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1
Kucheriavyi, Y. P. "TRANSMISSION ELECTRON MICROSCOPY FOR THE DIRECT ANALYSIS OF FIBRIN CLOT STRUCTURE." Biotechnologia Acta 16, no. 2 (April 28, 2023): 30–31. http://dx.doi.org/10.15407/biotech16.02.030.
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The aim of our study was to compare the structure of clots formed as a result of thrombin-induced fibrin polymerization in the presence or absence of monoclonal fibrin-specific antibodies fragments as factors that change the clot structure. We concentrated on the final stage of fibrin clot formation at maximal turbidity point for every sample. Methods. Fibrin polymerization was studied by transmission electron microscopy (TEM) of negatively contrasted samples on H-600 Transmission Electron Microscope (“Hitachi”,Japan); 1% water solution of uranyl acetate (“Merck”, Germany) was used as a negative contrast. For sample preparation, in sterile glass tubes were sequentially added 0.32 mg/mL human fibrinogen, 0.025 M CaCl2 in 0.05 M ammonium formiate buffer (pH 7.9), and a total sample volume was 0.22 mL. The polymerization of fibrin was initiated by the introduction of thrombin at a final concentration of 0.25 NIH/mL. After 180 s, aliquots were taken from the polymerization medium. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/mL; 0.01 mL probes of fibrinogen solution were transferred to a carbon lattice, which was treated with a 1% uranyl acetate solution after 2 minutes. Investigations were per-formed using an H-600 electron microscope at 75 kV. Electron microscopic images were obtained at magnification of 20,000 -50,000. Results. Two monoclonal antibodies fragments were obtained towards the mixture of separated Aα-, Bβ- and γ-chains of fibrinogen. Antibodies fragments that were marked as III-1D and I-4A, had different epitopes within fragment Аα105-206 of D-region of fibrinogen. It was shown that addition of antibody fragment I-4A lead to formation of abnormal fibrils that were thinner than in the control sample and were organized in the dense network (Figure). Control sample exhibited the thick fibrils with well-structured classically organized network. The difference between control and I-4A samples demonstrated that antibody I-4A disrupted the structure of polymerized fibrin. In the same time the fibrils obtained in the presence of antibody fragment III-1D were closer to the control ones. Conclusions. TEM is an informative method for the study of the fibrin network formation. Its application allows to estimate the disruption in fi brin formation directly. In a combination with turbidity study and other functional tests TEM can provide important information about molecular mechanisms of clot formation.
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Rosen, Arlene Miller. "Ancient Town and City Sites: A View from the Microscope." American Antiquity 54, no. 3 (July 1989): 564–78. http://dx.doi.org/10.2307/280783.
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Archaeological studies concerned with reconstructing activity areas, room functions, and site-formation processes can benefit greatly from analyses of the microartifacts found on and within occupation surfaces. These remains are often primary refuse directly related to activities, and can be used to identify such locations as food-preparation areas, flint-knapping stations, and storage facilities. In addition, certain microartifacts are informative about site-formation processes. For example, the grain-size distribution of charcoal may be indicative of primary vs. secondary refuse, high percentages of corroded and crushed bone from scavenger feces may indicate locations of secondary refuse, and many small sherds could point to heavily trampled areas. A case study from the Iron Age city site of Tel Miqne-Ekron in central Israel demonstrates the use of microarchaeology at a complex sedentary site.
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Demicheli, Romano, William Hrushesky, Michael Retsky, and Elia Biganzoli. "Interpreting Breast Cancer Survival Data by the Hazard Function: Remarkable Findings from Event Dynamics." Medicina 56, no. 9 (September 12, 2020): 468. http://dx.doi.org/10.3390/medicina56090468.
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The report addresses the role of the hazard function in the analysis of disease-free survival data in breast cancer. An investigation on local recurrences after mastectomy provided evidence that uninterrupted growth is inconsistent with clinical findings and that tumor dormancy could be assumed as working hypothesis to understand the clinical course of the disease. Additionally, it was deemed that the lag-time between primary tumor removal and tumor recurrence is dynamically dependent on the subclinical metastasis development within the host-tumor system and, therefore, may be informative about the biology of the disease. Accordingly, the hazard function, which estimates the event risk pattern through the time, was adopted to analyze survival data. The multipeak pattern of the hazard function suggested that the process metastasis development has discontinuous features. A new paradigm of breast cancer metastatic development was proposed, involving the notions of tumor homeostasis, tumor quiescence in specific metastatic microscopic phases and surgery-related acceleration of the metastatic process. All analyses by prognostic factors (e.g., by menopausal status) or treatment modalities (e.g., by adjuvant chemotherapy) or other parameters (e.g., site of metastasis), provided coherent data in agreement with the model. The hazard rate function allowed addressing several clinical questions including meaning of ipsilateral breast tumor recurrence (IBTR), oncologic effect of delayed breast reconstruction, surgery related metastasis acceleration, possible role of anti-inflammatory drugs and body mass index (BMI) to modulate the recurrence risk. We conclude that the hazard function is a powerful tool to investigate the post-surgical course of early breast cancer and other operable tumors and to make inferences on their biology.
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Iñiguez, Alena Mayo, Lorrayne Brito, Lucélia Guedes, and Sergio Augusto de Miranda Chaves. "Helminth infection and human mobility in sambaquis: Paleoparasitological, paleogenetic, and microremains investigations in Jabuticabeira II, Brazil (2890 ± 55 to 1805 ± 65 BP)." Holocene 32, no. 3 (November 30, 2021): 200–207. http://dx.doi.org/10.1177/09596836211060490.
