The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

Yindan Xinnaotong capsule has been used for treating cardio-cerebrovascular diseases for several decades in China. Exercise training can protect against the development of atherosclerosis. The aim of the present study is to evaluate the joint effect of YXC and exercise on atherosclerosis in rats.

Xueshuantong capsule (XST) is a patented traditional Chinese medicine used for the prevention and treatment of thrombosis. The molecular mechanism of anti-thrombotic effect of XST was investigated through the cross-talk among the platelets/leukocytes, endothelial cells (ECs), and flow shear stress. The Bioflux 1000 system was used to generate two levels of shear stress conditions: 0.1 and 0.9 Pa. Bioflux Metamorph microscopic imaging system was used to analyze the adhesion cell numbers. Protein expressions were detected by western blotting and flow cytometry. The flow-cytometry results showed that under 0.1 Pa flow, XST decreased ADP induced platelets CD62p surface expression in a concentration-dependent manner. Under 0.9 Pa flow, XST at a concentration of 0.15 g⋅L-1 reduced the platelets activation by 29.5%, and aspirin (ASA) showed no inhibitory effects. XST showed similar efficiency on monocytes adhesion both under 0.1 and 0.9 Pa flow conditions, and the inhibition rate was 30.2 and 28.3%, respectively. Under 0.9 Pa flow, the anti-adhesive effects of XST might be associated with the suppression of VE-cadherin and Cx43 in HUVECs. Blood flow not only acts as a drug transporter, but also exerts its effects to influence the pharmacodynamics of XST. Effects of XST on inhibiting platelets activation and suppressing platelets/leukocytes adhesion to injured ECs are not only concentration-dependent, but also shear stress-dependent. The mechanic forces combined with traditional Chinese medicine may be used as a precise treatment for cardiovascular diseases.

The adaptor protein CARD9 links detection of fungi by surface receptors to the activation of the NF-κB pathway. Mice deficient in CARD9 exhibit dysbiosis and are more susceptible to colitis. Here we examined the impact of Card9 deficiency in the development of colitis-associated colon cancer (CAC). Treatment of Card9-/- mice with AOM-DSS resulted in increased tumor loads as compared to WT mice and in the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissue. The impaired fungicidal functions of Card9-/- macrophages led to increased fungal loads and variation in the overall composition of the intestinal mycobiota, with a notable increase in C. tropicalis. Bone marrow cells incubated with C. tropicalis exhibited MDSC features and suppressive functions. Fluconazole treatment suppressed CAC in Card9-/- mice and was associated with decreased MDSC accumulation. The frequency of MDSCs in tumor tissues of colon cancer patients correlated positively with fungal burden, pointing to the relevance of this regulatory axis in human disease.

C-type lectin receptors (CLRs) play critical roles as pattern-recognition receptors (PRRs) for sensing Candida albicans infection, which can be life-threatening for immunocompromised individuals. Here we have shown that Dectin-3 (also called CLECSF8, MCL, or Clec4d), a previously uncharacterized CLR, recognized α-mannans on the surfaces of C. albicans hyphae and induced NF-κB activation. Mice with either blockade or genetically deleted Dectin-3 were highly susceptible to C. albicans infection. Dectin-3 constantly formed heterodimers with Dectin-2, a well-characterized CLR, for recognizing C. albicans hyphae. Compared to their respective homodimers, Dectin-3 and Dectin-2 heterodimers bound α-mannans more effectively, leading to potent inflammatory responses against fungal infections. Together, our study demonstrates that Dectin-3 forms a heterodimeric PRR with Dectin-2 for sensing fungal infection and suggests that different CLRs may form different hetero- and homodimers, which provide different sensitivity and diversity for host cells to detect various microbial infections.

Osteoimmunity plays an important role in the process of implant osseointegration. Autophagy is a conservative metabolic pathway of eukaryotic cells, but whether the interaction between autophagy and osteoimmunity plays a key role in osseointegration remains unclear. In this study, we prepared smooth titanium disks and micro-nano topography titanium disks, to study the immune microenvironment of RAW264.7 cells, and prepared the conditioned medium to study the effect of immune microenvironment on the osteogenesis and autophagy of MC3T3-E1 cells. Autophagy inhibitor 3-MA was used to inhibit autophagy to observe the change of expression of osteogenic markers. The results showed that the micro-nano topography titanium disks could stimulate RAW264.7 cells to differentiate into M2 type, forming an anti-inflammatory immune microenvironment; compared with the control group, the anti-inflammatory immune microenvironment promoted the proliferation and differentiation of osteoblasts better. The anti-inflammatory immune environment activated the autophagy level of osteoblasts, while the expression of osteogenic markers was down-regulated after inhibition of autophagy. These results indicate that anti-inflammatory immune microenvironment can promote cell proliferation and osteogenic differentiation, autophagy plays an important role in this process. This study further explains the mechanism of implant osseointegration in osteoimmune microenvironment, and provides reference for improving implant osseointegration.

