Illuminating protein space with a programmable generative model.

Three billion years of evolution has produced a tremendous diversity of protein molecules1, but the full potential of proteins is likely to be much greater. Accessing this potential has been challenging for both computation and experiments because the space of possible protein molecules is much larger than the space of those likely to have functions. Here we introduce Chroma, a generative model for proteins and protein complexes that can directly sample novel protein structures and sequences, and that can be conditioned to steer the generative process towards desired properties and functions. To enable this, we introduce a diffusion process that respects the conformational statistics of polymer ensembles, an efficient neural architecture for molecular systems that enables long-range reasoning with sub-quadratic scaling, layers for efficiently synthesizing three-dimensional structures of proteins from predicted inter-residue geometries and a general low-temperature sampling algorithm for diffusion models. Chroma achieves protein design as Bayesian inference under external constraints, which can involve symmetries, substructure, shape, semantics and even natural-language prompts. The experimental characterization of 310 proteins shows that sampling from Chroma results in proteins that are highly expressed, fold and have favourable biophysical properties. The crystal structures of two designed proteins exhibit atomistic agreement with Chroma samples (a backbone root-mean-square deviation of around 1.0 Å). With this unified approach to protein design, we hope to accelerate the programming of protein matter to benefit human health, materials science and synthetic biology.

Discovery of a novel class of rosmarinic acid derivatives as antibacterial agents: Synthesis, structure-activity relationship and mechanism of action.

Twenty-seven rosmarinic acid derivatives were synthesized, among which compound RA-N8 exhibited the most potent antibacterial ability. The minimum inhibition concentration of RA-N8 against both S. aureus (ATCC 29213) and MRSA (ATCC BAA41 and ATCC 43300) was found to be 6 µg/mL, and RA-N8 killed E. coli (ATCC 25922) at 3 µg/mL in the presence of polymyxin B nonapeptide (PMBN) which increased the permeability of E. coli. RA-N8 exhibited a weak hemolytic effect at the minimum inhibitory concentration. SYTOX Green assay, SEM, and LIVE/DEAD fluorescence staining assay proved that the mode of action of RA-N8 is targeting bacterial cell membranes. Furthermore, no resistance in wildtype S. aureus developed after incubation with RA-N8 for 20 passages. Cytotoxicity studies further demonstrated that RA-N8 is non-toxic to the human normal cell line (HFF1). RA-N8 also exerted potent inhibitory ability against biofilm formation of S. aureus and even collapsed the shaped biofilm.

A Cytoplasm-Specific Fluorescent Ligand for Selective Imaging of RNA G-Quadruplexes in Live Cancer Cells.

The development of site-specific, target-selective and biocompatible small molecule ligands as a fluorescent tool for real-time study of cellular functions of RNA G-quadruplexes (G4s), which are associated with human cancers, is of significance in cancer biology. We report a fluorescent ligand that is a cytoplasm-specific and RNA G4-selective fluorescent biosensor in live HeLa cells. The in vitro results show that the ligand is highly selective targeting RNA G4s including VEGF, NRAS, BCL2 and TERRA. These G4s are recognized as human cancer hallmarks. Moreover, intracellular competition studies with BRACO19 and PDS, and the colocalization study with G4-specific antibody (BG4) in HeLa cells may support that the ligand selectively binds to G4s in cellulo. Furthermore, the ligand was demonstrated for the first time in the visualization and monitoring of dynamic resolving process of RNA G4s by the overexpressed RFP-tagged DHX36 helicase in live HeLa cells.

WNT/ß-catenin modulates the axial identity of embryonic stem cell-derived human neural crest.

WNT/ß-catenin signaling is crucial for neural crest (NC) formation, yet the effects of the magnitude of the WNT signal remain ill-defined. Using a robust model of human NC formation based on human pluripotent stem cells (hPSCs), we expose that the WNT signal modulates the axial identity of NCs in a dose-dependent manner, with low WNT leading to anterior OTX+ HOX- NC and high WNT leading to posterior OTX- HOX+ NC. Differentiation tests of posterior NC confirm expected derivatives, including posterior-specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC exhibits a transient TBXT+/SOX2+ neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, which suggest a crucial role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected retinoic acid (RA) and FGF are able to modulate HOX expression in the posterior NC. Surprisingly, early RA supplementation prohibits NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.

Human neural crest induction by temporal modulation of WNT activation.

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/ß-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/ß-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/ß-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.

An adherent-cell depletion technique to generate human neural progenitors and neurons.

Existing methodologies to produce human neural stem cells and neurons from embryonic stem cells frequently involve multistep processes and the use of complex and expensive media components, cytokines or small molecules. Here, we report a simple technique to generate human neuroepithelial progenitors and neurons by periodic mechanical dissection and adherent-cell depletion on regular cell-culture grade plastic surfaces. This neural induction technique does not employ growth factors, small molecules or peptide inhibitors, apart from those present in serum-free supplements. Suggestive of their central nervous system origin, we found that neural progenitors formed by this technique expressed radial glia markers, and, when differentiated, expressed TUBB3, RBFOX3 (NeuN) and serotonin, but not markers for peripheral neurons. With these data, we postulate that incorporation of periodic mechanical stimuli and plastic surface-mediated cell selection could improve and streamline existing human neuron production protocols.

WNT/ß-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/ß-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/ß-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates.

Weightbearing Digital Tomosynthesis of Foot and Ankle Arthritis: Comparison With Radiography and Simulated Weightbearing CT in a Prospective Study.

OBJECTIVE: Foot and ankle arthritis is common and debilitating. Weightbearing radiography is the reference standard for evaluating alignment, but overlapping bones and hardware limit evaluation for osteoarthritic bony detail. The purpose of this study was to evaluate whether digital tomosynthesis (DTS) can yield reliable quantitative alignment values, as radiography does with its weightbearing capability, and good qualitative osteoarthritic detail, as CT does. SUBJECTS AND METHODS: Adults with foot or ankle arthritis referred for simulated weightbearing CT were recruited to undergo weightbearing radiography and DTS. Four readers independently evaluated radiographs and DTS images for foot and ankle alignment and severity of osteoarthritis in each joint. Two readers performed consensus readings of CT images. Agreement between modalities was assessed by intraclass correlation coefficient (ICC) and Cohen kappa statistics. RESULTS: Ninety-one ankles were analyzed. Most joints were significantly less obscured by overlapping bone when seen with DTS (11.2%) or CT (4.3%) compared with radiography (30.4%). For quantitative foot alignment measurements, DTS had good to excellent agreement with weightbearing radiography (ICC, 0.65-0.93), which performed significantly better than CT (ICC, 0.39-0.87). For qualitative osteoarthritic details of each joint, DTS had significantly better agreement with weightbearing radiography on joint space narrowing (κ = 0.38-0.67) than did CT (κ = 0.08-0.62). Weightbearing radiography and DTS had similar levels of agreement with CT on grading of osteophytes, subchondral cysts, and loose bodies. CONCLUSION: DTS is associated with less obscuration of joints than radiography and yields more reliable weightbearing quantitative foot and ankle alignment values than radiography does and more reliable osteoarthritic bony details than CT does.

Differences in definitive endoderm induction approaches using growth factors and small molecules.

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

The Molecular Pathway Regulating Bergmann Glia and Folia Generation in the Cerebellum.

Evolution of complex behaviors in higher vertebrates and primates require the development of sophisticated neuronal circuitry and the expansion of brain surface area to accommodate the vast number of neuronal and glial populations. To achieve these goals, the neocortex in primates and the cerebellum in amniotes have developed specialized types of basal progenitors to aid the folding of their cortices. In the cerebellum, Bergmann glia constitute such a basal progenitor population, having a distinctive morphology and playing a critical role in cerebellar corticogenesis. Here, we review recent studies on the induction of Bergmann glia and their crucial role in mediating folding of the cerebellar cortex. These studies uncover a key function of FGF-ERK-ETV signaling cascade in the transformation of Bergmann glia from radial glia in the ventricular zone. Remarkably, in the neocortex, the same signaling axis operates to facilitate the transformation of ventricular radial glia into basal radial glia, a Bergmann glia-like basal progenitor population, which have been implicated in the establishment of neocortical gyri. These new findings draw a striking similarity in the function and ontogeny of the two basal progenitor populations born in distinct brain compartments.

