Journal: Science Advances

Article Title: Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift

doi: 10.1126/sciadv.adv8656

Figure Lengend Snippet: ( A ) Membrane system facilitates mass transport primarily through a dominant convective mechanism, driven by a pressure gradient that directs the flow of ss-mRNA and dsRNA through the membrane pores allowing for adsorptive interaction with pore walls. ( B ) Primary amine ligands carry a positive charge, which can be tuned via mobile phase pH (to control ligand deprotonation) or salt concentration adjustments (to screen electrostatic interactions) creating a surface capable of anion exchange and hydrogen bonding. ( C ) These multimodal interactions allow ss-mRNA to bind to the membrane surface through not only electrostatic interaction with its negatively charged phosphate backbone but also hydrogen bonding with exposed hydrogen bond acceptor sites within unhybridized base pairs. ( D ) In contrast, dsRNA primarily binds to the membrane via electrostatic interactions, as its base pairs are naturally hybridized and thus inaccessible for hydrogen bonding. This fundamental difference in binding mechanisms creates a separation opportunity, where pH, salt adjustments, and surface charge density can selectively modulate electrostatic and hydrogen bonding interactions, facilitating ss-mRNA purification.

Article Snippet: The dsRNA content in feed and eluate samples was quantified using a dsRNA ELISA kit (Hzymes Biotech Co. Ltd, Wuhan, China).

Techniques: Membrane, Control, Concentration Assay, Binding Assay, Purification

Journal: Science Advances

Article Title: Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift

doi: 10.1126/sciadv.adv8656

Figure Lengend Snippet: ( A ) Overlay of pure feed ss-mRNA (black line) and pure feed dsRNA (red line) flow-through (0 to 10 min), wash (10 to 50 min), and elution (50 to 80 min) behavior of a membrane stack (1 8700 ± 260 nmol/m 2 + 1 unmodified). ( B ) Pure feed dsRNA behavior of membranes stacks (1 modified + 1 unmodified) modified with 8700 ± 260 nmol/m 2 (red line), 4100 ± 120 nmol/m 2 (blue line), and 2080 ± 60 nmol/m 2 (black line). ( C ) Overlay of pure feed ss-mRNA (black line) and pure feed dsRNA (blue line) flow-through behavior of a membrane stack (4100 ± 120 nmol/m 2 + 1 unmodified). [(A) to (C)] Dashed red line represents a trace of the increasing mobile phase pH, and the dashed blue line represents the trace of the concentration of NaCl (and EDTA) in the wash. Q = 0.5 ml/min

Article Snippet: The dsRNA content in feed and eluate samples was quantified using a dsRNA ELISA kit (Hzymes Biotech Co. Ltd, Wuhan, China).

Techniques: Membrane, Modification, Concentration Assay

Journal: Science Advances

Article Title: Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift

doi: 10.1126/sciadv.adv8656

Figure Lengend Snippet: ( A ) Overlay of pure feed dsRNA with 4.2 mM spermine (black line) and 0 mM spermine (red line) flow-through (0 to 10 min), wash (10 to 50 min), and elution (50 to 80 min) behavior of a RC membrane containing 7890 ± 240 nmol/m 2 AEMA. Representative curves ( n = 3 injections). ( B ) Overlay of pure feed dsRNA with 4.2 mM spermine (green line) and pure ss-mRNA with 0.4 mM spermine of a RC membrane containing 7890 ± 240 nmol/m 2 AEMA. ( C ) Overlay of pure feed dsRNA with 4.1 mM spermine (green line) and pure ss-mRNA with 4.1 mM spermine (black line) of a RC membrane containing 7890 ± 240 nmol/m 2 AEMA. Representative curves ( n = 3 injections). ( D ) Dynamic characterization of representative ss-mRNA and dsRNA mixture (6 wt %) feedstock with a modified membrane containing 7890 ± 240 nmol/m 2 AEMA. [(A) to (D)] Dashed red line represents a trace of the increasing mobile phase pH, and the dashed blue line represents the trace of the concentration of NaCl (and EDTA) in the wash. Q = 0.5 ml/min

Article Snippet: The dsRNA content in feed and eluate samples was quantified using a dsRNA ELISA kit (Hzymes Biotech Co. Ltd, Wuhan, China).

Techniques: Membrane, Modification, Concentration Assay

Journal: Journal of innate immunity

Article Title: Cross-talk between human dendritic cell subsets influences expression of RNA sensors and inhibits picornavirus infection.

doi: 10.1159/000300568

Figure Lengend Snippet: Fig. 2. Human DC subsets express different profiles of viral nu- cleic acid receptors. a Mean expression (+SD) of TLR3, TLR7 and TLR8 in human moDCs, freshly isolated mDCs and freshly iso- lated pDCs. RNA was isolated from unstimulated cells and TLR mRNA transcript levels determined using conventional qPCR. Expression levels relative to PBGD. * p ! 0.05; * * p ! 0.01. b Ex- pression levels of RIG-I, MDA5, PKR and LGP2 in resting human DC subsets were determined as described for a . Shown is mean expression + SD. * p ! 0.05; * * p ! 0.01. c Confocal laser scanning microscopy analysis of MDA5 expression in human moDCs and pDCs. Unstimulated cells were stained using antibodies against

Article Snippet: Anti-RIG-I and anti-PKR antibodies were purchased from ProSci Incorporated and Becton Dickinson Transduction Laboratories, respectively.

Techniques: Expressing, Isolation, Confocal Laser Scanning Microscopy, Staining

Journal: Translational Psychiatry

Article Title: Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration

doi: 10.1038/s41398-023-02429-y

Figure Lengend Snippet: a – e Western blot analysis of p -TrkB, BDNF, p -mTOR, mTOR, p -AKT, and AKT levels in the hippocampus. f Immunofluorescence of CPE, MAP2, DCX, and GFAP; and the relative fluorescent intensities of g CPE in Sub, h MAP2 in Sub, i MAP2 in hilius, j DCX in DG, k GFAP in DG of WT, CPE flox/− , and CPE flox/flox mice at 100× and 400× (square in the panel). n = 6; * P < 0.05 and ** P < 0.01 compared with WT; values are mean ± SEM.

Article Snippet: After blocking with 5% nonfat milk, the membrane was blotted with antibodies against mouse anti-β-actin antibody (1:1000, CST, 4967S), mouse anti-CPE antibody (1:1000; BD bioscience, 610758), mouse anti-BDNF antibody (1:600, Abcam, UK, ab108319), rabbit anti-p-TrkB antibody (1:1000; Boster, Wuhan, China, BM4437), rabbit anti-AKT (1:2000, Cell Signaling Technology, 4691S), and anti-p-AKT (1:2000, Cell Signaling Technology, 23430S) overnight at 4 °C.

Techniques: Western Blot, Immunofluorescence