Tissue Binding Research Articles

The Effects of Structural & Materials Design on the Mechanisms of Tissue Integration with the 3D Printed Polyether-Ether-Ketone Cranial Implants in Vivo

3D printed polyether-ether-ketone (PEEK) implant has become a popular clinical alternative to implants made of titanium alloy for cranial bone substitutes due to its bone-match properties, however its biological inert hindered the progress of clinical applications of such material. To enhance the tissue integration capability of PEEK implants and promote their long-term stability, cranial implants of gradient porous structures were designed and manufactured via fused filament fabrication (FFF) 3D printing technology in both PEEK and PEEK/HA composites materials, then functionally evaluation of the implants on the tissue in-growth and the osteointegration mechanisms was conducted via animal tests. The 3D printed PEEK scaffold was found to have 2–5 folds of the compressive strength to those of the natural cranial bone. The in vivo studies verified that the porous PEEK/HA scaffold could effectively induce the bone ingrowth to form a stable biointegration boundary surrounding the host bone tissue after 4 weeks of implantation. Moreover, the PEEK/HA scaffold showed no significant advantages in improving the soft tissue in-growth, only making its distribution more evenly. It is also interestingly to find out that the vertically connective pores throughout the implant did not enhance the tissue binding force even though it did promote the nutrient transportation. In conclusion, the use of PEEK/HA composite material and a well-designed porous structure was proved to be an effective approach to improve the biointegration between the implant and host tissues.

Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-β-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a β-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or β-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for β-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between β-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

A Tibetan window into the twentieth-century Himalayan world

This article examines interconnections between parts of the Indian Himalaya and Tibet in the period encompassing the escape of the Fourteenth Dalai Lama to India in 1959. While Buddhism served as connecting tissue binding together communities across recently drawn national borders, networks linking families in Sikkim, Bhutan and Tibet were also forged through monastic patronage, colonial education, intermarriage, seasonal migration and trade. Through a reading of colonial and postcolonial archives from Delhi, London and Gangtok, as well as the private papers of Indian political appointees in the Himalaya, the article shows how, far from blazing a trail, the Dalai Lama and the Tibetans who followed him into exile were treading well-worn migration routes and leaning on relationships forged over centuries. Archival evidence from press and parliamentary proceedings in the 1950s and 1960s reveals a shared religious history and information that India claimed to be the cradle of the subcontinent’s Buddhist heritage, which helped garner greater support for the Dalai Lama and his countrymen.

Novel melanin‐binding carbonic anhydrase II inhibitor for long‐acting topical glaucoma treatment

Aims/Purpose: We are developing new eye drop treatment for glaucoma with dosing frequency once a week by utilizing drug binding to ocular melanin. Carbonic anhydrase (CA) II enzyme inhibitors decrease intraocular pressure (IOP) and thus halt the progression of retinal damage by reducing the secretion of aqueous humour in the ciliary body. Since ciliary body is a melanin‐rich tissue, we hypothesize that long‐lasting CAII inhibition and IOP‐lowering effect may be achieved due to gradual drug release from melanin‐bound drug depot.Methods: Melanin binding affinity for about 30 novel CAII inhibitors was studied with microscale thermophoresis, and three structurally rather similar and equipotent molecules with different melanin binding properties were selected for in vivo efficacy studies. IOP‐lowering effect of these new molecules was studied with albino and pigmented rabbits with iCare® PRO rebound tonometer. For pharmacokinetic studies, the high melanin binding new CAI (molecule 184) was labelled with tritium, and the study was conducted with albino and pigmented rats with conventional methods. Bound drug was analysed from iris‐ciliary body pellet and unbound fraction from supernatants.Results: Melanin‐binding affinity of new CAII inhibitors varies from low or non‐binders (Kd > 650 μM) to high melanin binders (Kd < 65 μM). In vivo efficacy results show that the molecules decrease IOP in both albino and pigmented eyes at the level comparable with dorzolamide (Trusopt®). In pigmented eyes, melanin binding prolongs the effect of compound 184 even up to 2 weeks after single eye drop. In the pharmacokinetic rat study, molecule 184 shows remarkably prolonged tissue binding and high iris‐ciliary body exposure in pigmented eyes.Conclusions: After topical administration IOP‐lowering effect of high melanin‐binding CA II inhibitor is significantly prolonged in pigmented eyes.

