In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

A cell-autonomous requirement for neutral sphingomyelinase 2 in bone mineralization

A deletion mutation called fro (fragilitas ossium) in the murine Smpd3 (sphingomyelin phosphodiesterase 3) gene leads to a severe skeletal dysplasia. Smpd3 encodes a neutral sphingomyelinase (nSMase2), which cleaves sphingomyelin to generate bioactive lipid metabolites. We examined endochondral ossification in embryonic day 15.5 fro/fro mouse embryos and observed impaired apoptosis of hypertrophic chondrocytes and severely undermineralized cortical bones in the developing skeleton. In a recent study, it was suggested that nSMase2 activity in the brain regulates skeletal development through endocrine factors. However, we detected Smpd3 expression in both embryonic and postnatal skeletal tissues in wild-type mice. To investigate whether nSMase2 plays a cell-autonomous role in these tissues, we examined the in vitro mineralization properties of fro/fro osteoblast cultures. fro/fro cultures mineralized less than the control osteoblast cultures. We next generated fro/fro;Col1a1-Smpd3 mice, in which osteoblast-specific expression of Smpd3 corrected the bone abnormalities observed in fro/fro embryos without affecting the cartilage phenotype. Our data suggest tissue-specific roles for nSMase2 in skeletal tissues.     

The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX

Carbonic anhydrases (EC 4.2.1.1) catalyse the reversible hydration of CO₂ into bicarbonate and protons. As a hypoxia-sensitive and tumour-associated isoform, isoform CA IX, is significantly overexpressed in various malignancies, being a validated target for new anticancer/antimetastatic drugs. A multitude of studies has shown that CA IX inhibition decreases cancer cell proliferation and metastasis through pHe/pHi modulation and enhancement of ferroptosis among others. Numerous studies demonstrated increased efficacy of cytotoxic drugs combined with CA inhibitors (CAIs) in various cancer types. We tested the inhibitory effect of boric acid (BA), an inorganic Lewis acid, on CA IX as well as other isoforms (CA I, II, and XII). BA acted as a millimolar in vitro CAI, decreased proliferation of two cancer cell lines, although not strong correlations between the in vitro inhibition and in vivo effects were observed. The mechanism of antiproliferative action of BA should be investigated in more detail.     

Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.     

Sheehan syndrome: clinical and laboratory evaluation of 20 cases

Sheehan syndrome (SS) or post-partum pituitary necrosis is a pituitary insufficiency secondary to excessive post-partum blood losses. SS is a very significant cause of maternal morbidity and mortality in developing countries although it is a rarity in developed countries in which obstetrical care has been improved. In this study, we reviewed 20 cases retrospectively who were diagnosed as SS in our clinic. The patients aged 40 to 65 years with a mean age of 51.12 +/- 9.44 years (mean +/- SD). Time to make a definitive diagnosis of the disease ranged between 5 and 25 years with a mean of 16.35 +/- 4.74 years. Three of our patient (15%) had a previous diagnosis of SS. Three patients (15%) were referred to emergency service for hypoglycemia, three patients (15%) for hypothyroidism and one patient (5%) for hyponatremia. Dynamic examination of the pituitary revealed GH, Prolactin, FSH, TSH and ACTH insufficiency in all of the patients. One of our patients had a sufficient LH response to LHRH challenge. All of the patients were imaged with pituitary MRI. Eleven patients had empty sella and 9 patients had partial empty sella. SS is still a common problem in our country, especially in rural areas. Considering the duration of disease, important delays occur in diagnosis and treatment of the disease.     

Immobilization of tyrosinase in polysiloxane/polypyrrole copolymer matrices

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) was achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.     

U wave: an important noninvasive electrocardiographic diagnostic marker

Study of U waves exemplifies important clinical role of noninvasive electrocardiography in modern cardiology. Present article highlights significance of U waves with a clinical case and also summarizes in brief the history of the same.     

Plasmodium falciparum: evaluation of a quantitative nucleic acid sequence-based amplification assay to predict the outcome of sulfadoxine-pyrimethamine treatment of uncomplicated malaria

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay was employed to predict retrospectively the outcome of sulfadoxine-pyrimethamine (SP) treatment of uncomplicated malaria in children aged <6 years in an endemic region. Blood samples were collected at initial diagnosis and during follow-up. Mutation-specific nested PCR methods to analyse DHFR (Arg-59) and DHPS (Glu-540) mutations that are associated with SP drug resistance were applied. Parasite genotyping was performed to distinguish between re-infection and recrudescence. Eighty-six patients were recruited of which 66 were available for follow-up. Nine children were classified as early treatment failure, 13 cases were classified as late clinical failure, 32 as late parasitological failure, and only 12 children had an adequate clinical and parasitological response. DHFR and DHPS mutations conferring SP resistance were abundant in the Plasmodium population. Blood samples obtained 7 days after treatment were used to predict retrospectively the outcome of SP treatment. QT-NASBA was able to give a correct prediction of treatment outcome in 85.7% of the cases. Positive predictive value (PPV) of QT-NASBA case was 95% (95% confidence interval = 88.3-100) and negative predictive value (NPV) was 63% (95% CI = 39.5-86.5). In contrast, microscopy correctly predicted outcome in only 37.5% of the cases. PPV of microscopy was 100% (95% CI = 73.9-100) and the NPV was 25.5% (95% CI = 13.0-38.0). The analysis of a day 7 blood sample with QT-NASBA allows for the prediction of late clinical or parasitological treatment failure in the majority of the cases analysed in the present study.     

Control of misincorporation of serine for asparagine during antibody production using CHO cells

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed‐batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time‐course analysis of cell culture suggest that misincorporation was occurring midway through the fed‐batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench‐scale culture via controlled supplementation of asparagine‐containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales. Biotechnol. Bioeng. 2010;107: 116–123. © 2010 Wiley Periodicals, Inc.