Stereocontrolled Synthesis of Aryl C-Nucleosides under Ambient Conditions.

A stereocontrolled synthesis of an aryl C-nucleoside has been developed using D-ribals and arylboronic acids catalyzed by palladium without additional ligands in common solvents under an open-air atmosphere at room temperature. This protocol features very mild conditions, simplicity in operation, exclusive ß-stereoselectivity, broad substrate scopes, and good compatibility with reactive amino and hydroxyl groups. The functionalization of unsaturated C-nucleosides and the late-stage glycosylation of natural products/drugs demonstrated the high practicality of this strategy.

CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

Treg cells-derived exosomes promote blood-spinal cord barrier repair and motor function recovery after spinal cord injury by delivering miR-2861.

The blood-spinal cord barrier (BSCB) is a physical barrier between the blood and the spinal cord parenchyma. Current evidence suggests that the disruption of BSCB integrity after spinal cord injury can lead to secondary injuries such as spinal cord edema and excessive inflammatory response. Regulatory T (Treg) cells are effective anti-inflammatory cells that can inhibit neuroinflammation after spinal cord injury, and their infiltration after spinal cord injury exhibits the same temporal and spatial characteristics as the automatic repair of BSCB. However, few studies have assessed the relationship between Treg cells and spinal cord injury, emphasizing BSCB integrity. This study explored whether Treg affects the recovery of BSCB after SCI and the underlying mechanism. We confirmed that spinal cord angiogenesis and Treg cell infiltration occurred simultaneously after SCI. Furthermore, we observed significant effects on BSCB repair and motor function in mice by Treg cell knockout and overexpression. Subsequently, we demonstrated the presence and function of exosomes in vitro. In addition, we found that Treg cell-derived exosomes encapsulated miR-2861, and miR-2861 regulated the expression of vascular tight junction (TJs) proteins. The luciferase reporter assay confirmed the negative regulation of IRAK1 by miR-2861, and a series of rescue experiments validated the biological function of IRAKI in regulating BSCB. In summary, we demonstrated that Treg cell-derived exosomes could package and deliver miR-2861 and regulate the expression of IRAK1 to affect BSCB integrity and motor function after SCI in mice, which provides novel insights for functional repair and limiting inflammation after SCI.

Exosomal USP13 derived from microvascular endothelial cells regulates immune microenvironment and improves functional recovery after spinal cord injury by stabilizing IκBα.

Spinal cord injury (SCI) can result in irreversible sensory and motor disability with no effective treatment currently. After SCI, infiltrated macrophages accumulate in epicenter through destructed blood-spinal cord barrier (BSCB). Further, great majority of macrophages are preferentially polarized to M1 phenotype, with only a few transient M2 phenotype. The purpose of this study was to explore roles of vascular endothelial cells in microglia/macrophages polarization and the underlying mechanism. Lipopolysaccharide (LPS) was used to pretreat BV2 microglia and RAW264.7 macrophages followed by administration of conditioned medium from microvascular endothelial cell line bEnd.3 cells (ECM). Analyses were then performed to determine the effects of exosomes on microglia/macrophages polarization and mitochondrial function. The findings demonstrated that administration of ECM shifted microglia/macrophages towards M2 polarization, ameliorated mitochondrial impairment, and reduced reactive oxygen species (ROS) production in vitro. Notably, administration of GW4869, an exosomal secretion inhibitor, significantly reversed these observed benefits. Further results revealed that exosomes derived from bEnd.3 cells (Exos) promote motor rehabilitation and M2 polarization of microglia/macrophages in vivo. Ubiquitin-specific protease 13 (USP13) was shown to be significantly enriched in BV2 microglia treated with Exos. USP13 binds to, deubiquitinates and stabilizes the NF-κB inhibitor alpha (IκBα), thus regulating microglia/macrophages polarization. Administration of the selective IκBα inhibitor betulinic acid (BA) inhibited the beneficial effect of Exos in vivo. These findings uncovered the potential mechanism underlying the communications between vascular endothelial cells and microglia/macrophages after SCI. In addition, this study indicates exosomes might be a promising therapeutic strategy for SCI treatment.

USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m6A Modification of YAP1 mRNA.

RNA N6-methyladenosine (m6A) modification is involved in diverse biological processes. However, its role in spinal cord injury (SCI) is poorly understood. The m6A level increases in injured spinal cord, and METTL3, which is the core subunit of methyltransferase complex, is upregulated in reactive astrocytes and further stabilized by the USP1/UAF1 complex after SCI. The USP1/UAF1 complex specifically binds to and subsequently removes K48-linked ubiquitination of the METTL3 protein to maintain its stability after SCI. Moreover, conditional knockout of astrocytic METTL3 in both sexes of mice significantly suppressed reactive astrogliosis after SCI, thus resulting in widespread infiltration of inflammatory cells, aggravated neuronal loss, hampered axonal regeneration, and impaired functional recovery. Mechanistically, the YAP1 transcript was identified as a potential target of METTL3 in astrocytes. METTL3 could selectively methylate the 3'-UTR region of the YAP1 transcript, which subsequently maintains its stability in an IGF2BP2-dependent manner. In vivo, YAP1 overexpression by adeno-associated virus injection remarkably contributed to reactive astrogliosis and partly reversed the detrimental effects of METTL3 knockout on functional recovery after SCI. Furthermore, we found that the methyltransferase activity of METTL3 plays an essential role in reactive astrogliosis and motor repair, whereas METTL3 mutant without methyltransferase function failed to promote functional recovery after SCI. Our study reveals the previously unreported role of METTL3-mediated m6A modification in SCI and might provide a potential therapy for SCI.SIGNIFICANCE STATEMENT Spinal cord injury is a devastating trauma of the CNS involving motor and sensory impairments. However, epigenetic modification in spinal cord injury is still unclear. Here, we propose an m6A regulation effect of astrocytic METTL3 following spinal cord injury, and we further characterize its underlying mechanism, which might provide promising strategies for spinal cord injury treatment.

