scholarly journals Harnessing the Intrinsic Photochemistry of Isoxazoles for the Development of Chemoproteomic Crosslinking Methods

2021 ◽  
Author(s):  
Marshall G. Lougee ◽  
Vinayak Vishnu Pagar ◽  
Hee Jong Kim ◽  
Samantha X. Pancoe ◽  
Robert H. Mach ◽  
...  
Keyword(s):  
Protein Binding ◽  
Small Molecules ◽  
Functional Groups ◽  
Ex Vivo ◽  
Initial Report ◽  
Biologically Relevant ◽  
Fine Grained ◽  
Wide Range ◽  
Unique Method

Photo-crosslinking is a powerful technique for identifying both coarse- and fine-grained information on protein binding by small molecules. However, the scope of useful functional groups remains limited, with most studies focusing on diazirine, aryl azide, or benzophenone-containing molecules. Here, we report a unique method for photo-crosslinking, employing the intrinsic photochemistry of the isoxazole, a common heterocycle in medicinal chemistry, to offer an alternative to existing strategies using more perturbing, extrinsic crosslinkers. In this initial report, this technique is applied both in vitro and ex vivo, used in a variety of common chemoproteomic workflows, and validated across multiple proteins, demonstrating the utility of isoxazole photo-crosslinking in a wide range of biologically relevant experiments.

scholarly journals hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

2022 ◽  
Author(s):  
Homa Majd ◽  
Ryan M Samuel ◽  
Jonathan T Ramirez ◽  
Ali Kalantari ◽  
Kevin Barber ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Xenograft Model ◽  
Developmental Relationships ◽  
Drug Candidates ◽  
Effective Strategies ◽  
Wide Range ◽  
Functional Features ◽  
And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.  

scholarly journals In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
N. N. Parayath ◽  
S. B. Stephan ◽  
A. L. Koehne ◽  
P. S. Nelson ◽  
M. T. Stephan
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Human Leukemia ◽  
Antigen Receptors ◽  
Wide Range ◽  
Engineered T Cells ◽  
Circulating T Cells ◽  
The Cost

AbstractEngineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

scholarly journals Inhibition of Stimulated Interleukin-2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect

1999 ◽  
Vol 45 (9) ◽  
pp. 1477-1484 ◽  
Author(s):  
C Michael Stein ◽  
John J Murray ◽  
Alastair JJ Wood
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Major Effect ◽  
Biologically Relevant ◽  
Maximum Inhibition ◽  
Before And After ◽  
Dose Dependent Fashion ◽  
The Relationship

Abstract Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, measurement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic. Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated IL-2 production, measured by ELISA, was studied ex vivo in whole blood drawn before, and after subjects received 4 mg/kg oral CSA. Results: Four hours after CSA was administered, the mean (± SD) CSA concentration was 702 ± 196 μg/L and PHA-stimulated IL-2 production decreased by 68.7% ± 17.2% (P <0.0001; n = 17). Twenty-four hours after CSA was administered, concentrations were low (64 ± 24 μg/L), with no inhibition of IL-2 production. A rapid, concentration-dependent response occurred. Maximum CSA concentrations (944 ± 187 μg/L) and maximum inhibition of IL-2 production (86.9% ± 13.7%) occurred 90 min after subjects received CSA. In vitro, 32.5–1200 μg/L CSA also inhibited PHA-stimulated IL-2 production in whole blood in a dose-dependent fashion with a similar IC50 (∼300–400 μg/L) ex vivo and in vitro. Conclusion: In the search for a pharmacodynamic marker to better guide immunosuppressive therapy, the relationship between this simple, biologically relevant measure of CSA effect and clinical outcome should be determined.

scholarly journals A Novel PET Imaging Using64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
Kazuko Kobayashi ◽  
Takanori Sasaki ◽  
Fumiaki Takenaka ◽  
Hiromasa Yakushiji ◽  
Yoshihiro Fujii ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cancer Cells ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Chelating Agent ◽  
Pancreatic Cancer Cells ◽  
Positron Emission ◽  
Wide Range

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb toin vivoimaging to detect MSLN-expressing tumors. Inin vitroandex vivoimmunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with64Cu via a chelating agent DOTA and was used in bothin vitrocell binding assay andin vivopositron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

scholarly journals CLIPB10 is a Terminal Protease in the Regulatory Network That Controls Melanization in the African Malaria Mosquito Anopheles gambiae

2021 ◽  
Vol 10 ◽  
Author(s):  
Xin Zhang ◽  
Miao Li ◽  
Layla El Moussawi ◽  
Sally Saab ◽  
Shasha Zhang ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Serine Proteases ◽  
Ex Vivo ◽  
Protein A ◽  
Tumor Formation ◽  
Sub Saharan Africa ◽  
Humoral Immune ◽  
Wide Range ◽  
Sub Saharan

Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.

scholarly journals Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-β and α-Synuclein and Suppress the Toxicity of Their Oligomers

2021 ◽  
Vol 15 ◽  
Author(s):  
Ryan Limbocker ◽  
Roxine Staats ◽  
Sean Chia ◽  
Francesco S. Ruggeri ◽  
Benedetta Mannini ◽  
...  
Keyword(s):  
Small Molecules ◽  
Neuroblastoma Cells ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Parkinson’S Diseases ◽  
Wide Range ◽  
Opposing Effects

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer’s and Parkinson’s diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer’s and Parkinson’s diseases.

scholarly journals Pump-Free Microfluidic Hemofiltration Device

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 992
Author(s):  
Takahiro Ito ◽  
Takashi Ota ◽  
Rei Kono ◽  
Yoshitaka Miyaoka ◽  
Hidetoshi Ishibashi ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Small Molecules ◽  
Ex Vivo ◽  
Small Region ◽  
Transmembrane Pressure ◽  
In Vitro Experiments ◽  
Oncotic Pressure ◽  
Beagle Dogs ◽  
Electrical Components

