A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

In yeast, titration of an increasing number of molecules of the G1 cyclin Cln3 by a fixed number of DNA-bound molecules of the transcription factor SBF might underlie the dependence of cell cycle entry on cell size.

The asymmetric localization of cell fate determinants results in asymmetric cell cycle control in budding yeast.

The G1 cyclin Cln3 is a key activator of cell-cycle entry in budding yeast. Here we show that Whi3, a negative G1 regulator of Cln3, interacts in vivo with the cyclin-dependent kinase Cdc28 and regulates its localization in the cell. Efficient interaction with Cdc28 depends on an N-terminal domain of Whi3 that is also required for cytoplasmic localization of Cdc28, and for proper regulation of G1 length and filamentous growth. On the other hand, nuclear accumulation of Cdc28 requires the nuclear localization signal of Cln3, which is also found in Whi3 complexes. Both Cln3 and Cdc28 are mainly cytoplasmic during early G1, and become nuclear in late G1. However, Whi3-deficient cells show a distinct nuclear accumulation of Cln3 and Cdc28 already in early G1. We propose that Whi3 constitutes a cytoplasmic retention device for Cln3-Cdc28 complexes, thus defining a key G1 event in yeast cells.

With the rapid development of biotechnology and nanomedicine, extensive research has focused on the investigations of delivering large-cargo molecules using nanoparticles through the cell membrane for disease diagnosis and treatment. Various inorganic and polymeric nanoparticles with optimized surface properties have been developed to carry these active cargo molecules such as organic molecules, oligonucleotides and proteins. Phagocytosis and pinocytosis have been suggested as the two major uptake mechanisms for nanoparticles to enter into cellular interior, but such mechanisms are still under debate. In order to enhance the efficiency of cellular uptake of nanoparticles and further understand the physiological process, it is important to investigate detailed interaction mechanisms between nanoparticles and cell membranes. Here, we will review the recent advances of the effect of nanoparticle properties (e.g., nanoparticle shape, size, charge, surface modification, etc.) on cellular uptake mechanisms. These will aid in the future design and development of nanoparticles with improved surface properties for drug and biomolecule delivery. Up to now, novel analytical techniques have been used to examine nanoparticle-cell membrane interactions, but their detailed uptake mechanisms and pathways still need more in-depth research. It is suggested that developing appropriate analytical techniques to study cellular uptake mechanisms of nanoparticles in real time is urgently desired.

Whi3 is an RNA-binding protein associated with the endoplasmic reticulum (ER) that binds the CLN3 mRNA and plays a key role in the efficient retention of cyclin Cln3 at the ER. In the present work, we have identified new Whi3-associated mRNAs by a genomic approach. A large and significant number of these Whi3 targets encode for membrane and exocytic proteins involved in processes such as transport and cell wall biogenesis. Consistent with the genomic data, we have observed that cell wall integrity is compromised in Whi3-deficient cells and found strong genetic interactions between WHI3 and the cell integrity pathway. Whi3-associated mRNAs are enriched in clusters of the tetranucleotide GCAU, and mutation of the GCAU clusters in the CLN3 mRNA caused a reduction in its association to Whi3, suggesting that these sequences may act as cis-determinants for binding. Our data suggest that Whi3 is involved in the regulation and/or localization of a large subset of mRNAs functionally related to the ER and, since it is important for different molecular processes such as cytoplasmic retention or exocytic traffic of proteins, we propose that Whi3 is a general modulator of protein fate in budding yeast.

Copper-based nanomaterials have broad applications in electronics, catalysts, solar energy conversion, antibiotics, tissue imaging, and photothermal cancer therapy. However, it is challenging to prepare ultrasmall and ultrastable CuS nanoclusters (NCs) at room temperature. In this article, a simple method to synthesize water-soluble, monodispersed CuS NCs is reported based on the strategy of trapping the reaction intermediate using thiol-terminated, alkyl-containing short-chain poly(ethylene glycol)s (HS-(CH2)11-(OCH2CH2)6-OH, abbreviated as MUH). The MUH-coated CuS NCs have superior stability in solutions with varied pH values and are stable in pure water for at least 10 months. The as-prepared CuS NCs were highly toxic to A549 cancer cells at a concentration of higher than 100 μM (9.6 μg/mL), making them be potentially applicable as anticancer drugs via intravenous administration by liposomal encapsulation or by direct intratumoral injection. Besides, for the first time, CuS NCs were used for antibacterial application, and 800 μM (76.8 μg/mL) CuS NCs could completely kill the E. coli cells through damaging the cell walls. Moreover, the NCs synthesized here have strong near-infrared (NIR) absorption and can be used as a candidate reagent for photothermal therapy and photoacoustic imaging. The method of trapping the reaction intermediate for simple and controlled synthesis of nanoclusters is generally applicable and can be widely used to synthesize many metal-based (such as Pt, Pd, Au, and Ag) nanoclusters and nanocrystals.

Electrospun nanofibers were widely studied to be applied as potential materials for tissue engineering. A new technology to make poly-l-lactic acid/chitosan core/shell nanofibers from heterologous solution by coaxial electrospinning technique was designed for vascular gasket. Chitosan surface was cross-linked by genipin and modified by heparin. Different ratios of PLA/CS in heterologous solution were studied to optimize the surface morphology of fibers. Clean core-shell structures formed with a PLA/CS ratio at 1:3. Superior biocompatibility and mechanical properties were obtained by optimizing the core-shell structure morphology and surface cross-linking of chitosan. UE7T-13 cells grew well on the core-shell structure fibers as indicated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) results and scanning electron microscopy (SEM) images. Compared with the pure PLA fiber meshes and commercial vascular patch, PLA/CS core-shell fibers had better mechanical strength. The elastic modulus was as high as 117.18 MPa, even though the yield stress of the fibers was lower than that of the commercial vascular patch. Attachment of red blood cell on the fibers was evaluated by blood anticoagulation experiments and in vitro blood flow experiments. The activated partial thromboplastin time (APTT) and prothrombin time (PT) value from PLA/CS nanofibers were significantly longer than that of pure PLA fibers. SEM images indicated there were hardly any red blood cells attached to the fibers with chitosan coating and heparin modification. This type of fiber mesh could potentially be used as vascular gasket.