Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B)

Determination of micro-scale plastic strain caused by orthogonal cutting

An electron beam lithography technique has been used to produce microgrids in order to measure local plastic strains, induced during an orthogonal cutting process, at the microscopic scale in the shear zone and under the machined surface. Microgrids with a 10 μm pitch and a line width less than 1 μm have been printed on the polished surface of an aluminium alloy AA 5182 to test the applicability of the technique in metal cutting operations. Orthogonal cutting tests were carried out at 40 mm/s. Results show that the distortion of the grids could successfully be used to compute plastic strains due to orthogonal cutting with higher accuracy compared to other techniques reported in the literature. Strain maps of the machined specimens have been produced and show high-strain gradients very close to the machined surface with local values reaching 2.2. High-resolution strain measurements carried out in the primary deformation zone also provide new insight into the material deformation during the chip formation process

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

The expression of yoeb and pezt bacterial toxin genes in microalga Chlorella vulgaris and the effects on cell viability / Ng Shet Lee

Chlorella vulgaris is an oleaginous microalgae which is a potential candidate for the harvesting of valuable cellular contents, particularly for the production of biofuels. However, the microalgae’s rigid cell wall proved to be a challenge faced during microalgae transformation as well as the downstream harvesting of cellular contents. Toxin-antitoxin (TA) systems are genetic entities that are almost ubiquitous in prokaryotic genomes and have been implicated in programmed bacterial cell death and to date, no TA homologs are found in eukaryotic cells. A TA system is usually made up of two genes, an antitoxin gene that encodes a labile antitoxin, which can either be an untranslated RNA or a protein, and the toxin gene which encodes for the more stable toxin protein. The expression of several TA toxins were reported to be functionally lethal in several eukaryotic organisms such as zebrafish, Arabidopsis thaliana, yeast as well as human cell lines. In this study, the utility of a two-component chemical-inducible expression system which was originally developed for the Arabidopsis plant system was investigated in the green microalgae, C. vulgaris UMT-M1 for the expression of the YoeBSpn and PezT toxin from the Gram-positive bacterium, Streptococcus pneumoniae. Both the activator vector, pMDC150 that harbored the constitutive CaMV 35S promoter together with responder vector (pMDC221) cloned with translational fusions of either yoeBSpn-GFP or pezT-GFP, respectively, were co-transformed into C. vulgaris UMT-M1 via Agrobacterium tumefaciens-mediated transformation. The XVE transcription activator encoded on the pMDC150 vector was constitutively expressed under the control of CaMV 35S promoter. In the presence of 17-β-estradiol as the inducer, an XVE-responsive promoter (OlexTATA) readily initiates transcription of the yoeBSpn-GFP and pezT-GFP fusion genes that were cloned separately into the pMDC221 responder vector. Following Agrobacterium-mediated transformation, PCR analysis confirmed that the transgenes were present in the transformed C. vulgaris lines. Upon 17-β-estradiol treatment to express the yoeBSpn-GFP and pezT-GFP fusions, GFP signals were observed in transgenic C. vulgaris cells which showed signs of cellular damage and lysis. Expression of the YoeBSpn and PezT toxins greatly affected the cell viability of the transgenic C. vulgaris cells. This is the first report demonstrating the simultaneous transformation of two vectors into C. vulgaris as well as the functionality of the XVE-based two-component expression system in C. vulgaris. This is also the first demonstration of the lethality of bacterial TA toxins in eukaryotic microalgae as evidenced by the morphological changes and cell lysis of transgenic Chlorella vulgaris subsequent to the activation of YoeB and PezT toxins. The conditional expression of the bacterial toxin in microalgae can be used to develop novel means to efficiently harvest microalgal cellular contents through the lysis of transgenic microalgal cells triggered by toxin activation upon induction with the appropriate signal

Diagnostic and prognostic potential of extracellular vesicles in peripheral blood

AbstractPurposeExtracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood.MethodsSeveral techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined.FindingsAlthough EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood.ImplicationsOverall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential

Interplay between coagulation and vascular inflammation in sickle cell disease

Sickle cell disease is the most common inherited hematologic disorder that leads to the irreversible damage of multiple organs. Although sickling of red blood cells and vaso-occlusion are central to the pathophysiology of sickle cell disease the importance of hemolytic anemia and vasculopathy has been recently recognized. Hypercoagulation state is another prominent feature of sickle cell disease and is mediated by activation of both intrinsic and extrinsic coagulation pathways. Growing evidence demonstrates that coagulation may not only contribute to the thrombotic complications, but also to vascular inflammation associated with this disease. This article summarizes the role of vascular inflammation and coagulation activation, discusses potential mechanisms responsible for activation of coagulation and reviews recent data demonstrating the crosstalk between coagulation and vascular inflammation in sickle cell disease