Systemic delivery of mRNA-based therapeutics remains a challenging issue for preclinical and clinical studies. Here, we describe new lipid-like nanoparticles (TT-LLNs) developed through an orthogonal array design, which demonstrates improved delivery efficiency of mRNA encoding luciferase in vitro by over 350-fold with significantly reduced experimental workload. One optimized TT3 LLN, termed O-TT3 LLNs, was able to restore the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. Consequently, these mRNA based nanomaterials merit further development for therapeutic applications.
Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs.
Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered Acidaminococcus sp. Cpf1 (AsCpf1) CRISPR RNAs (crRNAs) and 5 types of AsCpf1 mRNAs, and show that the top-performing modified crRNA (cr3′5F, containing five 2′-fluoro ribose at the 3′ termini) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3′5F and ψ-modified AsCpf1 or Lachnospiraceae bacterium Cpf1 (LbCpf1) mRNAs augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologs enabled genome editing in the presence of AsCpf1. Engineered CRISPR-Cpf1 systems should facilitate a broad range of genome editing applications.
Small-molecule organic fluorophores spectrally active in the 800-950 nm region are sought-after for their broad potential in biomedical and material applications. We have developed a new family of brightly fluorescent dyes (ECX) to meet this challenge. ECX dyes are transparent to the visible region, while strongly absorbing in the NIR region at approximately 880 nm. They emit at around 915 nm with a fluorescence quantum yield up to 13.3 %. ECX dyes exhibit high chemostability, high photostability, and low tendency to aggregate. Other merits of ECX dyes include low degree of solvatochromism and facile post-synthetic derivatization. ECX dyes potentially make available the 800-950 nm region for spectroscopic and microscopic applications and are also expected to find broad material applications.
The input characteristics and distribution of climbing fibre field potentials evoked by electrical stimulation of various parts of the skin were investigated in the cerebellum of barbiturate anaesthetized rats. Climbing fibre responses were recorded in sagittally oriented microelectrode tracks across the mediolateral width of the anterior lobe.
Climbing fibres with similar response latencies and convergence patterns terminated in sagittal bands with widths of 0.5–1.5 mm. The principal organization of the anterior lobe with respect to input characteristics and locations of sagittal zones was similar to that in the cat and ferret. Hence, the sagittal bands in the rat were tentatively named the a, b, c1, c2 and d1 zones.
In contrast to the cat and ferret, the a zone of the rat was characterized by short latency ipsilateral climbing fibre input. Furthermore, it was divisible into a medial ‘a1′ zone with convergent, proximal input and a lateral ‘ax’ zone with somatotopically organized input. A forelimb area with similar location and input characteristics as the X zone of the cat was found, but it formed an integral part of the ax zone. A somatotopic organization of ipsilateral, short latency climbing fibre input was alsofound in the c1 zone.
Rostrally in the anterior lobe, climbing fibres activated at short latencies from the ipsilateral side of the body terminated in a somatotopically organized transverse band which extended from the midline to the lateral end of the anterior lobe.
The absence of the C3 and Y zones may be interpreted as a reflection of differences in the organization of the motor systems in the rat as compared with the cat. Skilled movements, which in the cat are controlled by the C1, C3 and Y zones via the anterior interposed nucleus, may in the rat be partly controlled by the ax zone via the rostrolateral part of the fastigial nucleus.
Efficient and safe delivery of the CRISPR/Cas system is one of the key challenges for genome-editing applications in humans. Herein, we designed and synthesized a series of biodegradable lipidlike compounds containing ester groups for the delivery of mRNA-encoding Cas9. Two lead materials, termed N-methyl-1,3-propanediamine (MPA)-A and MPA-Ab, showed a tunable rate of biodegradation. MPA-A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains in the presence of esterase or in wild-type mice. Most importantly, MPA-A and MPA-Ab demonstrated efficient delivery of Cas9 mRNA both in vitro and in vivo. Consequently, these biodegradable lipidlike nanomaterials merit further development as genome-editing delivery tools for biological and therapeutic applications.
Insulin-like peptide 5 (INSL5), a member of the insulin/relaxin superfamily, can activate the G-protein-coupled receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), but its precise biological functions are largely unknown. Recent studies suggest that INSL5/RXFP4 is involved in the control of food intake and glucose homoeostasis. We report in the present study that RXFP4 is present in the mouse insulinoma cell line MIN6 and INSL5 augments glucose-stimulated insulin secretion (GSIS) both in vitro and in vivo. RXFP4 is also expressed in the mouse intestinal L-cell line GLUTag and INSL5 is capable of potentiating glucose-dependent glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. We propose that the insulinotrophic effect of INSL5 is probably mediated through stimulation of insulin/GLP-1 secretion and the INSL5/RXFP4 system may be a potential therapeutic target for Type 2 diabetes.
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