A metabolic-dysfunction associated steatotic liver acinus biomimetic induces pancreatic islet dysfunction in a coupled microphysiology system.

Preclinical and clinical studies suggest that lipid-induced hepatic insulin resistance is a primary defect that predisposes to dysfunction in pancreatic islets, implicating a perturbed liver-pancreas axis underlying the comorbidity of T2DM and MASLD. To investigate this hypothesis, we developed a human biomimetic microphysiological system (MPS) coupling our vascularized liver acinus MPS (vLAMPS) with primary islets on a chip (PANIS) enabling MASLD progression and islet dysfunction to be quantitatively assessed. The modular design of this system (vLAMPS-PANIS) allows intra-organ and inter-organ dysregulation to be deconvoluted. When compared to normal fasting (NF) conditions, under early metabolic syndrome (EMS) conditions, the standalone vLAMPS exhibited characteristics of early stage MASLD, while no significant differences were observed in the standalone PANIS. In contrast, with EMS, the coupled vLAMPS-PANIS exhibited a perturbed islet-specific secretome and a significantly dysregulated glucose stimulated insulin secretion (GSIS) response implicating direct signaling from the dysregulated liver acinus to the islets. Correlations between several pairs of a vLAMPS-derived and a PANIS-derived secreted factors were significantly altered under EMS, as compared to NF conditions, mechanistically connecting MASLD and T2DM associated hepatic factors with islet-derived GLP-1 synthesis and regulation. Since vLAMPS-PANIS is compatible with patient-specific iPSCs, this platform represents an important step towards addressing patient heterogeneity, identifying complex disease mechanisms, and advancing precision medicine.

The underlying causes, treatment options of gut microbiota and food habits in type 2 diabetes mellitus: a narrative review.

Type 2 diabetes mellitus is a long-lasting endocrine disorder characterized by persistent hyperglycaemia, which is often triggered by an entire or relative inadequacy of insulin production or insulin resistance. As a result of resistance to insulin (IR) and an overall lack of insulin in the body, type 2 diabetes mellitus (T2DM) is a metabolic illness that is characterized by hyperglycaemia. Notably, the occurrence of vascular complications of diabetes and the advancement of IR in T2DM are accompanied by dysbiosis of the gut microbiota. Due to the difficulties in managing the disease and the dangers of multiple accompanying complications, diabetes is a chronic, progressive immune-mediated condition that plays a significant clinical and health burden on patients. The frequency and incidence of diabetes among young people have been rising worldwide. The relationship between the gut microbiota composition and the physio-pathological characteristics of T2DM proposes a novel way to monitor the condition and enhance the effectiveness of therapies. Our knowledge of the microbiota of the gut and how it affects health and illness has changed over the last 20 years. Species of the genus Eubacterium, which make up a significant portion of the core animal gut microbiome, are some of the recently discovered 'generation' of possibly helpful bacteria. In this article, we have focused on pathogenesis and therapeutic approaches towards T2DM, with a special reference to gut bacteria from ancient times to the present day.

Design and investigation of interactions of novel peptide conjugates of purine and pyrimidine derivatives with EGFR and its mutant T790M/L858R: an in silico and laboratory study.

Peptide-based therapeutics have been gaining attention due to their ability to actively target tumor cells. Additionally, several varieties of nucleotide derivatives have been developed to reduce cell proliferation and induce apoptosis of tumor cells. In this work, we have developed novel peptide conjugates with newly designed purine analogs and pyrimidine derivatives and explored the binding interactions with the kinase domain of wild-type EGFR and its mutant EGFR [L858R/ T790M] which are known to be over-expressed in tumor cells. The peptides explored included WNWKV (derived from sea cucumber) and LARFFS, which in previous work was predicted to bind to Domain I of EGFR. Computational studies conducted to explore binding interactions include molecular docking studies, molecular dynamics simulations and MMGBSA to investigate the binding abilities and stability of the complexes. The results indicate that conjugation enhanced binding capabilities, particularly for the WNWKV conjugates. MMGBSA analysis revealed nearly twofold higher binding toward the T790M/L858R double mutant receptor. Several conjugates were shown to have strong and stable binding with both wild-type and mutant EGFR. As a proof of concept, we synthesized pyrimidine conjugates with both peptides and determined the KD values using SPR analysis. The results corroborated with the computational analyses. Additionally, cell viability and apoptosis studies with lung cancer cells expressing the wild-type and double mutant proteins revealed that the WNWKV conjugate showed greater potency than the LARFFS conjugate, while LARFFS peptide alone showed poor binding to the kinase domain. Thus, we have designed peptide conjugates that show potential for further laboratory studies for developing therapeutics for targeting the EGFR receptor and its mutant T790M/L858R.

