Pathophysiology and Treatment of Septic and Traumatic Shock

Background. While various medical treatments have been proven effective in the treatment of sepsis in animal models, the only current clinically accepted treatment of human sepsis is limited to the use of activated protein C. The complex pathogenesis of human sepsis presents a challenge in precisely duplicating the process of disease development in the particular animal models which are currently employed for preclinical testing. Further research examining the pathogenesis of this deadly condition is essential for the implementation of novel therapies that target distinct disease mechanisms. The objective of this 48-hour study is to utilize a clinical model that accurately replicates severe human sepsis along with gut ischemia/reperfusion (I/R). I/R further leads to the injury of multiple organs within the time period most closely mirroring disease progression in humans. Methodology. The experimental protocol was approved by the Committee for the Humane Use of Animals at SUNY Upstate Medical University and complied with the National Institutes of Health Guidelines for the Use of Experimental Animals in Research. Five pigs were subjected to a “two-hit” injury involving the clamping of the superior mesenteric artery (SMA) for 30 minutes as well as a laparotomy used for insertion of a fecal clot, following appropriate administration of anesthetics and ventilation. A drain was inserted into this laparatomy wound twelve hours post injury. Monitoring of animals took place under a standard Intensive Care Unit setting over the course of 48 hours, with oxygen desaturation resolved by increasing FiO2. Hemodynamics were stabilized through administration of antibiotics and intravenous fluids as needed, while measurements of arterial and mixed venous blood gases as well as lung, kidney, liver, renal, and hemodynamic function measurements were recorded. Progression of the abdominal compartment syndrome was determined by monitoring bladder pressure changes. Serial measurements of peritoneal and plasma ascites were also taken for evaluation of cytokine concentration. Morphometric analysis was carried out using the organ tissues harvested and fixed at necropsy. Results. All animals presented with polymicrobial sepsis. Over the course of 48 hours the lung, liver, kidney, and intestine showed ongoing deterioration and histopathological as well as clinical damage. This was found to occur in conjunction with increased levels of cytokines within the peritoneal fluid and serum. Conclusion. In combining both sepsis and ischemia reperfusion injury, the animal model used in this study is valid for uncovering the intricate pathophysiological progression of septic shock and its transition to multiple organ dysfunction syndrome. This system mirrors the systemic inflammation and major organ systems dysfunction seen in humans. Through demonstrating the success of the current animal model, prospective treatments may be developed through conducting sophisticated preclinical trials

Ultrafast cooling reveals microsecond-scale biomolecular dynamics

The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10–15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations

Preclinical Efficacy of a Carboxylesterase 2-Activated Prodrug of Doxazolidine

Doxazolidine (Doxaz) is a functionally distinct formaldehyde conjugate of doxorubicin (Dox) that induces cancer cell death in Dox-sensitive and resistant cells. Pentyl PABC-Doxaz (PPD) is a prodrug of Doxaz that is activated by carboxylesterase 2 (CES2), which is expressed by liver, non-small cell lung, colon, pancreatic, renal, and thyroid cancer cells. Here, we demonstrate that in two murine models, PPD was effective at slowing tumor growth and demonstrated markedly reduced cardiotoxic and nephrotoxic effects, as well as better tolerance, relative to Dox. Hepatotoxicity, consistent with liver expression of the murine CES2 homolog, was induced by PPD. Unlike irinotecan, a clinical CES2-activated prodrug, PPD produced no visible gastrointestinal damage. Finally, we demonstrate that cellular response to PPD may be predicted with good accuracy using CES2 expression and Doxaz sensitivity, suggesting that these metrics may be useful as clinical biomarkers for sensitivity of a specific tumor to PPD treatment

A novel insertion mutation in the cartilage-derived morphogenetic protein-1 (CDMP1) gene underlies Grebe-type chondrodysplasia in a consanguineous Pakistani family

