Partial Unfolding of Tubulin Heterodimers Induced by Two-Photon Excitation of Bound <i>meso</i>-Tetrakis(sulfonatophenyl)porphyrin

The water-soluble porphyrin <i>meso</i>-tetrakis­(<i>p</i>-sulfonatophenyl)­porphyrin (TSPP) can be noncovalently bound to tubulin and used as a photosensitizer, which upon irradiation triggers photochemical reactions that lead to conformational changes of the protein. These conformational changes in turn inhibit tubulin’s primary function of polymerizing into microtubules. We explored the possibility of using two-photon excitation of the bound porphyrin to induce photosensitized protein unfolding. Although TSPP has a relatively low cross section (∼30 GM) our results did find that two-photon excitation of the ligand causes partial unfolding of the tubulin host and the inhibition of the <i>in vitro</i> formation of microtubules. Conversely, irradiating tubulin alone caused no such effects despite the large irradiance per pulse (97–190 GW/cm²). The conformational changes were characterized using spectroscopic studies and provide a promising protocol for the future application of non-native photosensitization of proteins

Resonance Raman and vibrational mode analysis used to predict ligand geometry for docking simulations of a water soluble porphyrin and tubulin

<p>The ability to modify the conformation of a protein by controlled partial unfolding may have practical applications such as inhibiting its function or providing non-native photosensitive properties. A water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin (TSPP), non-covalently bound to tubulin can be used as a photosensitizer, which upon irradiation can lead to conformational changes of the protein. To fully understand the mechanism responsible for this partial unfolding and determine the amino acid residues and atoms involved, it is essential to find the most likely binding location and the configuration of the ligand and protein. Techniques typically used to analyze atomic position details, such as nuclear magnetic resonance and X-ray crystallography, require large concentrations, which are incompatible with the dilute conditions required in experiments for photoinduced mechanisms. Instead, we develop an atomistic description of the TSPP–tubulin complex using vibrational mode analysis from density functional theory calculations correlated to resonance Raman spectra of the porphyrin paired with docking simulations. Changes in the Raman peaks of the porphyrin molecule correlate with changes in its structural vibrational modes when bound to tubulin. The data allow us to construct the relative geometry of the porphyrin when bound to protein, which are then used with docking simulations to find the most likely configuration of the TSPP–tubulin complex.</p

Systematic design, generation, and application of synthetic datasets for flow cytometry

Application of synthetic datasets in training and validation of analysis tools have led to improvements in many decision-making tasks in a range of domains from computer vision to digital pathology. Synthetic datasets overcome the constraints of real-world datasets, namely difficulties in collection and labelling, expense, time and privacy concerns. In flow cytometry, real cell-based datasets are limited by properties such as size, number of parameters, distance between cell populations and distributions, and are often focused on a narrow range of disease or cell types. Researchers in some cases have designed these desired properties into synthetic datasets, however operators have implemented them in inconsistent approaches and there is a scarcity of publicly available, high-quality synthetic datasets. In this research, we propose a method to systematically design and generate flow cytometry synthetic datasets with highly controlled characteristics. We demonstrate the generation of two-cluster synthetic datasets with specific degrees of separation between cell populations, and of non-normal distributions with increasing levels of skewness and orientations of skew pairs. We apply our synthetic datasets to test the performance of a popular automated cell populations identification software, SPADE3, and define the region where the software performance decreases as the clusters get closer together. Application of the synthetic skewed dataset suggests the software is capable of processing non-normal data. We calculate the classification accuracy of SPADE3 with robustness not achievable with real-world datasets. Our approach aims to advance research towards generation of high-quality synthetic flow cytometry datasets, and to increase their awareness among the community. The synthetic datasets can be utilised in benchmarking studies that critically evaluate cell population identification tools and help illustrate potential digital platform inconsistencies. These datasets have the potential to improve cell characterisation workflows that integrate automated analysis in clinical diagnostics and cell therapy manufacturing

Combination of Resonance Raman Spectroscopy and Docking Simulations to Study the Nonspecific Binding of a Free-Base Porphyrin to a Globular Protein