Development of Self-Assembled Biomimetic Nanoscale Collagen-like Peptide-Based Scaffolds for Tissue Engineering: An In Silico and Laboratory Study.

Development of biocomposite scaffolds has gained tremendous attention due to their potential for tissue regeneration. However, most scaffolds often contain animal-derived collagen that may elicit an immunological response, necessitating the development of new biomaterials. Herein, we developed a new collagen-like peptide,(Pro-Ala-His)10 (PAH)10, and explored its ability to be utilized as a functional biomaterial by incorporating it with a newly synthesized peptide-based self-assembled gel. The gel was prepared by conjugating a pectin derivative, galataric acid, with a pro-angiogenic peptide (LHYQDLLQLQY) and further functionalized with a cortistatin-derived peptide, (Phe-Trp-Lys-Thr)4 (FWKT)4, and the bio-ionic liquid choline acetate. The self-assembly of (PAH)10 and its interactions with the galactarate-peptide conjugates were examined using replica exchange molecular dynamics (REMD) simulations. Results revealed the formation of a multi-layered scaffold, with enhanced stability at higher temperatures. We then synthesized the scaffold and examined its physicochemical properties and its ability to integrate with aortic smooth muscle cells. The scaffold was further utilized as a bioink for bioprinting to form three-dimensional cell-scaffold matrices. Furthermore, the formation of actin filaments and elongated cell morphology was observed. These results indicate that the (PAH)10 hybrid scaffold provides a suitable environment for cell adhesion, proliferation and growth, making it a potentially valuable biomaterial for tissue engineering.

A computational and laboratory approach for the investigation of interactions of peptide conjugated natural terpenes with EpHA2 receptor.

CONTEXT: Ephrin type A receptor 2 (EphA2) is a well-known drug target for cancer treatment due to its overexpression in numerous types of cancers. Thus, it is crucial to determine the binding interactions of this receptor with both the ligand-binding domain (LBD) and the kinase-binding domain (KBD) through a targeted approach in order to modulate its activity. In this work, natural terpenes with inherent anticancer properties were conjugated with short peptides YSAYP and SWLAY that are known to bind to the LBD of EphA2 receptor. We examined the binding interactions of six terpenes (maslinic acid, levopimaric acid, quinopimaric acid, oleanolic, polyalthic, and hydroxybetulinic acid) conjugated to the above peptides with the ligand-binding domain (LBD) of EphA2 receptor computationally. Additionally, following the "target-hopping approach," we also examined the interactions of the conjugates with the KBD. Our results indicated that most of the conjugates showed higher binding interactions with the EphA2 kinase domain compared to LBD. Furthermore, the binding affinities of the terpenes increased upon conjugating the peptides with the terpenes. In order to further investigate the specificity toward EphA2 kinase domain, we also examined the binding interactions of the terpenes conjugated to VPWXE (x = norleucine), as VPWXE has been shown to bind to other RTKs. Our results indicated that the terpenes conjugated to SWLAY in particular showed high efficacy toward binding to the KBD. We also designed conjugates where in the peptide portion and the terpenes were separated by a butyl (C4) group linker to examine if the binding interactions could be enhanced. Docking studies showed that the conjugates with linkers had enhanced binding with the LBD compared to those without linkers, though binding remained slightly higher without linkers toward the KBD. As a proof of concept, maslinate and oleanolate conjugates of each of the peptides were then tested with F98 tumor cells which are known to overexpress EphA2 receptor. Results indicated that the oleanolate-amido-SWLAY conjugates were efficacious in reducing the cell proliferation of the tumor cells and may be potentially developed and further studied for targeting tumor cells overexpressing the EphA2 receptor. To test if these conjugates could bind to the receptor and potentially function as kinase inhibitors, we conducted SPR analysis and ADP-Glo assay. Our results indicated that OA conjugate with SWLAY showed the highest inhibition. METHODS: Docking studies were carried out using AutoDock Vina, v.1.2.0; Molecular Dynamics and MMGBSA calculations were carried out through Schrodinger Software DESMOND.

