Starch modification by annealing (ANN) and heat-moisture treatment (HMT) results in a lower crystallinity compared to native but the change of B crystalline type to A type is only observed in HMT starch. All starches possess two different digestion rate constants i.e. k1 (at rapid phase) and k2 (at slow phase) which may be linked to the preserved intact starch granule following thermal treatment. HMT starch contains higher content of slowly digestible starch (C2∞) compared to the C2∞ of the other starches. The lower enzyme binding to HMT starch (Kd value increases from 0.12 mg/mL in native starch to 0.83 mg/mL) may be linked to the increase in the degree of ordered structure of the granule surface (observed from the absorption band ratio of 1000 cm-1/1022 cm-1). The lower affinity may lead to a lower k1 value. This holds true for ANN and native starch which displays similar k1, Kd value and degree of ordered to disordered structure. Lower k2 in HMT starch compared to the corresponding k2 in the other starches may be linked to the slower enzyme diffusion into the core of starch granule due to the tightly packed structure of A crystalline type in HMT starch.

Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein-protein interactions. The main advantage of SPR is the ability of measuring binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the \"ligand,\" on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the \"analyte,\" over the surface containing the ligand. The binding is monitored by following changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 15 years, SPR has been intensively used in the study of bacterial secretion systems due to its ability of detecting highly dynamic complexes, which are difficult to investigate by other techniques. This chapter will guide users in setting up SPR experiments in order to identify protein complexes and to assess their binding affinity and/or kinetics. It will include detailed protocols for (i) immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetics measurements, and (iv) data analysis.

This article provides an ethnographic account of members of the Phellowship, a group of sober concert goers who connect and sometimes attend informal meetings at Phish concerts. The work attends to the ways this group is meaningful for people\'s sobriety and livelihoods. The overall purpose of this article is to provide a rich description of a novel approach to the creation and extension of a culture of recovery and sobriety through music and music communities.
This article utilizes two years of ethnographic data collected from interviews with roughly 20 participants and conversations from participant observation with people who are generally representative of the demographics of this group and fanbase (25-55, white, about half male and female presenting). The ethnographic data were physically coded into emergent themes and analyzed to highlight the important facets of participation and experience of members of the Phellowship. This method of qualitative analysis utilizes aspects of phenomenological inquiry, in that the subjective experiences of individuals are described and analyzed, to meet the larger aims of an ethnographic analysis that explicates the understandings and cultural practices of this community in their own words.
Thematic results include the performative and oral importance of narration at meetings, the semiotics of recovery and their community, the extension of sobriety beyond their \"programs\" or health-related arenas, and, most importantly, the role of connection and community. Each topic and result includes first-person accounts from interviews and participant observation to explicate how these thematic areas were chosen and have become meaningful for members of the Phellowship.
This nonprogrammatic approach to recovery and sobriety that focuses more on cultural practices and salient versions of people\'s everyday identities, rather than \"healing\" and process seems to be a promising and novel addition to better the lives of people with substance use disorders. This group, and the network of these groups that exists in the jam band music scene at large, could act as a model for person-first approaches to recovery that incorporate different elements of sociality, community, and creative expression.

Regardless of the promising use of nanoparticles (NPs) in biomedical applications, several toxic effects have increased the concerns about the safety of these nanomaterials. Although the pathways for NPs toxicity are diverse and dependent upon many parameters such as the nature of the nanoparticle and the biochemical environment, numerous studies have provided evidence that direct contact between NPs and biomolecules or cell membranes leads to cell inactivation or damage and may be a primary mechanism for cytotoxicity. In such a context, this work focused on developing a fast and accurate method to characterize the interaction between NPs, proteins and lipidic membranes by surface plasmon resonance imaging (SPRi) technique. The interaction of gold NPs with mimetic membranes was evaluated by monitoring the variation of reflectivity after several consecutive gold NPs injections on the lipidic membranes prepared on the SPRi biochip. The interaction on the membranes with varied lipidic composition was compared regarding the total surface concentration density of gold NPs adsorbed on them. Then, the interaction of gold and silver NPs with blood proteins was analyzed regarding their kinetic profile of the association/dissociation and dissociation constants (koff). The surface concentration density on the membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol (POPC/cholesterol) was 2.5 times higher than the value found after the injections of gold NPs on POPC only or with dimethyldioctadecylammonium (POPC/DDAB). Regarding the proteins, gold NPs showed preferential binding to fibrinogen resulting in a value of the variation of reflectivity that was 8 times higher than the value found for the other proteins. Differently, silver NPs showed similar interaction on all the tested proteins but with a variation of reflectivity on immunoglobulin G (IgG) 2 times higher than the value found for the other tested proteins.

