Biomedical subjects

Mark A Wilson

Publications and source records attributed to Mark A Wilson.

At least 19 recordsLinked to original sources

PubMed2026

Towards time-resolved MicroED grid preparation using mix-and-inject gas dynamic virtual nozzles.

Recent progress in gas dynamic virtual nozzle (GDVN) technologies in combination with high-brilliance synchrotron and X-ray free-electron lasers (XFELs) has allowed the visualization of protein dynamics in crystallo by mixing macromolecular protein crystals with a substrate using tunable mixing times on the order of milliseconds to seconds prior to serial X-ray diffraction data collection. This has become the method of choice for high-resolution structure determination of intermediate states. However, such experiments require large counts of crystals of proper sizes for high-resolution data collection, and premium beam times for screening efforts. Cryogenic microcrystal electron diffraction (MicroED) represents a complementary technique that may be a more accessible avenue for time-resolved nanocrystallography compared with serial X-ray diffraction experiments. MicroED can produce full diffraction datasets from just a few submicrometre-thick crystals, and the approach is more readily accessible, requiring standard cryogenic transmission electron microscopy (TEM) equipment available at many universities and institutes. Cryogenic MicroED, like other forms of cryo-EM, begins with rapidly freezing biological material on electron microscopy grids. In the case of MicroED, micro- to nano-crystals (<500 nm thick) are deposited onto electron microscopy grids and plunge-frozen for subsequent electron diffraction data collection. Here, we have incorporated GDVN technology developed originally for XFEL experiments into the freezing process as a first step towards time-resolved studies. We describe the limited deposition efficiency of the model MicroED protein proteinase K on TEM grids using GDVNs, preceding sample vitrification and successful MicroED data collection. We discuss both the initial results from such experiments and the methodological challenges in developing this approach into a reliable workflow for millisecond-to-second time-resolved structural studies of macromolecules. Our results promise a strategy to deposit crystals on grids using GDVNs and determine high-resolution structures by MicroED, constituting a first step towards development of time-resolved MicroED experiments.

MicroED↗

PubMed2006

Comparative approaches to facilitate the discovery of prolongevity interventions: effects of tocopherols on lifespan of three invertebrate species.

Many compounds hold promise for pharmacologic manipulation of aging. However, such claims are difficult to investigate due to time and budget constraints. Here, we took a comparative approach, using short-lived invertebrate species, to directly test the effects of two tocopherols (Vitamin E) on longevity. gamma-Tocopherol represents the most abundant tocopherol in the Western diet, while alpha-tocopherol is selectively enriched in human plasma. Both isoforms demonstrate antioxidant activity and are proposed to have anti-aging activities. We compared the effects of alpha- and gamma-tocopherol supplementation on lifespan in three invertebrate species. gamma-Tocopherol, but not alpha-tocopherol, slightly extended lifespan in nematodes, but neither significantly affected lifespan in two fly species. This study shows that a comparative approach, utilizing multiple invertebrate species, can increase the robustness of invertebrate-based pilot screens for prolongevity interventions.

Animals↗

PubMed2006

Dietary deprivation extends lifespan in Caenorhabditis elegans.

Dietary restriction (DR) is well known as a nongenetic intervention that robustly extends lifespan in a variety of species; however, its underlying mechanisms remain unclear. We have found in Caenorhabditis elegans that dietary deprivation (DD) during adulthood, defined as removal of their food source Escherichia coli after the completion of larval development, increased lifespan and enhanced thermotolerance and resistance to oxidative stress. DD-induced longevity was independent of one C. elegans SIRTUIN, sir-2.1, which is required for the effects of DR, and was independent of the daf-2/insulin-like signaling pathway that independently regulates longevity and larval diapause in C. elegans. DD did not significantly alter lifespan of fem-1(hc17); eat-2(ad465) worms, a genetic model of DR. These findings suggest that DD and DR share some downstream effectors. In addition, DD was detrimental for longevity when imposed on reproductively active young adults, suggesting that DD may only be beneficial in the absence of competing metabolic demands, such as fertility. Adult-onset DD offers a new paradigm for investigating dietary regulation of longevity in C. elegans. This study presents the first evidence that long-term DD, instead of being detrimental, can extend lifespan of a multicellular adult organism.

Aging↗

PubMed2006

Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans.

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blueberry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.

Aging↗

PubMed2005

The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein.

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.

Animals↗

PubMed2005

Hemorrhagic shock-activated neutrophils augment TLR4 signaling-induced TLR2 upregulation in alveolar macrophages: role in hemorrhage-primed lung inflammation.

