Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Mechanism of Allosteric Modulation of Nicotinamide Phosphoribosyltransferase to Elevate Cellular NAD.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.

Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy.

Presynaptic syntaxin binding protein 1 (STXBP1) is essential for neurotransmitter release. Heterozygous mutations in this protein cause STXBP1 encephalopathy (STXBP1-E), which is characterized by intellectual disabilities and epilepsies. Since nonhuman primates closely resemble humans, monkey models may advance studies on the pathogenesis and therapeutic treatments of STXBP1-E. We generated cynomolgus monkeys carrying STXBP1 (R292H) mutation through base editing of in vitro fertilized embryos to mimic a clinical condition. The newborn STXBP1-edited monkeys exhibited focal epilepsy, and the animal that survived beyond the first week postpartum presented typical EEG phenotypes. Biochemical analysis of brain biopsy samples showed reduced levels of STXBP1 (MUNC18-1) and SNARE complex proteins. Single-cell sequencing identified one specific cell cluster that may contribute to encephalopathy. Thus, our case report shows that base-edited STXBP1 mutant monkeys are a good animal model for STXBP1-E, and that a base-editing approach is useful for generating primate models of human genetic disorders.

Total Synthesis of Nucleoside Antibiotics Plicacetin and Streptcytosine A.

Disaccharide nucleoside antibiotics plicacetin and streptcytosine A (also named rocheicoside A) were effectively synthesized through the common precursor cytosamine. The amosamine and amicetose moieties were efficiently assembled through an α-selective O-glycosylation, and the cytosine nucleus was subsequently introduced through a ß-selective gold(I)-catalyzed N-glycosylation. Further microwave-assisted amidation reactions completed the modular syntheses.

Knockdown of ribosomal protein S15A induces human glioblastoma cell apoptosis.

BACKGROUND: RPS15A is a ribosome protein that is highly conserved in many organisms from yeast to human. A number of studies implied its role in promoting cancer cell growth. METHODS: Here, we firstly conducted RPS15A gene expression analysis in brain cancer using Oncomine database and found RPS15A was remarkably overexpressed in glioblastoma (GBM) compared with that in normal tissues. Then, the expression of RPS15A was specifically silenced in GBM cell line U251 using lentiviral-mediated RNA interference technique. We further investigated the effect of RPS15A knockdown in U251 cells using MTT assay, colony formation test, and flow cytometry analysis. We detected the protein level of Bcl-2 and poly (ADP-ribose) polymerase (PARP) as well as activation of caspase-3. RESULTS: Our results showed that the knockdown of RPS15A could inhibit cancer cell growth and colony formation in vitro, as well as induced cell cycle arrest at G0/G1 phase and cell apoptosis. In addition, Western blot analysis indicated that the knockdown of RPS15A could significantly inhibit bcl-2 and activate caspase-3 and PARP. CONCLUSIONS: Our findings suggest RPS15A may play an important role in the progression of GBM and lentiviral-mediated silencing of RPS15A could be an effective tool in GBM treatment.

Structure-Guided Design and Synthesis of Pyridinone-Based Selective Bromodomain and Extra-Terminal Domain (BET)-First Bromodomain (BD1) Inhibitors.

The bromodomain and extra-terminal domain (BET) proteins are epigenetic readers, regulating transcription via two highly homologous tandem bromodomains, BD1 and BD2. Clinical development of nonselective pan-BD BET inhibitors has been challenging, partly due to dose-limiting side effects such as thrombocytopenia. This has prompted the push for domain-selective BET inhibitors to achieve a more favorable therapeutic window. We report a structure-guided drug design campaign that led to the development of a potent BD1-selective BET inhibitor, 33 (XL-126), with a Kd of 8.9 nM and 185-fold BD1/BD2 selectivity. The high selectivity was first assayed by SPR, validated by a secondary time-resolved fluorescence energy transfer assay, and further corroborated by BROMOscan (∼57-373 fold selectivity). The cocrystal of 33 with BRD4 BD1 and BD2 demonstrates the source of selectivity: repulsion with His437 and lost binding with the leucine clamp. Notably, the BD1 selectivity of BET inhibitor 33 leads to both the preservation of platelets and potent anti-inflammatory efficacy.

