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WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana , such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 ( aprf1 ) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Background: Acute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies., Methods: To build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM's stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells., Results: The selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse., Conclusions: Our results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments., (© 2023. The Author(s).)

Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium-specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle., (© 2023. The Author(s).)

Lignin is a nontoxic and biocompatible biopolymer with many promising characteristics, including a high tensile strength and antioxidant properties. This natural polymer can be processed through several chemical methods and modified into lignin nanomaterials for potential biomedical applications. This review summarizes the latest developments in nanolignin (NL)-based biomaterials for cancer therapy; various NL applications related to cancer therapy are considered, including drug and gene delivery, biosensing, bioimaging, and tissue engineering. The manuscript also outlines the potential use of these materials to improve the therapeutic potency of chemotherapeutic drugs by decreasing their dose and reducing their adverse effects. Due to its high surface area-to-volume ratio and the easy modification of its chemical components, NL could serve as an appropriate matrix for the binding and controlled release of various pharmaceutical agents. Moreover, the challenges in the utilization of NL-based materials for cancer therapy are discussed, along with the prospects of advances in such nanomaterials for medical research applications., (© 2023 Wiley-VCH GmbH.)

Background: Nanotopographical cues play a critical role as drivers of mesenchymal stem cell differentiation. Nanowire scaffolds, in this regard, provide unique and adaptable nanostructured surfaces with focal points for adhesion and with elastic properties determined by nanowire stiffness., Results: We show that a scaffold of nanowires, which are remotely actuated by a magnetic field, mechanically stimulates mesenchymal stem cells. Osteopontin, a marker of osteogenesis onset, was expressed after cells were cultured for 1 week on top of the scaffold. Applying a magnetic field significantly boosted differentiation due to mechanical stimulation of the cells by the active deflection of the nanowire tips. The onset of differentiation was reduced to 2 days of culture based on the upregulation of several osteogenesis markers. Moreover, this was observed in the absence of any external differentiation factors., Conclusions: The magneto-mechanically modulated nanosurface enhanced the osteogenic differentiation capabilities of mesenchymal stem cells, and it provides a customizable tool for stem cell research and tissue engineering., (© 2022. The Author(s).)

The major impediments to the implementation of cancer immunotherapies are the sustained immune effect and the targeted delivery of these therapeutics, as they have life-threatening adverse effects. In this work, biomimetic metal-organic frameworks [zeolitic imidazolate frameworks (ZIFs)] are used for the controlled delivery of nivolumab (NV), a monoclonal antibody checkpoint inhibitor that was U.S. Food and Drug Administration-approved back in 2014. The sustained release behavior of NV-ZIF has shown a higher efficacy than the naked NV to activate T cells in hematological malignancies. The system was further modified by coating NV-ZIF with cancer cell membrane to enable tumor-specific targeted delivery while treating solid tumors. We envisage that such a biocompatible and biodegradable immunotherapeutic delivery system may promote the development and the translation of hybrid superstructures into smart and personalized delivery platforms., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In-vitro transfection of cells by electroporation is a widely used approach in cell biology and medicine. The transfection method is highly dependent on the cell culture's electrical resistance, which is strongly determined by differences in the membranes, but also on the morphology of the electrodes. Microneedle (MN)-based electrodes have been used to concentrate the electrical field during electroporation, and therefore maximize its effect on cell membrane permeability. So far, the methods used for the fabrication of MN electrodes have been relatively limited with respect to the needle design. In this work, we provide a method to fabricate MNs using 3D printing, which is a technology that provides a high degree of flexibility with respect to geometry and dimensions. Pyramidal-shaped MN designs were fabricated and tested on HCT116 cancer cells. Customization of the tips of the pyramids permits tailoring of the electrical field in the vicinity of the cell membranes. The fabricated device enables low-voltage (2 V) electroporation, eliminating the need for the use of specialized chemical buffers. The results show the potential of this method, which can be exploited and optimized for many different applications, and offer a very accessible approach for in-vitro electroporation and cell studies. The MNs can be customized to create complex structures, for example, for a multi-culture cell environment.

Directing the fate of human mesenchymal stem/stromal cells (hMSCs) toward bone formation using mechanical strain is a promising approach in regenerative medicine related to bone diseases. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically-based method for stimulating differentiation. Here, we study the differentiation of hMSCs cultured on elastic polydimethylsiloxane (PDMS) membrane, which is magnetically actuated to induce periodically varying strain. The strain distribution across the membrane was calculated by finite-element modeling and demonstrates three main areas of different strain amplitudes. The strain effect on the hMSCs was evaluated by measuring the mineralization of differentiated hMSCs using Alizarin S red stain. The results indicate a strain-dependent differentiation of hMSCs, where the highest region of strain on the membrane resulted in the most accelerated differentiation. Osteogenic differentiation was achieved as early as two weeks, which is significantly sooner than control hMSCs treated with osteogenic media alone.

Motivation: Leucine-aspartic acid (LD) motifs are short linear interaction motifs (SLiMs) that link paxillin family proteins to factors controlling cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood., Results: To enable a proteome-wide assessment of LD motifs, we developed an active learning based framework (LD motif finder; LDMF) that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome revealed a dozen new proteins containing LD motifs. We found that LD motif signalling evolved in unicellular eukaryotes more than 800 Myr ago, with paxillin and vinculin as core constituents, and nuclear export signal as a likely source of de novo LD motifs. We show that LD motif proteins form a functionally homogenous group, all being involved in cell morphogenesis and adhesion. This functional focus is recapitulated in cells by GFP-fused LD motifs, suggesting that it is intrinsic to the LD motif sequence, possibly through their effect on binding partners. Our approach elucidated the origin and dynamic adaptations of an ancestral SLiM, and can serve as a guide for the identification of other SLiMs for which only few representatives are known., Availability and Implementation: LDMF is freely available online at www.cbrc.kaust.edu.sa/ldmf; Source code is available at https://github.com/tanviralambd/LD/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15-39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P-glycoprotein (P-gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P-gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P-gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P-gps: All compounds were bioactive especially compound 1 c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P-gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR.