The Role of Hydrogen Bonds in Thermally Responsive Crystallization-Driven Template Autocatalysis.

Autocatalysis has been recognized to be involved in the emergence of life and intrinsic to biomolecular replication. Recently, an efficient template autocatalysis driven by solvent-free crystallization has been reported. Herein, we unveil the role of intermolecular hydrogen bonds formed by amides in crystallization-driven template autocatalysis (CDTA), which involves the autocatalytic activity, template selectivity, and thermal responsiveness. We found that the thermal-induced cis-trans isomerization of amides possibly affects the H-bonding-mediated template ability of products for autocatalytic transformation. As a result, CDTA can be reversibly inhibited and activated by tuning the reaction temperatures. Our work sheds light on the significance of noncovalent H-bonding interactions in artificial self-replicators.

Recent advances in the development of STING inhibitors: an updated patent review.

INTRODUCTION: STING is at the center of the cGAS-STING signaling and acts as the hub of the innate immune system. Hyper-activation of STING has been observed in various severe autoimmune diseases, such as AGS, SLE, and many other diseases including neurological and metabolic disorders. Therefore, STING has been considered as a promising target. In recent years, several STING inhibitors have been claimed in patents. AREAS COVERED: Small-molecule STING inhibitors reported in patents (disclosed before May 2022 through the public database at https://worldwide.espacenet.com) were summarized in this review and the available structure-activity relationships (SARs) and molecular mechanisms of action were presented. EXPERT OPINION: Compared with STING agonists, the development of STING inhibitors is still in its infancy and no candidates have entered clinical investigation stage. Fortunately, patent applications are appearing at an increasing rate and a few of them have been validated in vivo, thus providing valuable insights for further structural optimization. More efforts are urgently needed since it is not clear yet that inhibitors targeting STING can solely exert sufficient therapeutic effects on autoimmune diseases, and the toxicity profile of such inhibitors is unknown as well. Therefore, it is extremely important to identify a selective and efficacious STING inhibitor for clinical evaluation to provide proof-of-concept for this approach.

[Expression of the hemagglutinin and neuramidinase gene of influenza A virus H1N1 in Pichia methanolica].

On the basis of successful cloning the full length hemagglulinin (HA) and neuramidinase (NA) gene and sequence analysis of influenza virus H1N1, part of the gene was ligated into pMETA. Expression vectors pMETA/HA (52-1 557 bp) and pMETA/NA (121-1 263 bp) were constructed and expressed in pMAD16 induced by methanol. Recombinant protein was purified through Ni2+ affinity chromatography. Western blotting and ELISA were used to determine the antigenic activity of the recombinant protein. SDS-PAGE showed that the recombinant capsid gene could be overexpressed in Pichia methanolica. ELISA and Western blotting showed that the recombinant protein had antigenicity.

Cloning and analysis of IFRG (interferon responsive gene) in rabbit oocytes and preimplantation embryos.

Although there is more evidence that shows that IFNs (interferons) plays a very important role in the early development of the embryo, the mechanism of IFNs is still unclear. Our study showed that IFRG is expressed from oocytes- through to the preimplantation embryo in rabbits. This finding provides some clues for better understanding the role of IFNs in the development of the embryo. The full length of rabbit IFRG cDNA (Accession No. AJ584672), with a 2794bp encoding 131 amino acid sequence, was cloned IFRG expression can be detected in 8 different tissues: ovary, heart, lung, liver, kidney, spleen, cerebra, and the 18-day whole-body embryo. Whole-mount in situ hybridization showed that IFRG was highly expressed in the inner-cell mass of rabbit blastula. IFRG may play an important role in embryo development and tissue differentiation.

[Porcine follistatin cDNA cloning and expression in Escherichia coli].

The total RNA was extracted from porcine ovary. Porcine Follistatin cDNA was cloned by RT-PCR. Complete porcine follistatin cDNA coding sequences are presented including 1038 bp of open reading frame. The purified porcine follistatin cDNA was inserted into pGEX-4T-3 vector to construct the prokaryotic fusion protein expression vector. The recombinant expression plasmid was transformed into BL21 (DE3) and expression was induced by IPTG. Protein products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the yield of the Follistatin cDNA was a 63kD protein expression vector. Follistatin protein was expressed in the form of glutathione-S-transferase (GST) fusion protein in E. coli.

Effect of the Chinese traditional medicine "Bushen Yinao Pian" on the cerebral gene expression of the senescence-accelerated mouse prone 8/ta.

The effect of Chinese traditional medicine "Bushen Yinao Pian," a complex prescription used for anti-aging, on the cerebral gene expression of the senescence-accelerated mouse prone 8/Ta (SAMP8/Ta) had been studied with messenger ribonuclear acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR). Eight differential displayed bands had been discerned and sequenced. The sequences of those fragments are matched to adipocyte-specific protein-5; low density lipoprotein receptor-related protein associated protein-1; reticulon-3; cysteine and histidine-rich domain (CHORD)-containing, zinc-binding protein-1; cytochrome c oxidase subunit-2 (Cox-2); cytochrome c gene, MC1; DNA sequence from clone RP23-72M11 on chromosome X, respectively and a novel sequence fragment. Most of these genes are aging-related. It can be proved that the "Bushen Yinao Pian" truly has anti-aging function.

The Chinese traditional medicine 'Bushen Yinao Pian' increased the level of ageing-related gene LRPAP-1 expression in the cerebral tissue of accelerated senescence-prone mouse 8/Ta.