<p>Abstract</p> <p>Background</p> <p>Grebe-type chondrodysplasia (GCD) is a rare autosomal recessive syndrome characterized by severe acromesomelic limb shortness with non-functional knob like fingers resembling toes. Mutations in the cartilage-derived morphogenetic protein 1 (<it>CDMP1</it>) gene cause Grebe-type chondrodysplasia.</p> <p>Methods</p> <p>Genotyping of six members of a Pakistani family with Grebe-type chondrodysplasia, including two affected and four unaffected individuals, was carried out by using polymorphic microsatellite markers, which are closely linked to <it>CDMP1 </it>locus on chromosome 20q11.22. To screen for a mutation in <it>CDMP1 </it>gene, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the family and sequenced directly in an ABI Prism 310 automated DNA sequencer.</p> <p>Results</p> <p>Genotyping results showed linkage of the family to <it>CDMP1 </it>locus. Sequence analysis of the <it>CDMP1 </it>gene identified a novel four bases insertion mutation (1114insGAGT) in exon 2 of the gene causing frameshift and premature termination of the polypeptide.</p> <p>Conclusion</p> <p>We describe a 4 bp novel insertion mutation in <it>CDMP1 </it>gene in a Pakistani family with Grebe-type chondrodysplasia. Our findings extend the body of evidence that supports the importance of <it>CDMP1 </it>in the development of limbs.</p

Culturing Aerobic and Anaerobic Bacteria and Mammalian Cells with a Microfluidic Differential Oxygenator

In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen−nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated)

Brachydactyly

Brachydactyly ("short digits") is a general term that refers to disproportionately short fingers and toes, and forms part of the group of limb malformations characterized by bone dysostosis. The various types of isolated brachydactyly are rare, except for types A3 and D. Brachydactyly can occur either as an isolated malformation or as a part of a complex malformation syndrome. To date, many different forms of brachydactyly have been identified. Some forms also result in short stature. In isolated brachydactyly, subtle changes elsewhere may be present. Brachydactyly may also be accompanied by other hand malformations, such as syndactyly, polydactyly, reduction defects, or symphalangism

Microfluidic advantage : novel techniques for protein folding and oxygen control in cell cultures

The young field of microfluidics has been growing due to its utility in chemical and biological applications. Microfluidic devices can be rapidly and inexpensively fabricated from silicone elastomers, making them ideal for prototyping and subsequent production. Further, the behavior of fluid flows in micrometer-diameter channels can be accurately predicted - due to the properties of laminar flow and purely diffusive mixing - decreasing experimental uncertainties, while allowing access to a wide range of experiments impossible with traditional methods. The projects presented here fall into two separate areas of biophysics, although they are all facilitated by microfluidics. Chapter 2 deals with the control of the gas content in the medium of cell cultures. This is an important consideration, as the oxygen concentration, [O₂], available to cells has been shown to affect their metabolism, growth, and gene expression. The first project is a microfluidic chemostat supplying nine different [O₂] to bacteria growing in chambers beneath the gas channels. Here, we compared the growth rates of E. coli growing at nine different [O₂] simultaneously. Section 2.2 introduces a multi-channel, computer- controlled gas mixer that can provide up to ten arbitrary gas mixtures to a microfluidic device. Finally, Section 2.3 describes gas control strips for use with mammalian cell cultures in standard multiwell culture plates. These gas control strips allow cell culture media in different rows of wells to contain different [O₂]. Chapter 3 describes a novel system to rapidly heat and cool a small volume of solution of biological macromolecules using time -controlled deposition of heat into a small volume with a focused infrared laser beam. By fluorescently labeling the molecules, their conformational changes due to temperature shifts can be observed. This system improves the time resolution of the cooling transition over traditional methods by at least two orders of magnitude, down to one microsecond. Further, the temperature change from the laser heating pulse is several times larger than with other techniques. We used this system to measure the kinetics of fast DNA hairpin folding and unfolding under varying salt concentration

Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays