S. aureus (HA-MRSA) remains a significant cause of morbidity and mortality worldwide. We conducted a study to determine risk factors for HA-MRSA in order to inform control strategies in South Africa.Hospital-associated methicillin-resistant We used surveillance data collected from five tertiary hospitals in Gauteng and Western Cape provinces during 2014 for analysis. A case of HA-MRSA was defined as isolation of MRSA from a blood culture 48 hours after admission and/or if the patient was hospitalised in the six months prior to the current culture. Multivariable logistic regression modelling was used to determine risk factors for HA-MRSA.S. aureus bacteraemia (SAB). The overall prevalence of MRSA among those with SAB was 30.9% . HA-MRSA infections accounted for 28.3% of patients with SAB . Burns , age ≤1 month , residency at a long-term care facility , antibiotic use within two months of the current SAB episode , hospital stay of 13 days or more  and mechanical ventilation , were independent risk factors for HA-MRSA infection.Of the 9971 patients with positive blood cultures, 7.7% (772) had The prevalence of MRSA remains high in South African tertiary public hospitals. Several identified risk factors of HA-MRSA infections should be considered when instituting infection and prevention strategies in public-sector hospitals, including intensifying the implementation of antimicrobial stewardship programmes. There is an urgent need to strengthen infection prevention and control in burn wards, neonatal wards, and intensive care units which house mechanically ventilated patients. Staphylococcus aureus is the most common human pathogen causing both community- and hospital-associated infections, including pneumonia and bacteraemia [S. aureus (HA-MRSA) accounts for a high proportion of hospitalised patients infected with S. aureus [S. aureus infections [Nosocomial infections are a significant cause of morbidity, mortality, and excess healthcare costs globally . Staphylteraemia . Hospita. aureus . In mostfections .S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) infections are associated with higher morbidity and mortality [Since its emergence in the 1960s and recognition as a nosocomial pathogen, MRSA remains a significant cause of illness among the population today . Compareortality . A meta-ortality . MRSA inortality ,8.In order to control HA-MRSA infections, it is important to understand the burden of disease and risk factors associated with infection. Identification of risk factors for HA-MRSA is essential as it guides infection control and prevention policies, guidelines and related activities. In South Africa, there are limited reports on HA-MRSA and its associated risk factors. We aimed to describe the epidemiology of HA-MRSA and identify risk factors in comparison to HA-MSSA in five public sector hospitals in South Africa.We conducted a cross-sectional analysis on demographic, laboratory and clinical data collected through GERMS-SA, an active laboratory-based surveillance system for selected pathogens of public health importance. All hospitals taking part in the surveillance program and included in our study were tertiary academic facilities with diagnostic stewardship programs that includes clinical microbiology consultancy on specimen submission practices as one of the components. All patients presenting with clinical signs and symptoms such as temperature >38°C, increased white cell count, elevated C-reactive protein, erythrocyte sedimentation rate, urea and electrolytes, and lung abnormalities on a chest X-ray, qualify for a blood culture. Five tertiary public-sector hospitals, including Helen Joseph Hospital (HJH), Steve Biko Academic/Tshwane District Hospital (SBH), Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), Groote Schuur Hospital (GSH) and Tygerberg Hospital (TBH) participated in the GERMS-SA surveillance system during 2014. HJH (900 beds), SBH (832 beds) and CMJAH (900 beds) are in Gauteng Province, and GSH (975 beds) and TBH  are in the Western Cape Province. These hospitals are affiliated with universities and serve mainly urban populations.S. aureus bacteraemia (SAB) and captured data using standard case report forms. For patients who refused to give consent, only medical and laboratory records were used to obtain data. Isolates from these patients were submitted to the Antimicrobial Resistance Reference Laboratory at the National Institute for Communicable Diseases for confirmatory identification and antimicrobial susceptibility testing. Isolate identification was done using the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Flex analysis system, version 3.4 . Antimicrobial susceptibility testing was done using the automated Microscan Walkaway system (Beckman Coulter Inc.) and minimum inhibitory concentrations were interpreted using Clinical and Laboratory Standard Institute guidelines [Patient level and laboratory data were obtained from the GERMS-SA electronic database. GERMS-SA surveillance officers interviewed consenting patients who had idelines . Data weS. aureus positive blood culture isolated between 1st January 2014 and 31st December 2014 from patients admitted to any of the sentinel sites. Where a patient had additional isolates within 21 days of the first positive culture, only the first episode was included. MRSA was defined as non-susceptibility to oxacillin. Hospital-associated infection was defined as collection of a positive blood culture more than 48 hours after admission, or if a patient was hospitalised within a six-month period preceding the positive culture. SAB cases occurring within 48 hours of admission with no prior hospitalisation in the preceding six months were considered community-associated infections, and were not included in our analysis for risk factors.A case of SAB was defined as a S. aureus, the number of blood cultures positive for S. aureus was divided by the total number of blood cultures performed at hospital laboratories (one culture per patient per day). To determine the proportion of patients with SAB among patients with bloodstream infections , the number of SAB cases was divided by the total number of positive cultures. The corresponding 95% confidence intervals (CI) were also calculated for positivity rates and for the proportions. Descriptive statistics were used to describe characteristics of HA-MRSA and HA-MSSA cases. Associations between categorical variables were tested using Chi-square tests. Differences in non-normally distributed continuous variables between two groups were tested using the non-parametric Wilcoxon rank-sum test. Logistic regression was used to determine risk factors for HA-MRSA infection. The potential risk factors analysed included demographic characteristics, clinical characteristics such as presence of mechanical ventilation and central venous catheters, and underlying conditions such as heart disease, diabetes mellitus, smoking and malignancy. All variables with a p-value of ≤ 0.2 in the univariate analyses were entered into the multivariable model using the manual forward stepwise selection. Multi-collinearity tests were performed before entering variables in the multivariable model to ensure that only non-collinear variables were included. Variables with a p-value of ≤ 0.05 were kept in the model. The final model was tested using the Hosmer-Lemeshow Chi-square goodness-of-fit test. All statistical analyses were performed using STATA statistical software  and a p-value of ≤ 0.05 was considered statistically significant.To calculate positivity rates for S. aureus surveillance was granted by the Human Research Ethics Committee  of the University of Witwatersrand (No: M10464). Permission to use data from the National Health Laboratory Service Corporate Data Warehouse was obtained from the National Health Laboratory Service Research and Academic Committee. Extracted data were anonymised and stored on password protected computers.Ethical approval for this study was obtained from the Faculty of Health Sciences Research Ethics Committee of the University of Pretoria (458/2015). The protocol was also approved by the U.S Centers for Disease Control and Prevention (CDC)’s Human Subjects Research Office. Ethics approval for the GERMS-SA enhanced S. aureus bloodstream infections.There were 772 SAB cases diagnosed at the five sentinel sites during 2014 . Of thesS. aureus was isolated from 1.4%  of all blood cultures performed at sentinel sites, with ranges of 1.1%– 1.5% between hospitals  accounted for the highest proportion  of HA-MRSA infections across all hospitals, while none of the patients aged 6–14 years were infected with HA-MRSA , admission at hospital D  and hospital E , hospital stay of 5–12 days , hospitals stay of ≥13 days , burns , mechanical ventilation , abdominal surgery , residence in long-term care facilities (LTCFs) , and antibiotic use in the last two months  were significantly associated with HA-MRSA . PreviouIn the multivariable analysis, burns  and age ≤1 month  were the strongest risk factors for HA-MRSA . Living S. aureus bloodstream infections among patients admitted to tertiary public-sector hospitals in Gauteng and Western Cape. Patients infected with HA-MRSA had poorer outcomes such as higher mortality and longer hospital stays, compared to patients infected with HA-MSSA. Patients with burns, neonates and who lived at LTCFs were at highest risk of developing HA-MRSA infection, and the duration of hospitalisation, mechanical ventilation, and antibiotic exposure were also significant risk factors, but of lower magnitude.In our study, we found a high prevalence of MRSA, and HA-MRSA accounted for approximately a third of In our study, the prevalence of MRSA among patients with SAB was lower than previously reported in similar South African studies ,10. AlthSimilar to previous studies, we found that compared to patients with HA-MSSA infections, a higher proportion of patients with HA-MRSA had longer hospital stays and higher mortality rates ,15. ThesS. aureus was the major pathogen infecting patients in burn units, and MRSA accounted for 66% of these infections in KwaZulu-Natal Province and 58% in Eastern Cape Province [Control of MRSA in healthcare settings relies on understanding factors that predispose patients to the acquisition. Risk factors for HA-MRSA acquisition have been well described and include, invasive procedures, long hospital stay, antibiotic exposure and use of medical devices –19. The Province ,24. A siProvince . Taken tOur study showed that neonates represented the highest proportion of patients infected with HA-MRSA. In addition, neonates had eight times the odds of HA-MRSA infection compared to HA-MSSA. These findings are contrary to studies done in Scotland, Belgium and the USA that demonstrated a higher incidence of HA-MRSA infections among adults, and an association of HA-MRSA with older age –28. The Residents of LTCFs, which are often older frail patients with underlying conditions, were also identified as a subset of hospitalised individuals independently associated with HA-MRSA. Similar to our study, one laboratory-based study including patients of all ages found that MRSA positivity was strongly associated  with residents of LTCFs compared to patients in acute care hospitals or other facilities such as military hospitals . LTCFs aIn our study, previous antibiotic exposure independently increased the odds of HA-MRSA five-fold compared to HA-MSSA. Previous studies have similarly found that antibiotic exposure is a significant risk factor