Evaluation of an Image-Derived Input Function for Kinetic Modeling of Nicotinic Acetylcholine Receptor-Binding PET Ligands in Mice.

Positron emission tomography (PET) radioligands that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allow for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. Here, we investigate the use of an image-derived arterial input function and the cerebellum for kinetic analysis of radioligand binding in mice. Two radioligands were explored: 2-[18F]FA85380 (2-FA), displaying similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, displaying similar pKa and binding affinity to nicotine. Time-activity curves of the left ventricle of the heart displayed similar distribution across wild type mice, mice lacking the β2-subunit for ligand binding, and acute nicotine-treated mice, whereas reference tissue binding displayed high variation between groups. Binding potential estimated from a two-tissue compartment model fit of the data with the image-derived input function were higher than estimates from reference tissue-based estimations. Rate constants of radioligand dissociation were very slow for 2-FA and very fast for Nifene. We conclude that using an image-derived input function for kinetic modeling of nicotinic PET ligands provides suitable results compared to reference tissue-based methods and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

Sea-ATI unravels novel vocabularies of plant active cistrome.

The cistrome consists of all cis-acting regulatory elements recognized by transcription factors (TFs). However, only a portion of the cistrome is active for TF binding in a specific tissue. Resolving the active cistrome in plants remains challenging. In this study, we report the assay sequential extraction assisted-active TF identification (sea-ATI), a low-input method that profiles the DNA sequences recognized by TFs in a target tissue. We applied sea-ATI to seven plant tissues to survey their active cistrome and generated 41 motif models, including 15 new models that represent previously unidentified cis-regulatory vocabularies. ATAC-seq and RNA-seq analyses confirmed the functionality of thecis-elements from the new models, in that they are actively bound in vivo, located near the transcription start site, and influence chromatin accessibility and transcription. Furthermore, comparing dimeric WRKY CREs between sea-ATI and DAP-seq libraries revealed that thermodynamics and genetic drifts cooperatively shaped their evolution. Notably, sea-ATI can identify not only positive but also negative regulatory cis-elements, thereby providing unique insights into the functional non-coding genome of plants.

A New Version of the Tissue Composition-Based Model for Improving the Mechanism-Based Prediction of Volume of Distribution at Steady-State for Neutral Drugs

In-vitro models are available in the literature for predicting the volume of distribution at steady-state (Vdss) of drugs. The mechanistic model refers to the tissue composition-based model (TCM), which includes important factors that govern Vdss such as drug physiochemistry and physiological data. The recognized TCM published by Rodgers and Rowland (TCM-RR) and a subsequent adjustment made by Simulations Plus Inc. (TCM-SP) have been shown to be generally less accurate with neutral compared to ionized drugs. Therefore, improving these models for neutral drugs becomes necessary. The objective of this study was to propose a new TCM for improving the prediction of Vdss for neutral drugs. The new TCM included two modifications of the published models (i) accentuate the effect of the blood-to-plasma ratio (BPR) that should cover permeated molecules across the biomembranes, which is lacking in these models for neutral compounds, and (ii) use a different approach to estimate the binding in tissues. The new TCM was validated with a large dataset of 202 commercial and proprietary compounds including preclinical and clinical data. All scenario datasets were predicted more accurately with the TCM-New, whereas all statistical parameters indicate that the TCM-New showed significant improvements in terms of accuracy over the TCM-RR and TCM-SP. Predictions of Vdss were frequently more accurate for the TCM-new with 83% within twofold error versus only 50% for the TCM-RR. And more than 95% of the predictions were within threefold error and patient interindividual differences can be predicted with the TCM-New, greatly exceeding the accuracy of the published models. Overall, the new TCM incorporating BPR significantly improved the Vdss predictions in animals and humans for neutral drugs, and, hence, has the potential to better support the drug discovery and facilitate the first-in-human predictions.

Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.

Blood- and Urine-Based Liquid Biopsy for Early-Stage Cancer Investigation: Taken Clear Renal Cell Carcinoma as a Model

Liquid biopsy is a noninvasive technique that can provide valuable information for disease characterization by using biofluids as a source of biomarkers. Proteins found in biofluids can offer a wealth of information for understanding pathological processes. In this study, we used early-stage clear cell renal cell carcinoma (ccRCC) as a model to explore the proteomic relationships among tissue, plasma, and urine. We analyzed samples of tumor tissue, plasma, and urine from a cohort of 27 ccRCC patients with T1-2 stage and 27 matched healthy controls, using liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis. We integrated the differential proteins found in the three types of samples to explore ccRCC-associated molecular changes. Our results showed that both plasma and urine proteomes could reflect functional changes in tumor tissue. In plasma, cytoskeletal proteins and metabolic enzymes were differentially expressed, while in urine, adhesion molecules and defense proteins showed differential levels. The differential proteins found in plasma and urine both reflect the binding and catalytic activity of tumor tissue. Additionally, proteins only changed in biofluids could reflect body immune response changes, with plasma proteins involved in actin cytoskeleton and oxidative stress, and urine proteins involved in granulocyte adhesion and leukocyte extravasation signaling. Plasma and urine proteins could effectively distinguish RCC from control, with good performances (plasma/urine: 92.6%/92.6% specificity, 96.3%/92.6% sensitivity, and an area under the curve of 0.981/0.97). In conclusion, biofluids could not only reflect functional changes in tumor tissue but also reflect changes in the body's immune response. These findings will benefit the understanding of body biomarkers in tumors and the discovery of potential disease biomarkers.

Synaptic vesicle glycoprotein 2A (SV2A) levels in the cerebral cortex in patients with Alzheimer's disease: a radioligand binding study in postmortem brains

Histological and biochemical analyses in postmortem tissues have demonstrated neurodegenerative changes in the cerebral cortex in patients with Alzheimer's disease (AD), and it has been suggested that this represents a loss of synapses. PET imaging of the (pre)synaptic vesicular glycoprotein 2A (SV2A) has demonstrated a reduction in synapse density in AD in the hippocampus but not consistently in the neocortex. This investigation examines the level of [3H]UCB-J binding in postmortem cortical tissue from patients with AD and matched healthy controls using autoradiography. Among the neocortical areas examined, the binding was significantly lower only in the middle frontal gyrus in AD compared to matched controls. No differences were observed in the parietal, temporal, or occipital cortex. The binding levels in the frontal cortex in the AD cohort displayed large variability among subjects, and this revealed a highly significant negative association with the age of the patient. These results demonstrate low UCB-J binding in the frontal cortex of patients with AD, and this biomarker correlates negatively with age, which may further indicate that SV2A could be an important biomarker in AD patients.

Computational design and molecular modeling of morphine derivatives for preferential binding in inflamed tissue.

The opioid epidemic has impacted over 10million Americans in 2019. Opioids, like morphine, bind non-selectively in both peripheral tissue, leading to effective pain relief, and central tissue, resulting in dangerous side effects and addiction. The inflamed conditions of injured tissues have a lower pH (pH=6-6.5) environment than healthy tissue (pH=7.4). We aim to design a morphine derivative that binds selectively within inflamed tissue using molecular extension and dissection techniques. Morphine binds to the μ-opioid receptor (MOR) when the biochemically active amine group is protonated. Fluorination of a β-carbon from the tertiary amine group led to a reduced pKa of the derivative through induction. Through a decrease in the pKa, protonation is still statistically favored in lower pH environments of inflamed tissue but primarily deprotonated in healthy tissue. The cyclohexenol and N-methyl-piperidine rings of morphine are removed to increase conformational flexibility when binding while still maintaining the interactions required for analgesia. Electronic structure calculations were performed with Gaussian16 using the Keck Computational Research Cluster at Chapman University to determine the pKa. The theoretical pKa values are determined at the M06-2X(SMD)/aug-cc-pVDZ level of theory to calculate the ΔG°aq values for the amine deprotonation reactions. Fluoromorphine β-C2 was designed computationally and modeled within the MOR using Maestro: Schrödinger. This derivative exhibits a pKa reduction and enhanced ligand-protein interactions within the MOR. β-fluorination decreased the overall pKa values of the morphine derivatives (pKa: 6.1-7.83) relative to morphine, reducing binding within healthy, central tissue.

