cells. PMA similarly increased cholesterol efflux to a maximum of 256% in controls, 182% in FHD, and 191% in TD cells. This effect was inhibited by calphostin C. PA (100 microM) also increased cholesterol efflux by 25% in control, 44% in FHD, and 100% in TD cells. Conversely, LPA reduced cholesterol efflux in a dose-dependent fashion in control and FHD cells (-50%, 200 microM) but not in TD cells, where efflux was increased by 140%. Propranolol (100 microM) significantly increased cholesterol efflux at 24 h in all three cell lines. n-Butanol partially decreased the DOG-mediated increase in cholesterol efflux. The inhibitory effect of calphostin C on DOG-stimulated cholesterol efflux could be partially overcome by propranolol, suggesting that PA is a downstream mediator of PKC-stimulated cholesterol efflux.We conclude that PLC and PLD activities are required for apoA-I-mediated cellular cholesterol efflux, and modulating cellular PA concentration can correct, at least partially, the cholesterol efflux defect in FHD and TD.     

Evaluation of Gossypium species for resistance to cotton leaf curl Burewala virus

Cotton leaf curl disease (CLCuD), caused by cotton leaf curl Burewala virus (CLCuBV), has emerged as a major threat to cotton production in Pakistan. Resistance to CLCuBV was evaluated in cultivated and wild cotton genotypes representing six Gossypium species by visual symptom scoring and virus assessment using PCR tests. Considerable variation in responses was observed when using whitefly and graft transmission to inoculate Gossypium genotypes with CLCuBV in field and greenhouse experiments. Under field evaluation, all cultivated genotypes of Gossypium hirsutum and three genotypes of G. barbadense were susceptible. Eleven genotypes that represented six wild and cultivated Gossypium species were considered to be highly resistant as they were free from infection. Similar results were obtained when these genotypes were tested using whitefly transmission. To verify these findings, 132 cultivated and wild genotypes were tested by graft inoculation. All G. hirsutum genotypes (116 cultivated, 1 wild, 1 transgenic Coker-312 and 1 non-transgenic Coker-312), three G. barbadense genotypes and one G. thurberi genotype were highly susceptible and exhibited symptoms 9–12 days after grafting. Four genotypes of G. arboreum and one genotype of G. anomalum did not express symptoms but had a detectable level of virus. One genotype of G. herbaceum and three wild genotypes of G. hirsutum showed mild symptoms (severity indexes of 1–2) and exhibited delayed disease development. These genotypes were classified as moderately resistant to resistant. Resistant genotypes that were identified in this study will be useful sources for exploitation of breeding programmes aimed at developing CLCuBV-resistant varieties and increasing genetic diversity.     

Glucose-responsive insulin and glucagon delivery (dual-hormone artificial pancreas) in adults with type 1 diabetes: a randomized crossover controlled trial

Background:

Most patients with type 1 diabetes do not achieve their glycemic targets. We aimed to assess the efficacy of glucose-responsive insulin and glucagon closed-loop delivery for controlling glucose levels in adults with type 1 diabetes.

Methods:

We conducted a randomized crossover trial involving 15 adults with type 1 diabetes, comparing standard insulin-pump therapy with dual-hormone, closed-loop delivery. Patients were admitted twice to a clinical research facility and received, in random order, both treatments. Each 15-hour visit (from 1600 to 0700) included an evening exercise session, followed by a medium-sized meal, a bedtime snack and an overnight stay. During visits that involved closed-loop delivery, basal insulin and glucagon miniboluses were delivered according to recommendations based on glucose sensor readings and a predictive dosing algorithm at 10-minute intervals. During visits involving standard insulin-pump therapy (control visits), patients used conventional treatment.

Results:

Dual-hormone closed-loop delivery increased the percentage of time for which patients’ plasma glucose levels were in the target range (median 70.7% [interquartile range (IQR) 46.1%–88.4%] for closed-loop delivery v. 57.3% [IQR 25.2%–71.8%] for control, p = 0.003) and decreased the percentage of time for which plasma glucose levels were in the low range (bottom of target range [< 4.0 mmol/L], 0.0% [IQR 0.0%–3.0%] for closed-loop delivery v. 10.2% [IQR 0.0%–13.0%] for control, p = 0.01; hypoglycemia threshold [< 3.3 mmol/L], 0.0% [IQR 0.0%–0.0%] for closed-loop delivery v. 2.8% [IQR 0.0%–5.9%] for control, p = 0.006). Eight participants (53%) had at least 1 hypoglycemic event (plasma glucose < 3.0 mmol/L) during standard treatment, compared with just 1 participant (7%) during closed-loop treatment (p = 0.02).