for HA-MRSA infection ,19,29. AWe found that the odds of HA-MRSA infection were twice as likely as HA-MSSA when patients were mechanically ventilated, underlining the importance of the presence of medical devices in hospital acquisition of drug-resistant organisms. In the United States, ventilator-associated pneumonia accounts for 39.1% of pneumonia events . In addiSimilar to other studies, we also found that HA-MRSA infection is associated with an overall longer hospital stay ,7,27, anOne of the strengths of our study was the use of enhanced surveillance data that were extensive and of good quality. Additionally, we were able to identify important risk factors for HA-MRSA not described in previous South African studies. However, the risk factors described in our study may not reflect risk factors for patients admitted to non-tertiary public-sector hospitals in South Africa. Due to the small number of patients with burns, neonates and LTCFs residents who were found to be at high risk of HA-MRSA, our analysis lacked the statistical power to find any association with known hospital-related factors specific to these patients. Hospital-related factors such as overcrowding, hand hygiene practices, and the existence of antimicrobial stewardship programmes are important determinants of HA-MRSA that were not collected by the surveillance programmes from which we obtained data. Thus, we could not determine their contribution to HA-MRSA acquisition. This information is critical for prevention of HA-MRSA infection and other hospital-related infections.The prevalence of MRSA among patients with SAB hospitalised in the five tertiary-level public-sector hospitals in South Africa remains higher than expected. In view of our findings that patients with burns, neonates, those admitted in LTCFs, prolonged hospitalisation, mechanical ventilation, and antibiotic exposure are important risk factors for HA-MRSA infection, control strategies should employ use of “MRSA bundles”. These bundles may include optimum hand hygiene, educating healthcare workers on best practices for handling ventilated patients, isolation and cohorting of infected patients, antimicrobial stewardship programmes, surveillance screening, and decolonisation of high risk patients –41. In o


Augmented reality (AR), mixed reality (MR), and virtual reality devices are enabling technologies that may facilitate effective communication in healthcare between those with information and knowledge  and those seeking understanding and insight . Investigators initiated an exploratory program to enable the study of AR/MR use-cases in acute care clinical and instructional settings.Academic clinician educators, computer scientists, and diagnostic imaging specialists conducted a proof-of-concept project to 1) implement a core holoimaging pipeline infrastructure and open-access repository at the study institution, and 2) use novel AR/MR techniques on off-the-shelf devices with holoimages generated by the infrastructure to demonstrate their potential role in the instructive communication of complex medical information.The study team successfully developed a medical holoimaging infrastructure methodology to identify, retrieve, and manipulate real patients’ de-identified computed tomography and magnetic resonance imagesets for rendering, packaging, transfer, and display of modular holoimages onto AR/MR headset devices and connected displays. Holoimages containing key segmentations of cervical and thoracic anatomic structures and pathology were overlaid and registered onto physical task trainers for simulation-based “blind insertion” invasive procedural training. During the session, learners experienced and used task-relevant anatomic holoimages for central venous catheter and tube thoracostomy insertion training with enhanced visual cues and haptic feedback. Direct instructor access into the learner’s AR/MR headset view of the task trainer was achieved for visual-axis interactive instructional guidance.Investigators implemented a core holoimaging pipeline infrastructure and modular open-access repository to generate and enable access to modular holoimages during exploratory pilot stage applications for invasive procedure training that featured innovative AR/MR techniques on off-the-shelf headset devices. However, these worlds are accessible only when users wear opaque VR goggles and relinquish several essential and interactive aspects of the physical realm of reality. In contrast, augmented reality (AR) overlays a supplemental digital realm Beyond consumer-focused uses such as gaming and social media, AR/MR technologies harbor dramatic potential for meaningful scientific application due to their ability to radically shift the way individuals interact with other people, places, objects, and ideas. Specifically, the representation and sharing of informative data through sophisticated visualization approaches is likely to blossom into an explosive phenomenon. AR/MR is already catalyzing healthcare and medical education, as it embodies an innovative and accessible approach for clinicians, patients, researchers, and educators to see, discuss, study, experiment, implement, and share complex concepts. Established examples in diverse fields include anatomy education,Our goals were as follows: 1) Implement a basic holoimaging pipeline infrastructure with open-access repository to facilitate exploratory applications of AR/MR at the study institution and beyond; and 2) Use novel AR/MR techniques on off-the-shelf devices with holoimages generated by the infrastructure to demonstrate their potential role in the instructive communication of complex medical information.e.g., probe failure) and non-availability.Program investigators structured an exploratory research framework to examine AR/MR’s unique capabilities, implementation needs and barriers, and use characteristics in accessible, high-yield, best-case healthcare settings . For oneTo set up the envisioned training application, the team accessed two department-funded, off-the-shelf HoloLens headsets  at a hospital simulation center. Specifically, these devices were used to expand an existing hands-on procedural teaching curriculum in place for EM fellows, residents, advanced practice providers, and medical students. We designed a two-hour pilot session to accommodate 40 learners for the “blind insertion” procedural training using two distinct and complementary approaches: 1) modular AR/MR holoimaging overlay and registration of patient anatomic structures and pathology onto procedural task trainers; and 2) first-person view visual-axis interactive instructional guidance. The institutional review board approved the project protocol.Diagnostic imaging and computer science co-investigators collaborated with clinician educators to develop and assemble a medical holoimaging pipeline infrastructure with the following elements:Query mechanism to identify, locate, and retrieve radiographic imagesets with specified clinical findings in the hospital network’s Centricity Picture Archiving and Communication Systems  using mPower Imageset manipulation approach for de-identified, thin-slice, contrast-enhanced trauma “pan-scan” computed tomography (CT) volumetric imagesets to create modular and discrete regional segmentations of anatomic structures and pathologic findings of interest:2a. We extracted structures of interest with voxel intensity-based growing algorithms using an Advantage Workstation .2b. We converted 3D scalar fields of structures of interest into polygonal mesh isosurfaces as an .stl file  with a marching cubes algorithm, using iNtuition .2c. We managed mesh smoothness and vertex counts using open-source MeshLab  to fit within the processing capabilities of select AR/MR headset devices.Compositing, rendering, and packaging process with differentiation of mesh model layers by color and synthesis of integrating holoimages using Maya software for export in .fbx format Transfer mechanism to move holoimages onto institution-approved secure network storage, cloud storage solution, and/or an open-access repositoryRetrieval and display procedure to access holoimages on AR/MR headset devices.holocomposites that reflect patterns of pathology, e.g., combinations of multi-organ traumatic injury holoimages. At an administrative level, procedures and protocols for formalization of the infrastructure and dissemination mechanisms for institution-wide use are under development in parallel with discussions of staffing and funding arrangements. In the interim, approximately 20 of the developed holoimage .fbx files are accessibly stored on a storage cloud repository located at: https://repository.library.brown.edu/studio/collections/id_753/ .In its current implementation, the process pipeline required approximately 30 minutes for a typical head or chest CT imageset from the time of patient selection until holoimage display . One specific advantage of developing this type of infrastructure was the ever-expanding repository of modular holoimages that can be subsequently retrieved and displayed, either as originally rendered or in assembled ., sternal notch, laryngeal prominence, rib spaces. This permitted the stable overlay of task-relevant anatomy, e.g., internal jugular, carotid artery; ribcage, lung parenchyma, and pneumothorax, for training with enhanced visual cues and haptic feedback. A wirelessly networked laptop connected with the AR/MR device’s built-in webportal system to broadcast the learner’s view with superimposed holoimages for the viewer cohort.Overlaying real patients’ holoimages of key cervical and thoracic structures onto physical task trainers was essential to provide learners with the equivalent of a “visible human” for the explicit purpose of conveying the locations, shapes, sizes, and juxtapositions of internal vascular and thoracic anatomic structures with respect to surface features. This was accomplished by displaying the rendered holoimages on the HoloLens units with 3DViewer Beta (Microsoft) during the practice insertion of invasive therapeutic devices . The hol., the introduction of educator-inserted 3D visual cues such as digital pointers and arrows. By using the headset’s Skype functionality, the learner video-called an educator’s laptop Skype application with HoloLens Add-in (with screen-mirroring projection of the learner FPV). This resulted in lagless sharing of the learner’s procedural performance perspective along with the ability for both the learner and educator to “draw” into the shared view using an on-screen toolset, e.g., digital pencil tool or arrow tool. For environments without network connectivity or Skype, an alternative approach has been tested using an ad hoc device-laptop connection and Bluetooth-connected pointing device such as a wireless mouse to remotely direct the learner’s attention  between the learner’s HoloLens headset and an educator laptop with LCD projector. This arrangement was critical for accessing the learner’s AR/MR headset view of the task trainer for visual-axis interactive instructional guidance, i.ettention .Investigators successfully applied the described AR/MR holoimage visualization methodologies to a two-hour pilot session with approximately 40 learners. Four 30-minute breakout sessions (with two educators each instructing groups of 10 participants) were completed as part of a scheduled EM residency conference period; simulation and standardized patient scenarios were conducted in parallel sessions during the AR/MR-enhanced invasive procedural training. To