Hemorrhagic shock renders patients susceptible to the development of acute lung injury in response to a second inflammatory stimulus by as yet unclear mechanisms. We investigated the role of neutrophils (PMN) in alveolar macrophage (AMphi) priming, specifically, the role in mediating Toll-like receptor (TLR)4 and TLR2 cross talk in AMphi. Using a mouse model of hemorrhagic shock followed by intratracheal administration of LPS, we explored a novel function of shock-activated PMN in the mechanism of TLR2 upregulation induced by LPS-TLR4 signaling in AMphi. We showed that antecedent hemorrhagic shock enhanced LPS-induced TLR2 upregulation in AMphi. In neutropenic mice subjected to shock, the LPS-induced TLR2 expression was significantly reduced, and the response was restored upon repletion with PMN obtained from shock-resuscitated mice but not by PMN from sham-operated mice. These findings were recapitulated in mouse AMphi cocultured with PMN. The enhanced TLR2 upregulation in AMphi augmented the expression of macrophage inflammatory protein-2, TNF-alpha, and macrophage migration inhibitory factor in the AMphi in response to sequential challenges of LPS and peptidoglycan, a prototypical TLR2 ligand, which physiologically associated with amplified AMphi-induced PMN migration into air pouch and lung alveoli. Thus TLR2 expression in AMphi, signaled by TLR4 and regulated by shock-activated PMN, is an important positive-feedback mechanism responsible for shock-primed PMN infiltration into the lung after primary PMN sequestration.

Animals↗

PubMed2005

The atomic resolution crystal structure of the YajL (ThiJ) protein from Escherichia coli: a close prokaryotic homologue of the Parkinsonism-associated protein DJ-1.

The Escherichia coli protein YajL (ThiJ) is a member of the DJ-1 superfamily with close homologues in many prokaryotes. YajL also shares 40% sequence identity with human DJ-1, an oncogene and neuroprotective protein whose loss-of-function mutants are associated with certain types of familial, autosomal recessive Parkinsonism. We report the 1.1 angstroms resolution crystal structure of YajL in a crystal form with two molecules in the asymmetric unit. The structure of YajL is remarkably similar to that of human DJ-1 (0.9 angstroms C(alpha) RMSD) and both proteins adopt the same dimeric structure. The conserved cysteine residue located in the "nucleophile elbow" is oxidized to either cysteine sulfenic or sulfinic acid in the two molecules in the asymmetric unit, and a mechanism for this oxidation is proposed that may be valid for other proteins in the DJ-1 superfamily as well. Rosenfield difference matrix analysis of the refined anisotropic displacement parameters in the YajL structure reveals significant differences in the intramolecular flexibility of the two non-crystallographic symmetry-related molecules in the asymmetric unit. Lastly, a comparison of the crystal structures of the four different E.coli members of the DJ-1 superfamily indicates that the variable oligomerization in this superfamily is due to a combination of protein-specific insertions into the core fold that form specific interfaces while occluding others plus optimization of residues in the structurally invariant regions of the core fold that facilitate protein-protein interactions.

Crystallography, X-Ray↗

PubMed2005

Processing of pulse oximeter data using discrete wavelet analysis.

A wavelet-based signal processing technique was employed to improve an implantable blood perfusion monitoring system. Data was acquired from both in vitro and in vivo sources: a perfusion model and the proximal jejunum of an adult pig. Results showed that wavelet analysis could isolate perfusion signals from raw, periodic, in vitro data as well as fast Fourier transform (FFT) methods. However, for the quasi-periodic in vivo data segments, wavelet analysis provided more consistent results than the FFT analysis for data segments of 50, 10, and 5 s in length. Wavelet analysis has thus been shown to require less data points for quasi-periodic data than FFT analysis making it a good choice for an indwelling perfusion monitor where power consumption and reaction time are paramount.

Algorithms↗

PubMed2005

An autocorrelation-based time domain analysis technique for monitoring perfusion and oxygenation in transplanted organs.

In designing an implantable sensor for perfusion monitoring of transplant organs the ability of the sensor to gather perfusion information with limited power consumption and in near real time is paramount. The following work was performed to provide a processing method that is able to predict perfusion and oxygenation change within the blood flowing through a transplanted organ. For this application, an autocorrelation-based algorithm was used to reduce the acquisition time required for fast Fourier transform (FFT) analysis while retaining the accuracy inherent to FFT analysis. In order to provide data proving that the developed method is able to predict perfusion as accurately as FFT two experiments were developed isolating both periodic and quasi-periodic cardiac frequencies. It was shown that the autocorrelation-based method was able to perform comparably with FFT (limited to a sampling frequency of 300 Hz) and maintain accuracy down to acquisition times as low as 4 s in length.

