Hemoglobin A
(A1C) is used in assessment of patients for elective surgeries because hyperglycemia increases risk of adverse events. However, the interplay of A1C, glucose, and surgical outcomes ...remains unclarified, with often only two of these three factors considered simultaneously. We assessed the association of preoperative A1C with perioperative glucose control and their relationship with 30-day mortality.
Retrospective analysis on 431,480 surgeries within the Duke University Health System determined the association of preoperative A1C with perioperative glucose (averaged over the first 3 postoperative days) and 30-day mortality among 6,684 noncardiac and 6,393 cardiac surgeries with A1C and glucose measurements. A generalized additive model was used, enabling nonlinear relationships.
A1C and glucose were strongly associated. Glucose and mortality were positively associated for noncardiac cases: 1.0% mortality at mean glucose of 100 mg/dL and 1.6% at mean glucose of 200 mg/dL. For cardiac procedures, there was a striking U-shaped relationship between glucose and mortality, ranging from 4.5% at 100 mg/dL to a nadir of 1.5% at 140 mg/dL and rising again to 6.9% at 200 mg/dL. A1C and 30-day mortality were not associated when controlling for glucose in noncardiac or cardiac procedures.
Although A1C is positively associated with perioperative glucose, it is not associated with increased 30-day mortality after controlling for glucose. Perioperative glucose predicts 30-day mortality, linearly in noncardiac and nonlinearly in cardiac procedures. This confirms that perioperative glucose control is related to surgical outcomes but that A1C, reflecting antecedent glycemia, is a less useful predictor.
Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum ...are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.
•P. chrysogenum or D. hansenii reduce OTA production by Penicillium nordicum.•Indications of nutritional limitation of P. nordicum by P. chrysogenum•P. nordicum cell wall integrity protein levels altered by bioprotective cultures•PgAFP production is not essential for P. chrysogenum bio-protective mode of action.
BACKGROUNDThe Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the ...existing Myh11-CreERT2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreERT2 mouse (referred to as Myh11-CreERT2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. METHODSA Myh11-CreERT2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ERT2 after the Myh11 start codon. Myh11-CreERT2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. RESULTSMyh11-CreERT2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreERT2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreERT2 mice. Labeling was equivalent in both male and female Cre+ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. CONCLUSIONSWe generated and validated the function of an autosomal Myh11-CreERT2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.
CC97 and CC151 are two of the most common Staphylococcus aureus lineages associated with bovine intramammary infection. The genotype of the infecting S. aureus strain influences virulence and the ...progression of intramammary disease. Strains from CC97 and CC151 encode a distinct array of virulence factors. Identification of proteins elaborated in vivo will provide insights into the molecular mechanism of pathogenesis of these lineages, as well as facilitating the development of tailored treatments and pan-lineage vaccines and diagnostics. The repertoire of genes encoding cell wall-anchored (CWA) proteins was identified for S. aureus strains MOK023 (CC97) and MOK124 (CC151); MOK023 encoded more CWA proteins than MOK124. Serum collected during an in vivo challenge trial was used to investigate whether the humoral response to cell wall proteins was strain-specific. Immunoproteomic analysis demonstrated that the humoral response in MOK023-infected cows predominantly targeted high molecular weight proteins while the response in MOK124-infected cows targeted medium or low molecular weight proteins. Antigenic proteins were identified by two-dimensional serum blotting followed by mass spectometry-based identification of immunoreactive spots, with putative antigens subsequently validated. The CWA proteins ClfB, SdrE/Bbp and IsdA were identified as immunogenic regardless of the infecting strain. In addition, a number of putative strain-specific imunogens were identified. The variation in antigens produced by different strains may indicate that these strains have different strategies for exploiting the intramammary niche. Such variation should be considered when developing novel control strategies including vaccines, therapeutics and diagnostics.
Antifungal agents are essential in the fight against serious fungal disease, however emerging resistance is threatening an already limited collection of therapeutics. Proteomic analyses of effects of ...antifungal agents can expand our understanding of multifactorial mechanisms of action and have also proven valuable to elucidate proteomic changes associated with antifungal resistance.
This review covers the application of proteomic techniques to examine sensitivity and resistance to antifungals including commonly used therapeutics, amphotericin B, echinocandins and the azoles, based predominantly on studies involving Aspergillus fumigatus, Candida albicans and Candida glabrata from the last 10 years. In addition, non-clinical antimicrobial agents are also discussed, which highlight the potential of proteomics to identify new antifungal targets.
Fungal proteomics has evolved in the last decade with increased genome availability and developments in mass spectrometry. Collectively, these have led to the advancement of proteomic techniques, allowing increased coverage of the proteome. Gel-based proteomics laid the foundation for these types of studies, which has now shifted to the more powerful gel-free proteomics. This has resulted in the identification of key mediators and potential biomarkers of antifungal resistance, as well as elucidating the mechanisms of action of novel and established antifungal agents.
A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun ...proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Department of Molecular Physiology and Biophysics, Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia
Submitted 24 November 2008
; accepted in final form 22 January ...2009
There is clear evidence that the phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by the loss of SMC marker gene expression. The transcription factor Krüppel-like factor 4 (KLF4) is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing the expression of the serum response factor (SRF) myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate the expression of KLF4 in phenotypically modulated SMCs. The results demonstrated that the transcription factor stimulating protein-1 (Sp1) regulated the expression of KLF4 in SMCs. The KLF4 promoter contains three consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, overexpression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with small-interfering RNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, the results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, the results suggest a novel role for Sp1 in increasing the expression of KLF4 in phenotypically modulated SMCs.
Krüppel-like factor 4; stimulating protein-1; platelet-derived growth factor-BB; vascular injury
Address for reprint requests and other correspondence: G. K. Owens, Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia, PO Box 800736, Charlottesville, VA 22908-0736 (e-mail: gko{at}virginia.edu )
With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic ...alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.
OBJECTIVE:Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent ...macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO).
APPROACH AND RESULTS:Using Myh11-Cre eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP; and unintended proinflammatory consequences. Using newly generated Myh11-DretdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1 lymphatic endothelial cells in the epididymal AT.
CONCLUSIONS:Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.
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