emphasize anatomic visualization with AR/MR assistance and to review “blind insertion” techniques, the learners were intentionally not provided with ultrasound devices; standard approaches, equipment, and procedural kits were otherwise used for both procedures. All participants finished both task-training exercises without physical discomfort; formal objective metrics were not obtained during this proof-of-concept session. See Investigators successfully piloted the use of off-the-shelf AR/MR devices to integrate holoimaging-enhanced anatomic visualization with acute care invasive procedure training. We developed and implemented novel methodologies, including a core institutional holoimaging pipeline infrastructure, open-access storage repository of modular holoimage segmentations, and visual axis instruction techniques, for education and research purposes.Program researchers are continuing to explore AR/MR applications in acute care, surgical care, and medical science while concurrently working to formally study the utility and usability of the developed AR/MR methodologies in healthcare. As part of the research program’s multiphasic strategy, investigations of AR/MR devices at the live clinical bedside are being prepared in peri-procedural settings: anatomic and diagnostic imaging AR/MR visualizations will be shown to patients and family members to gauge how the intervention facilitates discussions regarding management options and whether it assists with shared decision-making. Simultaneous, shared holoimage visualization across multiple headsets/viewers has already been accomplished at the study site; automation of holoimage registration with the real world for improved user experiences is being actively pursued.., holoimage registration accuracy, stability, and usability) and longitudinal, checklist-based procedural performance assessments in simulated settings will need to be established and validated. Additional acute care procedural training that could uniquely benefit from AR/MR-enabled anatomic referencing and guidance are being reviewed, e.g., arthrocentesis, epistaxis control, lumbar puncture, orthopedic manipulations, and complex wound exploration. Well-designed trials will be necessary to ascertain the effectiveness of AR/MR-based training and treatment interventions on live-patient clinical outcomes.With respect to future directions, the program team is pursuing ongoing use of AR/MR supplementation during EM residency sessions to objectively examine its educational utility for acute care procedural training. Defined metrics such as operational quality markers  are anticipated to begin laying the foundation for meaningful and judicious use of AR/MR in healthcare.The program’s core holoimaging infrastructure was made possible by the significant in-kind efforts of co-investigators  with deep, authorized access into the institution’s clinical diagnostic imaging systems. The primary operational limitation encountered by the research team derived from the manual overlay and registration process, in that the spatial alignment between the holoimage and the task trainer was intermittently lost and required adjustments. We did not assess or compare the effects of the experimental AR/MR approach on learner training and performance against existing methods during this initial pilot application. As a novel visualization approach that has only recently become more accessible to healthcare providers and researchers, holoimaging is anticipated to progress through the “hype cycle” framework,., distortion of real-world perception,Investigators have focused on the technical aspects of core system implementation, such that continued efforts will need to address the substantial work that remains in applying human factors engineering, ensuring institutional acceptance, and conducting empirical investigations into the clinical utility of holoimaging’. Previous work has already identified a variety of emergent AR-specific adverse effects in the clinical environment, e.g


Alcohol-to-jet (ATJ) is one of the technical feasible biofuel technologies. It produces jet fuel from sugary, starchy, and lignocellulosic biomass, such as sugarcane, corn grain, and switchgrass, via fermentation of sugars to ethanol or other alcohols. This study assesses the ATJ biofuel production pathway for these three biomass feedstocks, and advances existing techno-economic analyses of biofuels in three ways. First, we incorporate technical uncertainty for all by-products and co-products though statistical linkages between conversion efficiencies and input and output levels. Second, future price uncertainty is based on case-by-case time-series estimation, and a local sensitivity analysis is conducted with respect to each uncertain variable. Third, breakeven price distributions are developed to communicate the inherent uncertainty in breakeven price. This research also considers uncertainties in utility input requirements, fuel and by-product outputs, as well as price uncertainties for all major inputs, products, and co-products. All analyses are done from the perspective of a private firm.The stochastic dominance results of net present values (NPV) and breakeven price distributions show that sugarcane is the lowest cost feedstock over the entire range of uncertainty with the least risks, followed by corn grain and switchgrass, with the mean breakeven jet fuel prices being $0.96/L , $1.01/L , and $1.38/L , respectively. The variation of revenues from by-products in corn grain pathway can significantly impact its profitability. Sensitivity analyses show that technical uncertainty significantly impacts breakeven price and NPV distributions.Technical uncertainty is critical in determining the economic performance of the ATJ fuel pathway. Technical uncertainty needs to be considered in future economic analyses. The variation of revenues from by-products plays a significant role in profitability. With the distribution of breakeven prices, potential investors can apply whatever risk preferences they like to determine an appropriate bid or breakeven price that matches their risk profile.The online version of this article (doi:10.1186/s13068-017-0702-7) contains supplementary material, which is available to authorized users. Similarly, the United States (US) Federal Aviation Administration  breakeven jet prices per liter of ATJ from corn grain, sugarcane, and switchgrass are $1.01 , $0.96 , and $1.38 , respectively. The mean values are the price for jet fuel at which investors have a 50% probability of earning more than their threshold discount rate. The breakeven price distributions are within the deterministic range calculated by Staples et al.  confirmit test to determine whether the three breakeven price distributions are statistically different from each other. The two-sample test assuming unequal variances, conducted for three pairwise breakeven price samples, confirms that the mean values of the three breakeven price distributions are significantly different from each other  we take technical uncertainty into account by linking conversion efficiency with input and output quantities through statistical methods; (2) in addition to NPV, we develop breakeven price distributions to provide potential investors the price level required to achieve their stipulated rate of return at each probability level; (3) price forecasts are based on case-by-case historical time-series analyses. Sugarcane is the lowest cost feedstock over the entire range of uncertainty with the least risks, followed by corn grain and switchgrass, with the mean breakeven jet fuel prices being $0.96/L , $1.01/L , and $1.38/L , respectively. The probability of loss given the future fuel market price projections for sugarcane, corn grain, and switchgrass ATJ are 88, 95, and 100%, respectively. Price support policies based on breakeven price distributions should be implemented to avoid potential losses and achieve targeted profitability.Incorporating both technical and economic uncertainty is critical in characterizing the economic performance of any new technology and needs to be considered in future economic analyses. We find that the variation of revenues from by-products can impact profitability differently at different probability levels.Additional file 1. Supplementary materials and descriptions of regression functions of each sub-process of ATJ production, first-order and second-order stochastic dominance, and Welch's t-test result for breakeven price distribution.


Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen  The ISS National Laboratory is a unique research environment whereby scientists from Academia, Government, and Industry seek to learn the effects of space on life and matter in their many forms. Although a great deal has already been learned about the effects of microgravity on biological systems, –3 the laAlthough there are increasing numbers of studies of gene expression in microgravity, there are no reports of any methodologies for RNA purification and RT-qPCR under microgravity conditions, effectively preventing on-orbit analysis of gene expression or PCR-based diagnostics from biological samples. Specifically, recent RNA isolation and PCR-based gene expression studies of biological samples on ISS, Space Shuttle and Biosatellites have been conducted on earth after preserved sample return from space –14. ReceTo address the pressing ISS research and diagnostics need for novel on-orbit end-to-end molecular biology gene expression analysis methods and tools, we devised a robust microgravity fluidic system that can intake diverse biological sample  and using temperature-stable reagents, allow for the isolation and purification of nucleic acids, such as RNA, for molecular biology analysis. In addition, the system includes a microgravity compatible thermal cycler, lyophilized reagents and enzymes for PCR assays and methods for reverse transcription of RNA into cDNA, and capabilities for heat inactivation of enzymes such as proteinase K and PCR inhibitors, plus RT-qPCR gene expression analysis using fluorescent TaqMan probes. The WetLab2 suite of instruments and reagents we describe here is novel and unique methodology, substantially distinct from all other existing RNA isolation and RT-qPCR gene expression analysis systems, because of its ability to function in microgravity, with altered fluid flow, hydrostatic pressure, convection, and surface tension conditions of space, while still providing full reagent containment for safe operation on ISS. Using this system, schematically shown in In its validation flight, WetLab-2 successfully demonstrated its capabilities to conduct molecular biology experimentation in microgravity. This consisted of first demonstrating that qPCR functions in microgravity conditions, followed by demonstration of RNA isolation from bacterial cell and mammalian tissue and gene expression analysis by RT-qPCR. When gases evolved by thermal cycling accumulated in PCR tubes due to microgravity conditions interfered with data uniformity, the system demonstrated its flexibility and adaptability to on-orbit experiment modification and iteration in real-time. WetLab-2 also used volunteer astronaut time and spare supplies to design and execute follow-up experiments while on-orbit to resolve the gas bubble problem. Below we describe in detail the design and scientific validation of the WetLab-2 suite of methods, reagents, and hardware for RNA purification and gene expression analysis by RT-qPCR, enabling future molecular biology