Spinosin inhibits activated hepatic stellate cell to attenuate liver fibrosis by targeting Nur77/ASK1/p38 MAPK signaling pathway.

AIM: Liver fibrosis remains a great challenge in the world. Spinosin (SPI), a natural flavonoid-C-glycoside, possesses various pharmacological activities including anti-inflammatory and anti-myocardial fibrosis effects. In this study, we investigate whether SPI can be a potential lead for the treatment of liver fibrosis and explore whether the orphan nuclear receptor Nur77, a negative regulator of liver fibrosis development, plays a critical role in SPI's action. METHODS: A dual luciferase reporter system of α-SMA was established to evaluate the effect of SPI on hepatic stellate cell (HSC) activation in LX2 and HSC-T6 cells. A mouse model of CCl4-induced liver fibrosis was used to test the efficacy of SPI against liver fibrosis. The expression levels of Nur77, inflammatory cytokines and collagen were determined by Western blotting and qPCR. Potential kinase pathways involved were also analyzed. The affinity of Nur77 with SPI was documented by fluorescence titration. RESULTS: SPI can strongly suppress TGF-ß1-mediated activation of both LX2 and HSC-T6 cells in a dose-dependent manner. SPI increases the expression of Nur77 and reduces TGF-ß1-mediated phosphorylation levels of ASK1 and p38 MAPK, which can be reversed by knocking out of Nur77. SPI strongly inhibits collagen deposition (COLA1) and reduces inflammatory cytokines (IL-6 and IL-1ß), which is followed by improved liver function in the CCl4-induced mouse model. SPI can directly bind to R515 and R563 in the Nur77-LBD pocket with a Kd of 2.14 µM. CONCLUSION: Spinosin is the major pharmacological active component of Ziziphus jujuba Mill. var. spinosa which has been frequently prescribed in traditional Chinese medicine. We demonstrate here for the first time that spinosin is a new therapeutic lead for treatment of liver fibrosis by targeting Nur77 and blocking the ASK1/p38 MAPK signaling pathway.

Non-Covalent Inhibitors of SARS-CoV-2 Papain-Like Protease (PLpro): In Vitro and In Vivo Antiviral Activity.

The SARS-CoV-2 papain-like protease (PLpro), essential for viral processing and immune response disruption, is a promising target for treating acute infection of SARS-CoV-2. To date, there have been no reports of PLpro inhibitors with both submicromolar potency and animal model efficacy. To address the challenge of PLpro's featureless active site, a noncovalent inhibitor library with over 50 new analogs was developed, targeting the PLpro active site by modulating the BL2-loop and engaging the BL2-groove. Notably, compounds 42 and 10 exhibited strong antiviral effects and were further analyzed pharmacokinetically. 10, in particular, showed a significant lung accumulation, up to 12.9-fold greater than plasma exposure, and was effective in a mouse model of SARS-CoV-2 infection, as well as against several SARS-CoV-2 variants. These findings highlight the potential of 10 as an in vivo chemical probe for studying PLpro inhibition in SARS-CoV-2 infection.

A rapid and stable spontaneous reprogramming system of Spermatogonial stem cells to Pluripotent State.

BACKGROUND: The scarcity of pluripotent stem cells poses a major challenge to the clinical application, given ethical and biosafety considerations. While germline stem cells commit to gamete differentiation throughout life, studies demonstrated the spontaneous acquisition of pluripotency by spermatogonial stem cells (SSCs) from neonatal testes at a low frequency (1 in 1.5 × 107). Notably, this process occurs without exogenous oncogenes or chemical supplementation. However, while knockout of the p53 gene accelerates the transformation of SSCs, it also increases risk and hampers their clinical use. RESULTS: We report a transformation system that efficiently and stably convert SSCs into pluripotent stem cells around 10 passages with the morphology similar to that of epiblast stem cells, which convert to embryonic stem (ES) cell-like colonies after change with ES medium. Epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and fresh mouse embryonic fibroblast feeder (MEF) are essential for transformation, and addition of 2i (CHIR99021 and PD0325901) further enhanced the pluripotency. Transcriptome analysis revealed that EGF activated the RAS signaling pathway and inhibited p38 to initiate transformation, and synergically cooperated with LIF to promote the transformation. CONCLUSION: This system established an efficient and safe resource of pluripotent cells from autologous germline, and provide new avenues for regenerative medicine and animal cloning.