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Sambaquis or shellmounds are archeological sites constructed by hunter-fisher-gatherers that inhabited the Brazilian coast about 10,000–2000 yrs BP. Jabuticabeira II (JABII: 2890 ± 55 to 1805 ± 65 BP) is one of dozens of contemporaneous sambaquis of the Santa Catarina state, South Brazil, and contains hundreds of neatly organized burials, indicating great population density. In order to gather information about the health, diet and way of life of people in JABII, a paleoparasitological, paleogenetic, and micro-human remains investigation was carried out. Pelvic region and environmental control samples from six individuals exhumed from JABII were submitted to microscopic and ancient DNA (aDNA) investigation. Paleoparasitological analyses based on light microscopy were negative. However, a variety of informative microremains were found. Diatoms, fish scales, and algae characterize the marine and estuarine environment. Ipomoea batatas and Zea mays starch grains suggested cultivated items as part of their diet in agreement with the literature. The finding of Podocarpus sp. pollen grain, characteristic of highlander vegetation, suggests human mobility of JABII individuals which were settlement in the coast. Paleogenetic analyses showed Ascaris sp. helminth infection based on nad1 gene fragment detected from an individual excavated at L3 FS7 burial (1826 ± 40 BP). This aDNA result places the antiquity of Ascaris sp. infection, and haplotypes that are circulating in humans and other animals nowadays, in Pre-Columbian South American times.
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Thuy, Ben, Larry Knox, Lea D. Numberger-Thuy, Nicholas S. Smith, and Colin D. Sumrall. "Ancient deep ocean as a harbor of biotic innovation revealed by Carboniferous ophiuroid microfossils." Geology 51, no. 2 (January 3, 2023): 157–61. http://dx.doi.org/10.1130/g050596.1.
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Abstract Fossil-informed molecular phylogenies have emerged as the most powerful tool for correlating biotic evolution and Earth history. The accuracy of these trees, however, depends on the completeness of fossil sampling. For most organismal clades, the available fossil record is insufficiently sampled. This is especially true for groups with a multi-element skeleton, such as echinoderms and vertebrates, where sampling efforts focus largely on rare finds of intact skeletons. For these groups, inconspicuous but informative skeletal fragments are commonly neglected. This sampling bias excludes the numerous paleoenvironments in which preservation of intact skeletons is extremely unlikely, in particular deep-water settings. We describe new finds of brittle-star, or ophiuroid, fossils retrieved from sieving residues of sediments deposited during the Atokan (Upper Carboniferous) on the deep shelf to upper slope of the Ardmore Basin in present-day southern Oklahoma, USA. Although preserved as disarticulated, microscopic ossicles, the pristine preservation of the skeletal microstructure allows for precise identification of the remains. Comparative anatomical and phylogenetic analyses confirm the presence of basal representatives of the extant ophiuroid orders Ophioscolecida and Amphilepidida. Our finds provide the first unambiguous fossil evidence that the early crown-group diversification of the Ophiuroidea was well under way long before the end-Permian mass extinction, and that a significant part of this diversification took place in deep-water settings, as previously predicted by molecular evidence.
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Boccacci, Giulia, Francesca Frasca, Chiara Bertolin, and Anna Maria Siani. "Influencing Factors in Acoustic Emission Detection: A Literature Review Focusing on Grain Angle and High/Low Tree Ring Density of Scots Pine." Applied Sciences 12, no. 2 (January 14, 2022): 859. http://dx.doi.org/10.3390/app12020859.
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Among non-destructive testing (NDT) techniques applied to structural health monitoring in existing timber structures, ranging from visual inspection to more sophisticated analysis, acoustic emission (AE) is currently seldomly used to detect mechanical stresses in wooden building assets. This paper presents the results from a systematic literature review on AE NDT applied to monitor micro and macro fracture events in softwood, specifically Scots pine. This survey particularly investigates its application with respect to the tree rings density and grain angle inspection, as influencing factors well correlated with physical and mechanical characteristics of wood. The literature review was performed in a three-step process defined by the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) flow diagram, leading to the selection of 31 documents from different abstract and citation databases (Scopus, Web of Science and Google Scholar). The outcomes have highlighted how laboratory experiments, including several types of tests (tensile, cutting, compressive, etc.), were conducted in most cases, while a very limited number of studies investigated on in situ monitoring. In addition, theoretical approaches were often explored in parallel with the experimental one. It emerges that—for tree ring density studies—a multi-technique approach, which may include microscopic observations, could be more informative. Indeed, although not widely investigated, high/low tree ring density and grain angle were found as influencing factors on the AE parameters detected by the sensors, during condition and structural health monitoring experiments.
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Burykina, S. I., S. P. Uzhevska, and N. V. Pylyak. "METHOD OF ASSESSING THE NEMATOCIDAL EFFECTIVENESS OF A BIOPREPA-RATION BASED ON PREDATORY MICROMYCETE AGAINST THE POTATO STEM NEMATODE." Microbiology&Biotechnology, no. 3(59) (December 20, 2023): 14–25. http://dx.doi.org/10.18524/2307-4663.2023.3(59).292392.