experimentation to be conducted end-to-end in real-time on ISS, and allowing data interpretation and experiment redesign while on-orbit.E. coli primer assays (ground), validation of lyophilized components (ground), validation of the SPM (ground), validation of all hardware (ground), validation of qPCR efficiency using pre-prepared assays (on-orbit), validation of RNA extracted from E. coli (on-orbit), and from mouse liver tissue (on-orbit) using the SPM. The methods used in these validation experiments are described in detail below.In order to validate the system, a number of ground-based and on-orbit tests were performed including: validation of E. coli genomic assay was used to demonstrate if qPCR was possible in microgravity and that the data matched 1 g controls. This was followed with a set of E. coli gene expression assays designed to validate the sample prep and RT-qPCR capabilities of the system. Finally, a set of mouse gene expression assays were used to validate the system with a mammalian tissue.This study used three types of assays for different portions of the validation of the system. First, an E. coli primers and probes used in this study were designed in-house to target both genomic and exonic regions. The E. coli genomic assay consisted of primers targeting the 16S ribosomal DNA sequence for rrnH gene while the gene expression assays targeted the following genes: heat shock protein 70–2 (dnaK), RNA polymerase (rpoA), and a glucitol operon repressor (srlR). For mouse assays, pre-designed intron spanning assays targeting genes for glyceraldehyde 3-phosphate dehydrogenase (gapdh) and 60S ribosomal protein L19 (rpl19) were used for normalization and fibronectin 1 (fn1) was used to measure gene expression analysis. All primers and probes were produced by Integrated DNA Technologies . In all cases, TaqMan based technology was used -1)*100.Cepheid SmartCycler 2.0d software was used to analyze all fluorescence intensities and generate Ct values. All pre-launch ground testing used the default software values , while microgravity to 1 g comparisons used slightly modified values with a Boxcar averaging of 3. Genomic DNA qPCR runs used: Background Subtraction: ON, Background Min: 5, Background Max: 40, Primary Curve Analysis, Manual Threshold: 30, Boxcar Averaging: 3; software values for the t-test as appropriate. In all cases, figures report the mean ± standard deviation (SD) and p ≤ 0.05 was considered the threshold for significance. Outliers were calculated using Grubb’s test with alpha = 0.05.Statistical analyses were performed using JMP10  and GraphPad Prism 5 . Comparisons made between microgravity and 1 g control data, or between lyophilized and benchtop control assays across different template concentrations were evaluated using ANOVA or Student’s E. coli DNA. The abundant target expression improved precision, sensitivity, and reproducibility, providing robust assay conditions.In order to assess the functionality of PCR in microgravity conditions and to establish the dynamic range and efficiency of amplification, we designed a robust quality control qPCR assay to measure these parameters. We targeted a 16S ribosomal region of genomic E. coli genomic DNA assay was performed using a standard thermal cycling protocol with controlled quantities of commercially available E. coli DNA as template. Assays were tested with template quantities between 1 pg and 1 μg, in duplicates  was compared to that of RNA extracted at 1 g on the same day (RIN 8) and is of comparable quality  from commercially available Claremont BioSolutions products . The res quality  showing E. coli sample, used to test the system’s capabilities with prokaryotic cells, was processed and analyzed on ISS as described previously. The RT-qPCR assays tested included singleplex  to assess PCR efficiency. Three replicate runs were conducted, of which two were successful in returning qPCR amplification curves that showed close correlation to 1 g controls. Furthermore, the microgravity Ct results were comparable to their respective 1 g controls . AlthougE. coli and mouse liver tissue on the ground. Results show that RNA was successfully isolated on-orbit using the SPM and that singleplex and multiplex RT-qPCR analyses from both E. coli cells and mouse liver tissues resulted in successful qPCR amplification Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 TableE. coli Genomic DNA validation, E. coli, and mouse experiments. Microgravity data are from on-orbit operations while 1 g control are from equivalent experiments run on the ground. Low, Mid, and High represent template concentrations of 0.01, 1.0, and 100 ng, respectively. Singleplex assays were amplified independently of one another, while Duplex and Triplex assays were amplified in multiplex assays, combined as shown. p-values are from t-test analyses, Ɨ denotes statistical outliers not included in t-test analyses.Complete Ct data from (DOCX)Click here for additional data file.


Malaria in pregnancy is associated with increased risk for both maternal and neonatal adverse outcomes, notably low birthweight and neonatal mortality.8The current WHO recommendation is for all HIV-positive women to receive daily co-trimoxazole  for prevention of opportunistic infections. The co-administration of co-trimoxazole and SP is contraindicated because of an increased risk of adverse effects. Co-trimoxazole has antimalarial activity; in one study, infant birthweight was similar among HIV-positive women taking daily co-trimoxazole and those taking IPTp-SP .10That SP, which has antibacterial activity, could improve birthweight independent of its antimalarial effect is perhaps unsurprising. A non-malaria-related beneficial effect of SP on birthweight could be due to several mechanisms, including anti-inflammatory effects or alterations in the bacterial flora of the gut or vagina leading to effects on maternal or infant weight gain, indirect metabolic effects, or a reduction in the impact of genitourinary tract organisms associated with adverse pregnancy outcomes.What is more surprising is that SP seemed to counteract the effects of antiretrovirals on pregnancy outcomes. Although administration of antiretroviral therapy (ART) to pregnant women, compared with not treating mothers, is associated with improved birthweight,http://www.who.int/malaria/publications/atoz/istp-and-act-in-pregnancy.pdf). The findings of Stoner and others provide evidence that in a setting where malaria was not likely an important factor affecting pregnancy outcome, IPTp-SP still appeared to confer benefit through as yet undefined pathways. Further research is needed to elucidate these other mechanisms, as well as to explore whether daily co-trimoxazole confers similar benefit.In light of concerns about the decreasing antimalarial efficacy of SP, recent studies have explored alternatives for IPTp, including dihydroartemisinin–piperaquine (DP), with demonstrated protection against malaria, but no clear benefit compared with SP with respect to birthweight.


Nanomaterial science and design have shifted from generating single passive nanoparticles to more complex and adaptive multi-component nanohybrids. These adaptive nanohybrids (ANHs) are designed to simultaneously perform multiple functions, while actively responding to the surrounding environment. ANHs are engineered for use as drug delivery carriers, in tissue-engineered templates and scaffolds, adaptive clothing, smart surface coatings, electrical switches and in platforms for diversified functional applications. Such ANHs are composed of carbonaceous, metallic or polymeric materials with stimuli-responsive soft-layer coatings that enable them to perform such switchable functions. Since ANHs are engineered to dynamically transform under different exposure environments, evaluating their environmental behavior will likely require new approaches. Literature on polymer science has established a knowledge core on stimuli-responsive materials. However, translation of such knowledge to environmental health and safety (EHS) of these ANHs has not yet been realized. It is critical to investigate and categorize the potential hazards of ANHs, because exposure in an unintended or shifting environment could present uncertainty in EHS. This article presents a perspective on EHS evaluation of ANHs, proposes a principle to facilitate their identification for environmental evaluation, outlines a stimuli-based classification for ANHs and discusses emerging properties and dynamic aspects for systematic EHS evaluation. Such hiThe necessity to achieve “on demand” control over material functionality and the ability to functionalize nanomaterials with unique combinations of organic polymer blocks have encouraged the design and synthesis of stimuli-responsive nanoparticles  or ANHs.i.e., of both covalent surface moieties and of soft polymeric/surfactant coatings, on aggregation, deposition, transformation and toxicity has also been evaluated [Stimuli-responsive ANHs are composed of well-studied nanostructures, e.g., carbonaceous ,32 and mvaluated ,44,45,46This article presents a perspective on EHS assessment of ANHs. The paper will first propose a principle for identifying ANHs and will outline a stimuli-based classification for these materials. Emerging properties and the dynamic aspects of the coatings and their influence in controlling nano-EHS will be discussed, which will allow for a critical analysis of the fate, transport, transformation, and toxicity assessment of these novel horizon materials in an aquatic environment.Since ANHs involve surface-coated metallic, carbonaceous or polymeric NMs, discerning the differences between passive and adaptive nanostructures is important in directing EHS efforts appropriately. Here is the first attempt to lay down the principle for identifying ANHs. This principle is derived from earlier NH foundational work .Conjugated nanostructures composed of carbonaceous, metallic, or polymeric materials when coated with a soft chemically bound exterior polymeric layer, resulting in core-shell type hybrids that respond to external stimuli that have enhanced properties or multifunctionality, can be identified as adaptive nanohybrids or ANHs.” This principle includes ANHs composed of metallic, carbonaceous or polymeric NMs and NHs functionalized with stimuli-responsive coronas or polymers [“polymers ,48,49,50polymers ,51,52  diblock copolymer [This principle would exclude selection of the following as ANHs; (i) NMs and NHs with coatings that are not stimuli responsive  porphyrin-functionalized single-walled carbon nanotubes (SWNTs) , NHs comopolymer  or at meopolymer ). This pThe behavior of ANHs is controlled by the external stimulus that can cause the exterior coatings to shrink/swell and to change the optical, mechanical or luminescence response of the nanostructure, to name a few. Although the field of ANHs is relatively new, there are numerous opportunities to design new nanosystems with single- or multi-stimuli-responsive attributes. The following discussion classifies ANHs on the basis of stimuli that invoke responses from the particles. A comprehensive literature search has been performed to identify relevant ANHs. A total of 812 publications from 1996 to 2014 were retrieved and classified using the Web of Science® search engine. After a list of relevant terms was identified, a search algorithm was designed using wildcards and Boolean operators, in combination with a title field tag, as the search criteria to limit the results to the most