Interactions of Nanoscale Self-Assembled Peptide-Based Assemblies with Glioblastoma Cell Models and Spheroids.

Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments.

An investigation of binding interactions of tumor-targeted peptide conjugated polyphenols with the kinase domain of ephrin B4 and B2 receptors.

Recent studies have shown that Ephrin receptors may be upregulated in several types of cancers including breast, ovarian and endometrial cancers, making them a target for drug design. In this work, we have utilized a target-hopping approach to design new natural product-peptide conjugates and examined their interactions with the kinase-binding domain of EphB4 and EphB2 receptors. The peptide sequences were generated through point mutations of the known EphB4 antagonist peptide TNYLFSPNGPIA. Their anticancer properties and secondary structures were analyzed computationally. Conjugates of most optimum of peptides were then designed by binding the N-terminal of the peptides with the free carboxyl group of the polyphenols sinapate, gallate and coumarate, which are known for their inherent anticancer properties. To investigate if these conjugates have a potential to bind to the kinase domain, we carried out docking studies and MMGBSA free energy calculations of the trajectories based on the molecular dynamics simulations, with both the apo and the ATP bound kinase domains of both receptors. In most cases binding interactions occurred within the catalytic loop region, while in some cases the conjugates were found to spread out across the N-lobe and the DFG motif region. The conjugates were further tested for prediction of pharmacokinetic properties using ADME studies. Our results indicated that the conjugates were lipophilic and MDCK permeable with no CYP interactions. These findings provide an insight into the molecular interactions of these peptides and conjugates with the kinase domain of the EphB4 and EphB2 receptor. As a proof of concept, we synthesized and carried out SPR analysis with two of the conjugates (gallate-TNYLFSPNGPIA and sinapate-TNYLFSPNGPIA). Results indicated that the conjugates showed higher binding with the EphB4 receptor and minimal binding to EphB2 receptor. Sinapate-TNYLFSPNGPIA showed inhibitory activity against EphB4. These studies reveal that some of the conjugates may be developed for further investigation into in vitro and in vivo studies and potential development as therapeutics.

Repopulation of decellularized organ scaffolds with human pluripotent stem cell-derived pancreatic progenitor cells.

Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.

Molecular dynamics simulations and in vitro studies of hybrid decellularized leaf-peptide-polypyrrole composites for potential tissue engineering applications.

