A new microdialysis probe for continuous lactate measurement during fetal monitoring:Proof of concept in an animal model

Introduction Cardiotocography (CTG) is currently the most commonly used method for intrapartum fetal monitoring during labor. However, a high false-positive rate of fetal acidosis indicated by CTG leads to an increase in obstetric interventions. We developed a microdialysis probe that is integrated into a fetal scalp electrode allowing continuous measurement of lactate subcutaneously, thus giving instant information about the oxygenation status of the fetus. Our aim was to establish proof of concept in an animal model using a microdialysis probe to monitor lactate subcutaneously. Material and methods We performed an in vivo study in adult male wild-type Wistar rats. We modified electrodes used for CTG monitoring in human fetuses to incorporate a microdialysis membrane. Optimum flow rates for microdialysis were determined in vitro. For the in vivo experiment, a microdialysis probe was inserted into the skin on the back of the animal. De-oxygenation and acidosis were induced by lowering the inspiratory oxygen pressure. Oxygenation and heart rate were monitored. A jugular vein cannula was inserted to draw blood samples for analysis of lactate, pH, pco(2), and saturation. Lactate levels in dialysate were compared with plasma lactate levels. Results Baseline blood lactate levels were around 1 mmol/L. Upon de-oxygenation, oxygen saturation fell to below 40% for 1 h and blood lactate levels increased 2.5-fold. Correlation of dialysate lactate levels with plasma lactate levels was 0.89 resulting in an R-2 of .78 in the corresponding linear regression. Conclusions In this animal model, lactate levels in subcutaneous fluid collected by microdialysis closely reflected blood lactate levels upon transient de-oxygenation, indicating that our device is suitable for subcutaneous measurement of lactate. Microdialysis probe technology allows the measurement of multiple compounds in the dialysate, such as glucose, albumin, or inflammatory mediators, so this technique may offer the unique possibility to shed light on fetal physiology during the intrapartum period

Systems-level interactions between insulin–EGF networks amplify mitogenic signaling

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal-regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin-induced increase in the phosphatidylinositol-3,4,5-triphosphate (PIP3) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin-induced amplification of mitogenic signaling is abolished by disrupting PIP3-mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non-linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues

Genetic Predisposition for Type 2 Diabetes, but Not for Overweight/Obesity, Is Associated with a Restricted Adipogenesis

BACKGROUND: Development of Type 2 diabetes, like obesity, is promoted by a genetic predisposition. Although several genetic variants have been identified they only account for a small proportion of risk. We have asked if genetic risk is associated with abnormalities in storing excess lipids in the abdominal subcutaneous adipose tissue. METHODOLOGY/PRINCIPAL FINDINGS: We recruited 164 lean and 500 overweight/obese individuals with or without a genetic predisposition for Type 2 diabetes or obesity. Adipose cell size was measured in biopsies from the abdominal adipose tissue as well as insulin sensitivity (HOMA index), HDL-cholesterol and Apo AI and Apo B. 166 additional non-obese individuals with a genetic predisposition for Type 2 diabetes underwent a euglycemic hyperinsulinemic clamp to measure insulin sensitivity. Genetic predisposition for Type 2 diabetes, but not for overweight/obesity, was associated with inappropriate expansion of the adipose cells, reduced insulin sensitivity and a more proatherogenic lipid profile in non-obese individuals. However, obesity per se induced a similar expansion of adipose cells and dysmetabolic state irrespective of genetic predisposition. CONCLUSIONS/SIGNIFICANCE: Genetic predisposition for Type 2 diabetes, but not obesity, is associated with an impaired ability to recruit new adipose cells to store excess lipids in the subcutaneous adipose tissue, thereby promoting ectopic lipid deposition. This becomes particularly evident in non-obese individuals since obesity per se promotes a dysmetabolic state irrespective of genetic predisposition. These results identify a novel susceptibility factor making individuals with a genetic predisposition for Type 2 diabetes particularly sensitive to the environment and caloric excess

Erythrocyte and Porcine Intestinal Glycosphingolipids Recognized by F4 Fimbriae of Enterotoxigenic Escherichia coli