Interpretation:

Dual-hormone, closed-loop delivery guided by advanced algorithms improved short-term glucose control and reduced the risk of hypoglycemia in a group of 15 adults with type 1 diabetes. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT01297946","term_id":"NCT01297946"}}NCT01297946.The Diabetes Control and Complications Trial has shown that intensive insulin therapy in type 1 diabetes with the aim of good glycemic control substantially reduces microvascular and macrovascular complications.^1,2 However, despite advances in insulin analogs, insulin pumps and continuous glucose-monitoring systems, glucose control remains problematic, and most patients with type 1 diabetes do not achieve their glycemic targets.³Hypoglycemia remains the major barrier to the intensification of insulin therapy.⁴ Intensive insulin therapy and lower levels of glycated hemoglobin are unfortunately associated with an increased risk of hypoglycemia.⁵ The frequency of patient-reported nonsevere hypoglycemia (blood glucose ≤ 3.5 mmol/L, with or without symptoms) is about 2.7 episodes/patient per week,⁶ with episodes commonly occuring during the night. In a recent continuous glucose-monitoring trial conducted by the Juvenile Diabetes Research Foundation,⁷ hypoglycemia (glucose sensor reading < 3.3 mmol/L) occurred during 8.5% of the nights included in the study period, with 47% of those nights involving at least 1 hour of hypoglycemia, 23% involving at least 2 hours, and 11% involving at least 3 hours.Advances in insulin infusion pumps and continuous glucose-monitoring systems could improve glycemic control;⁸ however, we still lack the ability to combine these devices in an automated manner. Closed-loop insulin delivery systems (i.e., the artificial pancreas) combine the 2 devices using a mathematical algorithm.⁹ These systems might improve glycemic control and reduce the risk of hypoglycemia compared with conventional insulin-pump therapy (i.e., continuous subcutaneous insulin infusion).^10,11 However, a clinically significant number of hypoglycemic events (blood glucose < 3.0 mmol/L) were still reported during tests of closed-loop delivery systems.^10,11Dual-hormone closed-loop delivery systems have also been proposed to regulate glucose levels. These systems combine insulin delivery with subcutaneous glucagon delivery to further reduce the risk of hypoglycemia.^12–14 However, their potential benefits to improve glycemic control are currently unknown. We sought to determine whether dual-hormone closed-loop delivery, compared with conventional insulin pump therapy, can improve glycemic control and reduce the risk of hypoglycemia in adults with type 1 diabetes.     

Sensitivity to Chronic Methamphetamine Administration and Withdrawal in Mice with Relaxin-3/RXFP3 Deficiency

Methamphetamine (METH) is a highly addictive psychostimulant, and cessation of use is associated with reduced monoamine signalling, and increased anxiety/depressive states. Neurons expressing the neuropeptide, relaxin-3 (RLN3), and its cognate receptor, RXFP3, constitute a putative ‘ascending arousal system’, which shares neuroanatomical and functional similarities with serotonin (5-HT)/dorsal raphe and noradrenaline (NA)/locus coeruleus monoamine systems. In light of possible synergistic roles of RLN3 and 5-HT/NA, endogenous RLN3/RXFP3 signalling may compensate for the temporary reduction in monoamine signalling associated with chronic METH withdrawal, which could alter the profile of ‘behavioural despair’, bodyweight reductions, and increases in anhedonia and anxiety-like behaviours observed following chronic METH administration. In studies to test this theory, Rln3 and Rxfp3 knockout (KO) mice and their wildtype (WT) littermates were injected once daily with saline or escalating doses of METH (2 mg/kg, i.p. on day 1, 4 mg/kg, i.p. on day 2 and 6 mg/kg, i.p. on day 3–10). WT and Rln3 and Rxfp3 KO mice displayed an equivalent sensitivity to behavioural despair (Porsolt swim) during the 2-day METH withdrawal and similar bodyweight reductions on day 3 of METH treatment. Furthermore, during a 3-week period after the cessation of chronic METH exposure, Rln3 KO, Rxfp3 KO and corresponding WT mice displayed similar behavioural responses in paradigms that measured anxiety (light/dark box, elevated plus maze), anhedonia (saccharin preference), and social interaction. These findings indicate that a whole-of-life deficiency in endogenous RLN3/RXFP3 signalling does not markedly alter behavioural sensitivity to chronic METH treatment or withdrawal, but leave open the possibility of a more significant interaction with global or localised manipulations of this peptide system in the adult brain.