Structural basis for the enhanced stability of highly fluorinated proteins

Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids.     

CD34(+)/CD38(-) stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin

Background

CD33 is a well-known stem cell target in acute myeloid leukemia. So far, however, little is known about expression of CD33 on leukemic stem cells in chronic leukemias.

Design and Methods

We analyzed expression of CD33 in leukemic progenitors in chronic myeloid leukemia by multi-color flow cytometry and quantitative polymerase chain reaction. In addition, the effects of a CD33-targeting drug, gemtuzumab/ozogamicin, were examined.

Results

As assessed by flow cytometry, stem cell-enriched CD34⁺/CD38⁻/CD123⁺ leukemic cells expressed significantly higher levels of CD33 compared to normal CD34⁺/CD38⁻ stem cells. Moreover, highly enriched leukemic CD34⁺/CD38⁻ cells (>98% purity) displayed higher levels of CD33 mRNA. In chronic phase patients, CD33 was found to be expressed invariably on most or all stem cells, whereas in accelerated or blast phase of the disease, the levels of CD33 on stem cells varied from donor to donor. The MDR1 antigen, supposedly involved in resistance against ozogamicin, was not detectable on leukemic CD34⁺/CD38⁻ cells. Correspondingly, gemtuzumab/ozogamicin produced growth inhibition in leukemic progenitor cells in all patients tested. The effects of gemtuzumab/ozogamicin were dose-dependent, occurred at low concentrations, and were accompanied by apoptosis in suspension culture. Moreover, the drug was found to inhibit growth of leukemic cells in a colony assay and long-term culture-initiating cell assay. Finally, gemtuzumab/ozogamicin was found to synergize with nilotinib and bosutinib in inducing growth inhibition in leukemic cells.

Conclusions

CD33 is expressed abundantly on immature CD34⁺/CD38⁻ stem cells and may serve as a stem cell target in chronic myeloid leukemia.     

First Clinical Experience with Intranasal Cooling for Hyperthermia in Brain-Injured Patients

Introduction

Hyperthermia is common in brain-injured patients and associated with a worse outcome. As brain rather than body temperature reduction, theoretically, is the most important in cerebral protection, there is logic in targeting cooling at the brain. Selective brain cooling can, in theory, be obtained by cooling the skull or by heat loss from the upper airways. In this preliminary safety and efficacy study, we report clinical data from brain-injured patients who because of hyperthermia were treated with intranasal cooling.

Methods

Nine intubated brain-injured patients with hyperthermia were treated using a prototype intranasal balloon system perfused with cold saline. Temperature in the cerebrum, esophagus, and bladder was monitored together with intracranial pressure.

Results

In only two of nine patients, normothermia was reached in the esophagus and in only four of nine patients it was reached in the bladder. When normothermia was reached, the time to normothermia was delayed. In the brain, normothermia was reached in two of five patients after approximately 72 h. Median temperature curves from the first 72 h of cooling showed that normothermia was not reached in any of the three compartments. The temperature in the brain and bladder were on average 0.6 and 0.5 °C higher than in the esophagus. ICP increased with increasing brain temperature. We found no signs of clinical important injury to the nasal mucosa from the cold saline or pressure in the balloons.

Conclusion

In brain-injured patients with hyperthermia, cooling with a prototype intranasal balloon system was clinically inadequate as the effect was delayed and not brain selective.     

Immunosenescence and organ transplantation

Increasing numbers of elderly transplant recipients and a growing demand for organs from older donors impose pressing challenges on transplantation medicine. Continuous and complex modifications of the immune system in parallel to aging have a major impact on transplant outcome and organ quality. Both, altered alloimmune responses and increased immunogenicity of organs present risk factors for inferior patient and graft survival. Moreover, a growing body of knowledge on age-dependent modifications of allorecognition and alloimmune responses may require age-adapted immunosuppression and organ allocation.Here, we summarize relevant aspects of immunosenescence and their possible clinical impact on organ transplantation.     

Motor subtypes of delirium: Past,present and future

Clinical subtyping of delirium according to motor-activity profile has considerable potential to account for the heterogeneity of this complex and multifactorial syndrome. Previous work has identified a range of clinically important differences between motor subtypes in relation to detection, causation, treatment experience and prognosis, but studies have been hampered by inconsistent methodology, especially in relation to definition of subtypes. This article considers research to date, including a number of recent studies that have attempted to address these issues and identify a means of achieving greater consistency in approaches to subtyping. Possibilities for future work are discussed and a research plan for the field is outlined.     