Tissue engineering (TE) aims to repair and regenerate damaged tissue by an assimilation of optimal combination of cells specific to the tissue with an appropriate biomaterial. In this work, a new biomaterial for potential cardiac TE applications was developed by utilizing a combination of in silico studies and in vitro experiments. Molecular dynamics (MD) simulations for the formation of the novel composite prepared from the decellularized leaf components cellulose and pectin along with the VEGF derived peptide (NYLTHRQ) and polypyrrole (PPy) was carried out to assess self-assembly, mechanical properties, and interactions with integrin and NPR-C receptors which are commonly found in cells of cardiac tissue. Results of molecular dynamics simulations indicated the successful formation of stable assemblies. MD simulations also revealed that the scaffold successfully interacted with integrin and NPR-C receptors. As a proof of concept, beet leaves were decellularized (DC) and cross-linked with NYLTHRQ and PPy using layer-by-layer assembly. Decellularization (DC) was confirmed by DNA and protein quantification. Incorporation of the NYLTHRQ peptide and polypyrrole was confirmed by FTIR spectroscopy and SEM imaging. The DC-NYLTHRQ-PPy scaffold was seeded with co-cultured cardiomyocytes and vascular smooth muscle cells. The scaffold promoted cell proliferation and adhesion. Actin and Troponin T immunofluorescence staining showed the presence of these critical cardiomyocyte markers. Thus, for the first time we have developed a decellularized leaf-peptide-PPy composite scaffold by a combination of in silico studies and laboratory analyses that may have potential applications in cardiac TE.Communicated by Ramaswamy H. Sarma.

Molecular dynamics, MMGBSA, and docking studies of natural products conjugated to tumor-targeted peptide for targeting BRAF V600E and MERTK receptors.

Recent studies have revealed that MERTK and BRAF V600E receptors have been found to be over-expressed in several types of cancers including melanoma, making these receptors targets for drug design. In this study, we have designed novel peptide conjugates with the natural products vanillic acid, thiazole-2-carboxylic acid, cinnamic acid, theanine, and protocatechuic acid. Each of these compounds was conjugated with the tumor targeting peptide sequence TAASGVRSMH, known to bind to NG2 and target tumor neovasculature. We examined their binding affinities and stability with MERTK and BRAF V600E receptors using molecular docking and molecular dynamics studies. Compared to the neat compounds, the peptide conjugates displayed higher binding affinity toward both receptors. In the case of MERTK, the most stable complexes were formed with di-theaninate-peptide, vanillate-peptide, and thiazole-2-amido peptide conjugates and binding occurred in the hinge region. Additionally, it was discovered that the peptide alone also had high binding ability and stability with the MERTK receptor. In the case of BRAF V600E, the peptide conjugates of protocatechuate, vanillate and thiazole-2-amido peptide conjugates showed the formation of the most stable complexes and binding occurred in the ATP binding cleft. Further analysis revealed that the number of hydrogen bonds and hydrophobic interactions played a critical role in enhanced stability of the complexes. Docking studies also revealed that binding affinities for NG2 were similar to MERTK and higher for BRAF V600E. MMGBSA studies of the trajectories revealed that the protocatechuate-peptide conjugate showed the highest binding energy with BRAF V600E while the peptide-TAASGVRSMH showed the highest binding energy with MERTK. ADME studies revealed that each of the compounds showed medium to high permeability toward MDCK cells and were not hERG blockers. Furthermore, the conjugates were not CYP inhibitors or substrates, but they were found to be Pgp substrates. Our results indicated that the protocatechuate-TAASGVRSMH, thiazole-2-amido-TAASGVRSMH, and vanillate-TAASGVRSMH conjugates may be furthered developed for in vitro and in vivo studies as novel tumor targeting compounds for tumor cells over-expressing BRAF V600E, while di-theaninate-amido-TAASGVRSMH and thiazole-2-amido-TAASGVRSMH conjugates may be developed for targeting MERTK receptors. These studies provide insight into the molecular interactions of natural product-peptide conjugates and their potential for binding to and targeting MERTK and BRAF V600E receptors in developing new therapeutics for targeting cancer.

Design of short peptides and peptide amphiphiles as collagen mimics and an investigation of their interactions with collagen using molecular dynamics simulations and docking studies.

