A Biomarker Panel (Bioscore) Incorporating Monocytic Surface and Soluble TREM-1 Has High Discriminative Value for Ventilator-Associated Pneumonia: A Prospective Observational Study

Introduction

Ventilator-associated pneumonia (VAP) increases mortality in critical illness. However, clinical diagnostic uncertainty persists. We hypothesised that measuring cell-surface and soluble inflammatory markers, incorporating Triggering Receptor Expressed by Myeloid cells (TREM)-1, would improve diagnostic accuracy.

Methods

A single centre prospective observational study, set in a University Hospital medical-surgical intensive Care unit, recruited 91 patients into 3 groups: 27 patients with VAP, 33 ventilated controls without evidence of pulmonary sepsis (non-VAP), and 31 non-ventilated controls (NVC), without clinical infection, attending for bronchoscopy. Paired samples of Bronchiolo-alveolar lavage fluid (BALF) and blood from each subject were analysed for putative biomarkers of infection: Cellular (TREM-1, CD11b and CD62L) and soluble (IL-1β, IL-6, IL-8, sTREM-1, Procalcitonin). Expression of cellular markers on monocytes and neutrophils were measured by flow cytometry. Soluble inflammatory markers were determined by ELISA. A biomarker panel (‘Bioscore’), was constructed, tested and validated, using Fisher’s discriminant function analysis, to assess its value in distinguishing VAP from non VAP.

Results

The expression of TREM-1 on monocytes (mTREM-1) and neutrophils (nTREM-1) and concentrations of IL-1β, IL-8, and sTREM-1 in BALF were significantly higher in VAP compared with non-VAP and NVC (p<0.001). The BALF/blood mTREM-1 was significantly higher in VAP patients compared to non-VAP and NVC (0.8 v 0.4 v 0.3 p<0.001). A seven marker Bioscore (BALF/blood ratio mTREM-1 and mCD11b, BALF sTREM-1, IL-8 and IL-1β, and serum CRP and IL-6) correctly identified 88.9% of VAP cases and 100% of non-VAP cases.

Conclusion

A 7-marker bioscore, incorporating cellular and soluble TREM-1, accurately discriminates VAP from non-pulmonary infection.     

Phosphorylation and Degradation of Tomosyn-2 De-represses Insulin Secretion

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here, we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mm glucose, ∼50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using ³²P labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2-binding proteins, we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1, and knockdown of Hrd-1 by short hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.     

Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.     

A double blind randomized controlled trial to evaluate the efficacy of green tea in gingivitis

Gingivitis is the most common form of oral disease especially among patients undergoing fixed orthodontic treatment. Green tea, which is extensively used in Asian countries, can help to improve the overall gingival health, which can be assessed by using the gingival indices. Evaluation of the effectiveness of green tea on the gingival health of patients undergoing Orthodontic treatment is of interest. 40 otherwise healthy patients undergoing fixed orthodontic treatment were randomly divided in two groups namely (1) study group and (2) control group. Gingival indices were scored for all the patients. Study group was given mouth rinse with green tea extract and control group was given placebo with no green tea extract. Gingival indices were measured for all the patients after 21 days. Mann Whitney U test and Wilcoxon test was used for statistical analysis. The gingival indices scoring in which the values before and after the use of mouthwash were compared. The p value was found to be statistically significant (p<0.05) in study group. But in control group statistical significant could not be reached.     

Effects of albumin supplementation on microvascular permeability in septic patients.

Albumin has a stabilizing effect on endothelium and helps maintain capillary permeability to macromolecules. Critically ill patients with sepsis may have profound hypoalbuminemia, but the effect of this hypoalbuminemia on microvascular permeability is unknown. To determine the degree and potential importance of this effect, we measured the transcapillary escape rate (TER) of (125)I-labeled albumin in 12 adult patients fulfilling American College of Chest Physicians/Society of Critical Care Medicine criteria for septic shock. We measured TER over a 90-min baseline period and then repeated these measurements immediately after the rapid infusion of 200 ml of 20% albumin. At baseline, patients had a mean serum albumin concentration of 10.3 +/- 3.8 g/l, which, at 30 min after the albumin infusion, was 18.5 +/- 3.7 g/l. The baseline TER was 6.7 +/- 1.5%/h, with a postinfusion TER of 6.4 +/- 2.1%/h (P = 0.550). Albumin supplementation sufficient to nearly double serum concentrations in profoundly hypoalbuminemic septic patients had no clinically significant effect in reducing microvascular permeability.     