Cuff tear arthropathy shoulder hemiarthroplasty: a radiographic outcome study.

OBJECTIVE: Cuff tear arthropathy (CTA) head prosthesis has recently become an alternative to standard shoulder hemiarthroplasty in patients with severe cuff disease by offering an increased surface area and decreased impingement. The purpose of this study is to evaluate the radiographic outcomes of CTA prosthesis and to correlate them with clinical outcomes. MATERIALS AND METHODS: In this retrospective study of CTA hemiarthroplasties over an 11-year period, two radiologists reviewed pre-/postoperative radiographs and clinical data. Radiographic complications were correlated with subsequent surgery using Cox regression models. Rates of surgical revision and radiographic complications over time were estimated using Kaplan-Meier curves. RESULTS: Ninety-seven CTA hemiarthroplasties were identified in 92 patients (5 bilateral) with a mean patient age of 68.7 years. Mean radiographic follow-up was 12 months with a mean of 3.3 radiographs per prosthesis. Twenty-six arthroplasties (26.8%) experienced at least one radiographic complication, including acromion remodeling (19.5%), anterior-posterior subluxation (5.2%), periprosthetic fracture (4.1%), glenoid remodeling (3.1%), hardware loosening (2.1%), superior subluxation (2.1%), and subsidence (1.0%). Eight cases underwent revision surgery (8.2%). The occurrence of a postoperative radiographic complication was associated with increased risk of surgical revision (hazard ratio 11.5, 95% CI: 2.4-55.7, p = 0.002); 73.5% of radiographic complications occurred by 3 months after the initial surgery (complication rate of 23.3%) based on Kaplan-Meier curve analysis. CONCLUSION: Radiographic complications after CTA head hemiarthroplasty are common with most occurring by 3 months after surgery and are highly associated with surgical revision.

Shp2-dependent ERK signaling is essential for induction of Bergmann glia and foliation of the cerebellum.

Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation.

Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells.

Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 µM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Structurally diverse G-quadruplexes as the noncanonical nucleic acid drug target for live cell imaging and antibacterial study.

The formation of G-quadruplex structures (G4s) in vitro from guanine (G)-rich nucleic acid sequences of DNA and RNA stabilized with monovalent cations, typically K+ and Na+, under physiological conditions, has been verified experimentally and some of them have high-resolution NMR or X-ray crystal structures; however, the biofunction of these special noncanonical secondary structures of nucleic acids has not been fully understood and their existence in vivo is still controversial at present. It is generally believed that the folding and unfolding of G4s in vivo is a transient process. Accumulating evidence has shown that G4s may play a role in the regulation of certain important cellular functions including telomere maintenance, replication, transcription and translation. Therefore, both DNA and RNA G4s of human cancer hallmark genes are recognized as the potential anticancer drug target for the investigation in cancer biology, chemical biology and drug discovery. The relationship between the sequence, structure and stability of G4s, the interaction of G4s with small molecules, and insights into the rational design of G4-selective binding ligands have been intensively studied over the decade. At present, some G4-ligands have achieved a new milestone and successfully entered the human clinical trials for anticancer therapy. Over the past few decades, numerous efforts have been devoted to anticancer therapy; however, G4s for molecular recognition and live cell imaging and for application as antibacterial agents and antibiofilms against antibiotic resistance have been obviously underexplored. The recent advances in G4-ligands in these areas are thus selected and discussed concentratedly in this article in order to shed light on the emerging role of G4s in chemical biology and therapeutic prospects against bacterial infections. In addition, the recently published molecular scaffolds for designing small ligands selectively targeting G4s in live cell imaging, bacterial biofilm imaging, and antibacterial studies are discussed. Furthermore, a number of underexplored G4-targets from the cytoplasmic membrane-associated DNA, the conserved promoter region of K. pneumoniae genomes, the RNA G4-sites in the transcriptome of E. coli and P. aeruginosa, and the mRNA G4-sites in the sequence for coding the vital bacterial FtsZ protein are highlighted to further explore in G4-drug development against human diseases.