The molecular mechanism of the Chinese traditional medicine 'Bushen Yinao Pian' (a complex prescription used for clinical anti-ageing in China for over 20 years) is elusive. In this study, the cDNA of low-density lipoprotein related-receptor associated protein-1 (LRPAP-1), an ageing-related gene, which functions as a chaperon or escort protein in the intracellular transport of low-density lipoprotein related-receptor, a transporter of amyloid beta protein (AbetaP), had been cloned by screening cDNA library based on analyzing the gene expression in cerebral tissue between the test and the control accelerated senescence-prone mouse 8/Ta (SAMP8/Ta). The result shows that this complex prescription increased the expression level of LRPAP-1. It indicated that the Chinese traditional medicine 'Bushen Yinao Pian' plays an important role in anti-ageing by increasing LRPAP-1 expression level.

Identification and analysis of stage-specific expression of lysosome-associated protein transmembrane 4alpha gene during development of preimplantation rabbit nuclear transfer embryo.

The stage-specific expression of Lysosome-associated protein transmembrane 4alpha (LAPTM4alpha) in preimplantation rabbit nuclear transfer (NT) embryo was identified with the DDRT-PCR and reverse Northern Blot. The full length (1,364 bp) cDNA of LAPTM4alpha was screened out from cDNA library constructed with rabbit ovary and in situ hybridization (ISH) was used to trace the distribution of the LAPTM4alpha mRNA in intra-ovary, especially the follicle which proved that the LAPTM4alpha gene expression is involved in the follicles development, maturation, ovulation, luteinization, and preimplantation development in the rabbit (Oryctolagus cuniculus domestica). To our knowledge, this is the first characterization of LAPTM4alpha gene expression and mRNA distribution in the rabbit ovary and first evidence for this gene involving in follicle development and rabbit preimplantation development.

A complex prescription for vitiligo activates mitochondrial ATP synthase-6 expression in B-16 murine melanoma cells.

A complex prescription (CP) used traditionally for the treatment of vitiligo was prepared from water extract of eight plant materials. To study the mechanism of this preparation its effect on gene expression of B-16 murine melanoma cells was estimated using differential display method (DDRT-PCR). The results showed that the complex prescription was effective in activating the mitochondrial ATP synthase-6 (ATPase-6) gene expression in B-16 murine melanoma cells. This activity may play an important role in its treatment of vitiligo.

Seeking for senile-related gene expression in cerebral tissue of senescence-accelerated mouse.

1. A better understanding of the molecular effect on aging in the brain may help reveal important aspects of organism aging, as well as the processes that lead to aging-related brain dysfunction. In this study, the aging-specific expression genes of the murine cerebrum were investigated by using the technique of DDRT-PCR in two senescence-accelerated mouse strains, SAMP10/Ta and SAMR1TA. 2. Through comparing gene expression profile among the age, 2, 4, 12, and 18 month of the SAMP10/Ta strain, four differential fragments have been found, and comparing gene expression profile between the two mouse strains, 24 fragments have been detected, 7 and 17 of them belong to SAMP10/Ta and SAMR1TA, respectively. 3. Sequencing analysis indicated that most of those fragments are homologous with some of certain gene cDNA that are related with senile. The data obtained from this study suggest that many genes are involved in the senile process and accelerate senescence phenotypic pathologies in SAMP10/Ta.

Homology of primate DNA fragments for estrous-associated oviductal glycoprotein.

In this study, the DNA fragments encoded for oviduct EGP were amplified by PCR method. Corresponding sequences from chimpanzee, orangutan and human were similarly amplified, but we failed to amplify the corresponding DNA sequences from: spider monkey, salmon and several different species of rodents, bandicoot, sheep and pig. Through analyzing the restriction enzyme map and the DNA sequence of the amplified fragments from primates, the data suggest that monkey and human DNA encoded for EGP are genetically more closely related to one another than each of them to other species.

Expression of stage-specific genes during zygotic gene activation in preimplantation mouse embryos.

The expression of mouse two-cell stage specific genes was studied using the modified DDRT-PCR method, which overcame the paucity of the experimental materials of preimplantation embryos. Embryo tissues equivalent to that of four blastomeres are sufficient for amplification of target genes as visualized using polyacrylamide gel. Sequence analyses and reverse Northern blots indicate that the genes of ATPase 6 and Ywhaz are expressed specifically in two-cell embryos. ATPase 6 is essential for one-cell to two-cell transition and plays an important role in establishment of oxidative phosphorylation, while Ywhaz is related to initiating cellular communication system.

[Epigenetic modifications and its impact on animal cloning].

Despite recent successes in cloning various mammals and amphibians, the low efficiency of animals production and abnormal symptoms in many cloned animals are crucial problems in cloning technology. To overcome these problems, scientists focus on mechanisms of cloning. A possible cause of the low success frequency of cloning is the insufficient dedifferentiation and the inadequate reprogramming of the high differentiated adult somatic nucleus in enucleated oocytes, which caused by incomplete methylation and premature de novo remethylation of donor DNA. In cloned embryos the methylation level is higher than normal embryos, and this may cause aberrant expression of several important genes, especially imprinting genes. Study on these mechanisms is very important to improve the rate of successful cloned animals.

[Isolation and identification of reprogramming genes related to the development of the rabbit reconstructed embryos].

By the method of single preimplantation embryos differential display polymerase chain reaction (SPEDDRT-PCR), 25 reprogramming cDNA fragments were obtained from single 2-cell, 8-cell embryos and blastula. After cloning and sequencing, five of them were identified by reverse-Northern and characterized with stage-specific expression during reconstructed embryo development. This results will help to isolate full length reprogramming genes and study their function during embryonic development.