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Aim – Studing of methods for determining the effectiveness of Nematophagin BT against potato stem nematode in the laboratory. Materials and methods. For the experiment, potato tubers with signs of damage by the stem nematode Ditylenchus destructor Thorne, 1945 were selected. The method of treatment of cut potato tubers with the preparation was used. It was compared with the method of treatment of whole tubers in the laboratory. The effect of the microbiological nematicidal preparation (Nematofagin BT) developed by ITI Biotechnology of NAAS was determined. The effectiveness of the microbiological preparation of nematicidal action (Nematofagin BT), developed by ITI Biotechnology of NAAS, was determined. The active ingredient of the drug is a microscopic fungus-predator Arthrobotrys oligospora strain 12, the initial culture of which is deposited in the collection of microorganism cultures of the D.K. Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine F100047. Results. The methods of laboratory evaluation of the nematicidal effect of the biological product against potato stem nematode are described. In laboratory conditions, to determine the effectiveness of the drug, it is more informative to use cut tubers that have been infected with stem nematode. This method requires much more time to set up, but the bioagent has bigger accessibility to the pest. The use of whole tubers is less labor-intensive, but there is a possibility of using uninfected material in the experiment. The expediency of using these methods in laboratory conditions for the selection of active strains and determination of their effectiveness is shown. Conclusion. The method of nematicidal treatment of cut tubers with established infection of stem nematode is more informative than the use of whole tubers. It is advisable to analyze for damage to potatoes by nematodes when using pieces of affected potatoes from 3 to 7 days after treatment with a 3% solution of the biological product with an exposure of 5 hours. In laboratory conditions, both methods of treatment of cut and whole tubers with the preparation can be used to test the effect of Nematophagin BT on potato stem nematode. Nematophagin BT showed nematicidal activity against potato stem nematode in laboratory conditions.
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Zeleneva, Yu V., Ph B. Gannibal, I. А. Kazartsev, and V. P. Sudnikova. "Molecular Identification, Effector genes and Virulence of Isolates of <i>Parastagonospora nodorum</i> from Altai Krai (Russia)." Микология и фитопатология 57, no. 5 (September 1, 2023): 362–71. http://dx.doi.org/10.31857/s0026364823050124.
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Septoria blotches represent some of the most harmful wheat diseases that encompass all grain-producing countries worldwide. They are caused mainly by fungi of the genera Parastagonospora and Zymoseptoria. The aims of the research were to analyze the nucleotide sequences of two phylogenetically informative DNA loci, ITS and tub2, of Parastagonospora nodorum isolates from the Altai Krai, to detect the presence of Tox1, Tox3, ToxA effector genes and to study the virulence of the isolates. Microscopic analysis of fragments of affected plant tissue was used for primary identification of the Septoria blotch. The causative agent from the studied plant samples was identified as P. nodorum. Fungal colonies cultivated on potato-glucose agar displayed a well-developed light brown velvety mycelium with dark periphery (mixed type) and numerous pycnidia exhibiting high sporulation capacity. Microbiological diagnostics were complemented by molecular genetic studies. Sequencing of ITS and tub2 loci isolates revealed complete genetic identity of all eight studied monoconidial isolates obtained from different samples. PCR-based detection of tree Tox-genes demonstrated that the only Tox3 was present in the geno-types of 80 tested P. nodorum monoconidial isolates. The ToxA and Tox1 genes were not found in the studied isolates. The virulence of P. nodorum isolates was evaluated in laboratory conditions using an isolated wheat leaves assay. Mixture of four isolates from spring wheat and one isolate from winter wheat, were characterized as virulent. When spring and winter wheat cultivars were infected with two isolates obtained from oats, pathogenic but avirulent properties were detected. The isolate from triticale was avirulent to winter wheat cultivars and virulent to spring wheat cultivars.
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Sokol, Ye I., K. V. Kolisnyk, and T. V. Bernads’ka. "Evaluation of metrological characteristics of spectral analysis method for determining erythrocyte morphology." Технология и конструирование в электронной аппаратуре, no. 3-4 (2021): 45–49. http://dx.doi.org/10.15222/tkea2021.3-4.45.
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Spectral photometry is currently widely used for quantitative and qualitative analysis of biological molecules in medical biology. The method is based on the ability of molecules to absorb electromagnetic radiation. Modern clinical laboratory diagnostics extensively uses optical methods of analysis that rely on these physical properties of semitransparent objects, such as blood components. Knowing the absorption spectra of blood and its components, it is possible to quantify the concentration of all the components by solving the mathematical system of equations corresponding to these spectra. However, the existing methods of optical analysis of erythrocytes do not allow quantifying their geometric parameters, which may also indicate certain diagnostic signs and be used to analyze the clinical condition of the patient's body. The aim of this work is to evaluate the metrological characteristics of the newly developed method of determining the geometric parameters of erythrocytes, which combines spectral analysis and double annealing. The input data for the 3D imaging of erythrocytes were taken from the images of the sample both made in natural light and illuminated by a coherent light source with different wavelengths. The latter, after some additional image correlation, increases the reliability of the result. The calculation results on the errors and the measuring channel resolution of the digital interference microscope indicate an acceptable accuracy of the method. The accuracy of the three-dimensional image obtained by the proposed method is more than 20% higher than that of other known methods. This allows determining the informative geometric parameters of the structure of erythrocytes more accurately and using them to obtain additional clinical diagnostic characteristics of the patient's body.
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Drozd, O. M., M. Yu Zhuravel, O. Ye Naidionova, I. P. Lezhenina, and N. Yu Polchaninova. "THE DETERMINATION OF LOCAL CHANGES IN THE SUPPORTING ECOSYSTEM SERVICE OF SOIL ON THE TERRITORY OF OIL AND GAS PRODUCTION." Ecological Safety and Balanced Use of Resources, no. 2(18) (December 3, 2018): 15–25. http://dx.doi.org/10.31471/2415-3184-2018-2(18)-15-25.