relevant studies in ANHs. i.e., for pH-, photo-, thermo-, ion-, chemical-, salt-, magneto-, acoustic-, redox-, glucose-, glutathione-, enzyme-, thiol-, radiation- and multi-stimuli-responsive materials. Results reveal the relative importance of each of the stimuli in the contemporary ANH literature. The distribution of ANH publications based on the most relevant stimuli is shown in Follow-up searches were performed for each individual stimulus of concern, Stimuli-responsive soft-layers enable ANHs to perform switchable functions and show considerable changes in their physical and chemical properties in response to small changes in their environment. The classification of ANHs can be done in a number of ways. Here, we present a classification scheme based on key environmental stimuli, namely pH-, thermo-, photo- and multi-stimuli-responsive ANHs.One of the most common stimuli is pH, where functional moieties on ANH surfaces respond to a specific pH range and perform desired functions. pH-responsive moieties are mostly acidic, which include carboxylic, amino acid or sulfonic acid groups. Depending on the pH, these polymers undergo a transition between protonation/deprotonation, relative to their pKa or pKb (equilibrium acidity or basicity constants). Weak acids are water-soluble via deprotonation, while weak bases become less soluble at these pHs, as protonation reduces surface potential and, hence, their relative polarity. Thus, in an aqueous environment, the ionization leads most commonly to swelling or shrinking of the polymeric shell of ANHs. For instance, at low pH, carboxylic functional groups are protonated and hydrophobic interactions dominate, leading to volume shrinkage of the polymer that contains them. On the other hand, at high pH, carboxylic groups dissociate, resulting in a high charge density in the polymer, resulting in swelling of the polymer. Another common pH-responsive functional group is pyridine, which responds in the opposite way to the carboxylic group with the changes in pH values .Representative polymers with pH-dependent conformational changes via protonation/deprotonation include poly(acrylic acid) (PAA) , poly molecules and a metal ion latch, assembled on MSNPs. This ANH forms a gating mechanism and is capable of storing and releasing metal ions and molecules trapped in the pores. Pore openings derivatized with IDA can be latched shut by forming a bis-IDA chelate complex with a metal ion. No cargo release is observed in a neutral aqueous environment; however, when the environment is acidic (pH = 5.0) and/or when a competitive binding ligand is introduced, cargo release is observed . SimilarBy altering the polymer shell structure, the ANH responsiveness to the stimulus can also be controlled. For example, a silica oxide core when modified with a poly(2-vinylpyridine) (P2VP) shell, and decorated with AuNPs, can be used as freestanding single-particle sensors in various miniaturized analytical systems. The P2VP polymer brush of the ANH undergoes reversible swelling/shrinking as pH changes from 2.5 to 5.7. Such dynamic change in the polymer brush conformation alters the access of the surrounding fluid to the metallic AuNP surfaces, and the plasmon resonance behavior is also modulated by a purple-blue shift (from red) . An inexDue to the simplicity of control, temperature is one of the most widely-used external stimulus in ANHs design. Temperature can trigger response from ANHs coated with thermo-responsive polymers that contain hydrophobic  moieties. The properties of thermo-responsive polymers are governed by the lower critical solution temperature (LCST), defined as the temperature at which the polymer undergoes a phase transition from a soluble to an insoluble state ,61. In gN-alkyl substituted acrylamides), e.g., poly(N-isopropylacrylamide) (PNIPA), with an LCST of 34.5 to 35 °C [N-vinylalkylamides), e.g., poly(N-vinylcaprolactam), with an LCST of about 31 to 38 °C, depending on the molecular weight and concentration of the polymer [Temperature-responsive polymers can be classified depending on the mechanism and chemistry of the polymer groups: (1) polyethyl methacrylate) (PMEO2MA). The thermo-responsive coating undergoes a phase transition from a hydrophilic water-swollen state to a hydrophobic globular state, when heated above its LCST. Such changes result in modification of the light scattering properties of the nano-system and cause a change in the turbidity of the gel network of PMEO2MA . Differethe ANHs . Metallithe ANHs .o-nitrobenzyl alcohol-poly(4-substituted-3-caprolactone) [Light stimulation and response is a particularly useful external trigger to efficiently manipulate ANH responses. A number of parameters  can be tuned for a specific target, which allows designing a wide selection of stimuli-responsive ANHs. Photo-responsive, particularly, photo-cleavable polymers, e.g., PNIPA-lactone) , poly(melactone)  and P4VPlactone) , have relactone)  and as plactone) .trans (E-isomer) and the bent cis (Z-isomer), when exposed to light at a certain wavelength  and UV (350 nm) range) or heating and can lead to considerable changes in molecular shape, size and dipole moments [trans-cis-trans isomerization of photo-responsive molecules containing an azobenzene moiety incorporated into networks of benzyldimethyl-stearyl-ammonium chloride and octadecylamine capped AuNPs results in changes from red to blue in the optical spectra of the surface plasmon peak position of the NH network [Photoisomerizable molecules, such as azobenzenes, have been incorporated to macromolecules to produce macroscopic changes in the polymeric material. Azobenzene is a well-known photo-responsive molecule that has been widely used in a diverse set of optical devices and to achieve multifunctionality; e.g., photo-switching , photo-o moments . Thus, t network . Similar network .i.e., from visible to UV). Aminopropyl-silsesquioxane (POSS-NH2) has been employed to functionalize graphene oxide (GO) sheets. The combination of the GO sheets with POSS-NH2 produces a hybrid silicon/graphite-based NP, which when exposed to visible light, exhibits dielectric or insulating behavior, rendering a photoconductive response [Other examples include bipyramidal DNA nanocapsules based on photo-responsive oligonucleotides that release AuNPs when photo-irradiated, via strand displacement mechanism . Such reresponse .i.e., thiolate synthesized via self-condensation of 3-mercaptopropyltrimethoxysilane) and light-sensitive azobenzenes that respond to pH, light and ionic strength, simultaneously [N-isopropylacrylamide-co-methacrylic acid)-coated magnetic SiNPs [Many ANHs are designed with polymer blocks that respond to more than one stimulus or are only responsive to biological stimuli a,b. pH aaneously . Such ANaneously . A triplic SiNPs . Similaric SiNPs .N-vinyl-2-pyrrolidone) with pendent phenylboronic acid moieties and applied for diabetes treatment. Similarly, glutathione [ANHs that are designed to respond to other stimuli include those that respond to biological molecules, such as glucose , where itathione  is used tathione . ANHs thi.e., chemical variability  [i.e., sand and sediment [Environmental implications of passive nanostructures have been extensively studied, where the roles of size , shape , surfaceditions) , presencditions) , heterogsediment , even thsediment , has beeWhen NMs are released in the natural environment, they undergo aggregation, primarily by the interplay of the inherent van der Waal’s attractive forces and surface charge-mediated electrostatic repulsive forces . AggregaThe following section will discuss the role of select environmental stimuli  in influencing the behavior and toxicity of emerging ANHs, with a focus on the aquatic environment. We will consider what we have learned from studying passive NMs in predicting how the environment should be considered with respect to ANH behavior and toxicity.i.e., it collapses onto a surface to avoid interaction with the surrounding polar medium in such conditions [The role of pH in nano-EHS has traditionally centered on the concept of protonation/deprotonation of surface moieties, which control the surface charge and, thus, the stability, transport and, in some cases, toxicity of the NMs. High salt concentrations and pHs close to the isoelectric point (IEP) promote NM aggregation by compressing electrical double-layer repulsion . IEP hasnditions . On the nditions .i.e., water column versus gastrointestinal (GI) tract versus lysosomes). For example, in the acidic stomach environment, single AgNPs can agglomerate and precipitate, while deposition of NHs composed of AgNPs and silicate clay, in the same acidic environment, is minimized [pH-responsive ANHs that combine PAA and pyridine soft layers and other NMs have great potential for application in drug delivery systems. Thus, in natural environments, the relative collapse or exfoliation of surface coatings on ANH surfaces will present variable aggregation and deposition (each polymer/polymer block has differing electrostatic and steric stabilization contribution), transformation  and toxicity . Studies performed to-date have shown that coatings like PAA influence NM solubility as a function of the ambient pH. For example, metal NPs coated with PAA are typically less soluble, but show enhanced ion release under acidic conditions , and sucinimized . The desinimized ,114. ThiIt is known that pH affects the hydrodynamic radii of NPs and that select size ranges are associated with observed toxic effects, such as lethality, reduced growth and reproduction rates ,116,117.i.e., 31 to 38 °C) [Temperature is not considered a key factor in assessing nano-EHS. However, the use of thermal-responsive polymers in decorating ANHs introduces new complexity in understanding their environmental and toxicological behavior. The temperature range at which these ANHs respond is within the range of physiological conditions (o 38 °C) . These to 38 °C) . PMAEMA,o 38 °C) . While to 38 °C) , and the2, which primarily shows that photo-oxidation increases ROS and toxicity. In fact, many studies have shown that cellular uptake of NMs is not necessary to induce toxicity; however, membrane damage by photo-oxidation-mediated oxidative stress controls the nanotoxicity mechanism [Transformation of passive NMs primarily focuses on studying the alteration of the particles’ photoactivity , reactivechanism ,122,123.The nano-EHS community needs to consider the modification of the strategies on assessing these dynamically-evolving stimuli-responsive ANHs. The underlying assumption of uniform surface properties in theoretical models and experimental assessment falls apart for these new sets of materials. The assumptions of potential transformation in the environment are also not applicable, as some of these ANHs will certainly undergo transformation, if exposed to the relevant stimulus. Furthermore, parameters that are otherwise ignored in nano-EHS, e.g., temperature, will require more attention in the case of these ANHs. New experimental tools are likely required to monitor the dynamic evolution of the coatings under changing stimuli conditions. The current state-of-the-art techniques mostly assess equilibrium processes and are not capable of evaluating time-dependent changes in surface properties.With the growing number and composition of NMs, including contemporary ANHs, it is evident that a shift to predictive modeling is needed. A number of models have been proposed to meet the growing demand to satisfy EHS testing ,125,126.


