Endocannabinoid Receptor Expression in Early Zebrafish Development.

The endocannabinoid system (ECS) is widely studied due to its interactions with cannabis and its role in modulating physiological responses. While most research has focused on the effects of cannabis on adult ECSs, recent studies have begun to investigate the role of the ECS in developing organisms. However, little is known about the spatial or temporal expression of these receptors during early development. This study combines reverse transcriptase-polymerase chain reaction (RT-PCR) with in situ hybridizations to compile a timeline of the developmental expression of six key cannabinoid receptors; cb1, cb2, trpv1, trpa1a, trpa1b, and gpr55 in zebrafish embryos, starting from as early as 6-h postfertilization (hpf) until 3 days pf (dpf). This time frame is roughly equivalent to 2-10 weeks in human embryonic development. All six genes were confirmed to be expressed within this time range and share similarities with human and rodent expression. Cb1 expression was first detected between 12 and 24 hpf in the retina and CNS, and its expression increased thereafter and was more evident in the olfactory bulb, tegmentum, hypothalamus, and gut. Cb2 expression was relatively high at the 6 and 24 hpf timepoints, as determined by RT-PCR but was undetectable at other times. Trpv1 was first detected at 1 dpf in the trigeminal (Tg) ganglia, Rohon-Beard neurons, and lateral line, and its expression increased in the first 3 dpf. Expression of trpa1a was first detected as late as 3 dpf in vagal (V) neurons, whereas trpa1b was first detected at 1 dpf associated with Tg, glossopharyngeal, and V ganglia. Expression of gpr55 was diffuse and widespread throughout the brain and head region but was undetectable elsewhere in the embryo. Thus, receptor expression was found to be enriched in the central nervous system and within sensory neurons. This work aims to serve as a foundation for further investigation on the role of cannabinoid and cannabinoid-interacting receptors in early embryonic development.

Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a mutation in SMN1 that stops production of SMN (survival of motor neuron) protein. Insufficient levels of SMN results in the loss of motor neurons, which causes muscle weakness, respiratory distress, and paralysis. A nearly identical gene (SMN2) contains a C-to-T transition which excludes exon 7 from 90% of the mature mRNA transcripts, leading to unstable proteins which are targeted for degradation. Although SMN2 cannot fully compensate for a loss of SMN1 due to only 10% functional mRNA produced, the discovery of the intronic splicing silencer (ISS-N1) opened a doorway for therapy. By blocking its function with antisense oligonucleotides manipulated for high specificity and efficiency, exon 7 can be included to produce full-length mRNA, which then compensates for the loss of SMN1. Nusinersen (Spinraza), the first FDA-approved antisense oligonucleotide drug targeting SMA, was designed based on this concept and clinical studies have demonstrated a dramatic improvement in patients. Novel chemistries including phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNAs), as well as peptide conjugates such as Pip that facilitate accurate targeting to the central nervous system, are explored to increase the efficiency of exon 7 inclusion in the appropriate tissues to ameliorate the SMA phenotype. Due to the rapid advancement of treatments for SMA following the discovery of ISS-N1, the future of SMA treatment is highly promising.