Delirium and long-term cognitive impairment

Delirium is a severe, acute neuropsychiatric syndrome that is highly prevalent in acute hospital populations. Delirium has noticeable effects on length of hospitalization, cost of care, mortality and morbidity. In addition to these well-established adverse consequences, there is increasing evidence linking delirium and a higher risk of long-term cognitive impairment (LTCI), including dementia. A prior review (Jackson, Gordon, Hart, Hopkins, & Ely, 2004), in which nine studies (total N = 1,885, years 1989–2003) were considered, concluded that there was evidence for an association between delirium and LTCI. Here we provide a review of studies published since Jackson's review. We included nine reports, with a total of 2,025 patients. The studies show diverse sample sizes, methodologies, designs and patient populations. However, taken together, the results of these new studies broadly confirm that there is a link between delirium and LTCI. We go on to discuss putative mechanisms and explanations. These include (1) delirium as a marker of chronic progressive pathology, but unrelated to any progression, (2) delirium as a consequence of acute brain damage which is also responsible for a ‘single hit’ or triggering of active processes causing LTCI, (3) delirium itself as a cause of LTCI, and (4) drug treatment of delirium or other conditions as a cause of LTCI. We conclude with suggestions for future research.     

Neural activity in the hippocampus predicts individual visual short‐term memory capacity

Although the hippocampus had been traditionally thought to be exclusively involved in long‐term memory, recent studies raised controversial explanations why hippocampal activity emerged during short‐term memory tasks. For example, it has been argued that long‐term memory processes might contribute to performance within a short‐term memory paradigm when memory capacity has been exceeded. It is still unclear, though, whether neural activity in the hippocampus predicts visual short‐term memory (VSTM) performance. To investigate this question, we measured BOLD activity in 21 healthy adults (age range 19–27 yr, nine males) while they performed a match‐to‐sample task requiring processing of object‐location associations (delay period = 900 ms; set size conditions 1, 2, 4, and 6). Based on individual memory capacity (estimated by Cowan's K‐formula), two performance groups were formed (high and low performers). Within whole brain analyses, we found a robust main effect of “set size” in the posterior parietal cortex (PPC). In line with a “set size × group” interaction in the hippocampus, a subsequent Finite Impulse Response (FIR) analysis revealed divergent hippocampal activation patterns between performance groups: Low performers (mean capacity = 3.63) elicited increased neural activity at set size two, followed by a drop in activity at set sizes four and six, whereas high performers (mean capacity = 5.19) showed an incremental activity increase with larger set size (maximal activation at set size six). Our data demonstrated that performance‐related neural activity in the hippocampus emerged below capacity limit. In conclusion, we suggest that hippocampal activity reflected successful processing of object‐location associations in VSTM. Neural activity in the PPC might have been involved in attentional updating. © 2013 Wiley Periodicals, Inc.     

Implant surface alters compartmental-specific contributions to fixation strength in rats

Mechanical fixation of the implant to host bone is an important contributor to orthopedic implant survivorship. The relative importance of bone-implant contact, trabecular bone architecture, and cortical bone geometry to implant fixation strength has never been directly tested, especially in the settings of differential implant surface properties. Thus, using a rat model where titanium rods were placed into the intramedullary canal of the distal femur, we determined the relative contribution of bone-implant contact and peri-implant bone architecture to the fixation strength in implants with different surface roughness: highly polished and smooth (as-received) and dual acid-etched (DAE) implants. Using a training set that maximized variance in implant fixation strength, we initially examined correlation between implant fixation strength and outcome parameters from microcomputed tomography and found that osseointegration volume per total volume (OV/TV), trabecular bone volume per total volume (BV/TV), and cortical thickness (Ct.Th) were the single best compartment-specific predictors of fixation strength. We defined separate regression models to predict implant fixation strength for as-received and DAE implants. When the training set models were applied to independent validation sets, we found strong correlations between predicted and experimentally measured implant fixation strength, with r² = .843 in as received and r² = .825 in DAE implants. Interestingly, for as-received implants, OV/TV explained more of the total variance in implant fixation strength than the other variables, whereas in DAE implants, Ct.Th had the most explanatory power, suggesting that surface topography of implants affects which bone compartment is most important in providing implant fixation strength.