Aging is a complex process that results from a combination of environmental, genetic, and epigenetic factors. A chronic pro-inflammatory status is a pervasive feature of aging. This chronic low-grade inflammation occurring in the absence of overt infection has been defined as “inflammaging” and represents a significant risk factor for morbidity and mortality in the elderly. The low-grade inflammation persists even after reversing pro-inflammatory stimuli such as LDL cholesterol and the renin–angiotensin system (RAS). Recently, several possible sources of chronic low-grade inflammation observed during aging and age-related diseases have been proposed. Cell senescence and dysregulation of innate immunity is one such mechanism by which persistent prolonged inflammation occurs even after the initial stimulus has been removed. Additionally, the coagulation factor that activates inflammatory signaling beyond its role in the coagulation system has been identified. This signal could be a new source of chronic inflammation and cell senescence. Here, we summarized the factors and cellular pathways/processes that are known to regulate low-grade persistent inflammation in aging and age-related disease. At present, chronic inflammation is thought to be a risk factor for a broad range of age-related diseases such as hypertension, diabetes, atherosclerosis, and cancer.  AlthoughIn this narrative review, we aimed to review the sources of chronic inflammation during aging . The celAcute inflammation is indispensable for immune responses to invading pathogens or acute traumatic injuries. This process enables repair and cell turnover in multiple tissues. In contrast, chronic inflammation normally causes low-grade and persistent inflammation, leading to tissue degeneration. . ChronicDebris and immunoglobulin accumulation due to inappropriate cell elimination systems in aging trigger the innate immune system activation leading to persist inflammation. Glycosylation represents the most frequent post-translational modification of proteins. Protein-linked sugar chains play a variety of specific roles in the “fine-tuning” of interactions between cells and between molecules , 12. HigClostridium cluster XIVa, Bifidobacterium spp., and F. prausnitzii are diminished in aged individuals (Bifidobacterium is inversely correlated with serum levels of inflammatory cytokines, such as TNF-α and IL-1β. Conversely, inflammatory and pathogenic microbiota, including Streptococcus spp., Staphylococcus spp., Enterococcus spp. and Enterobacter spp., are increased with age (The barrier of the oral and gut mucosa against bacterial invasion deteriorates with age. Periodontal disease has also demonstrated to cause chronic low-grade inflammation . The gutividuals . Toward with age . Changeswith age . These dCellular senescence is defined as irreversible cell cycle arrest driven by a variety of mechanisms, including telomere shortening, genotoxic stress, mitogen stimuli, and inflammatory cytokines, that result in the activation of the p53 tumor suppressor and/or the cyclin-dependent kinase inhibitor p16 . It is e“Immunosenescence”, which is the age-related dysregulation of an innate immune system, is characterized by persistent inflammatory responses . ImmunosIncreased coagulation and fibrinolysis activity in the elderly has recently been implicated in enhanced inflammation through the protease-activated receptor, PAR –37 leadiIdeally, inflammation should subside immediately after elimination of the pathogen and insult to allow normal tissue to be rebuilt. However, low-grade persistent inflammation occurs in the majority of older people, leading to degeneration of several organs. There is strong evidence that the development of age-related multi-factorial conditions such as cancer, cardiovascular disease, Alzheimer’s disease, type II diabetes, frailty, sarcopenia, and osteoporosis is associated with low-grade elevations of circulating inflammatory mediators. Considering that aging is a complex process that results from a combination of environmental, genetic, and epigenetic factors, focusing future work on interventions addressing selectively destroying senescent cells, namely, “senolytic therapies” in the aging host rather than by treating symptoms of disease or attempting to block the effects of the multi-source of inflammaging, will offer improved therapeutic opportunities –52.FS and YT performed experiment and organized manuscript. JM, HS, and RO performed experiments. HR and RM supervised this project.RM received research funding from Bayer Yakuhin, Ltd. Other authors have no conflicts of interests.


Caregivers of care-recipients with mild cognitive impairment (MCI) or dementia experience high caregiver burden; however, the psychiatric burden of caregivers of care-recipients with subjective cognitive decline (SCD) has not been investigated. We aimed to explore the prevalence of and risk factors for anxiety and depression symptoms among the caregivers of care-recipients with SCD and cognitive impairment.The Hospital Anxiety and Depression Scale (HADS) was used to examine the anxiety and depression symptoms among the caregivers of 343 care-recipients  treated at the Memory Clinic of Huashan Hospital in Shanghai, China from May 2012 to October 2014. A logistic regression was used to explore the factors associated with caregiver’s anxiety and depression symptoms.P = 0.1140), whereas 22.6, 24.2 and 20.9 %, respectively, had depression symptoms (P = 0.8165). The risk factors for caregiver’s anxiety symptoms were increased caregiver age as well as having care-recipients who were male, had higher Cohen Mansfield Agitation Inventory (CMAI) scores, and higher Geriatric Depression Scale (GDS) scores. The risk factors for caregiver’s depression symptoms were increased caregiver age as well as caring for care-recipients with MCI or SCD, those with lower Toronto Empathy Questionnaire (TEQ) scores, and those with higher GDS scores.In total, 26.5 % of caregivers had anxiety symptoms, and 22.4 % had depression symptoms. Totals of 17.9, 30.0 and 28.8 % of caregivers of care-recipients with SCD, MCI or dementia, respectively, had anxiety symptoms (Caregivers of care-recipients with SCD showed the same level of depression symptoms as those of care-recipients with MCI. Caregiver’s depression and anxiety symptoms were associated with their care-recipients’ psychiatric and behavioral syndromes. Caregiver burden is common in dementia. This condition is not only associated with adverse emotional states and psychiatric morbidity but also poorer physical condition and worse financial and social consequences. Factors in three other domains are also relevant: care-recipient characteristics, caregiver characteristics and living conditions –3. PreviSubjective cognitive decline (SCD) in older adults is increasingly recognized as a potential indicator of non-normative cognitive decline, and some people with this condition eventually progress to dementia –17. The Regarding the persistent cognitive decline and the high rate of its conversion, we assumed that both care-recipients and their caregivers would suffer from cognitive complaints. However, psychiatric symptoms among SCD-caregivers have been under-researched. This study explored the prevalence of and risk factors for anxiety and depression symptoms among caregivers of care-recipients with SCD and cognitive impairment using data from the Memory Clinic of Huashan Hospital in Shanghai.A hospital-based cross-sectional survey of 343 pairs of care-recipients and their caregivers was conducted at the Memory Clinic of Huashan Hospital in Shanghai from May 2012 to October 2014. A caregiver was defined as someone who spent at least 8 h per week caring for a care-recipient, regardless of whether they lived together. The inclusion criteria were: the care-recipient was diagnosed with mild or moderate dementia, MCI or SCD; one primary caregiver cared for one care-recipient; the principal family caregiver was over 18 years old and took care of or lived with the care-recipient; the caregiver’s relationship with the care-recipient and their caring time were also considered when recruiting participants, therefore, spouses, daughters, sons, daughters-in-law and other relatives were included, regardless of whether they lived with their care-recipients. Those who simultaneously provided care for another family member with a chronic medical condition were excluded.Demographic characteristics, including age, gender and education, were collected from the care-recipients and their caregivers. Additional information was collected regarding the caregiver such as their working status, experience in caring for care-recipients with dementia, familiarity with the care-recipient and their relationship with the care-recipient.For each care-recipient, cognitive function scales covering domains of global cognition  were administered, including the Mini Mental State Examination (MMSE) , the MemThe Hospital Anxiety and Depression Scale (HADS) is a popular clinical and research instrument used to screen for anxiety and depression symptoms in both hospital and community settings , 44. TheFour experienced neurologists and one neuropsychologist  who work at top institutions of neurology in China  reviewed the functional, medical, neurological, psychiatric, and neuropsychological data, and reached a consensus regarding the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria for dementia . Only caP < 0.05. The data were analyzed using SAS 9.3 .Continuous variables were expressed as mean and standard deviation (SD), and categorical variables were expressed as frequencies (%). We divided all care-recipients and caregivers into four groups according to the caregiver’s performance on the HADS: with anxiety [anxiety(+)], without anxiety [anxiety(−)], with depression [depression(+)], and without depression [depression(−)]. The Wilcoxon rank sum test and the Kruskal-Wallis H rank sum test were used to compare continuous variables, whereas Pearson