A top-down, three-scale numerical analysis of wafer-to-wafer metallic bonding

To study the sensitivity to micro-scale imperfections of the strength of a metallic, wafer-to-wafer MEMS bonding, we propose a three-scale numerical (finite element) approach. At the wafer level (macro-scale), accounting for the whole metallic sealing through nonlinear springs connecting the two silicon wafers modelled as thin plates, we link the force transferred by each single MEMS die to the external pressure applied to the wafers. This force is next used as an index for the input pressure at the die level (meso-scale), where the geometry of the metallic rings is accurately described: the local stress field at the interface between the upper and lower metallic rings is so obtained. Finally, a local (micro-scale) model is used to link the aforementioned local stress field in each die to the bonding strength: representative volumes of the rings getting into contact, accounting in a statistically way for the relevant surface roughness (which is on the order or tens of nanometers at most), are adopted to obtain the relationship between the external pressure and the percentage of sealed area. This information is exploited to assess the properties of the rings, in terms of expected bonding strength

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

BACKGROUND: In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. RESULTS: The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. CONCLUSION: We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome

Treatment of tuberculosis in a region with high drug resistance: Outcomes, drug resistance amplification and re-infection

Introduction: Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. Methods: We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. Results: At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/ 47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. Conclusion: In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.

Understanding transmission and control of Cystic Echinococcosis and other taeniid infections in the Falkland Islands

Cystic echinococcosis, caused by the larval form of the cestode parasite Echinococcus granulosus, has been identified as an important public health risk in the Falkland Islands since the early 1940s. This prompted the instigation of an intensive control scheme in the mid-1960s, comprised of regular dosing of domestic dogs with the anthelmintic praziquantel and education of local people about safe disposal of potentially infected offal. This scheme has remained in place to the current day and is generally considered to be a successful programme– resulting in a reduction in the prevalence of infection in sheep has reduced from >50% in the 1950s to less than 1 % now and there has not been a case of human hydatid disease for more than 20 years. However, concerns remain that hydatid cysts are still identified in a small number of sheep at slaughter (0.004% in 2017) and occurring every year subsequently suggesting transmission is still occurring. This is also supported by the observation that sheep continue to be infected at higher levels with the (non-zoonotic) cestode Taenia hydatigena, also transmitted by dogs. In 2010, all dogs on the Falkland Islands were tested by Copro-PCR, resulting in eight dogs (1.4%) testing positive. The dog population was tested again in 2012, where there were no cases but when tested in 2014 by Coproantigen testing, six (1.04%) were positive for E. granulosus coproantigens. This project used questionnaires, coproantigen and coproPCR analysis, abattoir data surveillance, DNA sequencing, environmental sample analysis and mathematical modelling to study Echinococcus granulosus and other taeniids endemic in the Falklands and investigate how their continued transmission can occur in the face of the prolonged intensive control programme. A questionnaire survey identified possible methods of disposal of offal that in a previous study, were associated with canine coproantigen positivity. The entire dog population was analysed via coproantigen techniques in 2018, and four (0.68%) dogs were coproantigen positive, though none of these were confirmed by PCR. From 2018 to 2020, five cases of CE were identified in sheep at the Sand Bay abattoir in the Falklands (0.01%), with one of the cases coming from a positive farm in 2018. There were two cases from farms with positive dogs in 2010 and one from a farm with a positive dog in 2014. To investigate environmental contamination on farms and potentially identify historical dog infections, soil samples taken from kennel sites were analysed for the presence of coproantigens, with five farms having positive results, one farm matching with a positive dog in 2018. To identify key processes fuelling the transmission of E. granulosus in the Falklands, a mechanically informed compartmental model was created, estimating the basic reproduction number (R0) for the parasite, and identifying scenarios where this estimate increased above one suggesting continued transmission could occur. Seven scenarios where lapses in control measures could result in the R0 estimate increasing above one and continued transmission of E. granulosus could occur. The results of this project show clear evidence of dogs still being involved in the transmission of taeniid parasites in the Falklands, with key areas of the eradication programme such as the inadequate disposal of offal and dogs gaining access to offal allowing the transmission cycle to be completed and transmission of E. granulosus and other taeniids to occur. Rectifying these lapses in control measures and focussing control and surveillance to a more localised control approach will help strengthen the control programme and move the Falklands closer towards the complete eradication of Cystic Echinococcosis

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-alpha concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection

People Living with HIV (PLHIV) are at an increased risk of pneumococcal pneumonia than HIV-uninfected adults, but the reasons for this are still not well understood. We investigated whether alveolar macrophages (AM) mediated control of pneumococcal infection is impaired in PLHIV compared to HIV-uninfected adults. We assessed anti-bactericidal activity against Streptococcus pneumoniae of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post ex vivo infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings, in vivo evidence showed that PLHIV had a higher propensity for harboring S. pneumoniae within their AMs than HIV-uninfected adults. Moreover, bacterial intracellular survival in AMs was associated with extracellular propagation of pneumococcal infection. Our data suggest that failure of AMs to eliminate S. pneumoniae intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV

Maximising the impact and reuse of citizen science data

Citizen science, the active participation of the public in scientific research projects, is a rapidly expanding field in open science and open innovation. It provides an integrated model of public knowledge production and engagement with science. As a growing worldwide phenomenon, it is invigorated by evolving new technologies that connect people easily and effectively with the scientific community. Catalysed by citizens’ wishes to be actively involved in scientific processes, as a result of recent societal trends, it also offers contributions to the rise in tertiary education. In addition, citizen science provides a valuable tool for citizens to play a more active role in sustainable development. This book identifies and explains the role of citizen science within innovation in science and society, and as a vibrant and productive science-policy interface. The scope of this volume is global, geared towards identifying solutions and lessons to be applied across science, practice and policy. The chapters consider the role of citizen science in the context of the wider agenda of open science and open innovation, and discuss progress towards responsible research and innovation, two of the most critical aspects of science today