Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

The Structure and Function of Next-Generation Gingival Graft Substitutes-A Perspective on Multilayer Electrospun Constructs with Consideration of Vascularization.

There is a shortage of suitable tissue-engineered solutions for gingival recession, a soft tissue defect of the oral cavity. Autologous tissue grafts lead to an increase in morbidity due to complications at the donor site. Although material substitutes are available on the market, their development is early, and work to produce more functional material substitutes is underway. The latter materials along with newly conceived tissue-engineered substitutes must maintain volumetric form over time and have advantageous mechanical and biological characteristics facilitating the regeneration of functional gingival tissue. This review conveys a comprehensive and timely perspective to provide insight towards future work in the field, by linking the structure (specifically multilayered systems) and function of electrospun material-based approaches for gingival tissue engineering and regeneration. Electrospun material composites are reviewed alongside existing commercial material substitutes', looking at current advantages and disadvantages. The importance of implementing physiologically relevant degradation profiles and mechanical properties into the design of material substitutes is presented and discussed. Further, given that the broader tissue engineering field has moved towards the use of pre-seeded scaffolds, a review of promising cell options, for generating tissue-engineered autologous gingival grafts from electrospun scaffolds is presented and their potential utility and limitations are discussed.

Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.

Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays.

Luciferase-based reporter assays are powerful tools for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. Here we describe the characterization and use of a luciferase reporter enzyme derived from the marine copepod Metridia luciferase family, referred to as TurboLuc luciferase (TurboLuc). To develop TurboLuc, the wild-type luciferase was modified to decrease its size, increase brightness, slow luminescent signal decay, and provide for efficient intracellular expression. To determine the enzyme susceptibility to compound inhibition and judge the suitability of using of TurboLuc as a reporter in screening assays, purified TurboLuc enzyme was screened for inhibitors using two different compound libraries. No inhibitors of this enzyme were identified in a library representative of typical diverse low molecular weight (LMW) compounds using a purified TurboLuc enzyme assay supporting that such libraries will show very low interference with this enzyme. We were able to identify a few inhibitors from a purified natural product library which can serve as useful tools to validate assays using TurboLuc. In addition to the inhibitor profile for TurboLuc we describe the use of this reporter in cells employing miniaturized assay volumes within 1536-well plates. TurboLuc luciferase is the smallest luciferase reporter enzyme described to date (16 kDa), shows bright luminescence and low interference by LMW compounds, and therefore should provide an ideal reporter in assays applied to high-throughput screening.

Mapping the interview transcript: Identifying spatial policy areas from daily working practices.

An interview transcript can be a rich source of geographical references whose potential are not always fully realised in their conventional analysis. Geo-referencing techniques can be used to assign a spatial footprint to place names, adding value to these data and allowing the geographic information within them to be exploited when coupled with GIS technology. This paper discusses a method of analysing and visualising interview transcripts in order to understand the spatial extent of public policy practitioners' activities. Through aggregation and statistical mapping it is possible to gain insight into the importance of space across a range of public policy themes and to understand the relationship between practitioner-defined policy themes and the formal administrative boundaries within which they typically work. The research demonstrates that spatial working practices rarely conform to formal administrative boundaries and that there are varying degrees of spatial focus between different policy themes within localities. It also reveals that spatial working practices can continue to be influenced by historic geographies and can be pulled in different directions, reflecting both the devolved nature of the sector and the particular geographical context of the setting. It concludes that mapping the interview transcript can add value and provide additional insights to more conventional analysis.

Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer.

According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) in the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, ß-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of ß-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or ß-Catenin siRNA can increase drug sensitivity of TKI-resistant cells.

Enamel surface roughness of preferred debonding and polishing protocols.

OBJECTIVE: This study investigated the surface roughness of enamel after debonding and instrumentation with commonly used methods. METHODS: Part I: a survey was sent to active members of the American Association of Orthodontists to determine popular bonding, debonding, and polishing protocols. Part II: brackets were bonded to the buccal surface of 30 extracted human premolar teeth. After debonding, residual adhesive was removed with 12-, 16-, and 20-fluted titanium carbide burs as based upon the survey results. The teeth were scanned with a surface profilometer for surface roughness. Part III: the teeth were further polished using a Reliance Renew polishing point or a prophy cup with pumice and rescanned for surface roughness. RESULTS: Part I: the majority of respondents used a generic bracket-removing plier to remove fixed appliances (53%) and a high-speed handpiece for adhesive removal (85%). The most popular bur was a 12-fluted carbide bur without water spray. The majority of respondents used pumice paste and/or Reliance Renew points after adhesive removal. Part II: there was a significant difference in enamel surface roughness when 12-, 16-, and 20-fluted carbide burs were compared via surface profilometry. Part III: further polishing with a Reliance Renew point or a prophy cup and pumice did not provide a significantly smoother surface. CONCLUSIONS: The results show large variation in debonding and polishing techniques. Creating a smooth enamel surface is equally possible with 12- or 20-fluted carbide burs. Further polishing with pumice and prophy cup or Renew point does not provide an enamel smoother surface.