Alleviating symptoms of neurodegenerative disorders by astrocyte-specific overexpression of TMEM164 in mice.

Neuroinflammatory microglia secrete cytokines to induce neurotoxic reactive astrocytes, which are one of the major causes of neuronal death. However, the intrinsic key regulators underlying neurotoxic reactive astrocytes induction are unknown. Here we show that the transmembrane protein 164 (TMEM164) is an early-response intrinsic factor that regulates neurotoxic astrocyte reactivity. TMEM164 overexpression inhibits the induction of neurotoxic reactive astrocytes, maintains normal astrocytic functions and suppresses neurotoxic reactive astrocyte-mediated neuronal death by decreasing the secretion of neurotoxic saturated lipids. Adeno-associated virus-mediated, astrocyte-specific TMEM164 overexpression in male and female mice prevents the induction of neurotoxic reactive astrocytes, dopaminergic neuronal loss and motor deficits in a Parkinson's disease model. Notably, brain-wide astrocyte-specific TMEM164 overexpression prevents the induction of neurotoxic reactive astrocytes, amyloid ß deposition, neurodegeneration and memory decline in the 5XFAD Alzheimer's disease mouse model, suggesting that TMEM164 could serve as a potential therapeutic target for neurodegenerative disorders.

CATI: an efficient gene integration method for rodent and primate embryos by MMEJ suppression.

The efficiency of homology-directed repair (HDR) plays a crucial role in the development of animal models and gene therapy. We demonstrate that microhomology-mediated end-joining (MMEJ) constitutes a substantial proportion of DNA repair during CRISPR-mediated gene editing. Using CasRx to downregulate a key MMEJ factor, Polymerase Q (Polq), we improve the targeted integration efficiency of linearized DNA fragments and single-strand oligonucleotides (ssODN) in mouse embryos and offspring. CasRX-assisted targeted integration (CATI) also leads to substantial improvements in HDR efficiency during the CRISPR/Cas9 editing of monkey embryos. We present a promising tool for generating monkey models and developing gene therapies for clinical trials.

Synthesis, Optimization, and Structure-Activity Relationships of Nicotinamide Phosphoribosyltransferase (NAMPT) Positive Allosteric Modulators (N-PAMs).

Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.

Cynomolgus monkey embryo model captures gastrulation and early pregnancy.

Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential is limited. Here, we construct cynomolgus monkey blastoids resembling blastocysts in morphology and transcriptomics using naive ESCs. These blastoids develop to embryonic disk with the structures of yolk sac, chorionic cavity, amnion cavity, primitive streak, and connecting stalk along the rostral-caudal axis through prolonged in vitro culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk sac endoderm, three germ layers, and hemato-endothelial progenitors in IVC cynomolgus monkey blastoids were observed by single-cell transcriptomics or immunostaining. Moreover, transferring cynomolgus monkey blastoids to surrogates achieves pregnancies, as indicated by progesterone levels and presence of early gestation sacs. Our results reveal the capacity of in vitro gastrulation and in vivo early pregnancy of cynomolgus monkey blastoids, providing a useful system to dissect primate embryonic development without the same ethical concerns and access challenges in human embryo study.

Oxytocin is expressed by both intrinsic sensory and secretomotor neurons in the enteric nervous system of guinea pig.

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.

Expression of P2X6 receptors in the enteric nervous system of the rat gastrointestinal tract.

Expression of P2X(4) and P2X(6) receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X(6) receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X(6) receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X(6) receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X(6) receptors. P2X(6) receptors do not form homomultimers, but rather heteromultimers with either P2X(2) or P2X(4) receptors. P2X(4) receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X(6) receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X(6) receptors probably in heteromeric combination with P2X(2) receptors, may be involved in regulating the physiological functions of these neurons.

