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RNA therapy appears to be a promising strategy to treat various diseases. In recent years, mRNA vaccines have shown notable efficacy in preclinical studies for cancer vaccines, autoimmune disease, and pandemic intervention. Internal ribosome entry sites (IRESs) are structured RNA elements to initiate translation independent of 5-cap recognition of mRNA, particularly show efficient activity under disease stress that causes global canonical translation repression. Studies on distinct structural properties and interaction with translational factors have revealed the mechanisms and regulation of IRES-mediated translation. This allowed the application of IRES for cap-independent translation and dynamic modulation of protein expression in response to cell signals. In this review, we discuss the current platforms and emerging strategies for employing IRES-mediated translation towards novel RNA therapeutics.

Macrophages are multifunctional immune cells whose functions depend on polarizable phenotypes and the microenvironment. Macrophages have two phenotypes, including the M1 proinflammatory phenotype and the M2 anti-inflammatory phenotype, which play important roles in many inflammatory responses and diseases. α-Ketoglutarate is a key metabolite of the TCA cycle and can regulate the phenotype of macrophage polarization to exert anti-inflammatory effects in many inflammation-related diseases. In this review, we primarily elucidate the metabolism, regulatory mechanism, and perspectives of α-ketoglutarate on macrophages. The regulation of macrophage polarization by α-ketoglutarate may provide a promising target for the prevention and therapy of inflammatory diseases and is beneficial to animal health.

NDH-1 is a key component of the cyclic-electron-transfer around photosystem I (PSI CET) pathway, an important antioxidant mechanism for efficient photosynthesis. Here, we report a 3.2-Å-resolution cryo-EM structure of the ferredoxin (Fd)-NDH-1L complex from the cyanobacterium Thermosynechococcus elongatus . The structure reveals three β-carotene and fifteen lipid molecules in the membrane arm of NDH-1L. Regulatory oxygenic photosynthesis-specific (OPS) subunits NdhV, NdhS and NdhO are close to the Fd-binding site whilst NdhL is adjacent to the plastoquinone (PQ) cavity, and they play different roles in PSI CET under high-light stress. NdhV assists in the binding of Fd to NDH-1L and accelerates PSI CET in response to short-term high-light exposure. In contrast, prolonged high-light irradiation switches on the expression and assembly of the NDH-1MS complex, which likely contains no NdhO to further accelerate PSI CET and reduce ROS production. We propose that this hierarchical mechanism is necessary for the survival of cyanobacteria in an aerobic environment. NDH-1 is a key component of the cyclic-electron-transfer around photosystem I pathway, an antioxidant mechanism for efficient photosynthesis. Here, authors report a cryo-EM structure of the ferredoxin (Fd)-NDH-1L complex from the cyanobacterium Thermosynechococcus elongatus .

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function. Successive fusion of yeast chromosomes is used to produce a single-chromosome strain that is viable, albeit with slightly reduced fitness.

CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.

Telomerase, a multi-subunit ribonucleoprotein complex, is a unique reverse transcriptase that catalyzes the processive addition of a repeat sequence to extend the telomere end using a short fragment of its own RNA component as the template. Despite recent structural characterizations of human and Tetrahymena telomerase, it is still a mystery how telomerase repeatedly uses its RNA template to synthesize telomeric DNA. Here, we report the cryo-EM structure of human telomerase holoenzyme bound with telomeric DNA at resolutions of 3.5 Å and 3.9 Å for the catalytic core and biogenesis module, respectively. The structure reveals that a leucine residue Leu980 in telomerase reverse transcriptase (TERT) catalytic subunit functions as a zipper head to limit the length of the short primer–template duplex in the active center. Moreover, our structural and computational analyses suggest that TERT and telomerase RNA (hTR) are organized to harbor a preformed active site that can accommodate short primer–template duplex substrates for catalysis. Furthermore, our findings unveil a double-fingers architecture in TERT that ensures nucleotide addition processivity of human telomerase. We propose that the zipper head Leu980 is a structural determinant for the sequence-based pausing signal of DNA synthesis that coincides with the RNA element-based physical template boundary. Functional analyses unveil that the non-glycine zipper head plays an essential role in both telomerase repeat addition processivity and telomere length homeostasis. In addition, we also demonstrate that this zipper head mechanism is conserved in all eukaryotic telomerases. Together, our study provides an integrated model for telomerase-mediated telomere synthesis.

