Combined optical trapping and single molecule fluorescence to study the force-dependent binding kinetics between filamentous actin and its partners

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.Includes bibliographical references (p. 75-77).Actin filaments are a major constituent of the cytoskeleton in most eukaryotic cells. They function as a connection between the cell body to the focal adhesions in order to transmit forces into and out of the cell. During the force transduction process, many proteins bind to actin filaments in order to initiate a signaling cascade that reaches the cell nucleus. However, the effects of forces in the binding kinetics between actin filaments and actin binding proteins are unknown. This work proposes an experimental setup to study the force-dependent binding kinetics of such proteins at the single molecule level by using an instrument that combines optical trapping with single molecule fluorescence. The main focus of this work was the design and construction of the experimental equipment. The results show position detection capabilities with a resolution of 5 nm. Also, the trap stiffness recorded was in the order of 0.05 pN/nm. With the combination of position and trap stiffness, the force resolution of the instrument is about 0.25 pN. Also, a photobleaching event for a single dye molecule was recorded, proving the single molecule fluorescence capabilities. In addition, a complete experimental assay is described in order to perform studies on how force application affects the binding of actin and actin binding proteins.by Jorge M. Ferrer.S.M

Mapping the actin and actin binding proteins interactions : from micromechanics to single molecule force spectroscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.Mechanical forces play an important role in cell morphology, orientation, migration, adhesion and can even induce apoptosis. The eukaryotic cell is equipped with a dynamic frame, known as the cytoskeleton, that provides the cell's structural integrity in order to sustain and react to such forces. Therefore, understanding the mechanical properties of the cytoskeleton is an important step towards building models describing cell behavior. Filamentous actin (F-actin), as one of the major constituents of the cytoskeleton, has been the target of extensive in vitro studies to determine its mechanical properties in bulk. However, there is still a lack in the understanding of how the molecular interactions between F-actin and the proteins that arrange these filaments into networks regulate the dynamic properties of the cytoskeleton Here we present a novel, single molecule assay to test the rupture force of a complex formed by an actin binding protein (ABP) linking two actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin, a crosslinker, and a-actinin, a bundler. We measured rupture forces of 28-73 pN and 30-56 pN for filamin/actin and a-actinin/actin respectively, suggesting that the former is a slightly stronger interaction. Moreover, since no ABP unfolding events were observed at our force levels, our results suggest that ABP unbinding is a more relevant mechanism than unfolding for the temporal regulation of the mechanical properties of the actin cytoskeleton. In addition, we explore the micro-scale properties of F-actin networks reconstituted in vitro.(cont.) Using imaging and microrheology techniques we characterized the effects of filament length and degree of crosslinking on the structural arrangement and mechanical properties of F-actin networks. We found that the mechanical properties of these networks are length-scale dependent. Also, when probed with active methods, the F-actin networks exhibited strain hardening followed by a gradual softening at forces -30 pN, in good agreement with the single molecule rupture force of 28-73 pN. Thus, with the combination of single molecule and network studies, we can expand the knowledge-base on the regulation and control of the cellular machinery starting from the molecular building blocks.by Jorge M. Ferrer.Ph.D

Passive and active microrheology for cross-linked F-actin networks in vitro

Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.Singapore-MIT Alliance for Research and Technology (SMART)National Institute of General Medical Sciences (U.S.) (NIGMS (GM076689))National Science Foundation (U.S.) (NSF Career Award (0643745))Nicholas Hobson Wheeles, Jr. (Fellowship)W. M. Keck FoundationWestaway Research Fun

Postoperative Corynebacterium macginleyi endophthalmitis

A 72-year-old man with chronic endophthalmitis who received steroid treatment for 3 months came to our center. Sterile endophthalmitis after cataract extraction had been diagnosed. Aqueous samples including smears, classic cultures, and polymerase chain reaction were taken for microbiological study. Amplified DNA was sequenced to identify the pathogen. Polymerase chain reaction amplification was positive for bacteria. Sequence analysis showed Corynebacterium macginleyi as the causal agent in 48 hours. The culture and smear stains from the ocular samples were negative. The patient was successfully treated with vancomycin. Polymerase chain reaction and subsequent DNA-typing were useful in detecting the microorganisms that caused the chronic endophthalmitis

Responsive and Minimalist App Based on Explainable AI to Assess Palliative Care Needs during Bedside Consultations on Older Patients

