The T Cell Repertoires from Nickel Sensitized Joint Implant Failure Patients

Nickel (Ni²⁺) is one of the most common allergens, affecting around 10–15% of the general population. As the demand for orthopedic implant surgery rises, the number of surgical revisions due to joint implant failure also increases. There is evidence that some patients develop joint failure due to an immune response to a component of the implant, and we have found that Ni²⁺ is an especially important cause. Hence, understanding the mechanisms by which Ni²⁺ allergy induces joint implant failure becomes a critical research question. The structural basis of Ni²⁺ activation of pathogenic T cells is still not clear. The purpose of this study was to characterize Ni²⁺-reactive T cell repertoires derived from the peripheral blood of joint failure patients due to Ni²⁺ sensitization using single-cell sequencing techniques. We stimulated the proliferation of Ni²⁺ -reactive T cells from two implant failure patients in vitro, and sorted them for single-cell VDJ sequencing (10× genomics). We identified 2650 productive V-J spanning pairs. Both TCR α chains and β chains were enriched. TRBV18 usage is the highest in the P7 CD4+ population (18.1%), and TRBV5-1 usage is the highest in the P7 CD8+ population (12.1%). TRBV19 and TRBV20-1 segments are present in a high percentage of both P7 and P9 sequenced T cells. Remarkably, the alpha and beta chain combination of TRAV41-TRBV18 accounts for 13.5% of the CD4+ population of P7 patient. Compared to current Ni specific T cell repertoire studies of contact dermatitis, the Vα and Vβ usages of these joint implant failure patients were different. This could be due to the different availability of self-peptides in these two different tissues. However, TRBV19 (Vβ17) was among frequently used TCR β chains, which are common in previous reports. This implies that some pathogenic T cells could be similar in Ni²⁺ hypersensitivities in skin and joints. The alignment of the TCR CDR3β sequences showed a conserved glutamic acid (Glu) that could potentially interact with Ni²⁺. The study of these Ni²⁺ specific TCRs may shed light on the molecular mechanism of T cell activation by low molecular weight chemical haptens.     

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2–c.378+1G>T) in the first patient and a nonsense mutation (DSG2–p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.     

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.     

Polylactide films formed by immersion precipitation: Effects of additives,nonsolvent, and temperature

The influence of nonsolvent, crystallinity of the polymer film, and addition of dodecane (a poor solvent for the polymer and for the nonsolvent) on the morphology of polylactides films has been investigated and was related to phase separation behavior. Both amorphous poly‐DL ‐lactide (PDLLA) and crystalline poly‐L ‐lactide (PLLA) were dissolved in dichloromethane, and subsequently films were made by immersion in nonsolvent baths. PDLLA gave dense films without any internal structure, since the structure was not solidified by crystallization or glassification. PLLA films show varying structure depending on the nonsolvent. With methanol, asymmetric morphologies were observed as a result from combined liquid‐liquid demixing and crystallization, while with water symmetric spherulitic structures were formed. As a next step, dodecane was added, which is not miscible with the nonsolvent, and we found it to have a strong influence on the morphology of the films. The PDLLA films with dodecane did not collapse: a closed cell structure was obtained. In PLLA films, dodecane speeds up phase separation and induces faster crystallization in the films, and the porosity, size of the pores, and interconnectivity increased. When the PLLA solutions were subjected to a heat pretreatment, crystallization could be postponed, which yielded a cellular structure around dodecane, which did not contain spherulites anymore. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 959–971, 2007     

High Throughput Screening of Low Temperature SCR and SCD De-NOx Catalysts in Scanning Mass Spectrometer

High-throughput synthesis and screening methods have been developed for the discovery of highly active lead compounds for the selective catalytic reduction as well as direct decomposition of NO in the temperature range 200–300 °C. The discovery libraries for primary screening consisted of 16 × 16 catalyst arrays on 4in. square quartz wafers. Catalysts were prepared by robotic liquid dispensing techniques and screened for catalytic activity in Symyx' scanning mass spectrometer. The scanning mass spectrometer is a fast serial screening tool that uses flat wafer catalyst surfaces, local laser heating, a scanning/sniffing nozzle and a quadrupolar mass spectrometer to compare relative catalytic activities. The feed consisted of NO/NH₃ mixtures with optional O₂ cofeed and Kr as the internal standard in Ar carrier gas. QMS detection allowed for tracking of H₂O, N₂, NO, O₂, N₂O and Kr. Screening protocols for catalytic materials encompassed metal precursors and carriers for supported vanadia systems, extensive doping of V₂O₅/TiO₂, and broad screening of mixed redox metal oxides and supported base and noble metal systems. More than 500 samples could be screened in a single day. Active hits (high NO consumption accompanied by corresponding N₂ production) identified in discovery libraries were re-synthesized as focus libraries for lead confirmation and further optimization. These libraries used shallower compositional gradients, for example 56 points (compositions) per ternary, with four 56-point ternaries per 4in. wafer. Broad screening ternaries were generally 8 or 15 points. The focus libraries more clearly reveal the trends and provide guidelines for secondary screening and scale-up. High conversions were achieved in scanning mass spectrometer so the scalability risk is small for the short contact time reactions.     