Algorithms↗

PubMed2005

Real-time separation of perfusion and oxygenation signals for an implantable sensor using adaptive filtering.

In this paper, an adaptive filtering algorithm to separate signals due to perfusion and oxygenation has been developed using an 810-nm source, in addition to 660-nm and 940-nm sources, as an internal reference due to its limited oxygen sensitivity. The newly developed algorithm was tested using Monte Carlo simulated data to prove the effectiveness of the 810-nm reference and adaptive algorithm. Following the simulation, an in vitro model was developed to test the algorithm that used a blood flow through system wrapped with tissue. The system had the ability to isolate the effects of perfusion and oxygenation and the algorithm accurately captured the changes in these signals with reliable consistency. Using the serosal surface of the swine jejunum, in vivo data was also taken to analyze the algorithms response to fluctuating perfusion levels like that seen in hemorrhaging or failing transplants. The algorithm was able to extract the perfusion information from the oxygenation information in this in vivo study. Overall, it was shown that an adaptive filtering algorithm using an 810-nm reference has provided a means to separate oxygenation and perfusion.

Algorithms↗

PubMed2004

The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.

Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.

1-Methyl-4-phenylpyridinium↗

PubMed2004

Anchoring a cationic ligand: the structure of the Fab fragment of the anti-morphine antibody 9B1 and its complex with morphine.

The crystal structures of an anti-morphine antibody 9B1 (to 1.6A resolution) and its complex with morphine (to 2.0 A resolution) are reported. The morphine-binding site is described as a shallow depression on the protein surface, an unusual topology for a high-affinity ( Ka approximately 10(9) M(-1)) antibody against a small antigen. The polar part of the ligand is exposed to solvent, and the cationic nitrogen atom of the morphine molecule is anchored at the bottom of the binding site by a salt-bridge to a glutamate side-chain. Additional affinity is provided by a double cation-pi interaction with two tryptophan residues. Comparison of the morphine complex with the structure of the free Fab shows that a domain closure occurs upon binding of the ligand.

Antibodies, Monoclonal↗

PubMed2004

The 1.8-A resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: a member of the DJ-1/ThiJ/PfpI superfamily.

The yeast gene YDR533C encodes a protein belonging to the DJ-1/ThiJ/PfpI superfamily. This family includes the human protein DJ-1, which is mutated in autosomal recessive early-onset Parkinson's disease. The function of DJ-1 and its yeast homologue YDR533Cp is unknown. We report here the crystal structure of YDR533Cp at 1.8-A resolution. The structure indicates that the closest relative to YDR533Cp is the Escherichia coli heat shock protein Hsp31 (YedU), which has both chaperone and protease activity. As expected, the overall fold of the core domain of YDR533Cp is also similar to that of DJ-1 and the bacterial protease PfpI. YDR533Cp contains a possible catalytic triad analogous to that of Hsp31 and an additional domain that is present in Hsp31 but is not seen in DJ-1 and other members of the family. The cysteine in this triad (Cys-138) is oxidized in this crystal structure, similar to modifications seen in the corresponding cysteine in the crystal structure of DJ-1. YDR533Cp appears to be a dimer both in solution and the crystal, but this dimer is formed by a different interface than that found in Hsp31 or other members of the superfamily.

Models, Molecular↗

PubMed2004

Management of adult blunt splenic injuries: comparison between level I and level II trauma centers.

BACKGROUND: The factors important in determining outcome when managing adult blunt splenic injuries continue to be debated. Whether trauma center level designation (Level I versus Level II) affects patient management has not been evaluated. STUDY DESIGN: We conducted a retrospective analysis of prospectively gathered data from the Pennsylvania Trauma Outcome Study database that collected information from 27 statewide trauma centers (Level I [15], Level II [17]). Adult patients (ages > or = 16 years) with blunt splenic injuries (ICD-9-CM 865) were evaluated. Demographic data, injury data, and trauma center level designation were collected, and patient management, length of stay, and mortality were analyzed. RESULTS: There were 2,138 adult patients who suffered blunt splenic injuries during the study period (1998-2000). Patients treated at Level II trauma centers (n = 772) had a higher rate of operative treatment (38.2% versus 30.7%) (p < 0.001), but a shorter mean length of stay (10.1 +/- 0.4 versus 12.0 +/- 0.4 days) (p < 0.01) compared with patients in Level I trauma centers (n = 1,366). The rate of failure of nonoperative treatment was lower at Level II trauma centers (13.0% versus 17.6%) (p < 0.05), but the mortality for patients managed nonoperatively was higher (8.4% versus 4.5%) (p < 0.05). Splenorrhaphy was performed more frequently in Level I trauma centers. CONCLUSIONS: Management differences exist in the treatment of adult blunt splenic injuries between institutions of different trauma center level designation. Multicenter studies should account for this finding in design and implementation.