Role of yeast domination in Indian idli batter fermentation

Adding Saccharomyces cerevisiae, along with natural bacterial flora of the ingredients, was the best method for standardizing idli termentation in terms of improved organoleptic characteristics, leavening and nutritional constituents.The authors are with the Department of Microbiology, Guru Nanak Dev University, Amristsar-143 005, India. GNDU IN. S.K. Soni is the corresponding author.     

Screening of the Philadelphia variant of galactokinase in racially unmixed black Africans: first results.

In previous reports, it was emphasized that the gene GALKA of galactokinase was the predominant allele in white populations and that another allele, GALKP, which reduces red blood cell activity (RBC GALK), was common in black people. In a group of black Americans living in Philadelphia, the frequency of GALKA was found to be very close to values expected from independent estimation of white admixture. The authors have suggested that the ancestors of these blacks might have been virtually all GALKP homozygous. We have looked for carriers of GALKP genotypes among 73 black Africans; only 33 probands were shown to have a low RBC GALK. To detect white admixture, immunoglobulin allotypes Km and Gm were investigated in 50 individuals of the sample; 15 GALKP carriers with low RBC GALK and 30 of 35 individuals with normal RBC GALK shared Gm phenotypes exclusive to blacks. Our work demonstrates for the first time the polymorphism of GALK in black Africans in the absence of white admixture.     

Solvent production from starch: effect of pH on α-amylase and glucoamylase localization and synthesis in synthetic medium

Summary The fermentation of starch by Clostridium acetobutylicum ATCC 824 has been reviewed in an optimised synthetic medium. A progressive increase of pH from 4.4 to 5.2 led to a higher production of extracellular -amylase whereas glucoamylase was poorly affected. A portion of these enzymes was cell-associated and on increasing the pH from 4.4 to 5.8 a decrease was noted in cell-bound enzymes. The association was higher for the glucoamylase than for the -amylase. The highest rate of starch consumption was at pH 5.2 whereas due to the earlier shift to solvent production at low pH, the highest solvent production was at pH 4.4. This study suggested that the level of -amylase and then the rate of starch hydrolysis was the limiting step of sugar catabolism in C. acetobutylicum ATCC 824. Correspondence to: P. Soucaille     

Nd3+‐Yb3+/Nd3+‐Yb3+‐Li+ co‐doped Gd2O3 phosphors for up and down conversion luminescence

Frequency up‐conversion (UC) emission from the Nd³⁺‐Yb³⁺/Nd³⁺‐Yb³⁺‐Li⁺ co‐doped gadolinium oxide (Gd₂O₃) phosphors prepared by the solution combustion technique in the visible range have been studied by using 980 nm near infrared (NIR) laser diode excitation. The crystalline structure and formation of the cubic phase has been confirmed with the help of X‐ray diffraction (XRD) studies. XRD peak shifts have been found towards the lower diffraction angle side in the case of the Nd³⁺‐Yb³⁺‐Li⁺ co‐doped phosphors. Surface morphology and particle size information have been observed by using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) analysis. Down‐conversion emission study under 351 nm excitation in the visible region for the Nd³⁺‐Yb³⁺/Nd³⁺‐Yb³⁺‐Li⁺ co‐doped phosphors has been performed. The UC emission bands lying in the green and red region arising from the Nd³⁺ ions have been enhanced by ~260 times, ~113 times due to incorporation of Li⁺ ions in the Nd³⁺‐Yb³⁺ co‐doped phosphors. Photometric characterization has been done for the Nd³⁺‐Yb³⁺/Nd³⁺‐Yb³⁺‐Li⁺ co‐doped phosphors. The present study suggests the capability of the synthesized phosphors in near‐infrared (NIR) to visible upconverter and luminescent device applications.