Articular Cartilage Wear Characterization With a Particle Sizing and Counting Analyzer

Quantitative measurements of cartilage wear have been challenging, with no method having yet emerged as a standard. This study tested the hypothesis that latest-generation particle analyzers are capable of detecting cartilage wear debris generated during in vitro loading experiments that last 24 h or less, by producing measurable content significantly above background noise levels otherwise undetectable through standard biochemical assays. Immature bovine cartilage disks (4 mm diameter, 1.3 mm thick) were tested against glass using reciprocal sliding under unconfined compression creep for 24 h. Control groups were used to assess various sources of contamination. Results demonstrated that cartilage samples subjected to frictional loading produced particulate volume significantly higher than background noise and contamination levels at all tested time points (1, 2, 6, and 24 h, p < 0.042). The particle counter was able to detect very small levels of wear (less than 0.02% of the tissue sample by volume), whereas no significant differences were observed in biochemical assays for collagen or glycosaminoglycans among any of the groups or time points. These findings confirm that latest-generation particle analyzers are capable of detecting very low wear levels in cartilage experiments conducted over a period no greater than 24 h.

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Articular Cartilage Wear Characterization With a Particle Sizing and Counting Analyzer

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80381 ◽

2012 ◽

Author(s):

Sevan R. Oungoulian ◽

Orian Bortz ◽

Kristin E. Hehir ◽

Kaicen Zhu ◽

Clark T. Hung ◽

...

Keyword(s):

Articular Cartilage ◽

Friction Coefficient ◽

Wear Debris ◽

Polyethylene Wear ◽

Quantitative Measurements ◽

Diarthrodial Joints ◽

Cartilage Wear ◽

Tangential Forces ◽

Fluid Pressurization

The primary function of articular cartilage is to serve as the bearing material in diarthrodial joints, transmitting loads while minimizing friction and wear. The friction coefficient of cartilage has been characterized extensively in the literature, using standard measurements of normal and tangential forces acting across a sliding interface [1]. However, quantitative measurements of cartilage wear have proven to be more challenging, with only a few studies having reported such measurements. The primary quantitative approaches proposed to date include biochemical assaying of cartilage and test solutions [2], and characterization of changing articular layer thickness [3] and surface roughness [4]. One study examining polyethylene wear debris in hip arthroplasty reported the use of an automated particle analyzer [5]. The aim of this study was to test the hypothesis that latest-generation particle analyzers are capable of detecting cartilage wear debris generated during in vitro loading experiments that last 24 h or less, by producing measurable content significantly above background noise levels. The longer-term objective of our studies is to test the hypothesis that elevated interstitial fluid pressurization, which is known to reduce the friction coefficient of cartilage [6], also reduces cartilage wear.

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ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

10.2196/preprints.25089 ◽

2020 ◽

Author(s):

Avik Sotira Scientific

Keyword(s):

Social Life ◽

Plaque Assay ◽

Host Cells ◽

Surface Glycoprotein ◽

Crystallographic Structure ◽

Therapeutic Implications ◽

Biochemical Assays ◽

Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

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PDGFRα: Expression and Function during Mitral Valve Morphogenesis

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd8030028 ◽

2021 ◽

Vol 8 (3) ◽

pp. 28

Author(s):

Kelsey Moore ◽

Diana Fulmer ◽

Lilong Guo ◽

Natalie Koren ◽

Janiece Glover ◽

...

Keyword(s):

Mitral Valve ◽

Primary Cilia ◽

Growth Factor Receptor ◽

Akt Phosphorylation ◽

Valve Development ◽

Biochemical Assays ◽

Factor Receptor Alpha ◽

Mesenchymal Transformation ◽

And Function

Mitral valve prolapse (MVP) is a common form of valve disease and can lead to serious secondary complications. The recent identification of MVP causal mutations in primary cilia-related genes has prompted the investigation of cilia-mediated mechanisms of disease inception. Here, we investigate the role of platelet-derived growth factor receptor-alpha (PDGFRα), a receptor known to be present on the primary cilium, during valve development using genetically modified mice, biochemical assays, and high-resolution microscopy. While PDGFRα is expressed throughout the ciliated valve interstitium early in development, its expression becomes restricted on the valve endocardium by birth and through adulthood. Conditional ablation of Pdgfra with Nfatc1-enhancer Cre led to significantly enlarged and hypercellular anterior leaflets with disrupted endothelial adhesions, activated ERK1/2, and a dysregulated extracellular matrix. In vitro culture experiments confirmed a role in suppressing ERK1/2 activation while promoting AKT phosphorylation. These data suggest that PDGFRα functions to suppress mesenchymal transformation and disease phenotypes by stabilizing the valve endocardium through an AKT/ERK pathway.

