Distribution Patterns of Astrocyte Populations in the Human Cortex.

Astrocytes are a major class of glial cell in the central nervous system that have a diverse range of types and functions thought to be based on their anatomical location, morphology and cellular properties. Recent studies highlight that astrocyte dysfunction contributes to the pathogenesis of neurological conditions. However, few studies have described the pattern, distribution and density of astrocytes in the adult human cortex. This study mapped the distribution and density of astrocytes immunolabelled with a range of cytoskeletal and membrane markers in the human frontal cortex. Distinct and overlapping astrocyte populations were determined. The frontal cortex from ten normal control cases (75 ± 9 years) was immunostained with glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase-1 L1 (ALDH1L1), connexin-43 (Cx43), aquaporin-4 (AQP4), and glutamate transporter 1 (GLT-1). All markers labelled populations of astrocytes in the grey and white matter, separate cortical layers, subpial and perivascular regions. All markers were informative for labelling different cellular properties and cellular compartments of astrocytes. ALDH1L1 labelled the largest population of astrocytes, and Cx43-immunopositive astrocytes were found in all cortical layers. AQP4 and GLT-1 labelled distal astrocytic process and end-feet in the same population of astrocytes (98% of GLT-1-immunopositive astrocytes contained AQP4). In contrast, GFAP, the most widely used marker, predominantly labelled astrocytes in superficial cortical layers. This study highlights the diversity of astrocytes in the human cortex, providing a reference map of the distribution of distinct and overlapping astrocyte populations which can be used for comparative purposes in various disease, inflammatory and injury states involving astrocytes.

Staphylococcus aureus Floating Biofilm Formation and Phenotype in Synovial Fluid Depends on Albumin, Fibrinogen, and Hyaluronic Acid.

Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria-protein interactions in vivo and the accompanying alterations in phenotype, virulence factor production, and ultimately antibiotic tolerance. We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus (MRSA or MSSA, respectively) and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein-bacteria interactions characteristic of physiological fluids.

Sterility of Ethyl Chloride Spray After Use in the Clinic.

Background: Ethyl chloride spray is used frequently in the outpatient setting as a local anesthetic for injections and aspirations with varying consensus about the sterility of the spray. We hypothesize that ethyl chloride spray remains sterile and would show no bacterial growth during routine clinical use. Methods: Thirteen ethyl chloride bottles were collected for testing. Two unopened bottles were used as controls. Eleven unopened bottles were placed in orthopedic clinics and recollected after varying duration of use. The final volume and duration of use were recorded. Each bottle was sprayed in a separate test tube and allowed to evaporate. Trypticase soy broth was added to each tube and incubated for 48 hours. Control test tubes with broth alone were prepared and incubated under the same conditions. Cultures were evaluated at 24 and 48 hours. Results: The mean duration of ethyl chloride bottle use prior to culturing was 26 days. The average volume used per day was 1.9 mL. Each ethyl chloride bottle had an initial volume of 103.5 mL. Using the average daily volume usage, an extrapolated lifespan of each bottle was estimated at 7.7 weeks. None of the samples showed bacterial or fungal growth at 24 or 48 hours. Conclusion: Ethyl chloride bottles used in the clinical settings showed no bacterial or fungal contamination through their shelf life and routine use. The duration and amount of use did not affect sterility. Although the antimicrobial activity of ethyl chloride spray on skin is debated, ethyl chloride itself remains sterile through clinical use.

Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

High-Throughput Proteomics Enabled by a Photocleavable Surfactant.

Mass spectrometry (MS)-based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high-throughput proteomics. Herein, we report a high-throughput bottom-up proteomic method enabled by a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS-analysis following UV degradation. Moreover, we developed an Azo-aided bottom-up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an "all-in-one" MS-compatible surfactant for both top-down and bottom-up proteomics, with streamlined workflows for high-throughput proteomics amenable to clinical applications.

A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Surgical Loupes Worn by Orthopaedic Surgeons Are a Reservoir for Microorganisms.

BACKGROUND: Surgeons frequently use optical loupes to magnify the surgical field; they are typically unprotected when positioned directly over the wound, where particulate shedding containing microorganisms could potentially lead to surgical site infections (SSIs). SSIs are rare in some orthopaedic subspecialties such as hand surgery; however, in other subspecialties, for example, the spine, where surgeons often use loupes, SSIs can have devastating consequences. QUESTIONS/PURPOSES: (1) What is the degree of bacterial and fungi organism colonization of surgical loupes and storage cases? (2) Is there a difference in the degree of colonization at the beginning and the end of a surgery day? (3) Does an alcohol swab reduce bacterial colonization of surgical loupes? METHODS: The surgical loupes of 21 orthopaedic surgeons from a large, regional orthopaedic practice were cultured over a 3-month period and form the basis of this study. Five loupe storage cases were also cultured. In two different subgroup comparisons, the presence of microorganisms was evaluated just before the start and immediately after the end of the surgical day (n = 9) and before and 1 minute after cleaning with an alcohol swab (n = 6). A total of 36 cultures were evaluated. Surgeons who declined to participate in the study were excluded. The number of loupes selected for all of the analyses were samples of convenience and limited by surgeon availability. The degree of bacterial and fungal presence was graded using a point system: 0 = no growth; 1 = limited growth (meaning few scattered colonies); 2 = moderate growth; 3 = extensive but scattered growth; and 4 = growth consuming the entire plate. Demographic data were assessed using descriptive statistics. Additionally, the Student's t and Wilcoxon signed-rank tests were used to detect differences in categorical bacterial growth between paired samples. A p value of 0.05 represented statistical significance. Kappa statistics of reliability were performed to evaluate interobserver agreement of microorganism growth in the culture plates. RESULTS: Bacteria were present in 19 of 21 (90%) sets of loupes. Five species of bacteria were noted. Fungi were present in 10 of 21 (48%) sets of loupes. Bacterial contamination was identified in two storage cases (40%) and fungi were present in five cases (100%). In a subset of nine loupes tested, the degree of bacterial presence had a median of 2 (range, 1-4; 95% confidence interval [CI], 1.0-2.6) in samples collected before starting the surgical day compared with 3 (range, 2-4; 95% CI, 2.0-3.3) at the end of the day (p = 0.004). In a separate study arm comprised of six loupes, 1 minute after being cleaned with an alcohol swab, bacterial presence on loupes decreased from a median of 2 (range, 2-3; 95% CI, 1.9-2.5) to a median of 1 (range, 0-2; 95% CI, 0.5-1.5; p = 0.012). CONCLUSIONS: Loupes are a common reservoir for bacteria and fungi. Given the use of loupes directly over the surgical field and the lack of a barrier, care should be taken to decrease the bacterial load by cleaning loupes and airing out storage cases, which may decrease the risk of surgical field contamination and iatrogenic wound infections. CLINICAL RELEVANCE: Routine cleaning and disinfecting of optical loupes with alcohol pads can reduce microorganism colonization and should be implemented by surgeons who regularly use loupes in the operating room. Theoretically, particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, although this has not been proven clinically.