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO3-3Galß1Cer), sulf-lactosylceramide (SO3-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer)

Knee instruments and rating scales designed to measure outcomes

In this article, the knee instruments and rating scales that are designed to measure outcomes are revised. Although the International Knee Documentation Committee Subjective Knee Form can be used as a general knee measure, no instrument is currently universally applicable across the spectrum of knee disorders and patient groups. Clinicians and researchers looking to use a patient-based score for measurement of outcomes must consider the specific patient population in which it has been evaluated. The Western Ontario and McMaster Universities Osteoarthritis Index is recommended for the evaluation of treatment effect in persons with osteoarthritis (OA). This is a generic health status questionnaire that contains 36 items, is widely used, and easy to complete. The Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire evaluates the functional status and quality of life (QoL) of patients with any type of knee injury who are at increased risk of developing OA; i.e., patients with anterior cruciate ligament (ACL) injury, meniscus injury, or chondral injury. So far, the KOOS questionnaire has been validated for several orthopedic procedures such as total knee arthroplasty, ACL reconstruction, and meniscectomy. The utilization of QoL questionnaires is crucial to the adequate assessment of a number of orthopedic procedures of the knee. The questionnaires are generally well accepted by the patients and open up new perspectives in the analysis of prognostic factors for optimal QoL of patients undergoing knee surgery

Role of urothelial cells in BCG immunotherapy for superficial bladder cancer

Intravesical instillation of Bacillus Calmette-Guérin (BCG) is used for the treatment of superficial bladder cancer, both to reduce the recurrence rate of bladder tumour and to diminish the risk of progression. Since its first therapeutic application in 1976, major research efforts have been directed to decipher the exact mechanism of action of the BCG-associated antitumour effect. Bacillus Calmette-Guérin causes an extensive local inflammatory reaction in the bladder wall. Of this, the massive appearance of cytokines in the urine of BCG-treated patients stands out. Activated lymphocytes and macrophages are the most likely sources of these cytokines, but at present other cellular sources such as urothelial tumour cells cannot be ruled out. Bacillus Calmette-Guérin is internalised and processed both by professional antigen-presenting cells and urothelial tumour cells, resulting in an altered gene expression of these cells that accumulates in the presentation of BCG antigens and secretion of particular cytokine

Domestication syndrome is investigated by proteomic analysis between cultivated cassava (Manihot esculenta Crantz) and its wild relatives

Cassava (Manihot esculenta Crantz) wild relatives remain a largely untapped potential for genetic improvement. However, the domestication syndrome phenomena from wild species to cultivated cassava remain poorly understood. The analysis of leaf anatomy and photosynthetic activity showed significantly different between cassava cultivars SC205, SC8 and wild relative M. esculenta ssp. Flabellifolia (W14). The dry matter, starch and amylose contents in the storage roots of cassava cultivars were significantly more than that in wild species. In order to further reveal the differences in photosynthesis and starch accumulation of cultivars and wild species, the globally differential proteins between cassava SC205, SC8 and W14 were analyzed using 2-DE in combination with MALDI-TOF tandem mass spectrometry. A total of 175 and 304 proteins in leaves and storage roots were identified, respectively. Of these, 122 and 127 common proteins in leaves and storage roots were detected in SC205, SC8 and W14, respectively. There were 11, 2 and 2 unique proteins in leaves, as well as 58, 9 and 12 unique proteins in storage roots for W14, SC205 and SC8, respectively, indicating proteomic changes in leaves and storage roots between cultivated cassava and its wild relatives. These proteins and their differential regulation across plants of contrasting leaf morphology, leaf anatomy pattern and photosynthetic related parameters and starch content could contribute to the footprinting of cassava domestication syndrome. We conclude that these global protein data would be of great value to detect the key gene groups related to cassava selection in the domestication syndrome phenomena

A Recombinant Vaccine Effectively Induces C5a-Specific Neutralizing Antibodies and Prevents Arthritis

OBJECTIVES: To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a. METHODS: We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology. RESULTS: Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered. CONCLUSIONS: Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity