Nanocrystals (NCs) exhibit potential in improving oral bioavailability for poorly water-soluble drugs. However, whether NCs improve oral absorption by quick dissolution or by endocytosis remains inconclusive because tracking of dissolved drugs and NCs particles cannot occur simultaneously. In this study, we aim to elucidate how NCs improve oral absorption by using coumarin 6 (C6), an aggregation-caused quenching fluorophore, and 2-((5-(4-(dip-tolylamino)phenyl)thiophen-2-yl)methylene)malononitrile (MeTTMN), an aggregation-induced emission fluorophore. C6 was used as a model drug to prepare NCs and MeTTMN was incorporated to construct fluorescence resonance energy transfer (FRET) pairs. Thus, the molecular absorption can be detected using the fluorescence signal of dissolved C6 and the NCs particles can be tracked simultaneously by monitoring FRET signals. The reliability of this tracking method was validated. Accordingly, in vitro dissolution, gastrointestinal traffic, and biodistribution studies were conducted. The results showed that dissolved C6 molecules were the main absorption mode of C6 NCs. Identification of such pathways bears considerable significance for the broad application of drug NCs in improving the druggability of insoluble drugs.

MIRAGE syndrome is a recently discovered rare genetic disease characterized by myelodysplasia (M), infection (I), growth restriction (R), adrenal hypoplasia (A), genital phenotypes (G), and enteropathy (E), caused by a gain-of-function mutation in the SAMD9 gene. We encountered a girl with molecularly-confirmed MIRAGE syndrome who developed steroid-resistant nephrotic syndrome.
She was born at 33 weeks gestational age with a birth weight of 1064 g. She showed growth failure, mild developmental delays, intractable enteropathy and recurrent pneumonia. She was diagnosed as MIRAGE syndrome by whole exome sequencing and a novel SAMD9 variant (c.4615 T > A, p.Leu1539Ile) was identified at age four. Biopsied skin fibroblast cells showed changes in the endosome system that are characteristic of MIRAGE syndrome, supporting the genetic diagnosis. Proteinuria was noted at age one, following nephrotic syndrome at age five. A renal biopsy showed focal segmental glomerulosclerosis (FSGS) with immune deposits. Steroid treatment was ineffective. Because we speculated that her nephrosis was a result of genetic FSGS, we decided not to introduce immunosuppressive agents and instead started enalapril to reduce proteinuria. Although her proteinuria persisted, her renal function was normal at age eight.
This is the first detailed report of a MIRAGE syndrome patient with nephrotic syndrome. Because patients with MIRAGE syndrome have structural abnormalities in the endosomal system, we speculate that dysfunction of endocytosis in podocytes might be a possible mechanism for proteinuria.

Oligonucleotides can be designed or evolved to bind to specific DNA, RNA, protein, or small molecule targets and thereby alter the biological function of the target. The therapeutic potential of oligonucleotides targeted to intracellular molecules will depend largely on their ability to be taken up by the cells of interest, as well as their subsequent subcellular distribution. Here we describe methods to characterize the extent and mechanism of cellular uptake of AS1411, an aptamer oligonucleotide that has progressed to human clinical trials and which is also widely used by researchers as a cancer-targeting ligand.

UNASSIGNED: Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.
UNASSIGNED: In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.
UNASSIGNED: The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.

The treatment efficacy of a nicotine vaccine largely relies on its ability to induce high titers of nicotine-specific antibodies. Due to its strong immune-potentiating effects, aluminum salt (Alum) has been commonly used as an adjuvant in various nicotine vaccine formulations. In this study, we attempted to improve the immunological performance of a hybrid nanoparticle-based nicotine vaccine (NanoNicVac) by co-administering it with Alum. It was found that Alum severely restricted the release of NanoNicVac at the site of injection. Moreover, Alum damaged the hybrid structure of the vaccine. In the animal trial, mice immunized with NanoNicVac alone achieved an anti-nicotine IgG titer of 3.5 ± 0.2 × 104 after three injections. Unexpectedly, Alum with quantities of 125, 250, 500, and 1000 μg did not enhance the immunogenicity of NanoNicVac. In addition, Alum did not improve the ability of the vaccine to reduce the entry of nicotine into the brain.

Nanotechnology is regarded as the enabling technology of the 21st century. However, only a relatively small number of nano-enabled medical and healthcare products finally made their way to the market. There are several reasons why such innovative approaches fail in translation, with one key factor being the uncertainty surrounding their safety assessment. Although well described, interference reactions of engineered nanomaterials (ENM) with classical cytotoxicity assays remain a major source of uncertainty. Flow cytometry is a powerful, widely used, in vitro technique. Its readout is based on the detection of refracted laser light and fluorescence signals. It is therefore susceptible to ENM interference. Here we investigated possible interferences of ENM in the Annexin V/propidium iodide (PI) assay, which quantifies apoptotic and necrotic cell populations by flow cytometry. Two case studies were conducted using either silica or gold nanoparticles differing in size, specific surface area and surface chemistry. Both ENM types were found to cause distinct interference reactions at realistic concentrations. Silica particles induced false-positive signals; however only in the absence of a protein corona and in conjunction with a particular fluorophore combination (FITC/PI). In contrast, gold particles led to complex quenching effects which were only marginally influenced by the presence of proteins and occurred for both fluorophore combinations analyzed. We present a versatile spike-in approach which is applicable to all ENM and cell types. It further allows for the identification of a broad range of different interference phenomena, thereby increasing the reliability and quality of flow cytometry and ENM hazard assessment.

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals\' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

OCTN2 (SLC22A5) is a Na+ -coupled absorption transporter for l-carnitine in small intestine. This study tests the potential of this transporter for oral delivery of therapeutic drugs encapsulated in l-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) and discloses the molecular mechanism for cellular endocytosis of transporter-targeting nanoparticles. Conjugation of l-carnitine to a surface of PLGA-NPs enhances the cellular uptake and intestinal absorption of encapsulated drug. In both cases, the uptake process is dependent on cotransporting ion Na+ . Computational OCTN2 docking analysis shows that the presence of Na+ is important for the formation of the energetically stable intermediate complex of transporter-Na+ -LC-PLGA NPs, which is also the first step in cellular endocytosis of nanoparticles. The transporter-mediated intestinal absorption of LC-PLGA NPs occurs via endocytosis/transcytosis rather than via the traditional transmembrane transport. The portal blood versus the lymphatic route is evaluated by the plasma appearance of the drug in the control and lymph duct-ligated rats. Absorption via the lymphatic system is the predominant route in the oral delivery of the NPs. In summary, LC-PLGA NPs can effectively target OCTN2 on the enterocytes for enhancing oral delivery of drugs and the critical role of cotransporting ions should be noticed in designing transporter-targeting nanoparticles.

The success of nanomedicine as a new strategy for drug delivery and targeting prompted the interest in developing approaches toward basic and clinical neuroscience. Despite enormous advances on brain research, central nervous system (CNS) disorders remain the world\'s leading cause of disability, in part due to the inability of the majority of drugs to reach the brain parenchyma. Many attempts to use nanomedicines as CNS drug delivery systems (DDS) were made; among the various non-invasive approaches, nanoparticulate carriers and, particularly, polymeric nanoparticles (NPs) seem to be the most interesting strategies. In particular, the ability of poly-lactide-co-glycolide NPs (PLGA-NPs) specifically engineered with a glycopeptide (g7), conferring to NPs\' ability to cross the blood brain barrier (BBB) in rodents at a concentration of up to 10% of the injected dose, was demonstrated in previous studies using different routes of administrations. Most of the evidence on NP uptake mechanisms reported in the literature about intracellular pathways and processes of cell entry is based on in vitro studies. Therefore, beside the particular attention devoted to increasing the knowledge of the rate of in vivo BBB crossing of nanocarriers, the subsequent exocytosis in the brain compartments, their fate and trafficking in the brain surely represent major topics in this field.

A phenomenological rate equation model is constructed to numerically simulate nanoparticle uptake and subsequent cellular response. Polyamidoamine dendrimers (generations 4-6) are modelled and the temporal evolution of the intracellular cascade of; increased levels of reactive oxygen species, intracellular antioxidant species, caspase activation, mitochondrial membrane potential decay, tumour necrosis factor and interleukin generation is simulated, based on experimental observations. The dose and generation dependence of several of these response factors are seen to well represent experimental observations at a range of time points. The model indicates that variations between responses of different cell-lines, including murine macrophages, human keratinocytes and colon cells, can be simulated and understood in terms of different intracellular antioxidant levels, and, within a given cell-line, varying responses of different cytotoxicity assays can be understood in terms of their sensitivities to different intracellular cascade events. The model serves as a tool to interpolate and visualise the range of dose and temporal dependences and elucidate the mechanisms underlying the in vitro cytotoxic response to nanoparticle exposure and describes the interaction in terms of independent nanoparticle properties and cellular parameters, based on reaction rates. Such an approach could be a valid alternative to that of effective concentrations for classification of nanotoxicity and may lay the foundation for future quantitative structure activity relationships and predictive nanotoxicity models.