UNUSUAL PRESENTATION OF LUPUS NEPHRITIS IN A 22 YEAR OLD MALE : A CASE REPORT

Systemic lupus erythematosus (SLE) is an autoimmune disease in which organ and cell undergo damage initially mediated by tissue binding autoantibodies & immune complexes.In most patients, autoantibodies are present for a few years before the rst clinical symptoms appears. It is ve to six times more prevalent in women than in men .Nephritis is usually the most serious manifestation of SLE, particularly because nephritis and infection are the leading cause of mortality in the rst decade of disease. We present a case of a 22 yr old male who came with complaint of bilateral pedal oedema and pufness of face since 1 month . On examination, patient was stable hemodynamically with pulse of 88 per minute, blood pressure of 110/80 millimeter of mercury with room air saturation of 99 percent and jugular venous pressure of 6-7 millimeters of water. He also had pallor in lower palpebral conjunctiva and bilateral pitting pedal oedema.There was no clubbing, icterus,cyanosis or lymphadenopathy. His routine blood investigations showed anaemia , leucocytosis , elevated creatinine, hyperkalaemia , hypoalbuminemia, iron deciency ,urine routine microscopy suggestive of RBC cast 60-70/high power eld . Complement C3 and C4 were low, ANA positive grade 3(1:1000) homogeneous and cytoplasmic speckled, ANA BLOT positive for dsDNA, nucleosome, Histones, Sm/RNP,Mi2b.His ultrasound (USG) revealed bilateral bulky kidney and heterogeneous with raised cortical echogenicity and normal size. His renal biopsy was done which showed diffuse lupus nephritis(ISN/RPS2018 modication)class IV and indices(modied NIH) of activity 15/24 and chronicity 2/12, necrotizing vascular lesions along with evidence of vascular immune deposits in diffuse immunoorescence studies and associated inammatory cells are noted. Patient received intravenous pulse therapy of methyl prednisolone 1gram for 5 days with monitoring of kidney function test. Patient was put on cyclophosphamide with hemodialysis for immunosuppression because patient was not responding to steroids, as seen by rising serum creatinine and urea levels and declining urine output. The patient improved dramatically, urine output increased and serum creatinine and urea decreased, therefore haemodialysis was discontinued, and the patient was managed conservatively and discharged on oral prednisolone with a monthly cyclophospmide injection.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Feasibility Study to Assess Canagliflozin Distribution and Sodium-Glucose Co-Transporter 2 Occupancy Using [18 F]Canagliflozin in Patients with Type 2 Diabetes.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors, including canagliflozin, reduce the risk of cardiovascular and kidney outcomes in patients with and without type 2 diabetes, albeit with a large interindividual variation. The underlying mechanisms for this variation in response might be attributed to differences in SGLT2 occupancy, resulting from individual variation in plasma and tissue drug exposure and receptor availability. We performed a feasibility study for the use of [18 F]canagliflozin positron emission tomography (PET) imaging to determine the association between clinical canagliflozin doses and SGLT2 occupancy in patients with type 2 diabetes. We obtained two 90-minute dynamic PET scans with diagnostic intravenous [18 F]canagliflozin administration and a full kinetic analysis in 7 patients with type 2 diabetes. Patients received 50, 100, or 300 mg oral canagliflozin (n = 2:4:1) 2.5 hours before the second scan. Canagliflozin pharmacokinetics and urinary glucose excretion were measured. The apparent SGLT2 occupancy was derived from the difference between the apparent volume of distribution of [18 F]canagliflozin in the baseline and post-drug PET scans. Individual canagliflozin area under the curve from oral dosing until 24-hours (AUCP0-24h ) varied largely (range 1,715-25,747 μg/L*hour, mean 10,580 μg/L*hour) and increased dose dependently with mean values of 4,543, 6,525, and 20,012 μg/L*hour for 50, 100, and 300 mg, respectively (P = 0.046). SGLT2 occupancy ranged between 65% and 87%, but did not correlate with canagliflozin dose, plasma exposure, or urinary glucose excretion. We report the feasibility of [18 F]canagliflozin PET imaging to determine canagliflozin kidney disposition and SGLT2 occupancy. This suggests the potential of [18 F]canagliflozin as a tool to visualize and quantify clinically SGLT2 tissue binding.

Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands.

The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647). Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue. All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex. Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans.

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.

Dual specificity and target gene selection by the MADS-domain protein FRUITFULL.

How transcription factors attain their target gene specificity and how this specificity may be modulated, acquiring different regulatory functions through the development of plant tissues, is an open question. Here we characterized different regulatory roles of the MADS-domain transcription factor FRUITFULL (FUL) in flower development and mechanisms modulating its activity. We found that the dual role of FUL in regulating floral transition and pistil development is associated with its different in vivo patterns of DNA binding in both tissues. Characterization of FUL protein complexes by liquid chromatography-tandem mass spectrometry and SELEX-seq experiments shows that aspects of tissue-specific target site selection can be predicted by tissue-specific variation in the composition of FUL protein complexes with different DNA binding specificities, without considering the chromatin status of the target region. This suggests a role for dynamic changes in FUL TF complex composition in reshaping the regulatory functions of FUL during flower development.

Characterization and Functional Analysis of OcomOBP7 in Ophraella communa Lesage

The olfactory system plays a key role in various insect behaviors, and odorant-binding proteins participate in the first step of the olfactory process. Ophraella communa Lesage is an oligophagous phytophagous insect that is a specific biological control agent for Ambrosia artemisiifolia L. The leaf beetle must identify and locate A. artemisiifolia through olfaction; however, its odorant-binding protein (OBP) function has not yet been reported. In this study, OcomOBP7 was cloned, and its tissue expression profile and binding ability were analyzed using RT-qPCR and fluorescence binding assays, respectively. Sequence analysis demonstrated that OcomOBP7 belongs to the classical OBP family. The RT-qPCR results showed that OcomOBP7 was specifically expressed in the antennae, indicating that OcomOBP7 may be involved in chemical communication. The fluorescence binding assay showed that OcomOBP7 has an extensive binding ability to alkenes. The electroantennography experiments showed that O. communa antennal response to α-pinene and ocimene decreased significantly after interference because the two odors specifically bound to OcomOBP7. In summary, α-pinene and ocimene are odorant ligands corresponding to OcomOBP7, indicating that OcomOBP7 is involved in the chemical recognition of A. artemisiifolia. Our study lays a theoretical foundation for research into O. communa attractants, which is helpful for the better biological control of A. artemisiifolia by O. communa.

Compartment model-based nonlinear unmixing for kinetic analysis of dynamic PET images.

When no arterial input function is available, quantification of dynamic PET images requires a previous step devoted to the extraction of a reference time-activity curve (TAC). Factor analysis is often applied for this purpose. This paper introduces a novel approach that conducts a new kind of nonlinear factor analysis relying on a compartment model, and computes the kinetic parameters of specific binding tissues jointly. To this end, it capitalizes on data-driven parametric imaging methods to provide a physical description of the underlying PET data, directly relating the specific binding with the kinetics of the non-specific binding in the corresponding tissues. This characterization is introduced into the factor analysis formulation to yield a novel nonlinear unmixing model designed for PET image analysis. This model also explicitly introduces global kinetic parameters that allow for a direct estimation of a binding potential that represents the ratio at equilibrium of specifically bound radioligand to the concentration of nondisplaceable radioligand in each non-specific binding tissue. The performance of the method is evaluated on synthetic and real data to demonstrate its potential interest.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects. This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.