Ferroptosis is a form of iron-dependent programmed cell death, which is distinct from apoptosis, necrosis, and autophagy. Mitochondria play a critical role in initiating and amplifying ferroptosis in cancer cells. Voltage-Dependent Anion Channel 1 (VDAC1) embedded in the mitochondrial outer membrane, exerts roles in regulation of ferroptosis. However, the mechanisms of VDAC1 oligomerization in regulating ferroptosis are not well elucidated. Here, we identify that a VDAC1 binding protein V-Set and Transmembrane Domain Containing 2 Like (VSTM2L), mainly localized to mitochondria, is positively associated with prostate cancer (PCa) progression, and a key regulator of ferroptosis. Moreover, VSTM2L knockdown in PCa cells enhances the sensitivity of RSL3-induced ferroptosis. Mechanistically, VSTM2L forms complex with VDAC1 and hexokinase 2 (HK2), enhancing their binding affinity and preventing VDAC1 oligomerization, thereby inhibiting ferroptosis and maintaining mitochondria homeostasis in vitro and in vivo. Collectively, our findings reveal a pivotal role for mitochondria-localized VSTM2L in driving ferroptosis resistance and highlight its potential as a ferroptosis-inducing therapeutic target for the treatment of PCa.

The purpose of this work was to develop biodegradable microspheres for long term delivery of a potent acetyl cholinesterase inhibitor, huperzine A (Hup-A), which is of interest in the palliative treatment of Alzheimer's disease. Microspheres were successfully prepared with specifically end-group uncapped poly(d,l-lactide acid) and poly(d,l-lactide-co-glycolide acid) using a simple o/w solvent evaporation method. The morphology, particle size and size distribution, drug loading capacity, drug entrapment efficiency (EE) and in vitro drug release were studied in detail. It was found that the terminal group and the inherent viscosity (IV) of the polymers played key role in the drug encapsulation: higher EE was achieved with end-group uncapped and low IV polymers. In vitro drug release from microspheres made from the selected three kinds of polymers revealed sustained release of Hup-A without significant burst release. Preliminary pharmacokinetic study following subcutaneous injection of Hup-A loaded microspheres illustrated the sustained release of the drug over 6—8 weeks at clinically relevant doses in vivo. The studies demonstrated the feasibility of long term delivery of Hup-A using biodegradable microspheres.

CRISPR/Cas9 is an efficient, accurate, and optimizable genome-editing tool. Here, we present a modified CRISPR/Cas9 genome-editing protocol for single nucleotide mutation in adherent cell lines. The protocol was adapted to focus on ease of use and efficiency. The protocol here describes how to generate a single nucleotide mutation in cultured 22Rv1 cells. We have also used the protocol in other adherent cell types. Thus, the protocol can be applied to assessing the effect of non-coding single nucleotide polymorphisms (SNPs) in a variety of cell types.

Background: Mostly mediated by STIM1 and Orai1, Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that has been linked with many types of diseases like myopathy, epilepsy, immune deficient diseases, and cancer. Currently many details about SOCE process have been revealed. However, the dissection of the molecular mechanisms underlying SOCE activation and the development possible treatments of SOCE related diseases are hindered, partly due to the lack of more specific pharmacological tools and poor understandings of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. Objective and methods: Using a combination of Ca2+ and förster resonance energy transfer (FRET) imaging, we aimed to systemically delineate the sites or domains needed for the inhibition of digitoxin on SOCE. Results: We revealed that the inhibitory effects of digitoxin are exerted on Orai1. If digitoxin were applied to resting Orai1, then its inhibition was dependent on S27-S30 residues of Orai1. While 8h-incubation of digitoxin with STIM1-prebound Orai1 or its constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27/S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. Conclusions: Our study identified two sites critical for the inhibition on Orai1 channels, providing valuable targets for future design of SOCE inhibitors.