Understanding the conformational changes induced by small ligands noncovalently bound to proteins is a central problem in biophysics. We focus on the binding location of the water-soluble porphyrin, meso-tetrakis (<i>p</i>-sulfonatophenyl) porphyrin, to a globular protein, <i>β-</i>lactoglobulin, which has been observed to partially unfold when irradiated by laser light. Identifying the binding location is necessary to determine the mechanism of action as well as the atoms and residues involved in the photoinduced partial unfolding. Such atomic details are typically investigated by nuclear magnetic resonance or X-ray crystallography. However, for biomolecules in solution at the low concentrations (μM) required to deliver uniform laser irradiation, these traditional techniques do not currently provide sufficient information, and one must rely upon less direct spectroscopic methods. We describe a method that uses resonance Raman spectroscopy and density functional theory (DFT) to select the most likely binding configuration among a set of solutions yielded by computational docking algorithms. This methodology may be generalized to use with other ligand–protein complexes where the ligand structure is amenable to DFT simulations

Sleep Apnea Severity between Control Subjects and Affected Patients.

<p>A depicts the individual apnea-hypopnea indices (average number of respiratory events with at least 4% oxygen desaturation per hour; AHI-4%) between control versus sleep apneic groups. B depicts the lowest percent oxygen saturation recorded by overnight pulse oximetry between groups. Horizontal lines within each group of data points indicate the group mean.</p

Individual Urinary NGAL-to-Creatinine Ratios (ng/mg) between Control versus Untreated Sleep Apneic (OSA) Groups.

<p>Horizontal lines within each group of data points indicate the group median.</p

Patient Characteristics.

<p>OSA = obstructive sleep apnea, M = male, F = female, Afr Amer = African American, BMI = body mass index, DM = diabetes mellitus, HTN = hypertension, SBP = systolic blood pressure, DBP = diastolic blood pressure, AHI-4% = apnea-hypopnea index of respiratory events per hour with at least 4% oxygen desaturation, RDI = respiratory disturbance index, O2 sat % = percentage oxygen saturation recorded, Stage N1% = percentage of total sleep in non-REM sleep stage I, Stage N2% = percentage of total sleep in non-REM sleep stage II, Stage N3% = percentage of total sleep in non-REM sleep stage III, REM% = percentage of total sleep in rapid eye movement (REM) sleep. Table shows mean (+/- standard deviation) or N = number of patients falling into category.</p

Individual NGAL-to-Creatinine Ratios (NGAL/Cr; ng/mg) versus Apnea-Hypopnea Indices (AHI-4%; events/hour) among All Subjects (Untreated Sleep Apnea and Control).

<p>Individual NGAL-to-Creatinine Ratios (NGAL/Cr; ng/mg) versus Apnea-Hypopnea Indices (AHI-4%; events/hour) among All Subjects (Untreated Sleep Apnea and Control).</p

Urinary NGAL-to-Creatinine Ratios (NGAL/Cr; ng/mg) among 11 Sleep Apneic Patients (OSA) before (Pre-Tx) and after (Post-Tx) Successful Treatment with Continuous Positive Airway Pressure.

<p>Urinary NGAL-to-Creatinine Ratios (NGAL/Cr; ng/mg) among 11 Sleep Apneic Patients (OSA) before (Pre-Tx) and after (Post-Tx) Successful Treatment with Continuous Positive Airway Pressure.</p

Model-averaged parameter estimates (estimates of fixed effects included in models with ΔAICc<i>_i</i> ≤2 with contributions to average weighted by <i>w</i>AICc<i>_i</i> of model), unconditional standard errors and 95% confidence intervals.

<p>Interactions are indicated by x.</p><p>Hatching date (1 =  1^st May) and date found (date nest discovered; 1 =  1^st May) were standardized before being input in models as fixed factors.</p><p>Model-averaged parameter estimates (estimates of fixed effects included in models with ΔAICc<i>_i</i> ≤2 with contributions to average weighted by <i>w</i>AICc<i>_i</i> of model), unconditional standard errors and 95% confidence intervals.</p