ZP3 and AIPL1 participate in GVBD of mouse oocytes by affecting the nuclear membrane localization and maturation of farnesylated prelamin A.

The low maturation rate of oocytes is an important reason for female infertility and failure of assisted pregnancy. The germinal vesicle breakdown (GVBD) is a landmark event of oocyte maturation. In our previous studies, we found that zona pellucida 3 (ZP3) was strongly concentrated in the nuclear region of germinal vesicle (GV) oocytes and interacted with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) and lamin A to promote GVBD. In the current study, we found that lamin A is mainly concentrated in the nuclear membrane. When ZP3 is knocked down, lamin A will be partially transferred to the nucleus of oocytes. The prelamin A is increased in both the nuclear membrane and nucleus, while phosphorylated lamin A (p-lamin A) is significantly reduced. AIPL1 was also proved to accumulate in the GV region of oocytes, and ZP3 deletion can significantly inhibit the aggregation of AIPL1 in the nuclear region. Similar to ZP3 knockdown, the absence of AIPL1 resulted in a decrease in the occurrence of GVBD, an increase in the amount of prelamin A, and a significant decrease in p-lamin A in oocytes developed in vitro. Finally, we propose the hypothesis that ZP3 can stabilize farnesylated prelamin A on the nuclear membrane of AIPL1, and promote its further processing into mature lamin A, therefore promoting the occurrence of GVBD. This study may be an important supplement for the mechanism of oocyte meiotic resumption and provide new diagnostic targets and treatment clues for infertility patients with oocyte maturation disorder.

Stereoselective Synthesis of 2-Deoxythiosugars from Glycals.

2-deoxythiosugars are more stable than 2-deoxysugars occurring broadly in bioactive natural products and pharmaceutical agents. An effective and direct methodology to stereoselectively synthesize α-2-deoxythioglycosides catalyzed by AgOTf has been developed. Various alkyl thiols and thiophenols were explored and the desired products were formed in good yields with excellent α-selectivity. This method was further applied to the syntheses of S-linked disaccharides and late-stage 2-deoxyglycosylation of estrogen, L-menthol, and zingerone thiols successfully.

Metal enhanced fluorescence improved protein and DNA detection by zigzag Ag nanorod arrays.

As metal nano-arrays show great potential on metal enhanced fluorescence (MEF) than random nanostructures, MEF of Ag zigzag nanorod (ZNR) arrays made by oblique angle deposition has been studied for biomolecule-protein interaction and DNA hybridization. By changing the folding number and the deposition substrate temperature, a 14-fold enhancement factor (EF) is obtained for biotin-neutravidin detection. The optimal folding number is decided as Z=7, owing to the high scattering intensity of Ag ZNRs. The substrate temperature T=25°C and 0°C slightly alters the morphology of Ag ZNRs but has no big difference in EF. Further, Ag ZNRs deposited on a layer of Ag film have been introduced to the DNA hybridization and a significant signal enhancement has been observed through the fluorescence microscope. Through a detailed quantitative EF analysis, which excludes the enhancing effect from the increased surface area of ZNRs and only considers the contribution of MEF, an EF of 28 is achieved for the hybridization of two single-stranded oligonucleotides with 33 bases. Furthermore, a limit of detection is determined as 0.01pM. We believe that the Ag ZNR arrays can serve as a universal and sensitive bio-detection platform.

Microfluidic-based metal enhanced fluorescence for capillary electrophoresis by Ag nanorod arrays.

As metal nanorods show much higher metal enhanced fluorescence (MEF) than metal nanospheres, microfluidic-based MEF is first explored with Ag nanorod (ND) arrays made by oblique angle deposition. By measuring the fluorescein isothiocyanate (FITC) solution sandwiched between the Ag NDs and a piece of cover slip, the enhancement factors (EFs) are found as 3.7 ± 0.64 and 6.74 ± 2.04, for a solution thickness at 20.8 µm and 10 µm, respectively. Because of the strong plasmonic coupling between the adjacent Ag NDs, only the emission of the fluorophores present in the three-dimensional NDs array gets enhanced. Thus, the corresponding effective enhancement factors (EEFs) are revealed to be relatively close, 259 ± 92 and 340 ± 102, respectively. To demonstrate the application of MEF in microfluidic systems, a multilayer of SiO2 NDs/Ag NDs is integrated with a capillary electrophoresis device. At a microchannel depth of 10 µm, an enhancement of 6.5 fold is obtained for amino acids separation detection. These results are very encouraging and open the possibility of MEF applications for the Ag ND arrays decorated microchannels. With the miniaturization of microfluidic devices, microfluidic-based MEF by Ag ND arrays will likely find more applications with further enhancement.