Human severe sepsis cytokine mixture increases ß2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

INTRODUCTION: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. METHODS: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). RESULTS: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either ß2 (CD18), αL/ß2 (CD11α/CD18; LFA-1) or αM/ß2 (CD11ß/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. CONCLUSIONS: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a ß2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

A soluble α-synuclein construct forms a dynamic tetramer.

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134.

FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn(2+) coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn(2+) coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD(+) dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD(+) to NADH that is subsequently used for furfural reduction.

A Missed Case of Synovial Osteochondromatosis.

Synovial osteochondromatosis or synovial chondromatosis is a benign joint pathology characterized by the development of multiple cartilaginous nodules or loose bodies in the synovial membrane that typically arise in the larger joints of the body. It usually presents as joint pain and, as seen in the present case, can occasionally be missed. Diagnosis involves a combination of clinical evaluation, imaging studies, and histopathological evaluation (which is confirmatory). Treatment depends on the severity of the disease, symptoms, and the patient's social situation, and may include monitoring for asymptomatic cases, non-surgical management (pain medications and physical therapy), or surgical intervention. Surgical intervention may include arthroscopic removal of loose bodies, synovectomy, or reconstruction/replacement in severe situations. Prognosis has a direct relationship to how early the disease is diagnosed. Early intervention with appropriate management can help alleviate symptoms; however, if left unmanaged, it can lead to joint damage and osteoarthritis or very rarely, malignant transformation into chondrosarcoma. This report describes the case of a 62-year-old female with complaints of bilateral knee pain who was originally diagnosed with osteoarthritis based on clinical exam and X-rays. Two magnetic resonance imaging (MRI) scans were done three years apart, with synovial osteochondromatosis being on the differential after the second scan. A left knee major synovectomy was conducted after the second MRI reading, where rubbery masses of tissue along with loose fragments were removed. Fluid from the tissue masses was sent to culture and pathology for interpretation. Two weeks post the surgery, the patient's pain improved tremendously, with adequate ambulation independently. Histopathology came back positive for synovial osteochondromatosis. This case report highlights the importance of keeping this joint pathology on the differential when treating patients with joint pain and interpreting imaging.

A novel multiplex biomarker panel for profiling human acute and chronic kidney disease.

Acute and chronic kidney disease continues to confer significant morbidity and mortality in the clinical setting. Despite high prevalence of these conditions, few validated biomarkers exist to predict kidney dysfunction. In this study, we utilized a novel kidney multiplex panel to measure 21 proteins in plasma and urine to characterize the spectrum of biomarker profiles in kidney disease. Blood and urine samples were obtained from age-/sex-matched healthy control subjects (HC), critically-ill COVID-19 patients with acute kidney injury (AKI), and patients with chronic or end-stage kidney disease (CKD/ESKD). Biomarkers were measured with a kidney multiplex panel, and results analyzed with conventional statistics and machine learning. Correlations were examined between biomarkers and patient clinical and laboratory variables. Median AKI subject age was 65.5 (IQR 58.5-73.0) and median CKD/ESKD age was 65.0 (IQR 50.0-71.5). Of the CKD/ESKD patients, 76.1% were on hemodialysis, 14.3% of patients had kidney transplant, and 9.5% had CKD without kidney replacement therapy. In plasma, 19 proteins were significantly different in titer between the HC versus AKI versus CKD/ESKD groups, while NAG and RBP4 were unchanged. TIMP-1 (PPV 1.0, NPV 1.0), best distinguished AKI from HC, and TFF3 (PPV 0.99, NPV 0.89) best distinguished CKD/ESKD from HC. In urine, 18 proteins were significantly different between groups except Calbindin, Osteopontin and TIMP-1. Osteoactivin (PPV 0.95, NPV 0.95) best distinguished AKI from HC, and ß2-microglobulin (PPV 0.96, NPV 0.78) best distinguished CKD/ESKD from HC. A variety of correlations were noted between patient variables and either plasma or urine biomarkers. Using a novel kidney multiplex biomarker panel, together with conventional statistics and machine learning, we identified unique biomarker profiles in the plasma and urine of patients with AKI and CKD/ESKD. We demonstrated correlations between biomarker profiles and patient clinical variables. Our exploratory study provides biomarker data for future hypothesis driven research on kidney disease.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

Structural and catalytic differences between two FADH(2)-dependent monooxygenases: 2,4,5-TCP 4-monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-monooxygenase (TcpA) from Cupriavidus necator JMP134.

2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH(2))-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

Preparation and Characterization of Antibacterial Films with Eggshell-Membrane Biopolymers Incorporated with Chitosan and Plant Extracts.