Short peptide sequences and bolaamphiphiles derived from natural proteins are gaining importance due to their ability to form unique nanoscale architectures for a variety of biological applications. In this work, we have designed six short peptides (triplet or monomeric forms) and two peptide bolaamphiphiles that either incorporate the bioactive collagen motif (Gly-X-Y) or sequences where Gly, Pro, or hydroxyproline (Hyp) are replaced by Ala or His. For the bolaamphiphiles, a malate moiety was used as the aliphatic linker for connecting His with Hyp to create collagen mimics. Stability of the assemblies was assessed through molecular dynamics simulations and results indicated that (Pro-Ala-His)3 and (Ala-His-Hyp)3 formed the most stable structures, while the amphiphiles and the monomers showed some disintegration over the course of the 200 ns simulation, though most regained structural integrity and formed fibrillar structures, and micelles by the end of the simulation, likely due to the formation of more thermodynamically stable conformations. Multiple replica simulations (REMD) were also conducted where the sequences were simulated at different temperatures. Our results showed excellent convergence in most cases compared to constant temperature molecular dynamics simulation. Furthermore, molecular docking and MD simulations of the sequences bound to collagen triple helix structure revealed that several of the sequences had a high binding affinity and formed stable complexes, particularly (Pro-Ala-His)3 and (Ala-His-Hyp)3. Thus, we have designed new hybrid-peptide-based sequences which may be developed for potential applications as biomaterials for tissue engineering or drug delivery.

De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids.

Hematopoietic humanized (hu) mice are powerful tools for modeling the action of human immune system and are widely used for preclinical studies and drug discovery. However, generating a functional human T cell compartment in hu mice remains challenging, primarily due to the species-related differences between human and mouse thymus. While engrafting human fetal thymic tissues can support robust T cell development in hu mice, tissue scarcity and ethical concerns limit their wide use. Here, we describe the tissue engineering of human thymus organoids from inducible pluripotent stem cells (iPSC-thymus) that can support the de novo generation of a diverse population of functional human T cells. T cells of iPSC-thymus-engrafted hu mice could mediate both cellular and humoral immune responses, including mounting robust proinflammatory responses on T cell receptor engagement, inhibiting allogeneic tumor graft growth and facilitating efficient Ig class switching. Our findings indicate that hu mice engrafted with iPSC-thymus can serve as a new animal model to study human T cell-mediated immunity and accelerate the translation of findings from animal studies into the clinic.

Identification of Clinical Phenotypes in Septic Patients Presenting With Hypotension or Elevated Lactate.

Introduction: Targeted therapies for sepsis have failed to show benefit due to high variability among subjects. We sought to demonstrate different phenotypes of septic shock based solely on clinical features and show that these relate to outcome. Methods: A retrospective analysis was performed of a 1,023-subject cohort with early septic shock from the ProCESS trial. Twenty-three clinical variables at baseline were analyzed using hierarchical clustering, with consensus clustering used to identify and validate the ideal number of clusters in a derivation cohort of 642 subjects from 20 hospitals. Clusters were visualized using heatmaps over 0, 6, 24, and 72 h. Clinical outcomes were 14-day all-cause mortality and organ failure pattern. Cluster robustness was confirmed in a validation cohort of 381 subjects from 11 hospitals. Results: Five phenotypes were identified, each with unique organ failure patterns that persisted in time. By enrollment criteria, all patients had shock. The two high-risk phenotypes were characterized by distinct multi-organ failure patterns and cytokine signatures, with the highest mortality group characterized most notably by liver dysfunction and coagulopathy while the other group exhibited primarily respiratory failure, neurologic dysfunction, and renal dysfunction. The moderate risk phenotype was that of respiratory failure, while low-risk phenotypes did not have a high degree of additional organ failure. Conclusions: Sepsis phenotypes with distinct biochemical abnormalities may be identified by clinical characteristics alone and likely provide an opportunity for early clinical actionability and prognosis.

Molecular dynamics simulations, docking and MMGBSA studies of newly designed peptide-conjugated glucosyloxy stilbene derivatives with tumor cell receptors.