VEGF microsphere technology to enhance vascularization in fat grafting.

Vascularization is crucial for implantation of engineered tissues in reconstructive surgery. Polypeptides encapsulated in microspheres can be efficiently transported to their site of action and released in a sustained dosage. We evaluated the effect of delivering vascular endothelial growth factor (VEGF)-encapsulated microspheres in a lipoaspirate scaffold on vascularization and tissue survival. The VEGF-loaded (n=6) and empty (n=6) poly(lactic-co-glycolic acid) microspheres in human lipoaspirate and the human lipoaspirate alone (n=6) were injected subcutaneously into the flanks of athymic nude mice. Three mice from each group were killed, and grafts were explanted at weeks 3 and 6. Increases in mass and volume of VEGF samples, as well as decreases in empty and lipoaspirate-only samples, were observed at 3 and 6 weeks, reaching statistical significance at 6 weeks. Hematoxylin and eosin and CD31+ imaging demonstrated significantly greater vascularization in VEGF samples than in both the empty and lipoaspirate-only groups at both 3 and 6 weeks.

NDM-1 Zn1-binding residue His116 plays critical roles in antibiotic hydrolysis.

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of ß-lactam antibiotics. The enzyme has a di­zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (kcat/Km) of the mutants followed the order: WT > > H116Q (decreased by 4-20 fold) > H116A (decreased by 20-700 fold) ≥ H116N (decreased by 6-800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of Km values, which informs the weakening of substrate binding, were: H116A by 5-45 fold; H116N by 6-100 fold; H116Q by 2-10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development.

Loss of procollagen IIA from the anterior mesendoderm disrupts the development of mouse embryonic forebrain.

Morphogenesis of the mammalian forebrain is influenced by the patterning activity of signals emanating from the anterior mesendoderm. In this study, we show that procollagen IIA (IIA), an isoform of the cartilage extracellular matrix protein encoded by an alternatively spliced transcript of Col2a1, is expressed in the prechordal plate and the anterior definitive endoderm. In the absence of IIA activity, the null mutants displayed a partially penetrant phenotype of loss of head tissues, holoprosencephaly, and loss of mid-facial structures, which is associated with reduced sonic hedgehog (Shh) expression in the prechordal mesoderm. Genetic interaction studies reveal that IIA function in forebrain and face development does not involve bone morphogenetic protein receptor 1A (BMPR1A)- or NODAL-mediated signaling activity.

Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems: Possibilities and Limitations.

The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell-matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

Rational structural modification of the isatin scaffold to develop new and potent antimicrobial agents targeting bacterial peptidoglycan glycosyltransferase.

A series of isatin derivatives bearing three different substituent groups at the N-1, C-3 and C-5 positions of the isatin scaffold were systematically designed and synthesized to study the structure-activity relationship of their inhibition of bacterial peptidoglycan glycosyltransferase (PGT) activity and antimicrobial susceptibility against S. aureus, E. coli and methicillin-resistant Staphylococcus aureus (MRSA (BAA41)) strains. The substituents at these sites are pointing towards three different directions from the isatin scaffold to interact with the amino acid residues in the binding pocket of PGT. Comparative studies of their structure-activity relationship allow us to gain better understanding of the direction of the substituents that contribute critical interactions leading to inhibition activity against the bacterial enzyme. Our results indicate that the modification of these sites is able to maximize the antimicrobial potency and inhibitory action against the bacterial enzyme. Two compounds show good antimicrobial potency (MIC = 3 µg mL-1 against S. aureus and MRSA; 12-24 µg mL-1 against E. coli). Results of the inhibition study against the bacterial enzyme (E. coli PBP 1b) reveal that some compounds are able to achieve excellent in vitro inhibitions of bacterial enzymatic activity (up to 100%). The best half maximal inhibitory concentration (IC50) observed among the new compounds is 8.9 µM.