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- ItemPodosome-Driven Defect Development in Lamellar Bone under the Conditions of Senile Osteoporosis Observed at the Nanometer Scale(Washington, DC : ACS Publications, 2021) Simon, Paul; Pompe, Wolfgang; Bobeth, Manfred; Worch, Hartmut; Kniep, Rüdiger; Formanek, Petr; Hild, Anne; Wenisch, Sabine; Sturm, ElenaThe degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 μm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC. © 2021 The Authors. Published by American Chemical Society.
- ItemHigh spatial and temporal resolution cell manipulation techniques in microchannels(Cambridge : Royal Society of Chemistry, 2016) Novo, Pedro; Dell’Aica, Margherita; Janasek, Dirk; Zahedi, René P.The advent of microfluidics has enabled thorough control of cell manipulation experiments in so called lab on chips. Lab on chips foster the integration of actuation and detection systems, and require minute sample and reagent amounts. Typically employed microfluidic structures have similar dimensions as cells, enabling precise spatial and temporal control of individual cells and their local environments. Several strategies for high spatio-temporal control of cells in microfluidics have been reported in recent years, namely methods relying on careful design of the microfluidic structures (e.g. pinched flow), by integration of actuators (e.g. electrodes or magnets for dielectro-, acousto- and magneto-phoresis), or integrations thereof. This review presents the recent developments of cell experiments in microfluidics divided into two parts: an introduction to spatial control of cells in microchannels followed by special emphasis in the high temporal control of cell-stimulus reaction and quenching. In the end, the present state of the art is discussed in line with future perspectives and challenges for translating these devices into routine applications.
- ItemHigh-Sensitivity Rheo-NMR Spectroscopy for Protein Studies(Columbus, Ohio : American Chemical Society, 2017) Morimoto, Daichi; Walinda, Erik; Iwakawa, Naoto; Nishizawa, Mayu; Kawata, Yasushi; Yamamoto, Akihiko; Shirakawa, Masahiro; Scheler, Ulrich; Sugase, KenjiShear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.
- ItemSecondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies(Columbus, Ohio : American Chemical Society, 2016) Davies, Heather S.; Singh, Prabha; Deckert-Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline E.; Dowd, Sarah E.; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
- ItemObject detection networks and augmented reality for cellular detection in fluorescence microscopy(New York, NY : Rockefeller Univ. Press, 2020) Waithe, Dominic; Brown, Jill M.; Reglinski, Katharina; Diez-Sevilla, Isabel; Roberts, David; Eggeling, ChristianObject detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al.
- ItemGlobal, regional, and national burden of mortality associated with non-optimal ambient temperatures from 2000 to 2019: a three-stage modelling study(Amsterdam : Elsevier, 2021) Zhao, Qi; Guo, Yuming; Ye, Tingting; Gasparrini, Antonio; Tong, Shilu; Overcenco, Ala; Urban, Aleš; Schneider, Alexandra; Entezari, Alireza; Vicedo-Cabrera, Ana Maria; Zanobetti, Antonella; Analitis, Antonis; Zeka, Ariana; Tobias, Aurelio; Nunes, Baltazar; Alahmad, Barrak; Armstrong, Ben; Forsberg, Bertil; Pan, Shih-Chun; Íñiguez, Carmen; Ameling, Caroline; De la Cruz Valencia, César; Åström, Christofer; Houthuijs, Danny; Dung, Do Van; Royé, Dominic; Indermitte, Ene; Lavigne, Eric; Mayvaneh, Fatemeh; Acquaotta, Fiorella; de'Donato, Francesca; Di Ruscio, Francesco; Sera, Francesco; Carrasco-Escobar, Gabriel; Kan, Haidong; Orru, Hans; Kim, Ho; Holobaca, Iulian-Horia; Kyselý, Jan; Madureira, Joana; Schwartz, Joel; Jaakkola, Jouni J. K.; Katsouyanni, Klea; Hurtado Diaz, Magali; Ragettli, Martina S.; Hashizume, Masahiro; Pascal, Mathilde; de Sousa Zanotti Stagliorio Coélho, Micheline; Valdés Ortega, Nicolás; Ryti, Niilo; Scovronick, Noah; Michelozzi, Paola; Matus Correa, Patricia; Goodman, Patrick; Nascimento Saldiva, Paulo Hilario; Abrutzky, Rosana; Osorio, Samuel; Rao, Shilpa; Fratianni, Simona; Dang, Tran Ngoc; Colistro, Valentina; Huber, Veronika; Lee, Whanhee; Seposo, Xerxes; Honda, Yasushi; Guo, Yue Leon; Bell, Michelle L.; Li, ShanshanBackground: Exposure to cold or hot temperatures is associated with premature deaths. We aimed to evaluate the global, regional, and national mortality burden associated with non-optimal ambient temperatures. Methods: In this modelling study, we collected time-series data on mortality and ambient temperatures from 750 locations in 43 countries and five meta-predictors at a grid size of 0·5° × 0·5° across the globe. A three-stage analysis strategy was used. First, the temperature–mortality association was fitted for each location by use of a time-series regression. Second, a multivariate meta-regression model was built between location-specific estimates and meta-predictors. Finally, the grid-specific temperature–mortality association between 2000 and 2019 was predicted by use of the fitted meta-regression and the grid-specific meta-predictors. Excess deaths due to non-optimal temperatures, the ratio between annual excess deaths and all deaths of a year (the excess death ratio), and the death rate per 100 000 residents were then calculated for each grid across the world. Grids were divided according to regional groupings of the UN Statistics Division. Findings: Globally, 5 083 173 deaths (95% empirical CI [eCI] 4 087 967–5 965 520) were associated with non-optimal temperatures per year, accounting for 9·43% (95% eCI 7·58–11·07) of all deaths (8·52% [6·19–10·47] were cold-related and 0·91% [0·56–1·36] were heat-related). There were 74 temperature-related excess deaths per 100 000 residents (95% eCI 60–87). The mortality