Release of methylene blue from graphene oxide-coated electrospun nanofibrous scaffolds to modulate functions of neural progenitor cells.

Transplantation of neural progenitor cells (NPCs) can repair the damaged neurons and therefore holds significant promise as a new treatment strategy for Alzheimer's disease (AD). Development of functional scaffolds for the growth, proliferation, and differentiation of NPCs offers a useful approach for AD therapy. In our study, the functional scaffolds were obtained by fabrication of a poly(lactic-co-glycolic acid) (PLGA) nanofibrous mat by the electrospinning technique, followed by coating of a layer of graphene oxide (GO) and then physisorption of methylene blue (MB) under mild conditions. The precoating of GO on the nanofibrous scaffolds allows efficient loading and release of MB from the substrate for regulating the functions of NPCs. The NPCs cultured on the scaffolds remained in the quiescence phase due to the activation of autophagy signaling pathway by MB. Moreover, the MB-loaded nanofibrous scaffolds diminish tau phosphorylation and protect NPCs from apoptosis. Definitely, more work, especially the in vivo experiment, is highly desired to demonstrate the feasibility of the current strategy for AD treatment. STATEMENT OF SIGNIFICANCE: Transplantation of neural progenitor cells (NPCs) can repair the damaged neurons and hold significant promise as a new treatment strategy for Alzheimer's disease (AD). Development of functional scaffolds for the growth, proliferation, and differentiation of NPCs offers a novel and useful approach for AD therapy. In this work, we have developed a GO and MB sequentially coated PLGA nanofibrous mat as a new scaffold for NPC transplantation and tauopathy inhibition. The coating of GO that we have demonstrated significantly enhanced the loading and release of MB on the scaffolds. Furthermore, NPCs cultured on the nanofibrous scaffolds entered quiescence phase through the activation of autophagy signaling pathway, leading to improved performance of NPCs to cope with stressors of disease. More importantly, the release of MB from the scaffolds leads to attenuation of tauopathy and protection of NPCs, which may represent a novel, versatile, and effective therapeutic approach for AD and other neurodegenerative diseases.

A single factor induces neuronal differentiation to suppress glioma cell growth.

AIM: Glioma, with fast growth and progression features, is the most common and aggressive tumor in the central nervous system and is essentially incurable. This study is aimed at inducing neuronal differentiation to suppress glioma cell growth with a single transcription factor. METHODS: Overexpression of transcription factor SRY (sex determining region Y)-box 11 (SOX11) and Zic family member 1 (ZIC1) was, respectively, performed in glioma cells with lentivirus infection. CRISPR/Cas9 technology was used to knock out ZIC1 in U87 cells, and knockout efficiency was identified by Western blotting and Sanger sequencing. Cell cycle and apoptosis were detected by flow cytometry. The downstream targets of SOX11 were analyzed by Affymetrix GeneChip microarrays. qRT-PCR and immunofluorescence technique were used to verify gene targets of genetically modified U87 cells. All the cells were imaged by a fluorescence microscope. Gene expression correlation analysis and overall survival analysis based on TCGA dataset are performed by GEPIA. RESULTS: We induced glioma cells into neuron-like cells to suppress cell growth using a single transcription factor, SOX11 or ZIC1. Besides, we proved that there is a strong correlation between SOX11 and ZIC1. Our study revealed that SOX11 upregulates ZIC1 expression by binding with ZIC1 promoter, and ZIC1 partially mediates SOX11-induced neuronal differentiation in U87 cells. However, SOX11 expression is not regulated by ZIC1. Moreover, high MAP2 expression means better overall survival in TCGA lower grade glioma. CONCLUSION: This study revealed that glioma cells can be reprogrammed into neuron-like cells using a single factor ZIC1, which may be a potential tumor suppressor gene for gliomas treatment.

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus.

Expression of P2X(1), P2X(2), P2X(3), P2X(4), P2X(5) and P2X(6) receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X(2), P2X(4), P2X(5) and P2X(6) receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X(2) and P2X(6) on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.