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The temporary withdrawal of agricultural land for the construction or overhaul of wells and pipelines laying is, in essence, an interference with the natural soil-forming process, the result of which is a change in the implementation of the ecosystem functions of the soil and, consequently, ecosystem services of the soil.
The article analyses the influence of industrial activity and soils reclamation on the implementation of the supporting ecosystem service of soil. Such indicators as agrophysical, physico-chemical, chemical indices of soil, species composition, trophic structure and the state of the indicator groups of the invertebrates (rainworms, enchitrides, larvae of elaterids), the number of the microorganisms of the main ecofunctional and taxonomic groups (bacteria that absorb organic and mineral nitrogen, actinomycetes, microscopic fungi, oligotrophic bacteria), enzymatic activity and soil toxicity were selected as indicators of anthropogenic change. Four soil samples were taken at each experimental and background site for the reckoning of mesofauna (50×50×30 cm). Thus, 1 m² of land was surveyed on each experimental area. The final evaluation of the soil condition at different points was carried out using the integral index of the biological state of the soil (IIBS).
Following the research of the agro-ecological state of the soil on the territory of two rehabilitated sites of oil and gas production wells local changes in the supporting ecosystem service are defined within the spread of gray forest soil. It is established that the general unfavorable property of background and recultivated soils is high density indices. Unfavorable agrophysical properties determine the differentiation of the parameters of the analyzed indicators of the supporting ecosystem service of soil. The article proves the expediency of using the complex of the most informative biological indicators to assess the quality of reclamation and the state of reclaimed soils.
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Oliveira, P. P. T., and Eduardo Galembeck. "Applying a Design-Based Research Framework in the Production of Web Content to Disseminate the Remote Laboratory Uses and Development." Revista de Ensino de Bioquímica 17 (September 13, 2019): 112. http://dx.doi.org/10.16923/reb.v17i0.892.
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INTRODUCTION. The adoption of teaching methods that highlight student's autonomy is a long term process and requires students to be involved with hands-on activities such as lab classes. Lab activities can vary in methodological approaches from demonstration to the authentic research, and requires the use of equipment, reagents, and time. A lot of low-cost experiments alternatives have been developed as an alternative to the expensive equipment not always available in didactic labs. The availability of low-cost microcontrollers and sensors has made possible not only having students do run experiments but also having them to build equipment to be used in their investigative labs. It is also possible to build equipment to be controlled over the internet and to build low-cost remote labs able to provides a real experience of controlling experiment online. OBJECTIVE. This work aims the production of a series of informative and explanatory videos about the use and development of low-cost equipment for remote operation, and to analyze the impact this strategy in biochemistry education, and knowledge dissemination. MATERIALS AND METHODS. Equipment and experiments already developed and in operation have been the starting point to produce a videos series for YouTube. The whole process is structured as Design-Based Research framework, which involves developing iterations of the material, each one incorporating changes based on the feedback taken about the source material. RESULTS AND DISCUSSION. We have produced 12 videos that have been released weekly on our YouTube channel. The topics covered so far are titration, photosynthesis and microscopic diversity. All videos are linked to a survey, which will serve to collect data regarding the utility and quality of the videos, reproducibility of the experiments, and demographic data from the viewers. CONCLUSION. The first two videos released have received positive feedback and a reasonable number of views.
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Ueno, Hideki, Megumi Ishiguro, Eiji Nakatani, Toshiaki Ishikawa, Hiroyuki Uetake, Kenta Murotani, Shigeyuki Matsui, et al. "Prognostic impact of tumor budding in stage II colon cancer: A prospective study (SACURA trial)." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 3609. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.3609.
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3609 Background: Growing number of studies indicate tumor budding is a significant prognostic factor in colorectal cancer (van Wyk, et al. Cancer Treat Rev 2015), but this has been shown only in retrospective studies. We prospectively evaluated prognostic factors in stage II colon cancer to determine their prognostic value in a multi-institutional phase III study (SACURA trial, ASCO2016 abst#3617). Methods: A total of 991 patients with curatively resected stage II colon cancer (2006–2010; 136 institutions) were included in the study. Tumor budding was defined as an isolated cancer cell or a cluster composed of fewer than five cells in the invasive frontal region, and was graded based on its number within a microscopic field of a 20x objective lens (0.785 mm2) in the hotspot. Tumors with < 5, 5–9, and ≥10 budding foci were classified as grades G1, G2, and G3, respectively. All clinical and pathological data including the grade of budding were prospectively recorded and prognostic analyses were performed at 5 years after the completion of registration. Results: According to budding grading, 376, 331 and 284 tumors were classified as G1, G2, and G3, and 5-year relapse-free survival (RFS) rate was 90.9%, 85.1%, and 74.4%, respectively ( P < 0.0001). Budding grade was significantly associated with the incidence of recurrence in the liver, lung, lymph node, and peritoneum ( P < 0.0001–0.01). Among conventional factors, T stage and the serum CEA levels were associated with RFS, however, tumor grade, lymphatic and venous invasions, and the number of lymph node examined were not significant factors. Multivariate analysis for RFS showed budding, along with T stage, exerted an independent influence on prognostic outcome. Budding grade surpassed T stage and tumor grade in the ability to stratify patients by RFS (Harrell’s c-index, 0.63, 0.59, and 0.54, respectively). Conclusions: Our prospective study indicates that the grade of tumor budding is more informative for prognostic prediction than conventional prognostic factors in stage II colon cancer. The role of this prognostic factor should be highlighted in the adjuvant treatment setting, and conversely, some of prognostic factors adopted in clinical guidelines may need to be reconsidered. Clinical trial information: NCT00392899.