Chi-squared test was used to compare categorical variables. Backward logistic regression models were used to explore risk factors for anxiety and depression symptoms among caregivers. Risk factors were presented as odds ratio (OR) with 95% confidence interval (95 % CI). The possible risk factors we adjusted for care-recipients included their gender, age, education, diagnosis, and presence of stroke as well as their IQCODE, TEQ, CMAI, GDS, ZSAS and AES scores; those for caregivers included their gender, age, education level, working status, and experience in caring for care-recipients with dementia as well as their familiarity with the care-recipient and their relationship with the care-recipient. Two-tailed tests were used for all analyses at a significance level of The demographic, medical history, neurology tests, psychiatric assessment and neuropsychological assessment data of the 343 pairs of care-recipients and caregivers are shown below.P = 0.1140), respectively; 22.6, 24.2 and 20.9 % of these caregivers had depression symptoms (P = 0.8165), respectively. Although caregivers of care-recipients with SCD had relatively low anxiety symptoms and relatively high depression symptoms, anxiety or depression symptoms were not associated with cognitive impairment severity  care-recipients had SCD, 120 (35.0 %) had MCI, and 139 (40.5 %) had mild to moderate dementia , we found that possible risk factors for caregiver’s anxiety symptoms were having a male care-recipient , a higher CAMI score , a higher GDS score  and increased caregiver age . The risk factors for caregiver’s depression symptoms were having a lower TEQ score , a care-recipient with MCI or SCD compared with one with dementia, a higher GDS score  and increased caregiver age   was 23 %  [The prevalence of depression symptoms among caregivers of care-recipients with SCD or MCI was higher than that among caregivers of care-recipients with dementia. The prevalence of anxiety and depression symptoms among caregivers of care-recipients with dementia was in accordance with that in previous studies , 6, 52. studies) , which istudies) . Howeverstudies) –56. Anotstudies) . One posWe found that care-recipient GDS scores were a risk factor for both anxiety and depression symptoms among caregivers; however, we did not find that care-recipient ZSAS or AES scores were associated with caregiver’s anxiety or depression symptoms. Most studies have found that depression and anxiety symptoms in caregivers of people with dementia are strongly associated with patients’ psychiatric symptoms, particularly, depression symptoms , 58–62. r = 0.59, p = 0.0002) as well as between depression and caregiver’s depression  [Our study showed that care-recipient CMAI scores were only related to caregiver’s anxiety symptoms. A study of 35 patient-caregiver pairs evaluated at two university-affiliated dementia clinics found highly significant correlations between patient agitation and caregiver burden ( 0.0001) . A Chine 0.0001) . The rea 0.0001) . Our resWe also found that care-recipient TEQ scores were a risk factor for caregiver’s depression symptoms. Previous studies have not investigated the relationship between empathy and depression symptoms. One possible reason might be that cognitive impairment is difficult to treat with either behavioral or pharmacologic methods, leading to embarrassment among care-recipients, which might contribute to caregivers’ feelings of social isolation.In our study, some characteristics of caregiver  were not associated with caregiver’s anxiety and depression symptoms. Having less education was significantly associated with depression of caregivers of care-recipients with MCI in two , 69 of tTo our knowledge, this study is the first to report the prevalence of and risk factors for anxiety and depression symptoms among the caregivers of care-recipients with SCD. The first limitation of our study is that the cross-sectional design limits any conclusions in a causal or predictive direction. Future work should examine these relationships longitudinally to more fully characterize the direction of cause and effect. Second, we adjusted for as many risk factors as possible in the logistic regression model; however, we can’t exclude the possible influence of uncollected risk factors such as the specific care time of caregivers, alcohol drinking habits, disease history , economic status, physical and psychological illness among caregivers, and drugs that affect mental health. Third, there are only care-recipients with mild or moderate dementia, not the severe dementia in our study, but care-recipients with the severe dementia may cause serious impacts on their caregivers.Our study suggests that clinicians should be aware of high rates of anxiety and depression symptoms among caregivers of care-recipients with SCD or MCI. We should also attend to the risk factors for anxiety and depression symptoms among caregivers such as care-recipient depressions, empathy and agitation. Furthermore, many caregivers require more social support via training as well as physical and mental health care. Such programs should provide caregivers with support to address their burden and educate them learn about cognitive impairment.


The advances in biotechnologies and efficiency in computational resources have provided unprecedented opportunities to study and analyze the genomics of human diseases. Over the last decade, high-throughput experiments studying -omics  have been used to generate informative data researchers can use to test different data-driven hypotheses. A big promise of such high-dimensional -omics data is the advancement of biomedicine by effectively translating findings from basic science research into clinical application. Designing and conducting genomic experiments in biomedical research aim to enhance the diagnosis, treatment, and prevention of human diseases. Translating -omics findings into clinical practice requires a flexible framework to incorporate different -omics data types to predict clinical outcomes in an integrated fashion.Developing rigorous statistical approaches and implementing innovative computational tools play essential roles in translating the findings based on high-dimensional -omics data into accurate and informative medical decisions. To equip readers with updated analytical approaches, this special issue covers a wide range of analytical approaches and pipelines. J. D. Hintzsche et al. provided a comprehensive review of computational tools to analyze and interpret the whole exome sequencing (WES) data, including alignment, variant calling, and annotation approaches developed for “pre-VCF ” analyzes, as well as major approaches to conduct downstream analysis after VCF file has been generated: pathway analysis, somatic prediction, copy number estimation, and so forth. Robustness and the ability to replicate findings in independent datasets are also critical in analyzing high-dimensional data. For analyzing transcriptomic data, A. E. Berglund et al. proposed a principal component analysis- (PCA-) based technique to reveal gene expression signatures that are robust in replicated datasets. The method can also identify complex signatures from independent biological components. Beyond traditional data analysis, W. Wei et al. demonstrated that visualization is a key component to translate -omics data into useful information. The study utilized the GPA  and MDS  techniques to illustrate genetic relationships between different human traits/diseases, revealing the underlying shared genetic architecture.Data integration is essential for robust modeling of complex or heterogeneous conditions. By integrating gene expression data with prior biological knowledge such as tissue of origin or mutation status, V. P. Kamath et al. illustrated enhanced performance on radiation sensitivity. Their results provide a proof of concept on how accounting for biological heterogeneity can lead to robust modeling of clinical response. D. M. Rotroff and A. A. Motsinger-Reif reviewed current data integration techniques for joint-analysis of multiple -omics data and discussed future directions and challenges for applying these integrative approaches in personalized medicine. K. Raja et al. then discussed how researchers can utilize the large volume of data from the literature to develop biological inference for -omics analysis by providing an in-depth review of text-mining approaches that can be used to synthesize biomedical or clinical information and also highlighted the applications of text-mining in genomic, proteomic, and transcriptomic studies.A. Muthiah et al. proposed a novel inference technique, called Module Anchored Network Inference (MANI), to reveal gene-gene relationships and provide inference on disease mechanism by using time-series gene expression data on adipocyte differentiation. Instead of utilizing all candidate genes from data, the MANI approach constructs small network modules, which is shown to outperform other in silico network inference techniques. Many human diseases are heterogeneous in nature and thus are challenging to provide accurate diagnosis and monitoring. Using systemic lupus erythematosus as a disease model, E. Zollars et al. applied a genomic technique to develop robust biomarker signature to better monitor disease activity. The approach utilized gene expression profiles to guide the classification of patients with different disease activities.This special issue presents and discusses technological and methodological developments in biomedical research leading to advances in biomedicine through analysis and evaluation of -omics data. The research and review articles provide a comprehensive collection of approaches and studies for translating biological information from high-dimensional data to clinical applications. With the explosion of big data, we believe that innovative techniques, rigorous analytical approaches, and pipelines are keys to provide robust findings that can advance their clinical applications.