BACKGROUND: CD123 is overexpressed on AML blasts compared to healthy myeloid progenitor cells. There is opportunity to design anti-CD123 therapy to discriminate on antigen density. Our goal is to study affinity modification of the single chain variable fragment (scFv) in the context of chimeric antigen receptor (CAR)-modified NK cell activation and target killing. We hypothesize that lower affinity CAR-NK cells will demonstrate improved discrimination between tumor and healthy tissue.
METHODS: We designed NK-cell specific CARs to contain anti-CD123 scFvs binding distinct target epitopes (26292 or 7G3) fused to intracellular portions of NK-activating receptors 4-1BB.CD3Z and 2B4.CD3Z. We synthesized novel 26292 and known 7G3 variants to capture a spectrum of CD123 binding affinities (26292: Kd=15nM; variant Kd range 0.29-160nM; 7G3: Kd=8.75nM; variant Kd range 23.9-64.9nM). These scFv were generated using site-directed mutagenesis of the wild type sequences and subcloned into CAR backbones. Retroviral transduction of primary human NK cells was performed, and CAR expression was measured using FACS (range 23.9%-89.6% CAR+ NK cells). Raji target cells (CD123neg) were engineered and sorted to obtain model cell lines with stable low (Raji.Low) and high (Raji. High) CD123 expression, comparable to healthy cells (CD123.Low) and AML blasts (CD123.High). We tested CAR-NK cell activation and antigen-specific cytotoxicity using in vitro co-culture assays. We established a mouse model of disease using implantation of Raji cells engineered to express firefly Luciferase (Raji.ffLuc).
RESULTS: Antitumor cytotoxicity of 26292 and 7G3-based CAR-NK cells with a range of binding affinities showed CAR-specific activation but did not differentiate on target antigen density. Similarly, no differences were seen in CAR-NK activation as measured by TNFa and IFNy secretion following coculture with CD123-positive cells. NSG mice implanted with Raji.ffLuc had median survival of 42 days without treatment.
CONCLUSIONS: There are no significant differences across a broad range of variable affinity 26292 and 7G3-based anti-CD123 CAR-NK cells in effector cell activation or target killing when assayed short-term. These data support the value of additional long-term investigation of the immune synapse and CAR-NK antitumor activity in vivo. This work is supported by the St. Baldrick\'s Foundation Fellowship Award and the Johns-Hopkins-Allegheny Health Network Cancer Research Fund.

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer\'s and Parkinson\'s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP-TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP-TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the -27G>A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP-TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p <0.01).
Гены GRIN1, ASCL3 и NOS1 связаны с различными фенотипами нервно-психических расстройств. Эти гены делают вклад в развитие шизофрении, болезней Альцгеймера и Паркинсона, эпилепсии и др. и ассоциируются также с различными онкологическими заболеваниями. Например, повышенная экспрессия ASCL3 наблюдается при раке молочной железы, NOS1 – в клеточных линиях рака яичников. Ранее на основе наших и литературных данных мы получили результаты, свидетельствующие в пользу того, что SNP, нарушающие эритропоэз, с большой вероятностью могут быть связаны с когнитивными и нервно-психическими расстройствами у человека. В настоящей работе исследовано влияние выявленных с помощью SNP_TATA_Z-tester неаннотированных SNP ТАТА-боксов промоторов генов GRIN1, ASCL3 и NOS1, участвующих в нервно-психических расстройствах и онкологических заболеваниях, на взаимодействие ТАТА-связывающего белка (ТВР). Для изучения in vitro кинетических характеристик образования комплексов ТВР/ТАТА и аффинности с помощью метода задержки ДНК в геле использованы двуцепочечные олигодезоксирибонуклеотиды, идентичные ТАТА-содержащим участкам промоторов генов GRIN1, ASCL3 и NOS1 (референсным и минорным аллелям), и рекомбинантный ТВР человека. Показано, например, что аллель «A» rs1402667001 промотора гена GRIN1 повышает аффинность ТВР/ТАТА в 1.4 раза, а аллель «С» ТАТА-бокса промотора гена ASCL3 снижает аффинность в 1.4 раза; при этом время жизни комплексов в обоих случаях уменьшается примерно на 20 % за счет изменения скоростей образования и диссоциации комплексов (ka и kd соответственно). Наши экспериментальные результаты согласуются с литературными данными, показывающими низкую экспрессию гена GRIN1 при шизофренических расстройствах и повышенный риск возникновения рака шейки матки, мочевого пузыря, почек и лимфомы при пониженной экспрессии гена АSCL3. Влияние аллеля «А» SNP –27G>A (rs1195040887) промотора гена NOS1 гипотетически может свидетельствовать о повышенном риске возникновения ишемического повреждения мозга у носителей. Сравнение экспериментальных значений аффинности (KD) ТВР/ТАТА «диких» (WT) и минорных аллелей c прогнозируемыми показало, что данные хорошо коррелируют друг с другом: коэффициент линейной корреляции r = 0.94 ( p < 0.01).

