Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30 degrees C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H(2)O(2) compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

The Practice of Cranial Neurosurgery and the Malpractice Liability Environment in the United States

Object

The potential imbalance between malpractice liability cost and quality of care has been an issue of debate. We investigated the association of malpractice liability with unfavorable outcomes and increased hospitalization charges in cranial neurosurgery.

Methods

We performed a retrospective cohort study involving patients who underwent cranial neurosurgical procedures from 2005-2010, and were registered in the National Inpatient Sample (NIS) database. We used data from the National Practitioner Data Bank (NPDB) from 2005 to 2010 to create measures of volume and size of malpractice claim payments. The association of the latter with the state-level mortality, length of stay (LOS), unfavorable discharge, and hospitalization charges for cranial neurosurgery was investigated.

Results

During the study period, there were 189,103 patients (mean age 46.4 years, with 48.3% females) who underwent cranial neurosurgical procedures, and were registered in NIS. In a multivariable regression, higher number of claims per physician in a state was associated with increased ln-transformed hospitalization charges (beta 0.18; 95% CI, 0.17 to 0.19). On the contrary, there was no association with mortality (OR 1.00; 95% CI, 0.94 to 1.06). We observed a small association with unfavorable discharge (OR 1.09; 95% CI, 1.06 to 1.13), and LOS (beta 0.01; 95% CI, 0.002 to 0.03). The size of the awarded claims demonstrated similar relationships. The average claims payment size (ln-transformed) (Pearson’s rho=0.435, P=0.01) demonstrated a positive correlation with the risk-adjusted hospitalization charges but did not demonstrate a correlation with mortality, unfavorable discharge, or LOS.

Conclusions

In the present national study, aggressive malpractice environment was not correlated with mortality but was associated with higher hospitalization charges after cranial neurosurgery. In view of the association of malpractice with the economics of healthcare, further research on its impact is necessary.     

Surgical Clipping versus Endovascular Intervention for the Treatment of Subarachnoid Hemorrhage Patients in New York State

Object

Randomized trials have demonstrated a survival benefit for endovascular treatment of ruptured cerebral aneurysms. We investigated the association of surgical clipping and endovascular coiling with outcomes in subarachnoid hemorrhage (SAH) patients in a real-world regional cohort.

Methods

We performed a cohort study involving patients with ruptured cerebral aneurysms, who underwent surgical clipping, or endovascular coiling from 2009–2013 and were registered in the Statewide Planning and Research Cooperative System (SPARCS) database. An instrumental variable analysis was used to investigate the association of treatment technique with outcomes.

Results

Of the 4,098 patients undergoing treatment, 2,585 (63.1%) underwent coiling, and 1,513 (36.9%) underwent clipping. Using an instrumental variable analysis, we did not identify a difference in inpatient mortality [marginal effect (ME), -0.56; 95% CI, -1.03 to 0.02], length of stay (LOS) (ME, 1.72; 95% CI, -3.39 to 6.84), or the rate of 30-day readmissions (ME, -0.30; 95% CI, -0.82 to 0.22) between the two treatment techniques for patients with SAH. Clipping was associated with a higher rate of discharge to rehabilitation (ME, 0.63; 95% CI, 0.24 to 1.01). In sensitivity analysis, mixed effect regression, and propensity score adjusted regression models demonstrated identical results.

Conclusions

Using a comprehensive all-payer cohort of patients in New York State presenting with aneurysmal SAH we did not identify an association of treatment method with mortality, LOS or 30-day readmission. Clipping was associated with a higher rate of discharge to rehabilitation.     

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.     

Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases

S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington''s disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.     

A conserved role for SNX9-family members in the regulation of phagosome maturation during engulfment of apoptotic cells

Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution.     

Predictors of new atherosclerotic carotid plaque development in patients with rheumatoid arthritis: a longitudinal study

Introduction

Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality attributed to both classical risk factors and chronic inflammation. We assessed longitudinally the factors associated with new carotid plaques in nondiabetic RA patients and apparently healthy individuals.

Methods

In our present prospective observational study, carotid plaques were identified by ultrasonography at baseline and follow-up end, separated by an average of 3.6 ± 0.2 years, in 64 patients (mean age 59.2 ± 12.0 and disease duration at baseline 7.8 ± 6.2 years, 83% women, clinical and laboratory evaluation every 3 to 6 months). In a substudy, 35 of the patients were matched 1:1 for traditional cardiovascular risk factors with 'healthy' controls and were studied in parallel.

Results

New atherosclerotic plaques formed in 30% of patients (first plaque in 9%) who were significantly older than the remaining patients. Tobacco use, blood pressure, body mass index, average cumulative low-density lipoprotein, high-sensitivity C-reactive protein, erythrocyte sedimentation rate level, RA stage, functional class, disease duration and treatment modalities during follow-up did not differ significantly between subgroups after application of the Bonferroni correction. RA was in clinical remission, on average, for approximately 70% of the follow-up time and was not different between subgroups. Multivariate analysis including all the above parameters revealed that age (P = 0.006), smoking (P = 0.009) and duration of low-dose corticosteroid use (P = 0.016) associated independently with new plaque formation. RA patients displayed similar numbers of newly formed carotid plaques to the tightly matched for traditional cardiovascular risk factors 'healthy' controls, although more patients than controls had carotid plaques at baseline.

Conclusions

Formation of new atherosclerotic plaques in this small cohort of patients with well-controlled RA depended mainly on traditional cardiovascular risk factors and corticosteroid use, whereas an adverse effect of residual systemic inflammation was not readily detectable.     

Palmitoylation-induced Aggregation of Cysteine-string Protein Mutants That Cause Neuronal Ceroid Lipofuscinosis

Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.