Sex determination is crucial for the transmission of genetic information through generations. In mammal, this process is primarily regulated by an antagonistic network of sex-related genes beginning in embryonic development and continuing throughout life. Nonetheless, abnormal expression of these sex-related genes will lead to reproductive organ and germline abnormalities, resulting in disorders of sex development (DSD) and infertility. On the other hand, it is possible to predetermine the sex of animal offspring by artificially regulating sex-related gene expression, a recent research hotspot. In this paper, we reviewed recent research that has improved our understanding of the mechanisms underlying the development of the gonad and primordial germ cells (PGCs), progenitors of the germline, to provide new directions for the treatment of DSD and infertility, both of which involve manipulating the sex ratio of livestock offspring.

Abstract Univariate or bivariate animal models were used to estimate the variance components and co-variance components for eight reproductive traits: total number born (TNB), number born alive (NBA), total litter weight of piglets born alive (BALWT), number of healthy births (NHB), number of weak births (NWB), number of deformed fetuses (NDF), number of stillborn (NSB), and number of mummified pigs (MUMM). In addition, the phenotypic and genetic correlations between traits at different parities were also estimated. The results showed that the heritabilities of the eight reproductive traits were lower than 0.10. Genetic correlations between NHB and TNB, NBA, or BALWT were 0.68, 0.84 and 0.89 respectively; whereas genetic correlations between NHB and NWB, NDF, NSB or MUMM were negative or close to 0, ranging from −0.28 to 0.13. NHB was relatively identified as an ideal informative trait for selection for improved reproduction. Furthermore, genetic correlations between different parities for all traits, except for NDF were strongly positive, showing that it was reasonable to consider different parities as the same trait. For NDF, genetic correlations between the first and the other parities were low, indicating that it was probably unreasonable to cull pigs according to the NDF at first parity. Optimum reproductive traits were observed at the third parity, and reinforcing the management of sows in the first and > 4 parities can be a practical method for improving reproductive traits.

Cryopreservation techniques have been widely used, especially in biomedical applications and preservation of germplasm resources. Ideally, biological materials would maintain functional integrity as well as a normal structure and can be recovered when needed. However, this tool does not work all the time. Ice formation and growth are the key challenges. The other major reason is that the cryoprotective agents (CPAs) currently used do not meet these needs and are always accompanied by their cytotoxicity. A comprehensive and synergistic approach that focuses on the overall frozen biological system is crucial for the evolution of cryopreservation methods. In this review, we first summarize the fundamental damage mechanisms during cryopreservation, as well as common cryoprotectants and their limitations. Next, we discuss materials that interact with ice to improve cryopreservation outcomes. We evaluated natural and synthetic materials, including sugars and polymers, AFPs and mimics, ice nucleators, and hydrogels. In addition, biochemical regulation, which enhances the tolerance of biosamples to cryopreservation-induced stresses, was also mentioned. Nanotechnology, cell encapsulation, cryomesh, and isochoric freezing, such scalable approaches, are further discussed for cryopreservation. Finally, future research directions in this field for efficient cryopreservation are proposed. We emphasized the need for multidisciplinary progress to address these challenges. The combination of cryobiology mechanisms with technologies, such as synthetic biology, nanotechnology, microfluidics, and 3D bioprinting, is highlighted. Graphical Abstract

RNA polymerase III (Pol III) synthesizes structured, essential small RNAs, such as transfer RNA, 5S ribosomal RNA and U6 small nuclear RNA. Pol III, the largest nuclear RNA polymerase, is composed of a conserved core region and eight constitutive regulatory subunits, but how these factors jointly regulate Pol III transcription remains unclear. Here, we present cryo-EM structures of human Pol III in both apo and elongating states, which unveil both an orchestrated movement during the apo-to-elongating transition and an unexpected apo state in which the RPC7 subunit tail occupies the DNA–RNA-binding cleft of Pol III, suggesting that RPC7 plays important roles in both autoinhibition and transcription initiation. The structures also reveal a proofreading mechanism for the TFIIS-like subunit RPC10, which stably retains its catalytic position in the secondary channel, explaining the high fidelity of Pol III transcription. Our work provides an integrated picture of the mechanism of Pol III transcription regulation. High-resolution cryo-EM structures of human RNA Pol III in both apo and elongating states provide insights into an autoinhibitory mechanism controlling the transition to transcription elongation unique to the mammalian holoenzyme.