Background: Extracorporeal membrane oxygenation (ECMO) is a complex life-saving support for acute cardio-respiratory failure, unresponsive to medical treatment. Emergency events on ECMO are rare but require immediate and proficient management. Multidisciplinary ECMO team members need to acquire and maintain over time cognitive, technical and behavioral skills, to safely face life-threatening clinical scenarios.Methods: A multistep educational program was delivered in a 4-year period to 32 ECMO team members, based on guidelines from the Extracorporeal Life Support Organization. A first traditional module was provided through didactic lectures, hands-on water drills, and laboratory animal training. The second phase consisted of a multi-edition high-fidelity simulation-based training on a modified neonatal mannequin (SimNewB®). In each session, participants were called to face, in small groups, ten critical scenarios, followed by debriefing time. Trainees underwent a pre-test for baseline competency assessment. Once completed the full training program, a post-test was administered. Pre- and post-test scores were compared. Trainees rated the educational program through survey questionnaires.Results: 28 trainees (87.5%) completed the full educational program. ECMO staff skills improved from a median pre-test score of 7.5/18 (IQR = 6–11) to 14/18 (IQR = 14–16) at post-test . All trainees highly rated the educational program and its impact on their practice. They reported high-fidelity simulations to be beneficial to novice learners as it increased self-confidence in ECMO-emergencies (according to 100% of surveyed), theoretical knowledge (61.5%) and team-work/communicative skills (58%).Conclusions: The multistep ECMO team training increased staff' knowledge, technical skills, teamwork, and self-confidence, allowing the successful development of a neonatal respiratory ECMO program. Conventional training was perceived as relevant in the early phase of the program development, while the active learning emerged to be more beneficial to master ECMO knowledge, specific skills, and team performance. Extracorporeal membrane oxygenation (ECMO) is a life-saving, high-risk support for acute cardio-respiratory failure, unresponsive to conventional treatment , 2. AbouProficiency in a high-tech procedure requires extensive training, which includes comprehensive knowledge of ECMO principles and the technical ability to perform a time-critical and well-coordinated emergency troubleshooting . As neoncrew resource management (CRM), developed by NASA and aviation industry . All subjects gave written informed consent in accordance with the Declaration of Helsinki.A multistep educational ECMO curriculum was delivered during a 4-year period for 32 ECMO team members, based on guidelines from the ELSO . A firstNeonatal Intensive Care Unit admits about 450 neonates per year. It is a national referral center for fetal and neonatal surgery with an average of 15 congenital diaphragmatic hernias treated each year. The ECMO program follows the “dual provider” model, the critical care nurse takes care of the patient, and the cardiovascular perfusionist is responsible for the ECMO circuit management , covering the main clinical aspects of respiratory ECMO: physiology, clinical indications, support modalities , equipment, cannulation strategies, coagulation management during the entire ECMO procedure, pharmacotherapy, mechanical and clinical complications, weaning, follow-up, ethics, and ECMO withdrawal. In-house and national trainers were invited for lectures on specific topics. Teaching resources included the ELSO guidelines, the “ELSO Redbook” and the “ELSO ECMO Specialist Training Manual.”The Wet labs or water drills included hands-on training on closed-loop ECMO circuit filled with water, used to address technical issues and to familiarize the practitioners with equipment and procedures .The animal laboratory consisted of establishing a veno-arterial ECMO on anesthetized cannulated piglets and reproducing a daily ECMO management . The software for SimNewB® Laerdal was used to remotely plan clinically relevant scenarios and simulate changes of vital signs. The features of simulations adhere to the principles of best educational practice .We conducted high-fidelity simulation sessions in a dedicated training area at our Institution. Our mobile simulation set-up included a modified high fidelity neonatal mannequin  with an  Figures , 3. Tubepractice . The traDuring each session, 3 participants at a time were faced with 10 critical scenarios. Attention was paid to both technical and behavioral factors that would impact patient's outcome. Our team configured the mannequin, the simulation setting, and the scenarios based on veno-venous or veno-arterial modality. The plots were derived from typical ECMO emergency cases and aimed at reaching specific learning outcomes  and subjected them to a written pre-curriculum test for baseline competency assessment. Once completed the training program, a written post-test with an 18-items checklist was administered . All preAll participants were asked to provide their perception of the educational programme. Trainees rated the educational program using a survey, based on a 5-point Likert scale, from one (strongly disagree) through five (strongly agree). We evaluated each step of the educational program: didactic sessions, wet labs, animal labs, simulation and debriefing sessions; inquiries were focused on the teaching methodology, i.e., knowledge, procedural confidence, teamwork, and real-life fidelity. Participants were given the opportunity to provide open-ended feedback to improve the educational curriculum. The research team designed the survey for the present study.p < 0.05 was considered significant. Statistical analyses were performed using R version 3.4.3 .We provided a descriptive analysis of the demographic aspects of the participants. Continuous variables that follow an approximately normal distribution were reported as mean (and standard deviation), skewed continuous variables will be summarized using medians and inter-quartile ranges and categorical variables as overall counts and percentage. We compared the average number of correct answers between pre- and post- “curriculum test” with the Wilcoxon rank test; a P < 0.001, Wilcoxon rank test). A focused analysis of the individual questions shows the performance of the ECMO team in relation to every single item  completed the full educational program, by attending at least 60% of didactic lectures and one of each practice session. Four trainees (12.5%) did not complete the training for non-compliance with the full schedule. Table em Table . In 3 quAll trainees have highly rated the educational program and its impact on their practice. They reported high fidelity simulations to be beneficial, as it increased self-confidence in ECMO-emergencies (according to 100% of surveyed), theoretical knowledge (61.5%) and team-work/communicative skills (58%). Results are presented in Table In the open-ended comment section, participants expressed enthusiasm toward the realism and the benefits of the simulations, asking for more frequent mannequin-based sessions and wet labs to maintain the acquired skills in the long-term.The delivery of a comprehensive ECMO curriculum is essential to succeed in the development of new programs. According to our findings, a multistep team training model that includes high-fidelity simulations emerged as a feasible and efficient educational tool to launch a new neonatal respiratory ECMO program. The first introductory module provided the necessary knowledge through didactic lectures. Next, the water drills allowed the trainees to better familiarize with the circuit, thus acquiring technical skills. Once theoretical and practical basis was acquired, animal-based, and high-fidelity mannequin-based simulations provided a protected environment to practice and optimize technological, cognitive and behavioral skills. In more detail, as emphasized by our pediatric surgeons, hands-on animal lab showed to be crucial to enhance cannulation ability in our surgical context. Overall, our educational model increased knowledge and self-reported confidence of novice ECMO providers. The evaluation of the team performance in relation to every single item suggests areas of strengths and limits of the training for further improvement of our ECMO curriculum, by highlighting the topics to further address versus those which emerge to be well retained by the team.Neonatal Intensive Care Unit environment without previous experience with cardiac surgery patients. A similar training model was previously reported in a pediatric intensive care unit, reinforcing the role of full dedicated practice, especially valuable for novice learners  the learners  (3) the learners , 12, 14,learners . Consistlearners .Although traditional methods of learning were associated with poor retention of knowledge and procedural skills , our resThis multistep team training was based on a multidisciplinary involvement of the members, even though masterful examples of training have been delivered to selected providers, to reach specific learning outcomes . ECMO emOur study had a few limitations. Firstly, as it is a single-center study, the results cannot be generalized. Nevertheless, the training of a novice ECMO team, especially in the early steps, needs to be customized to every single center. By taking into account the center-specific background knowledge, available facilities and goals of the program, the education may be tailored to allow the optimization of a resource-intense effort. Secondly, the efficacy of our training model should be demonstrated in clinical practice. However, the primary focus of this work was to describe the implementation of a multistep team training while establishing a non-cardiac neonatal ECMO program. The evaluation of patients' outcomes was therefore beyond the scope, but we have planned to evaluate the training model and the skills maintenance over time, by repeating a trainees' assessment on a yearly basis. Thirdly, the tests used for pre- and post-curriculum competency assessment were not externally validated. Nevertheless, as they were formulated by trained ECMO physicians, according to ELSO guidelines and tailored to our clinical context, we deemed them suitable for the scope of this report. Lastly, the design of the survey questions might have posed potential response bias in favor of the training program. A more effective wording could have improved the validity of self-reported measurements .The multistep team training increased staff's confidence, technical expertise, and teamwork in ECMO emergency scenarios. High-fidelity simulation-based training was the most appreciated educational tool by the trainees. Although our survey suggests that the active learning is relevant for ECMO team proficiency, passive learning may maintain its role in the early establishment of the team knowledge background. Further studies should focus on the clinical impact of this educational program and the development of training and educational standards for health-providers in charge of ECMO neonates.GC, GR, SG, LP, and FM contributed conception and design of the study; GR, FCa, FCo, and CB organized the database and collected data; NP performed the statistical analysis; GR and GC wrote the first draft of the manuscript. SG provided extensive critical revision. All authors contributed to manuscript critical revision, read and approved the submitted version.FCa and FCo were employed by company Betamed Perfusion Service srl. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Super-resolution fluorescence microscopy revolutionizes cell biology research and provides novel insights on how proteins are organized at the nanoscale and in the cellular context. In order to extract a maximum of information, specialized tools for image analysis are necessary. Here, we introduce the LocAlization Microscopy Analyzer (LAMA), a comprehensive software tool that extracts quantitative information from single-molecule super-resolution imaging data. LAMA allows characterizing cellular structures by their size, shape, intensity, distribution, as well as the degree of colocalization with other structures. LAMA is freely available, platform-independent and designed to provide direct access to individual analysis of super-resolution data. Super-resolution microscopy is about to expand the toolbox of microscopy techniques in many biological and biomedical research laboratories135Software that generate single-molecule localization lists from SMLM data910131415161118LocAlization Microscopy Analyzer (LAMA), an open source, platform-independent software tool that solely concentrates on extracting quantitative information from SMLM data that is relevant for biological interpretation  algorithms commonly used for other super-resolution techniques, but allows for powerful coordinate-based algorithms. LAMA analyzes protein organization at a near-molecular scale utilizing algorithms for all-distance second-order statistics like Ripley’s K, L, and H function, as well as density-based data sorting into clustered regions (e.g. DBSCAN and OPTICS)24An ultimate goal in super-resolution data analysis is to determine protein complex stoichiometries. LAMA is the first software that allows extracting molecular stoichiometries by using an automated Markov-chain analysis of single-molecule photoswitching kinetics. With this feature, the oligomeric state of protein complexes can easily be determined from SMLM dataAn exemplary workflow of LAMA will be illustrated with a two-color SMLM data set recorded for the HIV proteins gag and env in the plasma membrane of a T-cell .LAMA was developed over several years in close cooperation with biology research groups along specific questions and is How to cite this article: Malkusch, S. and Heilemann, M. Extracting quantitative information from single-molecule super-resolution imaging data with LAMA – LocAlization Microscopy Analyzer. Sci. Rep.6, 34486; doi: 10.1038/srep34486 (2016).