In this work, for the first time, we designed derivatives of beta-D-glucosyloxy-3-hydroxy-trans-stiblene-2-carboxylic acid (GHS), by conjugating GHS with tumor targeting peptides RPARPAR and GGKRPAR to target over-expressed receptors in tumor cells. The sequences RPARPAR and GGKRPAR are known to target the neuropilin1 (NRP1) receptor due to the C-terminal Arg domain; however, their effectiveness has never been examined with other commonly over-expressed receptors in tumor cells, particularly of chronic lymphocytic leukemia that include integrin α1ß1 and CD22. By conjugating these peptides with GHS, which is known for its inherent anti-cancer properties, the goal is to further enhance tumor cell targeting by developing compounds that can target multiple receptors. The physicochemical properties of the conjugates and individual peptides were analyzed using Turbomole and COSMOthermX20 in order to determine their hydrogen bond accepting and donating capabilities. The web server POCASA was used in order to determine the surface cavities and binding pockets of the three receptors. To explore the binding affinities, we conducted molecular docking studies with the peptides and the conjugates with each of the receptors. After molecular docking, the complexes were analyzed using Protein-Ligand Interaction Profiler to determine the types of interactions involved. Molecular dynamics simulation studies were conducted to explore the stability of the receptor-ligand complexes. Our results indicated that in most cases the conjugates showed higher binding and stability with the receptors. Additionally, highly stable complexes of conjugates were obtained with CD22, NRP1 and in most cases with the integrin α1ß1 receptor as well. The binding energies were calculated for each of the receptor ligand complexes through trajectory analysis using MMGBSA studies. SwissADME studies revealed that the compounds showed low GI absorption and were not found to be CYP inhibitors and had bioavailability score that would allow them to be considered as potential drug candidates. Overall, our results for the first time show that the designed conjugates can target multiple over-expressed receptors in tumor cells and may be potentially developed as future therapeutics for targeting tumor cells.

Exploring the Interactions of Ionic Liquids with Bio-Organic Amphiphiles Using Computational Approaches.

Bio-organic amphiphiles have been shown to effectively impart unique physicochemical properties to ionic liquids resulting in the formation of versatile hybrid composites. In this work, we utilized computational methods to probe the formation and properties of hybrids prepared by mixing three newly designed bio-organic amphiphiles with 14 ionic liquids containing cholinium or glycine betaine cations and a variety of anions. The three amphiphiles were designed such that they contain unique biological moieties found in nature by conjugating (a) malic acid with the amino acid glutamine, (b) thiomalic acid with the antiviral, antibacterial pyrazole compound [3-(3,5-dimethyl-1H-pyrazol-1-yl)benzyl]amine, and (c) Fmoc-protected valine with diphenyl amine. Conductor-like screening model for real solvents (COSMO-RS) was used to obtain sigma profiles of the hybrid mixtures and to predict viscosities and mixing enthalpies of each composite. These results were used to determine optimal ionic liquid-bio-organic amphiphile mixtures. Molecular dynamics simulations of three optimal hybrids were then performed, and the interactions involved in the formation of the hybrids were analyzed. Our results indicated that cholinium-based ILs interacted most favorably with the amphiphiles through a variety of inter- and intramolecular interactions. This work serves to illustrate important factors that influence the interactions between bio-organic amphiphiles and bio-ILs and aids in the development of novel ionic liquid-based composites for a wide variety of potential biological applications.

Alzheimer's Disease: Current Perspectives and Advances in Physiological Modeling.

Though Alzheimer's disease (AD) is the most common cause of dementia, complete disease-modifying treatments are yet to be fully attained. Until recently, transgenic mice constituted most in vitro model systems of AD used for preclinical drug screening; however, these models have so far failed to adequately replicate the disease's pathophysiology. However, the generation of humanized APOE4 mouse models has led to key discoveries. Recent advances in stem cell differentiation techniques and the development of induced pluripotent stem cells (iPSCs) have facilitated the development of novel in vitro devices. These "microphysiological" systems-in vitro human cell culture systems designed to replicate in vivo physiology-employ varying levels of biomimicry and engineering control. Spheroid-based organoids, 3D cell culture systems, and microfluidic devices or a combination of these have the potential to replicate AD pathophysiology and pathogenesis in vitro and thus serve as both tools for testing therapeutics and models for experimental manipulation.

