Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer''s disease, and head injury.Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.Click here to view the related article Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.Download video file.^{(53M, mov)}     

Low-dose hydrocortisone in patients with cirrhosis and septic shock: a randomized controlled trial

Background

Recent studies have reported a high prevalence of relative adrenal insufficiency in patients with liver cirrhosis. However, the effect of corticosteroid replacement on mortality in this high-risk group remains unclear. We examined the effect of low-dose hydrocortisone in patients with cirrhosis who presented with septic shock.

Methods

We enrolled patients with cirrhosis and septic shock aged 18 years or older in a randomized double-blind placebo-controlled trial. Relative adrenal insufficiency was defined as a serum cortisol increase of less than 250 nmol/L or 9 μg/dL from baseline after stimulation with 250 μg of intravenous corticotropin. Patients were assigned to receive 50 mg of intravenous hydrocortisone or placebo every six hours until hemodynamic stability was achieved, followed by steroid tapering over eight days. The primary outcome was 28-day all-cause mortality.

Results

The trial was stopped for futility at interim analysis after 75 patients were enrolled. Relative adrenal insufficiency was diagnosed in 76% of patients. Compared with the placebo group (n = 36), patients in the hydrocortisone group (n = 39) had a significant reduction in vasopressor doses and higher rates of shock reversal (relative risk [RR] 1.58, 95% confidence interval [CI] 0.98–2.55, p = 0.05). Hydrocortisone use was not associated with a reduction in 28-day mortality (RR 1.17, 95% CI 0.92–1.49, p = 0.19) but was associated with an increase in shock relapse (RR 2.58, 95% CI 1.04–6.45, p = 0.03) and gastrointestinal bleeding (RR 3.00, 95% CI 1.08–8.36, p = 0.02).

Interpretation

Relative adrenal insufficiency was very common in patients with cirrhosis presenting with septic shock. Despite initial favourable effects on hemodynamic parameters, hydrocortisone therapy did not reduce mortality and was associated with an increase in adverse effects. (Current Controlled Trials registry no. ISRCTN99675218.)Cirrhosis is a leading cause of death worldwide,¹ often with septic shock as the terminal event.^2–9 Relative adrenal insufficiency shares similar features of distributive hyperdynamic shock with both cirrhosis and sepsis^10,11 and increasingly has been reported to coexist with both conditions.^11,12 The effect of low-dose hydrocortisone therapy on survival of critically ill patients in general with septic shock remains controversial, with conflicting results from randomized controlled trials^13–17 and meta-analyses.^18,19 The effect of hydrocortisone therapy on mortality among patients with cirrhosis, who are known to be a group at high risk for relative adrenal insufficiency, has not been studied and hence was the objective of our study.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.     

Histone methyltransferase SUV39H1 participates in host defense by methylating mycobacterial histone‐like protein HupB

Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone‐like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1‐mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell–cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell–cell adhesion ability.     

Information theoretic perspective on genome clustering

Shannon’s information theoretic perspective of communication helps one to understand the storage and processing of information in one-dimensional sequences. An information theoretic analysis of 937 available completely sequenced prokaryotic genomes and 238 eukaryotic chromosomes is presented. Information content (Id) values were used to cluster these chromosomes. Chargaff’s second parity rule i.e compositional self-complementarity, an empirical fact is observed in all the genomes, except for the proteobacteria Candidatus Hodgkinia cicadicola. High information content, arising out of biased base composition in all the 14 chromosomes of Plasmodium falciparum is found among two other genomes of prokaryotes viz. Buchnera aphidicola str. Cc (Cinara cedri) and Candidatus Carsonella ruddii PV. Despite size and compositional variations, both prokaryotic and eukaryotic genomes do not deviate significantly from an equiprobable and random situation. Eukaryotic chromosomes of an organism tend to have similar informational restraints as seen when a simple distance based method is used to cluster them. In eukaryotes, in certain cases, Id values are also similar for the two arms (p and q arm) of the chromosomes. The results of this current study confirm that the information content can provide insights into the clustering of genomes and the evolution of messaging strategies of the genomes. An efficient and robust Perl CGI standalone tool is created based on this information theory algorithm for the analysis of the whole genomes and is made available at https://github.com/AlagurajVeluchamy/InformationTheory.     

Large scale association analysis identifies three susceptibility loci for coronary artery disease

Genome wide association studies (GWAS) and their replications that have associated DNA variants with myocardial infarction (MI) and/or coronary artery disease (CAD) are predominantly based on populations of European or Eastern Asian descent. Replication of the most significantly associated polymorphisms in multiple populations with distinctive genetic backgrounds and lifestyles is crucial to the understanding of the pathophysiology of a multifactorial disease like CAD. We have used our Lebanese cohort to perform a replication study of nine previously identified CAD/MI susceptibility loci (LTA, CDKN2A-CDKN2B, CELSR2-PSRC1-SORT1, CXCL12, MTHFD1L, WDR12, PCSK9, SH2B3, and SLC22A3), and 88 genes in related phenotypes. The study was conducted on 2,002 patients with detailed demographic, clinical characteristics, and cardiac catheterization results. One marker, rs6922269, in MTHFD1L was significantly protective against MI (OR=0.68, p=0.0035), while the variant rs4977574 in CDKN2A-CDKN2B was significantly associated with MI (OR=1.33, p=0.0086). Associations were detected after adjustment for family history of CAD, gender, hypertension, hyperlipidemia, diabetes, and smoking. The parallel study of 88 previously published genes in related phenotypes encompassed 20,225 markers, three quarters of which with imputed genotypes The study was based on our genome-wide genotype data set, with imputation across the whole genome to HapMap II release 22 using HapMap CEU population as a reference. Analysis was conducted on both the genotyped and imputed variants in the 88 regions covering selected genes. This approach replicated HNRNPA3P1-CXCL12 association with CAD and identified new significant associations of CDKAL1, ST6GAL1, and PTPRD with CAD. Our study provides evidence for the importance of the multifactorial aspect of CAD/MI and describes genes predisposing to their etiology.     

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.