Altering heparan sulfate suppresses cell abnormalities and neuron loss in Drosophila presenilin model of Alzheimer Disease.

We examined the function of heparan-sulfate-modified proteoglycans (HSPGs) in pathways affecting Alzheimer disease (AD)-related cell pathology in human cell lines and mouse astrocytes. Mechanisms of HSPG influences on presenilin-dependent cell loss were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss-of-function of sulfateless (sfl), a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in human cell lines, Drosophila, and mouse astrocytes. RNA interference (RNAi) of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. Neuron-directed knockdown of Psn in Drosophila produced apoptosis and cell loss in the brain, phenotypes suppressed by reductions in sfl expression. Abnormalities in mitochondria, liposomes, and autophagosome-derived structures in animals with Psn knockdown were also rescued by reduction of sfl. These findings support the direct involvement of HSPGs in AD pathogenesis.

Exosomes derived from tumor adjacent fibroblasts efficiently target pancreatic tumors.

The application of extracellular vesicles, particularly exosomes (EXs), is rapidly expanding in the field of medicine, owing to their remarkable properties as natural carriers of biological cargo. This study investigates utilization of exosomes derived from stromal cells of tumor adjacent normal tissues (NAF-EXs) for personalized medicine, which can be derived at the time of diagnosis by endoscopic ultrasound. Herein, we show that exosomes (EXs) derived from NAFs demonstrate differential bio-physical characteristics, efficient cellular internalization, drug loading efficiency, pancreatic tumor targeting and delivery of payloads. NAF-derived EXs (NAF-EXs) were used for loading ormeloxifene (ORM), a potent anti-cancer and desmoplasia inhibitor as a model drug. We found that ORM maintains normal fibroblast cell phenotype and renders them incompatible to be triggered for a CAF-like phenotype, which may be due to regulation of Ca2+ influx in fibroblast cells. NAF-EXs-ORM effectively blocked oncogenic signaling pathways involved in desmoplasia and epithelial mesenchymal transition (EMT) and repressed tumor growth in xenograft mouse model. In conclusion, our data suggests preferential tropism of NAF-EXs for PDAC tumors, thus imply feasibility of developing a novel personalized medicine for PDAC patients using autologous NAF-EXs for improved therapeutic outcome of anti-cancer drugs. Additionally, it provides the opportunity of utilizing this biological scaffold for effective therapeutics in combination with standard therapeutic regimen.

A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessments.

Assessing the development and function of the sympathetic nervous system in diseases on a large scale is challenging. Here, we present a protocol to generate human pluripotent stem cell (hPSC)-derived postganglionic sympathetic neurons (symNs) differentiated via neural crest cells (NCCs), which can be cryopreserved. We describe steps for hPSC replating, NCC replating and cryobanking, and symN differentiation. We then demonstrate the functionality of the hPSC-derived symNs, focusing on electrophysiological activity, calcium flux, and norepinephrine dynamics. For complete details on the use and execution of this protocol, please refer to Wu et al.1,2.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation.

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses.

Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.