Self‐regulation during e‐learning: using behavioural evidence from navigation log files

The current paper examined the relationship between perceived characteristics of the learning environment in an e‐module in relation to test performance among a group of e‐learners. Using structural equation modelling, the relationship between these variables is further explored in terms of the proposed double mediation as outlined by Ning and Downing. These authors initially proposed that motivation and self‐regulation strategies are mediators between the perception of the learning environment and performance. In our replication and extension study, we substituted self‐reported self‐regulation with behavioural indicators of self‐regulation using navigation log files and focused on test‐taking rather than general motivation. We proposed that navigational patterns captured using log files can also help deduce self‐regulation in e‐modules and provide information in the absence of self‐reports. Path analyses provide partial support for our navigational hypotheses and the model. Implications of our results for the use of e‐module data and conclusions based on navigation are discussed.     

Synthesizing robust systems

Systems should not only be correct but also robust in the sense that they behave reasonably in unexpected situations. This article addresses synthesis of robust reactive systems from temporal specifications. Existing methods allow arbitrary behavior if assumptions in the specification are violated. To overcome this, we define two robustness notions, combine them, and show how to enforce them in synthesis. The first notion applies to safety properties: If safety assumptions are violated temporarily, we require that the system recovers to normal operation with as few errors as possible. The second notion requires that, if liveness assumptions are violated, as many guarantees as possible should be fulfilled nevertheless. We present a synthesis procedure achieving this for the important class of GR(1) specifications, and establish complexity bounds. We also present an implementation of a special case of robustness, and show experimental results.     

Synthesis and characterization of cleavable surfactants derived from poly(ethylene glycol) monomethyl ether

A series of noncyclic acetal-linked cleavable surfactants were simply prepared by condensation of aldehydes with poly(ethylene glycol) monomethyl ethers. All of the products were characterized by¹H nuclear magnetic resonance. Their hydrophile-lipophile balance, surface tension, cloud point, critical micelle concentration, and foam height were determined. Hydrolysis kinetic studies, followed by gas chromatography, showed that they had higher hydrolytic reactivity in acidic solution than cyclic acetal-linked cleavable surfactants.     

Carbohydrate type and amount alter intravascular processing and catabolism of plasma lipoproteins in guinea pigs

To test the effects of exchanging dietary complex and simple carbohydrate for fat calories on lipoprotein metabolism, guinea pigs were fed two different fat/carbohydrate ratios: 2.5∶58% (w/w) or 25∶29% (w/w) with either sucrose or starch as the carbohydrate source. Animals fed high-fat had higher plasma low-density lipoprotein (LDL) and hepatic cholesterol concentrations than animals fed low-fat diets (P<0.01). The cholesteryl ester content per particle was higher, and the number of triacylglycerol (TAG) molecules was lower in very low density lipoprotein (VLDL) and LDL from animals fed high-fat diets. Intake of high-fat/sucrose resulted in higher plasma LDL concentrations than intake of high-fat/starch, and animals fed low-fat/starch had the highest plasma TAG concentrations associated with VLDL particles containing more TAG molecules, as well as a TAG-enriched LDL. The activity of plasma lecithin cholesteryl:acyl transferase (LCAT) was highest in animals fed high-fat/sucrose, and heart lipoprotein lipase (LPL) activity was higher in animals fed high-fat diets. Hepatic apoprotein B/E (apo B/E) receptor number (B_max) was increased 21% with low-fat diets (P<0.01). These results suggest that the hypercholesterolemia induced by high-fat and by sucrose intake are associated with a higher plasma LCAT activity which results in a cholesteryl ester-enriched VLDL which, by the action of LPL, might be more readily converted to LDL through the delipidation cascade leading to downregulation of hepatic apo B/E receptors. The hypertriglyceridemia associated with low-fat intake may result from increased production of VLDL TAG, which would explain the increased TAG content and the higher TAG/CE ratio of VLDL from animals fed the low-fat/starch diet.