There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs.

Interactions between commensal fungi and gut immune system are critical for establishing colonic homeostasis. Here we found that mice deficient in Dectin-3 (Clec4d-/-), a C-type lectin receptor that senses fungal infection, were more susceptible to dextran sodium sulfate (DSS)-induced colitis compared with wild-type mice. The specific fungal burden of Candida (C.) tropicalis was markedly increased in the gut after DSS treatment in Clec4d-/- mice, and supplementation with C. tropicalis aggravated colitis only in Clec4d-/- mice, but not in wild-type controls. Mechanistically, Dectin-3 deficiency impairs phagocytic and fungicidal abilities of macrophages, and C. tropicalis-induced NF-κB activation and cytokine production. The conditioned media derived from Dectin-3-deficient macrophages were defective in promoting tissue repairing in colonic epithelial cells. Finally, anti-fungal therapy was effective in treating colitis in Clec4d-/- mice. These studies identified the role of Dectin-3 and its functional interaction with commensal fungi in intestinal immune system and regulation of colonic homeostasis.

We propose a Leaky-Wave Antenna (LWA) based on one-way yttrium-iron-garnet (YIG)-air-metal waveguide. We first analyze the dispersion of the LWA, showing the one-way feature and the radiation loss. Owing to the unique one-way dispersive property, the beam radiated from the LWA can have very narrow beam width, at the same time having large scanning angle. The main beam angle obtained by full-wave simulation is consistent with our theoretical prediction with the aid of the dispersion. For a given frequency, we can realize continuous beam scanning by varying the magnetic field, where the 3 dB beam width is much narrower than previously demonstrated. Our results pave a new way to realize continuous angle scanning at a fix frequency for modern communications.

XueShuanTong (XST) comprising therapeutically active ginsenosides, a lyophilized extract of Panax notoginseng roots, is extensively used in traditional Chinese medicine to treat ischemic heart and cerebrovascular diseases. Our recent study shows that treatment with XST inhibits shear-induced thrombosis formation but the underlying mechanism remained unclear. This study aimed to investigate the hypothesis that XST inhibited shear-induced platelet aggregation via targeting the mechanosensitive Ca2+-permeable Piezo1 channel by performing platelet aggregation assay, Ca2+ imaging and Western blotting analysis. Exposure to shear at physiologically (1,000-2000 s-1) and pathologically related rates (4,000-6,000 s-1) induced platelet aggregation that was inhibited by treatment with GsMTx-4. Exposure to shear evoked robust Ca2+ responses in platelets that were inhibited by treatment with GsMTx-4 and conversely enhanced by treatment with Yoda1. Treatment with XST at a clinically relevant concentration (0.15 g L-1) potently inhibited shear-induced Ca2+ responses and platelet aggregation, without altering vWF-mediated platelet adhesion and rolling. Exposure to shear, while resulting in no effect on the calpain-2 expression in platelets, induced calpain-2-mediated cleavage of talin1 protein, which is known to be critical for platelet activation. Shear-induced activation of calpain-2 and cleavage of talin1 were attenuated by treatment with XST. Taken together, our results suggest that XST inhibits shear-induced platelet aggregation via targeting the Piezo1 channel to prevent Piezo1-mediated Ca2+ signaling and downstream calpain-2 and talin1 signal pathway, thus providing novel insights into the mechanism of the therapeutic action of XST on platelet aggregation and thrombosis formation.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F- ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin- Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f- ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f- ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f- ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin-Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1+/- heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing.

Currently, targeted alpha therapy (TAT) is a new therapy involving the administration of a therapeutic drug that combines a substance of α-emitting nuclides that kill cancer cells and a drug that selectively accumulates in cancer cells. It is known to be effective against cancers that are difficult to treat with existing methods, such as cancer cells that are widely spread throughout the whole body, and there are high expectations for its early clinical implementation. The nuclides for TAT, including 149Tb, 211At, 212/213Bi, 212Pb (for 212Bi), 223Ra, 225Ac, 226/227Th, and 230U, are known. However, some nuclides encounter problems with labeling methods and lack sufficient preclinical and clinical data. We labeled the compounds targeting prostate specific membrane antigen (PSMA) with 211At and 225Ac. PSMA is a molecule that has attracted attention as a theranostic target for prostate cancer, and several targeted radioligands have already shown therapeutic effects in patients. The results showed that 211At, which has a much shorter half-life, is no less cytotoxic than 225Ac. In 211At labeling, our group has also developed an original method (Shirakami Reaction). We have succeeded in obtaining a highly purified labeled product in a short timeframe using this method.

Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)-1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti-inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL-1β-induced inflammation-activated endothelial cell (EC)-smooth muscle cell (SMC)-mononuclear cell (MC) co-culture model was established, based on the changes in a series of atherosclerosis-associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low-density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti-inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed.

The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products.

The scanning accuracy of intraoral scanners' data collection plays a key role in the success of the final treatment. However, few studies start from scanning technology itself to directly evaluate it. The aim of this study was to evaluate the scanning accuracy of three intraoral scanners, to provide a reference for relevant research and clinical applications.

Yindanxinnaotong capsule (YDXNT), a traditional Chinese formula, has been used to treat cardio-cerebrovascular diseases for several decades. Previous research has focused on evaluating the pharmacological properties and main compounds of YDXNT in vitro and in vivo. However, the multiple bioactive compounds in vivo remain poorly understood. In the present research, an integrative strategy using UPLC-Q-TOF-MS combined with UPLC-QqQ-MS was employed to detect the absorbed constituents and investigate the pharmacokinetics of main compounds in the plasma after oral administration of YDXNT. UPLC-Q-TOF-MS was developed to detect the absorbed constituents and their metabolites in the plasma after oral administration in rats. A total of 52 constituents, including 44 prototype compounds and 8 metabolites, were identified or tentatively characterized. Then, nine main compounds (quercetin, isorhamnetin, kaempferol, ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, tanshinone IIA, and salvianolic acid B) were chosen to further investigate the pharmacokinetic behavior of YDXNT using UPLC-QqQ-MS. The concentration of nine main constituents were in the range of 27.85-76.54 ng mL-1. This research provides a systematic approach for rapid qualitative analysis of absorbed constituents and for evaluating the pharmacokinetics of the main ingredients of YDXNT following its oral administration. More importantly, this work provides key information on the identification of bioactive compounds and the clarification of their action mechanisms, as well as on the pharmacological actions of YDXNT.

Endothelial cells are sensitive to changes in both blood components and mechanical stimuli. Endothelial cells may undergo phenotypic changes, such as changes in adhesion protein expression, under different shear stress conditions. Such changes may impact platelet and monocyte adhesion to endothelial cells. This phenomenon is linked to chronic vascular inflammation and the development of atherosclerosis. In the present study, we investigated the effects of ginkgolide B on platelet and monocyte adhesion to human umbilical vein endothelial cells (HUVECs) under different conditions of laminar shear stress.

Targeted alpha therapy (TAT) has garnered significant interest as an innovative cancer therapy. Owing to their high energy and short range, achieving selective α-particle accumulation in target tumor cells is crucial for obtaining high potency without adverse effects. To meet this demand, we fabricated an innovative radiolabeled antibody, specifically designed to selectively deliver 211At (α-particle emitter) to the nuclei of cancer cells. The developed 211At-labeled antibody exhibited a superior effect compared to its conventional counterparts. This study paves the way for organelle-selective drug delivery.

Objective. To investigate the absorption property of the representative hydrolyzable tannin, namely corilagin, and its hydrolysates gallic acid (GA) and ellagic acid (EA) from the Fructus Phyllanthi tannin fraction (PTF) in vitro. Methods. Caco-2 cells monolayer model was established. Influences of PTF on Caco-2 cells viability were detected with MTT assay. The transport across monolayers was examined for different time points, concentrations, and secretory directions. The inhibitors of P-glycoprotein (P-gp), multidrug resistance proteins (MRPs), organic anion transporting polypeptide (OATP) and sodium/glucose cotransporter 1 (SGLT1), and tight junction modulators were used to study the transport mechanism. LC-MS method was employed to quantify the absorption concentration. Results. The apparent permeability coefficient (Papp) values of the three compounds were below 1.0 × 10-6 cm/s. The absorption of corilagin and GA were much lower than their efflux, and the uptake of both compounds was increased in the presence of inhibitors of P-gp and MRPs. The absorption of EA was decreased in the company of OATP and SGLT1 inhibitors. Moreover, the transport of corilagin, GA, and EA was enhanced by tight junction modulators. Conclusion. These observations indicated that the three compounds in PTF were transported via passive diffusion combined with protein mediated transport. P-gp and MRPs might get involved in the transport of corilagin and GA. The absorption of EA could be attributed to OATP and SGLT1 protein.