Hemofiltration removes water and small molecules from the blood via nanoporous filtering membranes. This paper discusses a pump-free hemofiltration device driven by the pressure difference between the artery and the vein. In the design of the filtering device, oncotic pressure needs to be taken into consideration. Transmembrane pressure (TMP) determines the amount and direction of hemofiltration, which is calculated by subtracting the oncotic pressure from the blood pressure. Blood pressure decreases as the channels progress from the inlet to the outlet, while oncotic pressure increases slightly since no protein is removed from the blood to the filtrate in hemofiltration. When TMP is negative, the filtrate returns to the blood, i.e., backfiltration takes place. A small region of the device with negative TMP would thus result in a small amount of or even zero filtrates. First, we investigated this phenomenon using in vitro experiments. We then designed a hemofiltration system taking backfiltration into consideration. We divided the device into two parts. In the first part, the device has channels for the blood and filtrate with a nanoporous membrane. In the second part, the device does not have channels for filtration. This design ensures TMP is always positive in the first part and prevents backfiltration. The concept was verified using in vitro experiments and ex vivo experiments in beagle dogs. Given the simplicity of the device without pumps or electrical components, the proposed pump-free hemofiltration device may prove useful for either implantable or wearable hemofiltration.

scholarly journals An Overview of Biological and Computational Methods for Designing Mechanism-Informed Anti-biofilm Agents

2021 ◽  
Vol 12 ◽  
Author(s):  
Andy Y. An ◽  
Ka-Yee Grace Choi ◽  
Arjun S. Baghela ◽  
Robert E. W. Hancock
Keyword(s):  
Antibiotic Resistance ◽  
Small Molecules ◽  
In Silico ◽  
Biofilm Formation ◽  
Stringent Response ◽  
Antibiotic Resistant ◽  
Biologically Relevant ◽  
Biofilm Model

Bacterial biofilms are complex and highly antibiotic-resistant aggregates of microbes that form on surfaces in the environment and body including medical devices. They are key contributors to the growing antibiotic resistance crisis and account for two-thirds of all infections. Thus, there is a critical need to develop anti-biofilm specific therapeutics. Here we discuss mechanisms of biofilm formation, current anti-biofilm agents, and strategies for developing, discovering, and testing new anti-biofilm agents. Biofilm formation involves many factors and is broadly regulated by the stringent response, quorum sensing, and c-di-GMP signaling, processes that have been targeted by anti-biofilm agents. Developing new anti-biofilm agents requires a comprehensive systems-level understanding of these mechanisms, as well as the discovery of new mechanisms. This can be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics, which can also be integrated to better understand biofilm biology. Guided by mechanistic understanding, in silico techniques such as virtual screening and machine learning can discover small molecules that can inhibit key biofilm regulators. To increase the likelihood that these candidate agents selected from in silico approaches are efficacious in humans, they must be tested in biologically relevant biofilm models. We discuss the benefits and drawbacks of in vitro and in vivo biofilm models and highlight organoids as a new biofilm model. This review offers a comprehensive guide of current and future biological and computational approaches of anti-biofilm therapeutic discovery for investigators to utilize to combat the antibiotic resistance crisis.

scholarly journals ANTIEPILEPTIC RECTAL HYDROGEL LOADED WITH CARBAMAZEPINE – RICE BRAN WAX MICROSPHERES

2017 ◽  
Vol 10 (3) ◽  
pp. 264 ◽  
Author(s):  
Sreeja C Nair ◽  
Krishnapriya M ◽  
Karthika Ramesh
Keyword(s):  
Controlled Release ◽  
Drug Release ◽  
Rice Bran ◽  
Ex Vivo ◽  
Rectal Mucosa ◽  
Natural Polymers ◽  
Rice Bran Wax ◽  
Release Pattern ◽  
Wide Range

ABSTRACTObjectives: The objective behind the study is to develop a mucoadhesive rectal hydrogel from carbamazepine (CBZ) – rice bran wax (RBW)microspheres for the purpose of controlled release for the treatment of epilepsy.Methods: The study was conducted to formulate controlled release rectal hydrogel loaded with CBZ – RBW microspheres in two different natural polymers,RBW and collagen which are prepared by modified cooling induced solidification method and gel preparation along with their evaluation studies.Results: A thorough analysis of the optimized gel revealed that all the evaluation parameters evaluated are within the acceptable limits. Further, theoptimized microsphere formulation (M5) was used to formulate it as rectal hydrogel using polymer collagen and was characterized. The mucoadhesiontime of 25% w/w collagen hydrogel (H4) was 565 minutes, allowing the loaded microspheres to be attached on rectal mucosa. In vitro drug releasefrom the mucoadhesive hydrogel formulations showed controlled drug release pattern with a maximum drug release of 96.45±0.35% for optimizedH4 formulation after 12 hr, followed zero order release pattern with diffusion mediated Higuchi model. Ex vivo permeation studies using bovine rectalmucosa revealed that H4 formulation showed greater permeability compared to control. Histopathological findings revealed that H4 formulation issafer for rectal administration without any signs of rectal irritancy. The stability studies of optimized formulation (H4) proved that hydrogel remainedstable over a wide range of temperature condition.Conclusion: Hence, the developed rectal hydrogel formulation seems to be a viable alternative to conventional drug delivery system for the effectivemanagement of epilepsy.Keywords: Carbamazepine, Rice bran wax, Rectal hydrogel, Sustainability.

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