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Hepatic pyruvate kinase. Quantitative measurements of phosphorylation in vitro and in the isolated rat hepatocyte.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)34421-1 ◽

1982 ◽

Vol 257 (13) ◽

pp. 7595-7602 ◽

Cited By ~ 2

Author(s):

J B Blair ◽

M A Cimbala ◽

M E James

Keyword(s):

Pyruvate Kinase ◽

Rat Hepatocyte ◽

Quantitative Measurements ◽

Isolated Rat

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Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells

Scientific Reports ◽

10.1038/s41598-021-89921-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Regan J. Hayward ◽

Michael S. Humphrys ◽

Wilhelmina M. Huston ◽

Garry S. A. Myers

Keyword(s):

Epithelial Cells ◽

Multiplicity Of Infection ◽

Rna Seq ◽

Infection Rates ◽

Time Points ◽

High Infection ◽

Infection Cycles ◽

Transcriptomic Responses ◽

Rna Depletion

AbstractDual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host–pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

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Synaptic background noise controls the input/output characteristics of single cells in an in vitro model of in vivo activity

Neuroscience ◽

10.1016/j.neuroscience.2003.08.027 ◽

2003 ◽

Vol 122 (3) ◽

pp. 811-829 ◽

Cited By ~ 162

Author(s):

J.-M Fellous ◽

M Rudolph ◽

A Destexhe ◽

T.J Sejnowski

Keyword(s):

Background Noise ◽

In Vitro Model ◽

Single Cells ◽

Input Output ◽

Vitro Model ◽

Output Characteristics

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In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01058-08 ◽

2009 ◽

Vol 53 (4) ◽

pp. 1377-1385 ◽

Cited By ~ 135

Author(s):

Tse-I Lin ◽

Oliver Lenz ◽

Gregory Fanning ◽

Thierry Verbinnen ◽

Frédéric Delouvroy ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Intestinal Tract ◽

Pharmacokinetic Profile ◽

Absolute Bioavailability ◽

Time Curve ◽

Biochemical Assays ◽

Single Oral Administration ◽

Half Maximal Effective Concentration

ABSTRACT The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC50) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

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670 Delayed Enzymatic Debridement of Burn Wounds using the Proteolytic Gel SN514

Journal of Burn Care & Research ◽

10.1093/jbcr/irab032.316 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

pp. S191-S192

Author(s):

Angela R Jockheck-Clark ◽

Randolph Stone ◽

Michelle Holik ◽

Lucy Schaffer ◽

Shanmugasundaram Natesan ◽

...

Keyword(s):

Burn Injury ◽

Vehicle Control ◽

Surrounding Tissue ◽

Burn Wound ◽

Burn Wounds ◽

Delayed Treatment ◽

Medical Evacuation ◽

Contamination Levels

Abstract Introduction Thermal burns account for 5–10% of casualties sustained in present-day conflicts and are expected to be one of the most common wounds to occur in future conflicts. In prolonged field care (PFC) situations, medical evacuation could be delayed for days. During this time, burn wounds can become infected, detrimentally impact neighboring tissue, and cause systemic immune responses. Therefore, it is essential to test and evaluate non-surgical debridement agents that could be implemented prior to reaching a Role 3 military treatment facility. This work details how the proprietary proteolytic gel SN514 impacts burn debridement when applied within a PFC-like timeline. SN514 contains an enzyme formulation that is thermostable, easy to apply, and selectively degrades non-viable tissue in vitro and in vivo. Methods Deep-partial thickness contact burns were created using an established porcine model and covered with gauze or an antimicrobial incise drape. Four days later, the burns were treated with one of five treatments: 0.2% SN514, 0.8% SN514, a vehicle control, gauze, or an antimicrobial silver dressing. Treatments were re-applied every 24 hours for 72 to 96 hours. The effects of the treatment regiments were compared histologically. Biopsies were also taken to monitor bacterial contamination levels. Results Burns treated with SN514 were partially debrided and visually distinct from those treated with gauze, the silver dressing, or the vehicle control. Preliminary analyses suggest that SN514-treated burns that had been covered with “dry” gauze had a much lower debridement efficiency than those treated with the incise drape. This suggests that SN514 debridement efficiency may depend on the presence of a moist eschar. Preliminary analyses also suggest that there was little difference in burn wound bacterial counts among the five treatment groups. Conclusions SN514 is able to debride burns that experienced delayed treatment, without any evidence of harm to the surrounding tissue or evidence of exacerbating the original burn injury. SN514-treated wounds displayed little to no blood loss and did not increase burn wound infection levels compared to wounds treated with gauze or an antimicrobial silver dressing.

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Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.566-573.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 566-573

Author(s):

C C Chou ◽

R A Gatti ◽

M L Fuller ◽

P Concannon ◽

A Wong ◽

...

Keyword(s):

Cell Line ◽

Terminal Differentiation ◽

Amino Acid Sequences ◽

Multigene Families ◽

Cdna Clones ◽

Macrophage Differentiation ◽

Time Points ◽

Human Ferritin ◽

Complex Manner

HL-60 is a human promyelocytic cell line with the capability of differentiating in vitro to give neutrophils, macrophages, or eosinophils. We screened libraries of HL-60 cDNA clones representing different time points during these differentiation processes to isolate clones corresponding to mRNAs whose expression is regulated during terminal differentiation. Upon sequencing this group of regulated clones, one clone encoding the heavy subunit and two clones encoding the light subunit of human ferritin were identified by reference to published amino acid sequences. Southern blot analyses showed that these clones are encoded by distinct multigene families. These clones identify two mRNAs whose ratios vary in a complex manner during both neutrophil and macrophage differentiation.

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