Thyroid hormones (TH), T4 and T3, mediate pro-mitogenic effects in cancer cells through binding the membrane receptor αvβ3 integrin. The deaminated analogue tetrac effectively blocks TH binding to this receptor and prevents their action. While computational data on TH binding to the αvβ3 integrin was published, a comprehensive analysis of additional TH metabolites is lacking.
In-silico docking of 26 TH metabolites, including the biologically active thyroid hormones (T3 and T4) and an array of sulfated, deiodinated, deaminated or decarboxylated metabolites, to the αvβ3 receptor binding pocket was performed using DOCK6, based on the three-dimensional representation of the crystallographic structure of the integrin. As the TH binding site upon the integrin is at close proximity to the well-defined RGD binding site, linear and cyclic RGD were included as a reference. Binding energy was calculated for each receptor-ligand complex using Grid score and Amber score with distance movable region protocol.
All TH molecules demonstrated negative free energy, suggesting affinity to the αvβ3 integrin. Notably, based on both Grid and Amber scores sulfated forms of 3,3\' T2 (3,3\' T2S) and T4 (T4S) demonstrated the highest binding affinity to the integrin, compared to both cyclic RGD and an array of examined TH metabolites. The major thyroid hormones, T3 and T4, showed high affinity to the integrin, which was superior to that of linear RGD. For all hormone metabolites, decarboxylation led to decreased affinity. This corresponds with the observation that the carboxylic group mediates binding to the integrin pocket via divalent cations at the metal-ion-dependent adhesion (MIDAS) motif site. A similar reduced affinity was documented for deaminated forms of T3 (triac) and T4 (tetrac). Lastly, the reverse forms of T3, T3S, and T3AM showed higher Amber scores relative to their native form, indicating that iodination at position 5 is associated with increased binding affinity compared to position 5\'.
Three-dimensional docking of various TH metabolites uncovered a structural basis for a differential computational free energy to the αvβ3 integrin. These findings may suggest that naturally occurring endogenous TH metabolites may impact integrin-mediate intracellular pathways in physiology and cancer.

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.

The structures of several aquaglyceroporins have been resolved to atomic resolution showing two or more glycerols bound inside a channel and confirming a glycerol-facilitator\'s affinity for its substrate glycerol. However, the kinetics data of glycerol transport experiments all point to unsaturated transport that is characteristic of low substrate affinity in terms of the Michaelis-Menten kinetics. In this article, we present an in silico-in vitro research focused on AQP3, one of the human aquaglyceroporins that is natively expressed in the abundantly available erythrocytes. We conducted 2.1 μs in silico simulations of AQP3 embedded in a model erythrocyte membrane with intracellular-extracellular asymmetries in leaflet lipid compositions and compartment salt ions. From the equilibrium molecular dynamics (MD) simulations, we elucidated the mechanism of glycerol transport at high substrate concentrations. From the steered MD simulations, we computed the Gibbs free-energy profile throughout the AQP3 channel. From the free-energy profile, we quantified the kinetics of glycerol transport that is unsaturated due to glycerol-glycerol interactions mediated by AQP3 resulting in the concerted movement of two glycerol molecules for the transport of one glycerol molecule across the cell membrane. We conducted in vitro experiments on glycerol uptake into human erythrocytes for a wide range of substrate concentrations and various temperatures. The experimental data quantitatively validated our theoretical-computational conclusions on the unsaturated glycerol transport through AQP3 that has high affinity for glycerol.