[EN] Palliative care is an alternative to standard care for gravely ill patients that has demonstrated many clinical benefits in cost-effective interventions. It is expected to grow in demand soon, so it is necessary to detect those patients who may benefit from these programs using a personalised objective criterion at the correct time. Our goal was to develop a responsive and minimalist web application embedding a 1-year mortality explainable predictive model to assess palliative care at bedside consultation. A 1-year mortality predictive model has been trained. We ranked the input variables and evaluated models with an increasing number of variables. We selected the model with the seven most relevant variables. Finally, we created a responsive, minimalist and explainable app to support bedside decision making for older palliative care. The selected variables are age, medication, Charlson, Barthel, urea, RDW-SD and metastatic tumour. The predictive model achieved an AUC ROC of 0.83 [CI: 0.82, 0.84]. A Shapley value graph was used for explainability. The app allows identifying patients in need of palliative care using the bad prognosis criterion, which can be a useful, easy and quick tool to support healthcare professionals in obtaining a fast recommendation in order to allocate health resources efficiently.This work was supported by the InAdvance project (H2020-SC1-BHC-2018-2020 grant agreement number 825750.) and the CANCERLEss project (H2020-SC1-2020-Single-Stage-RTD grant agreement number 965351), both funded by the European Union's Horizon 2020 research and innovation programme.Blanes-Selva, V.; Doñate-Martínez, A.; Linklater, G.; Garcés-Ferrer, J.; Garcia-Gomez, JM. (2021). Responsive and Minimalist App Based on Explainable AI to Assess Palliative Care Needs during Bedside Consultations on Older Patients. Sustainability. 13(17):1-11. https://doi.org/10.3390/su13179844111131

Biological treatment of the organic fibre from the autoclaving of municipal solid wastes : preliminary results

Commingled municipal solid waste (MSW) was autoclaved in the presence of saturated steam for 30 min at 145 °C and 600 kPa. The organic fibre fraction from the autoclaved resulting material was examined for biodegradability. Aerobic and anaerobic tests were carried out to characterise the fibre in terms of biodegradation potential, which was moderate (biogas production potential of 251 ± 22 l [biogas] kg⁻¹ [total solids (TS)] and dynamic respiration index (DRI) of 1575 ± 116 mg [O2] kg⁻¹ [TS] h⁻¹). Manual and chemical characterisations were also performed to organic fibre. Following this characterisation, a laboratory-scale thermophilic anaerobic digestion process and a pilot-scale composting process were carried out to determine the possibilities of these biological treatments. In the anaerobic digestion process the biogas yield values obtained were within 0.15-0.21 m³ [biogas] kg⁻¹ [volatile solids (VS)] with an organic loading rate (OLR) of 3 kg [VS] m⁻³ d⁻¹. However, it was difficult to reach the steady state in the anaerobic thermophilic process for the different organic loads tested. Further experiments are necessary to determine the optimal biogas production and performance under these conditions. The composting process performed correctly and the final material was stable (DRI of 504 ± 74 mg [O2] kg⁻¹ [TS] h⁻¹) and with good properties for its application to soil regarding heavy metal contents that corresponding to class B compost, with the exception of some metals that corresponded to class A

Rapid Molecular Diagnosis of Posttraumatic Keratitis and Endophthalmitis Caused by Alternaria infectoria

The first case of Alternaria infectoria ocular infection is reported. Keratitis and endophthalmitis developed after eye-perforating trauma from a lemon tree branch. Two months after surgery and empirical steroid and antibiotic treatment, diagnosis by molecular methods was performed. PCR amplification was positive for a fungus after 4 h. Antifungal treatment with amphotericin B and fluconazole was initiated immediately. DNA sequence analysis showed Alternaria infectoria to be the causal agent. After topical and systemic administration of antifungal treatment, ocular inflammation disappeared and visual acuity improved. DNA typing was found to be a useful tool to achieve early identification of the causal agen

Transcriptional signatures of synaptic vesicle genes define myotonic dystrophy type I neurodegeneration

Aim: To delineate the neurogenetic profiles of brain degeneration patterns in myotonic dystrophy type I (DM1). Methods: In two cohorts of DM1 patients, brain maps of volume loss (VL) and neuropsychological deficits (NDs) were intersected to large-scale transcriptome maps provided by the Allen Human Brain Atlas (AHBA). For validation, neuropathological and RNA analyses were performed in a small series of DM1 brain samples. Results: Twofold: (1) From a list of preselected hypothesis-driven genes, confirmatory analyses found that three genes play a major role in brain degeneration: dystrophin (DMD), alpha-synuclein (SNCA) and the microtubule-associated protein tau (MAPT). Neuropathological analyses confirmed a highly heterogeneous Tau-pathology in DM1, different to the one in Alzheimer's disease. (2) Exploratory analyses revealed gene clusters enriched for key biological processes in the central nervous system, such as synaptic vesicle recycling, localization, endocytosis and exocytosis, and the serotonin and dopamine neurotransmitter pathways. RNA analyses confirmed synaptic vesicle dysfunction. Conclusions: The combination of large-scale transcriptome interactions with brain imaging and cognitive function sheds light on the neurobiological mechanisms of brain degeneration in DM1 that might help define future therapeutic strategies and research into this condition

A Novel Imaging System Permits Real-time in Vivo Tumor Bed Assessment After Resection of Naturally Occurring Sarcomas in Dogs

Background Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery. Questions/purposes To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin? Methods We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9–15 months). Results Fluorescence was apparent from all 10 tumors and ranged from 3 × 107 to 1 × 109 counts/millisecond/cm2. During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months. Conclusions These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections. Clinical Relevance The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control