Adult↗

PubMed2003

Skin and soft-tissue infections: impact of resistant gram-positive bacteria.

Surgical site infections remain significant causes of postoperative morbidity and mortality despite an improved understanding of risk factors and an increasing armamentarium of antimicrobial agents. The overall rates of surgical site infections have been slowly decreasing, but the proportion of infections with gram-positive bacteria continues to increase, and drug-resistant species continue to become more prevalent. Although new antibiotic classes have efficacy against such organisms, these agents are only short-term solutions to the problem of multiple-drug resistance. Surgeons must focus on prevention of infections by appropriately using antibiotics, emphasizing principles of infection control, and understanding nontraditional measures that may decrease infection risk.

Anti-Bacterial Agents↗

PubMed2003

Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals.

Collection of quality data on high-energy synchrotron sources requires the selection of a cryoprotectant that allows protein crystals to be cryocooled without damage to the crystal and with suppression of ice formation. The use of sodium malonate as a versatile cryoprotectant for salt-grown protein crystals is presented here. In addition to its useful cryoprotectant properties, sodium malonate can also function as a versatile stabilizing solution that allows the manipulation, derivatization and ligand soaking of crystals grown from salt that may not be possible in the crystal mother liquor.

Bacterial Proteins↗

PubMed2003

Domain flexibility in the 1.75 A resolution structure of Pb2+-calmodulin.

Calmodulin (CaM) regulates a variety of cellular processes by interacting with a large number of proteins in a Ca(2+)-dependent manner. Conformational flexibility plays a key role in CaM function, although the full extent and detailed features of this flexibility are not fully characterized. Here, the 1.75 A resolution crystal structure of Pb(2+)-bound Paramecium tetraurelia CaM crystallized in a previously unobserved monoclinic lattice is reported. Pb(2+)-CaM is disordered in this new lattice and only a portion of each of the two molecules in the asymmetric unit can be modeled. Comparison of the structures of Ca(2+)-CaM and Pb(2+)-CaM show close agreement in the C-terminal domain but significant structural differences in the N-terminal domain. In addition, translation-libration-screw (TLS) refinement and Rosenfield difference analysis reveal inter-helical flexibility in the metal-bound N-terminal domain of the protein that is absent in the metal-bound C-terminal domain and indicates that the two structurally similar domains of CaM are dynamically distinct. These results demonstrate that TLS refinement and Rosenfield difference analysis allow detailed information about macromolecular flexibility to be extracted from X-ray diffraction data even when the crystal lattice prohibits full manifestation of this flexibility.

Animals↗

PubMed2003

The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.

Mutations in DJ-1, a human gene with homologues in organisms from all kingdoms of life, have been shown to be associated with autosomal recessive, early onset Parkinson's disease (PARK7). We report here the three-dimensional structure of the DJ-1 protein, determined at a resolution of 1.1 A by x-ray crystallography. The chain fold of DJ-1 resembles those of a bacterial protein, PfpI, that has been annotated as a cysteine protease, and of a domain of a bacterial catalase whose role in the activity of that enzyme is uncertain. In contrast to PfpI, a hexameric protein whose oligomeric structure is essential for its putative proteolytic activity, DJ-1 is a dimer with completely different intersubunit contacts. The proposed catalytic triad of PfpI is absent from the corresponding region of the structure of DJ-1, and biochemical assays fail to detect any protease activity for purified DJ-1. A highly conserved cysteine residue, which is catalytically essential in homologues of DJ-1, shows an extreme sensitivity to radiation damage and may be subject to other forms of oxidative modification as well. The structure suggests that the loss of function caused by the Parkinson's-associated mutation L166P in DJ-1 is due to destabilization of the dimer interface. Taken together, the crystal structure of human DJ-1 plus other observations suggest the possible involvement of this protein in the cellular oxidative stress response and a general etiology of neurodegenerative diseases.

Archaeal Proteins↗