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Grygorian, E., V. Olkhovsky, M. Gubin, and V. Shishkin. "FORENSIC MEDICAL ASSESSMENT OF MORPHOLOGICAL CHANGES AT DIFFERENT POSTMORTEM INTERVAL." Inter Collegas 8, no. 3 (December 9, 2021): 177–81. http://dx.doi.org/10.35339/ic.8.3.177-181.
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Purpose: The postmortem interval (PMI) evaluation is one of priorities while performing a forensic medical examination of corpse. To date, there is lack of information of morphological postmortem changes of some internal organs. Considering the persistent need to develop the method for a precise assessment of PMI, postmortem changes in these potentially informative organs were evaluated. The aim of study was to analyze morphological postmortem changes in prostate and uterus. Materials and Methods: histological samples of 40 prostate tissues and 40 uterus (n=80) from corpses of deceased aged 18-75 years. Only cases with known time of death were included to study, the time of death was taken from police reports. Exclusion criteria were cases of violent death, cases of death with massive blood loss, tumors of studied internal organs, cases when diagnosis was not made by a forensic medical examiner. The PMI of studied cases ranged from 1 to 6 days. Histological slides were made with a staining by hematoxylin and eosin, x200 magnification, using Olympus ВХ41 and Olympus ВХ46 microscopes, Olympus SC50 camera. Postmortem morphological changes were evaluated by a calculation of blank spaces percentage in microscopical structures using a JS-based program. Connection between PMI and morphological changes was calculated by the Spearman’s rank correlation. Results: the average percentage of blank spaces in uterus tissues was smaller than in prostate tissues (1,99 and 9,65 relatively). The slower growing of blank spaces was in uterus. In prostate samples, a notable increase of blank spaces was observed between 48 and 72 hours after the death. After this period, the increase slowed down and then an increase was observed again between 120 and 144 hours after the death. In uterus samples, a slight acceleration observed between 72 and 120 hours after the death and then slowing down between 120 and 144 hours after the death. Blank spaces in evaluated histological slides were increasing directly proportional to the PMI, a statistically significant interconnection was defined (p < 0.05). Conclusions: The morphological postmortem changes in prostate and uterus were developing at certain time frames. Blank spaces percentage, in studied histological slides, were increasing directly proportional to the PMI increase, a statistically significant interconnection was defined. Therefore, the results of study show the possibility of the evaluation of a postmortem time interval by assessing such morphological changes in these organs, which could be used in forensic medical cases.
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Williamson, Helen, Edward Mountjoy, Hashem Shihab, Jack Bartram, Mike Hubank, Nicholas Goulden, Ian Day, Eileen Roberts, John Moppett, and Jeremy Hancock. "Development Of Minimal Residual Disease (MRD) Analysis In Childhood Acute Lymphoblastic Leukaemia (ALL) By Next Generation Sequencing (NGS)." Blood 122, no. 21 (November 15, 2013): 2569. http://dx.doi.org/10.1182/blood.v122.21.2569.2569.
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Abstract Detection of sub-microscopic levels of disease (minimal residual disease; MRD) in childhood acute lymphoblastic leukaemia (ALL) during treatment is an important prognostic factor. Currently, stratification of therapy for the new frontline trial in childhood ALL (UKALL 2011) is provided by MRD analysis using real time quantitative PCR (RQ-PCR) to identify and quantitate the patient specific rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes. The current methodology is expensive, time-consuming and complex to perform. Although MRD has proven to be a powerful and essential tool in stratification of ALL patients, 8% of individuals in the current UKALL 2011 trial do not have an informative MRD result. Recently, Next Generation Sequencing (NGS) has led to the opportunity to improve the sensitivity and specificity of Ig/TCR based MRD analysis. In this study, we focussed on the IgH locus using BIOMED 2 primers (van Dongen et al., 2003) modified to allow target identification and quantitation by deep sequencing on the Illumina MiSeq platform. We developed a novel pipeline to automate the clustering and classification of sequencing reads leading to characterisation of the clonal subtypes present. In a sample of 12 patients, the method correctly identified all the major clones revealed by current methodologies, and also detected many related and unrelated low-frequency clones. Additional targets were also identified in patients in which no IgH targets were detectable by current methodologies. These NGS-identified targets were subsequently used to monitor MRD by RQ-PCR to the desired quantitative range required for stratification of therapy according to UKALL 2011 guidelines (Figure 1). In addition, we were able to delineate patterns of IgH rearrangements in two patients previously shown to have oligoclonal (>2) rearrangements. Such patients represent a time consuming and technical challenge for current technologies as it is important that all targets at the locus are followed by RQ-PCR to provide an informative and robust MRD result. Furthermore, by clustering similar sequences, we identified diagnostic samples where multiple V regions are attached to the same N1-D-N2-J region. This may allow for the study of clonal evolution in follow-up samples. Altogether, NGS sequencing has the potential to significantly reduce false negative results, as multiple evolved clones can be identified. This methodology also represents a significant time saving (5-7 days) in comparison to established methods (3-4 weeks).Figure 1.