In silico studies of interactions of peptide-conjugated cholesterol metabolites and betulinic acid with EGFR, LDR, and N-terminal fragment of CCKA receptors.

In this work, we designed three new ligands by conjugating cholesterol metabolites 3-hydroxy-5-cholestenoic acid (3-HC) and 3-oxo-4-cholestenoic acid (3-OC) and the natural tri-terpenoid betulinic acid with the tumor-targeting peptide YHWYGYTPQNVI. Molecular interactions with the unconjugated peptide and the conjugates were examined with three receptors that are commonly overexpressed in pancreatic adenocarcinoma cells using ligand docking and molecular dynamics. This study demonstrated the utility of the designed conjugates as a valuable scaffold for potentially targeting EGFR and LDLR receptors. Our results indicate that the conjugates showed strong binding affinities and formation of stable complexes with EGFR, while the unconjugated peptide, BT-peptide conjugate, an 3-HC-peptide conjugate showed the formation of fairly stable complexes with LDLR receptor. For EGFR, two receptor kinase domains were explored. Interactions with the N-terminal domain of CCKA-R were relatively weaker. For LDLR, binding occurred in the beta-propeller region. For the N-terminal fragment of CCKA-R, the conjugates induced significant conformational changes in the receptor. The molecular dynamic simulations for 100 ns demonstrate that BT-peptide conjugates and the unconjugated peptide had the highest binding and formed the most stable complexes with EGFR. RMSD and trajectory analyses indicate that these molecules transit to a dynamically stable configuration in most cases within 60 ns. NMA analysis indicated that amongst the conjugates that showed relatively higher interactions with the respective receptors, the highest potential for deformability was seen for the N-terminal-47 amino acid region of the CCKA-R receptor with and the lowest for the LDLR-receptor. Thus, the newly designed compounds may be evaluated in the future toward developing drug delivery materials for targeting tumor cells overexpressing LDLR or EGFR.

Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy.

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary α2ß2 complex between the catalytic (α2) and radical generating (ß2) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the ß subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in ß results in formation of an unusual ß4 tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the α4ß4 octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical α2ß2 complex.

Enhancing Kidney Vasculature in Tissue Engineering-Current Trends and Approaches: A Review.

Chronic kidney diseases are a leading cause of fatalities around the world. As the most sought-after organ for transplantation, the kidney is of immense importance in the field of tissue engineering. The primary obstacle to the development of clinically relevant tissue engineered kidneys is precise vascularization due to the organ's large size and complexity. Current attempts at whole-kidney tissue engineering include the repopulation of decellularized kidney extracellular matrices or vascular corrosion casts, but these approaches do not eliminate the need for a donor organ. Stem cell-based approaches, such as kidney organoids vascularized in microphysiological systems, aim to construct a kidney without the need for organ donation. These organ-on-a-chip models show complex, functioning kidney structures, albeit at a small scale. Novel methodologies for developing engineered scaffolds will allow for improved differentiation of kidney stem cells and organoids into larger kidney grafts with clinical applications. While currently, kidney tissue engineering remains mostly limited to individual renal structures or small organoids, further developments in vascularization techniques, with technologies such as organoids in microfluidic systems, could potentially open doors for a large-scale growth of whole engineered kidneys for transplantation.

Engineering Biophysical Cues for Controlled 3D Differentiation of Endoderm Derivatives.

Biophysical cues synergize with biochemical cues to drive differentiation of pluripotent stem cells through specific phenotypic trajectory. Tools to manipulate the cell biophysical environment and identify the influence of specific environment perturbation in the presence of combinatorial inputs will be critical to control the development trajectory. Here we describe the procedure to perturb biophysical environment of pluripotent stem cells while maintaining them in 3D culture configuration. We also discuss a high-throughput platform for combinatorial perturbation of the cell microenvironment, and detail a statistical procedure to extract dominant environmental influences.