burden varied geographically. Of all excess deaths, 2 617 322 (51·49%) occurred in Asia. Eastern Europe had the highest heat-related excess death rate and Sub-Saharan Africa had the highest cold-related excess death rate. From 2000–03 to 2016–19, the global cold-related excess death ratio changed by −0·51 percentage points (95% eCI −0·61 to −0·42) and the global heat-related excess death ratio increased by 0·21 percentage points (0·13–0·31), leading to a net reduction in the overall ratio. The largest decline in overall excess death ratio occurred in South-eastern Asia, whereas excess death ratio fluctuated in Southern Asia and Europe. Interpretation: Non-optimal temperatures are associated with a substantial mortality burden, which varies spatiotemporally. Our findings will benefit international, national, and local communities in developing preparedness and prevention strategies to reduce weather-related impacts immediately and under climate change scenarios. Funding: Australian Research Council and the Australian National Health and Medical Research Council. © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license
- ItemHighly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films(Cambridge : Soc., 2015) Srivastava, Sachin K.; Hamo, Hilla Ben; Kushmaro, Ariel; Marks, Robert S.; Grüner, Christoph; Rauschenbach, Bernd; Abdulhalim, IbrahimA nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample.
- ItemImpact of unseasonable flooding on women's food security and mental health in rural Sylhet, Bangladesh: a longitudinal observational study(Amsterdam : Elsevier, 2022) Gepp, Sophie; Waid, Jillian L; Brombierstäudl, Dagmar; Kader, Abdul; Müller-Hauser, Anna A; Wendt, Amanda S; Dame, Juliane; Gabrysch, SabineBackground Climate change will lead to more frequent and intensive flooding. In April, 2017, unseasonably early flooding led to the inundation of low-lying cropland before the rice harvest in northeastern Bangladesh. We describe coping strategies and quantify short-term and medium-term effects of flooding events on food security and depressive symptoms of women. Methods This observational study is part of the cluster-randomised Food and Agricultural Approaches to Reducing Malnutrition trial (FAARM; NCT02505711). Women self-reported flooding exposure on behalf of their households when surveyed (approximately 6 months after the event). Remote sensing analysis was used to detect the extent of the flooding. We collected data on household food security at baseline, depressive symptoms 4–5 months before the flooding, and coping strategies immediately after the event. We followed up on these outcome measurements for depressive symptoms and food security for up to 2·5 years after the flooding event. We used multilevel regression adjusting for intervention allocation and pre-flooding measures to quantify the flood's effect on household food security and women's mental health. Findings The FAARM trial included 2700 young women in 96 settlements in rural Sylhet, Bangladesh. 1335 (56%) of 2405 women reported that their household being greatly affected, with many losing a large part of their rice harvest. Borrowing money with interest was the most common coping strategy, with households paying back on average 1·5 times the borrowed amount. Greatly affected households had higher odds of food insecurity, with a decreasing effect with increasing time after the flood (odds ratio: 2·4 [p<0·0001] 0·5 years after; 1·6 [p<0·0001] 2·0 years after]; and 1·3 [p=0·012] 2·5 years after). Women in such households also had 1·45 times higher odds of depression (p=0·0001) 2·5 years after the flooding event. Interpretation The 2017 flooding event negatively affected food security and the mental health of women in rural Sylhet, Bangladesh, and few affected women received formal government support. To reduce the impact of future floods, livelihood adaptations and expansion of financial protection programmes are essential measures to pursue. Funding German Federal Ministry for Education and Research (Berlin, Germany) and UK Department for International Development (London, UK).
- ItemFiber-based SORS-SERDS system and chemometrics for the diagnostics and therapy monitoring of psoriasis inflammatory disease in vivo(Washington, DC : Optica, 2021-1-28) Schleusener, Johannes; Guo, Shuxia; Darvin, Maxim E.; Thiede, Gisela; Chernavskaia, Olga; Knorr, Florian; Lademann, Jürgen; Popp, Jürgen; Bocklitz, Thomas W.Psoriasis is considered a widespread dermatological disease that can strongly affect the quality of life. Currently, the treatment is continued until the skin surface appears clinically healed. However, lesions appearing normal may contain modifications in deeper layers. To terminate the treatment too early can highly increase the risk of relapses. Therefore, techniques are needed for a better knowledge of the treatment process, especially to detect the lesion modifications in deeper layers. In this study, we developed a fiber-based SORS-SERDS system in combination with machine learning algorithms to non-invasively determine the treatment efficiency of psoriasis. The system was designed to acquire Raman spectra from three different depths into the skin, which provide rich information about the skin modifications in deeper layers. This way, it is expected to prevent the occurrence of relapses in case of a too short treatment. The method was verified with a study of 24 patients upon their two visits: the data is acquired at the beginning of a standard treatment (visit 1) and four months afterwards (visit 2). A mean sensitivity of ≥85% was achieved to distinguish psoriasis from normal skin at visit 1. At visit 2, where the patients were healed according to the clinical appearance, the mean sensitivity was ≈65%.
- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.