(a) Polyacrylamide electrophoresis could not recognise a target to use in current MRD methodologies (well 1 containing the products from a PCR reaction that would amplify VH1 and VH7, and wells 2-6 amplifying VH2-6, respectively), while the NGS pipeline could identify a VH7 rearrangement (b). (c) ASOs were designed to amplify the NGS-identified VH7-81*01 DH3-9*01 JH4*02 rearrangement and optimised to correctly identify 10-2, 10-3, 10-4 dilutions with a single NAC (non-amplification control; monocytes from 20 normal individuals) replicate amplified, therefore meeting current guidelines for a MRD target.Figure 1. (a) Polyacrylamide electrophoresis could not recognise a target to use in current MRD methodologies (well 1 containing the products from a PCR reaction that would amplify VH1 and VH7, and wells 2-6 amplifying VH2-6, respectively), while the NGS pipeline could identify a VH7 rearrangement (b). (c) ASOs were designed to amplify the NGS-identified VH7-81*01 DH3-9*01 JH4*02 rearrangement and optimised to correctly identify 10-2, 10-3, 10-4 dilutions with a single NAC (non-amplification control; monocytes from 20 normal individuals) replicate amplified, therefore meeting current guidelines for a MRD target. Having established NGS for identifying clonal targets in ALL, we are currently assessing the ability of the method and pipeline to quantify disease levels in end of induction and relapse samples, previously analysed by RQ-PCR, to determine the concordance between the methodologies. Indeed, logarithmic dilution series of patient DNA in a normal background revealed that stratification based on a clinical threshold of 1 in 1,000,000 is possible using this methodology. Further investigation into the clinical utility of NGS for MRD analysis will focus on analysing earlier time points in treatment and studying the potential use of blood rather than bone marrow. Altogether, this will further improve the predictive value and specificity of MRD testing. Disclosures: No relevant conflicts of interest to declare.
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Mittal, Kush Mohan, Marc Timme, and Malte Schröder. "Efficient self-organization of informal public transport networks." Nature Communications 15, no. 1 (June 8, 2024). http://dx.doi.org/10.1038/s41467-024-49193-1.
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AbstractThe Global South, encompassing more than 80% of the world population, heavily relies on informal paratransit services with ad-hoc routes. Yet, it remains unclear how efficiently such informal public transport services organize and operate. Here, we analyze and compare the structural efficiency of more than 7000 formal and informal bus service routes in 36 cities across 22 countries globally. Intriguingly, informal transport self-organizes in ways at or above efficiency levels of centralized services. They exhibit fewer detours, more uniform paths, and comparable interconnectivities, all while remaining profitable without the major subsidies common in the Global North. These insights challenge the global perception of informal transport as an inferior alternative to centrally organized services. More generally, analyzing large-scale microscopic transport data and condensing them into informative macroscopic observables may qualitatively improve system understanding and reveal specific options to create more accessible, efficient, and sustainable public transport solutions.
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Villeneuve, Thomas, Grégoire Prévot, Grégory Pugnet, Gavin Plat, Valentin Héluain, Stanislas Faguer, and Nicolas Guibert. "Role of bronchoscopy for respiratory involvement in granulomatosis with polyangiitis and microscopic polyangiitis." ERJ Open Research, August 31, 2023, 00141–2023. http://dx.doi.org/10.1183/23120541.00141-2023.
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ObjectivesThis study describes data from bronchoscopy performed at the diagnosis of ANCA-associated vasculitis (AAV).MethodsWe conducted a retrospective study between 2004 and 2019 in patients older than 18 years old with a diagnosis of microscopic polyangiitis (MPA) or granulomatosis with polyangiitis (GPA) who underwent bronchoscopy at onset of the disease. We collected bronchoalveolar lavage (BAL) and histological findings obtained during bronchoscopy.ResultsTwo hundred and seventy-four patients with AAV were identified. Among 92 bronchoscopies, 62 were performed at diagnosis and 58 procedures were finally analysed. Cough was more frequent in patients with MPA than GPA (p=0.02). The presence of endobronchial lesions (24.1%) was found to be significantly associated with GPA (p<0.0001) and PR3-ANCA (p=0.01). The most frequent endobronchial lesions were inflammation and hyperaemia of the bronchial mucosa (50%), followed by stenoses (28%), ulcerations (21%) and mass-like granulomatosis (7%). The diagnostic yield of bronchial biopsies was useful for visible lesions (66.6%versus0%; p=0.006). On BAL, diffuse alveolar haemorrhage (DAH) was detected in 31 patients (53.4%) and was more frequent in MPA patients (70.4%versus38.7%; p=0.016). In 16.1% of DAH, BAL confirms the diagnosis despite the absence of clinical or biological arguments. The incidence of microbial infections on BAL (38%) was similar between MPA and GPA (p=0.54).ConclusionBronchoscopy is an informative procedure at the onset of AAV disease in patients with respiratory manifestations. Endobronchial lesions are more frequently found in GPA and should be biopsied. BAL can be used to confirm DAH or diagnose superadded infection.
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Choi, Bokyung, Yeojin Hwang, Scott A. M. McAdam, and Tae‐Soo Jang. "Comparative microscopic investigations of leaf epidermis in four Ajuga species from Korea." Microscopy Research and Technique, November 2023. http://dx.doi.org/10.1002/jemt.24450.
