Force spectroscopy of substrate molecules en route to the proteasome's active sites

We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (β-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome.     

Impact of improved potassium accumulation on pH homeostasis, membrane potential adjustment and survival of Corynebacterium glutamicum

Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this study the uptake and the impact of the most abundant internal cation, potassium, were investigated in Actinobacteria, a group of high G+C Gram-positives with a number of prominent biotechnologically and medically important members. Genome analyses revealed a variety of different potassium uptake systems in this monophyletic group ranging from potassium channels common in virtually all Actinobacteria to different active carriers that were present predominantly in pathogenic members able to cope with various stress conditions. By applying Corynebacterium glutamicum as model system we provide experimental evidence that under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however, viability of C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB potassium transporter from the pathogen Corynebacterium jeikeium was heterologously expressed. We provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved maintenance of internal pH and membrane potential. The results indicate that the occurrence of active potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis and survival of bacterial cells under stress conditions.     

Longitudinal study of prevalence and spatio-temporal distribution of Borrelia burgdorferi sensu lato in ticks from three defined habitats in Latvia, 1999–2010

Members of the Borrelia burgdorferi sensu lato (s.l.) species complex are known to cause human Lyme borreliosis. Because of longevity of some reservoir hosts and the Ixodes tick vectors' life cycle, long-term studies are required to better understand species and population dynamics of these bacteria in their natural habitats. Ticks were collected between 1999 and 2010 in three ecologically different habitats in Latvia. We used multilocus sequence typing utilizing eight chromosomally located housekeeping genes to obtain information about species and population fluctuations and/or stability of B. burgdorferi s.l. in these habitats. The average prevalence over all years was 18.9%. From initial high-infection prevalences of 25.5%, 33.1% and 31.8%, from 2002 onwards the infection rates steadily decreased to 7.3%. Borrelia afzelii and Borrelia garinii were the most commonly found genospecies but striking local differences were obvious. In one habitat, a significant shift from rodent-associated to bird-associated Borrelia species was noted whilst in the other habitats, Borrelia species composition was relatively stable over time. Sequence types (STs) showed a random spatial and temporal distribution. These results demonstrated that there are temporal regional changes and extrapolations from one habitat to the next are not possible.     

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.     

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Background

For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.

Methodology and Principle Findings

We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients'' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.

Conclusion

The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.     

Short-Term Azithromycin Treatment Promotes Cornea Allograft Survival in the Rat

Background

Any inflammatory response following corneal transplantation may induce rejection and irreversible graft failure. The purpose of this study is to analyze the anti-inflammatory effect of azithromycin (AZM) following experimental keratoplasty in rats.

Methods

Corneal transplants were performed between Fisher-donor and Lewis-recipient rats. Recipients were postoperatively treated three times daily with AZM, miglyol, ofloxacin or dexamethasone eye drops. As an additional control, AZM was applied following syngeneic keratoplasty. Furthermore, short-term treatments with AZM for seven days perioperatively or with AZM only three days prior to the transplantation were compared to appropriate controls. All transplants were monitored clinically for opacity, edema, and vascularization. Infiltrating CD45⁺, CD4⁺, CD8⁺, CD25⁺, CD161⁺ and CD163⁺ cells were quantified via immunohistochemistry.

Results

AZM significantly promoted corneal graft survival compared with miglyol or ofloxacin treatment. This effect was comparable to topical dexamethasone. No adverse AZM effect was observed. Histology confirmed a significant reduction of infiltrating leukocytes. The short-term application of AZM for three days prior to transplantation or for seven days perioperatively reduced corneal graft rejection significantly compared with the controls.

Conclusions

Along with antibiotic properties, topical AZM has a strong anti-inflammatory effect. Following keratoplasty, this effect is comparable to topical dexamethasone without the risk of steroid-induced adverse effects. Short-term treatment with AZM three days prior to the transplantation was sufficient to promote graft survival in the rat keratoplasty model. We therefore suggest further assessing the anti-inflammatory function of topical AZM following keratoplasty in humans.     

Affective State Influences Retrieval-Induced Forgetting for Integrated Knowledge

Background

Selectively testing parts of learned materials can impair later memory for nontested materials. Research has shown that such retrieval-induced forgetting occurs for low-integrated materials but may be prevented for high-integrated materials. However, previous research has neglected one factor that is ubiquitous in real-life testing: affective state.

Methodology/Principal Findings

We investigated whether affect influences the resistance of integrated materials to retrieval-induced forgetting by inducing neutral, positive, or negative affect immediately before selectively testing previously learned textbook passages containing interrelated facts and concepts. As negative affect is known to promote a detail-oriented local processing style, we hypothesized that experiencing negative affect during testing may decrease the protective effects of integration and lead to reoccurrence of forgetting. By contrast, as positive affect is known to promote a relation-oriented global processing style, we hypothesized that experiencing positive affect may support effects of integration and prevent forgetting. Our findings are consistent with these predictions. No subsequent forgetting occurred when testing memories for integrated text materials in affectively neutral and positive states, whereas forgetting occurred when testing in negative states. A correlation analysis showed that forgetting decreased with higher positive affect, with participants experiencing high positive affect even showing facilitation instead of forgetting.

Conclusions/Significance

These findings indicate that affect can moderate the memory consequences of test taking and suggest that educators should use testing as a tool to improve memory with care.     

Assessment of Urinary Betaine as a Marker of Diabetes Mellitus in Cardiovascular Patients

Abnormal urinary excretion of betaine has been demonstrated in patients with diabetes or metabolic syndrome. We aimed to identify the main predictors of excretion in cardiovascular patients and to make initial assessment of its feasibility as a risk marker of future diabetes development. We used data from 2396 patients participating in the Western Norway B-vitamin Intervention Trial, who delivered urine and blood samples at baseline, and in the majority at two visits during follow-up of median 39 months. Betaine in urine and plasma were measured by liquid-chromatography-tandem mass spectrometry. The strongest determinants of urinary betaine excretion by multiple regression were diabetes mellitus, age and estimated glomerular filtration rate; all p<0.001. Patients with diabetes mellitus (n = 264) had a median excretion more than three times higher than those without. We found a distinct non-linear association between urinary betaine excretion and glycated hemoglobin, with a break-point at 6.5%, and glycated hemoglobin was the strongest determinant of betaine excretion in patients with diabetes mellitus. The discriminatory power for diabetes mellitus corresponded to an area under the curve by receiver-operating characteristics of 0.82, and betaine excretion had a coefficient of reliability of 0.73. We also found a significant, independent log-linear relation between baseline betaine excretion and the risk of developing new diabetes during follow-up. The good discriminatory power for diabetes, high test-retest stability and independent association with future risk of new diabetes should motivate further investigation on the role of betaine excretion in risk assessment and long-term follow-up of diabetes mellitus.