A series of films containing chitosan (CS), eggshell membrane (ESM), soluble eggshell membrane (SEP), and plant extracts from Thymus vulgaris and Origanum valgare were prepared with varying concentrations and compositions. These novel films were characterized extensively with respect to film thickness and uniformity, solution absorption, degradation, microenvironmental pH, and antibacterial properties. All the films were flexible with appropriate mechanical stability. After 48 h of soaking in a lysozyme solution, all the films degraded 64 ± 4%, which would be expected to allow for the release of the plant extracts. The plant extracts on their own showed a pH of approximately 4, with the blended films having microenvironmental pHs from approximately 6.4-7.0, which would be expected to promote wound healing. A CS-ESM-SEP film with 5% of each plant extract inhibited almost all E. coli growth in liquid cultures and had no detriments to fluid absorption. Fluid absorption was approximately 100-150% by weight for all the films. The incorporation of SEP and plant extracts to a CS-ESM film provides a promising and novel method for the incorporation of SEP and antibacterial agents in a film with no detriment to wound fluid absorption or film degradation.

Implementation of a Preoperative Huddle at a Level 1 Trauma Center.

OBJECTIVE: Medical errors resulting in patient harm still occur at an alarmingly high rate. Surgery is a high-risk area that can frequently result in patient harm if errors occur. There is a need for standardization of communication and processes to decrease errors. We sought to determine whether the implementation of a preoperative huddle at our hospital could standardize communication and decrease medical errors. METHODS: A unique preoperative huddle was developed and implemented at a level 1 trauma center. We reviewed data before and after the implementation of the preoperative huddle including patient surveys and comments, the percentage of on-time operating room (OR) starts, OR turnover times, and the number of sentinel events. RESULTS: After huddle implementation, we observed a trend of improvement in our patient survey results on patient's perception that the doctors/nurses explained the procedure understandably after huddle implementation. There was a statistically yet limited clinically significant increase in OR turnover time from 37 to 40 minutes (P < 0.001). There was also an increase in on-time OR starts from 37% to 45% (P < 0.001). We observed a decrease in the number of sentinel events, with only 1 occurring each year since implementation. CONCLUSIONS: A preoperative huddle was successfully implemented at a level 1 trauma hospital and helped standardize communication without significantly disrupting workflow. To our knowledge, this method of preoperative huddling has not been previously described in the literature.

Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.

Bicinchoninic acid (BCA) assay in low volume.

The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 µL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.

A prospective analysis of interscalene brachial plexus blocks performed under general anesthesia.

BACKGROUND: The purpose of this prospective study was to assess the safety and efficacy of interscalene brachial plexus block anesthesia when performed on patients who were anesthetized with a general anesthetic prior to the performance of the block. METHODS: Patients were assessed postoperatively through surveys, interviews, and physical examinations to document block success, duration of anesthesia, block side effects, and persistent neurological complications. Nine-hundred fifty-one patients were available for the analysis. RESULTS: The overall block success rate was 97% and the mean duration of anesthesia provided by the blocks was 23.9 hours. Immediate postoperative block side effects occurred in 16% (142 of 910), persistent neurological complications occurred in 4.4% (40 of 910) of patients, and long-term neurologic complications occurred in 0.8% (8 of 910). CONCLUSION: Our study results suggest that the rates of success and complications associated with the performance of interscalene block regional anesthesia performed after induction of general anesthesia are similar to the results demonstrated in prior studies in which brachial plexus block was performed on nonanesthetized patients. Although significant complications were not common, this procedure is not without risk and can result in long-term neurologic complications.

Diabetes as a Risk Factor for Orthopedic Implant Surface Performance: A Retrieval and In Vitro Study.

Orthopedic devices are often associated with increased risk for diabetic patients due to impaired wound healing capabilities. Adverse biological responses for immunocompromised patients at the implant-tissue interface can lead to significant bone resorption that may increase failure rates. The goal of this study was to characterize the surface of implants removed from diabetic patients to determine underlying mechanisms of diabetes-induced impaired osseointegration. Thirty-nine retrieved titanium and stainless-steel orthopedic devices were obtained from diabetic and non-diabetic patients, and compared to non-implanted controls. Optical Microscopy, Scanning Electron Microscopy, Energy Dispersive X-ray Spectroscopy, and X-ray Photoelectron Spectroscopy revealed changes in morphology, chemical composition, oxidation state, and oxide thickness of the retrieval specimens, respectively. Additionally, titanium disks were immersed for 28 days in simulated in vitro diabetic conditions followed by Inductively Coupled Plasma-Optical Emission Spectroscopy to quantify metal dissolution. Electrochemical testing was performed on specimens from retrievals and in vitro study. Aside from biological deposits, retrievals demonstrated surface discoloration, pit-like formations and oxide thinning when compared to non-implanted controls, suggesting exposure to unfavorable acidic conditions. Cyclic load bearing areas on fracture-fixation screws and plates depicted cracking and delamination. The corrosion behavior was not significantly different between diabetic and non-diabetic conditions of immersed disks or implant type. However, simulated diabetic conditions elevated aluminum release. This elucidates orthopedic implant failures that potentially arise from diabetic environments at the implant-tissue interface. Design of new implant surfaces should consider specific strategies to induce constructive healing responses in immunocompromised patients while also mitigating corrosion in acidic diabetic environments.