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AbstractThe genus Ajuga is widely distributed in temperate to subtropical regions, and four species are currently recognized in Korea (A. decumbens, A. multiflora, A. nipponensis, and A. spectabilis), but epidermal anatomical differences across these species have never been described. A comparative study of the leaf micromorphological characteristics of Korean Ajuga species was performed using light microscopy (LM) and scanning electron microscopy (SEM) to elucidate their taxonomic usefulness and to assess leaf micromorphological diversity. Considerable diversity in epidermal and stomatal anatomy was observed across Korean Ajuga species. Species had both hypostomatic or amphistomatic leaves, with anomocytic, anisocytic, diactyic, or actinocytic stomatal complexes. Guard cell length across species ranged from 17.66 ± 0.57 μm to 32.50 ± 2.38 μm and correlated with genome size. Abnormal stomata were frequently observed in three species (A. decumbens, A. multiflora, and A. nipponensis) but not in A. spectabilis. Three types of glandular trichomes were found: peltate in all species, short‐stalked in all species, and long‐stalked glandular trichomes in A. multiflora. Among the investigated leaf micromophological characters, trichome type, epidermal cell shape, and stomatal morphology were all taxonomically informative traits at a species level.Research Highlights A comprehensive micromorphological description of the leaf surface is provided for Korean Ajuga species using scanning electron microscopic (SEM) and light microscopic (LM) analyses. The diverse range of stomatal development and the occurrence of polymorphic stomatal types are documented for the first time in Korean Ajuga species. The great diversity in stomatal and trichome morphology in Korean Ajuga species are taxonomically useful traits for species identification.
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Musa, Sandrine, Theo Hemberle, Staffan Bensch, Vaidas Palinauskas, Laima Baltrūnaitė, Friederike Woog, and Ute Mackenstedt. "Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites." Frontiers in Cellular and Infection Microbiology 14 (April 29, 2024). http://dx.doi.org/10.3389/fcimb.2024.1385599.
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Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.
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Thuy, Ben, Larry Knox, Lea D. Numberger-Thuy, Nicholas S. Smith, and Colin D. Sumrall. "Ancient deep ocean as a harbor of biotic innovation revealed by Carboniferous ophiuroid microfossils." Geology, January 3, 2023. http://dx.doi.org/10.1130/g50596.1.
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Fossil-informed molecular phylogenies have emerged as the most powerful tool for correlating biotic evolution and Earth history. The accuracy of these trees, however, depends on the completeness of fossil sampling. For most organismal clades, the available fossil record is insufficiently sampled. This is especially true for groups with a multi-element skeleton, such as echinoderms and vertebrates, where sampling efforts focus largely on rare finds of intact skeletons. For these groups, inconspicuous but informative skeletal fragments are commonly neglected. This sampling bias excludes the numerous paleoenvironments in which preservation of intact skeletons is extremely unlikely, in particular deep-water settings. We describe new finds of brittle-star, or ophiuroid, fossils retrieved from sieving residues of sediments deposited during the Atokan (Upper Carboniferous) on the deep shelf to upper slope of the Ardmore Basin in present-day southern Oklahoma, USA. Although preserved as disarticulated, microscopic ossicles, the pristine preservation of the skeletal microstructure allows for precise identification of the remains. Comparative anatomical and phylogenetic analyses confirm the presence of basal representatives of the extant ophiuroid orders Ophioscolecida and Amphilepidida. Our finds provide the first unambiguous fossil evidence that the early crown-group diversification of the Ophiuroidea was well under way long before the end-Permian mass extinction, and that a significant part of this diversification took place in deep-water settings, as previously predicted by molecular evidence.
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Persson, Dennis Krog, Kenneth Agerlin Halberg, Ricardo Cardoso Neves, Aslak Jørgensen, Reinhardt Møbjerg Kristensen, and Nadja Møbjerg. "Comparative myoanatomy of Tardigrada: new insights from the heterotardigrades Actinarctus doryphorus (Tanarctidae) and Echiniscoides sigismundi (Echiniscoididae)." BMC Evolutionary Biology 19, no. 1 (November 6, 2019). http://dx.doi.org/10.1186/s12862-019-1527-8.
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Abstract Background Tardigrada is a group of microscopic invertebrates distributed worldwide in permanent and temporal aquatic habitats. Famous for their extreme stress tolerance, tardigrades are also of interest due to their close relationship with Arthropoda and Cycloneuralia. Despite recent efforts in analyzing the musculature of a number of tardigrade species, data on the class Heterotardigrada remain scarce. Aiming to expand the current morphological framework, and to promote the use of muscular body plans in elucidating tardigrade phylogeny, the myoanatomy of two heterotardigrades, Actinarctus doryphorus and Echiniscoides sigismundi, was analyzed by cytochemistry, scanning electron and confocal laser scanning microscopy and 3D imaging. We discuss our findings with reference to other tardigrades and internal phylogenetic relationships of the phylum. Results We focus our analyses on the somatic musculature, which in tardigrades includes muscle groups spanning dorsal, ventral, and lateral body regions, with the legs being musculated by fibers belonging to all three groups. A pronounced reduction of the trunk musculature is seen in the dorsoventrally compressed A. doryphorus, a species that generally has fewer cuticle attachment sites as compared to E. sigismundi and members of the class Eutardigrada. Interestingly, F-actin positive signals were found in the head appendages of A. doryphorus. Our analyses further indicate that cross-striation is a feature common to the somatic muscles of heterotardigrades and that E. sigismundi—as previously proposed for other echiniscoidean heterotardigrades—has relatively thick somatic muscle fibers. Conclusions We provide new insights into the myoanatomical differences that characterize distinct evolutionary lineages within Tardigrada, highlighting characters that potentially can be informative in future phylogenetic analyses. We focus our current analyses on the ventral trunk musculature. Our observations suggest that seven paired ventromedian attachment sites anchoring a large number of muscles can be regarded as part of the ground pattern of Tardigrada and that fusion and reduction of cuticular attachment sites is a derived condition. Specifically, the pattern of these sites differs in particular details between tardigrade taxa. In the future, a deeper understanding of the tardigrade myoanatomical ground pattern will require more investigations in order to include all major tardigrade lineages.
