Biology subjects

Gin, A.

Publications and source records attributed to Gin, A..

3 recordsLinked to original sources

bioRxiv2024

Preferential Release of microRNAs via Extracellular Vesicles is Associated with Ductal Carcinoma In Situ to Invasive Breast Cancer Progression

Ductal carcinoma in situ (DCIS) is a benign "pre-cancer" that increases the risk of invasive breast cancer (IBC). Not all DCIS progress to IBC, and the primary factors driving progression remain unclear. Small extracellular vesicles (sEVs) or exosomes are known to play a role in advanced cancers, but their involvement in DCIS is poorly understood. This study examined the role of sEVs and their RNA content in DCIS progression. Rab27A, which regulates exosome release, is elevated in DCIS and IBC tissues compared with normal breast tissues. Inhibition of sEV release via Rab27A knockdown alters pro-invasive pathways and reduces invasion in a DCIS mouse model. Using the isogenic MCF10 breast cancer progression series, we found a significant increase in microRNAs (miRNAs) in sEVs from normal to malignant states, with the highest number of differentially expressed miRNAs in IBC sEVs compared with DCIS sEVs. In vivo, DCIS invasive progression elevated circulating sEV miRNA levels, which decreased upon Rab27A knockdown. Re-expression of miR-205, preferentially loaded into IBC sEVs, reduced proliferation, invasion, and EMT marker expression in DCIS cells. Combined Rab27A knockdown and miR-205 expression repressed TGF-{beta} signaling, activated p38, and induced cell cycle arrest and cell death. These findings illustrate that sEVs and their miRNAs promote DCIS progression, and the reintroduction of miR-205 in DCIS cells can inhibit invasive progression.

cancer biology↗

bioRxiv2023

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+ cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+ cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+ cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

cell biology↗

bioRxiv2023

Label-free, real-time monitoring of membrane binding events at zeptomolar concentrations using frequency-locked optical microresonators

Binding events to elements of the cell membrane act as receptors which regulate cellular function and communication and are the targets of many small molecule drug discovery efforts for agonists and antagonists. Conventional techniques to probe these interactions generally require labels and large amounts of receptor to achieve satisfactory sensitivity. Whispering gallery mode microtoroid optical resonators have demonstrated sensitivity to detect single-molecule binding events. Here, we demonstrate the use of frequency-locked optical microtoroids for characterization of membrane interactions in vitro at zeptomolar concentrations using a supported biomimetic membrane. Arrays of microtoroids were produced using photolithography and subsequently modified with a biomimetic membrane, providing high quality (Q) factors (>106) in aqueous environments. Fluorescent recovery after photobleaching (FRAP) experiments confirmed the retained fluidity of the microtoroid supported-lipid membrane with a diffusion coefficient of 3.38 {+/-} 0.26 m2 {middle dot}s-1. Utilizing this frequency-locked membrane-on-a-chip model combined with auto-balanced detection and non-linear post-processing techniques, we demonstrate zeptomolar detection levels The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside (GM1) was monitored in real-time, with an apparent equilibrium dissociation constant (kd) = 1.53 nM. The measured affiny of the agonist dynorphin A 1-13 to the {kappa}-opioid receptor revealed a kd = 3.1 nM using the same approach. Radioligand binding competition with dynorphin A 1-13 revealed a Kd in agreement (1.1 nM) with the unlabeled method. The biosensing platform reported herein provides a highly sensitive real-time characterization of membrane embedded protein binding kinetics, that is rapid and label-free, for toxin screening and drug discovery, among other applications.

biophysics↗