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Orsulic, Sandra, Joshi John, Ann E. Walts, and Arkadiusz Gertych. "Computational pathology in ovarian cancer." Frontiers in Oncology 12 (July 29, 2022). http://dx.doi.org/10.3389/fonc.2022.924945.
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Histopathologic evaluations of tissue sections are key to diagnosing and managing ovarian cancer. Pathologists empirically assess and integrate visual information, such as cellular density, nuclear atypia, mitotic figures, architectural growth patterns, and higher-order patterns, to determine the tumor type and grade, which guides oncologists in selecting appropriate treatment options. Latent data embedded in pathology slides can be extracted using computational imaging. Computers can analyze digital slide images to simultaneously quantify thousands of features, some of which are visible with a manual microscope, such as nuclear size and shape, while others, such as entropy, eccentricity, and fractal dimensions, are quantitatively beyond the grasp of the human mind. Applications of artificial intelligence and machine learning tools to interpret digital image data provide new opportunities to explore and quantify the spatial organization of tissues, cells, and subcellular structures. In comparison to genomic, epigenomic, transcriptomic, and proteomic patterns, morphologic and spatial patterns are expected to be more informative as quantitative biomarkers of complex and dynamic tumor biology. As computational pathology is not limited to visual data, nuanced subvisual alterations that occur in the seemingly “normal” pre-cancer microenvironment could facilitate research in early cancer detection and prevention. Currently, efforts to maximize the utility of computational pathology are focused on integrating image data with other -omics platforms that lack spatial information, thereby providing a new way to relate the molecular, spatial, and microenvironmental characteristics of cancer. Despite a dire need for improvements in ovarian cancer prevention, early detection, and treatment, the ovarian cancer field has lagged behind other cancers in the application of computational pathology. The intent of this review is to encourage ovarian cancer research teams to apply existing and/or develop additional tools in computational pathology for ovarian cancer and actively contribute to advancing this important field.
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Mendik, Caitlyn. "Redesigning Peppers RNA Imaging System." University of Colorado Honors Journal, April 30, 2024. http://dx.doi.org/10.33011/cuhj20242421.
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Abstract RNA is a central component of biochemistry as it carries genetic information, has enzymatic activity, mediates protein biodiversity, and supports other cellular functions. Scientists are continually developing new tools for studying RNA and applying it to fields of research such as medicine and molecular biology. RNA imaging provides one avenue for characterizing RNA by looking at trafficking, localization, and lifetime in living cells. Imaging tools have been on the rise in the past 20 years, providing innovative methods for looking at this nucleic acid under a microscope. Peppers is one of these tools designed by Chen et al. at the East China University of Science and Technology. Peppers is an RNA aptamer engineered to bind a fluorescent probe in a stem loop. The 8Peppers aptamer is the optimized form of their probe and is not repeating units of one aptamer, but a long hairpin turn with eight binding spots for probe; this 8Peppers aptamer has been shown to have difficulties folding into native conformation. This paper aims to redesign Peppers RNA imaging system to produce an effective imaging system with enhanced folding capabilities. This paper looks at its in vitro capabilities by fluorescence assay of RNA binding to the probe, HBC620. The redesigned Peppers imaging system is further tested by live cell imaging to determine the efficacy of redesigning the structure, stem, and synonymization of non-consequential regions. The experiments did not show an enhanced fluorescence turn-on or folding capabilities of redesigned Peppers compared to Chen’s original Peppers imaging system. However, the original Peppers imaging tool did not have reproducible results and illustrated the need for a comprehensive and standardized characterization system for RNA imaging tools to be dependable. Lay Summary Ribonucleic acid (RNA) is an integral part of any living organism, just like DNA, or your genetic code. RNA’s primary function is to convey genetic information from instruction to the actual product; it acts like the recipe for cells to make proteins. It has many other functions and can contribute to disease, thus it is important to develop more informative metrics of studying RNA. Imaging RNA involves using high powered microscopes that look at cells. The RNA can be seen by tagging it with a molecule that makes it light up under certain wavelengths of light, also known as fluorescence. This allows researchers to track where a molecule of RNA is moving within a cell, how it moves, and how long it lasts. This is invaluable for better understanding the complicated, yet essential, role RNA plays in biology. In this experiment, I set out to optimize an RNA imaging system named Peppers by redesigning the structure of the RNA into something that forms more reliably. Peppers did not have a strong fluorescence signal and it wasn’t clear if the imaging was actually showing RNA. After testing this redesigned system in cells and in a test tube, it became clear that many RNA imaging platforms do not work as well as they boast. I found that the redesigned Peppers, original Peppers, and controls performed the same and that none of the results were statistically significant; this was consistent with other researchers’ frustrations with these tools. Thus, I call to action a more standardized system for characterizing RNA imaging as the field quickly grows. Researchers should be able to make an educated choice on which tool to use and how to analyze their data accurately. For the full text, please visit https://scholar.colorado.edu/concern/undergraduate_honors_theses/ms35tb04h.