Forskolin-driven conversion of human somatic cells into induced neurons through regulation of the cAMP-CREB1-JNK signaling.

Human somatic cells can be reprogrammed into neuron cell fate through regulation of a single transcription factor or application of small molecule cocktails. Methods: Here, we report that forskolin efficiently induces the conversion of human somatic cells into induced neurons (FiNs). Results: A large population of neuron-like phenotype cells was observed as early as 24-36 h post-induction. There were >90% TUJ1-, >80% MAP2-, and >80% NEUN-positive neurons at 5 days post-induction. Multiple subtypes of neurons were present among TUJ1-positive cells, including >60% cholinergic, >20% glutamatergic, >10% GABAergic, and >5% dopaminergic neurons. FiNs exhibited typical neural electrophysiological activity in vitro and the ability to survive in vitro and in vivo more than 2 months. Mechanistically, forskolin functions in FiN reprogramming by regulating the cAMP-CREB1-JNK signals, which upregulates cAMP-CREB1 expression and downregulates JNK expression. Conclusion: Overall, our studies identify a safer and efficient single-small-molecule-driven reprogramming approach for induced neuron generation and reveal a novel regulatory mechanism of neuronal cell fate acquisition.

Palladium-catalyzed ring-opening [5+2] annulation of vinylethylene carbonates (VECs) and C5-substituted Meldrum's acids: rapid synthesis of 7-membered lactones.

A novel approach for the synthesis of unsaturated 7-membered lactones by Pd-catalyzed [5+2] dipolar cycloaddition of vinylethylene carbonates (VECs) and C5-substituted Meldrum's acid derivatives has been developed. Various Meldrum's acid derivatives worked well in this reaction under mild reaction conditions. A variety of 7-membered lactones can be accessed in a facile manner in moderate to good yields by employing easily prepared Meldrum's acid derivatives.

Effects of intracellular Ca2+ on developmental potential and ultrastructure of cryopreserved-warmed oocyte in mouse.

Maintaining appropriate intracellular calcium of oocytes is necessary to prevent ultrastructure and organelle damage caused by freezing and cryoprotectants. The present study aimed to investigate whether cryoprotectant-induced changes in the calcium concentrations of oocytes can be regulated to reduce damage to developmental potential and ultrastructure. A total of 33 mice and 1381 oocytes were used to explore the effects of intracellular calcium on the development and ultrastructures of oocytes subjected to 2-aminoethoxydiphenyl borate (2-APB) inhibition or thapsigargin (TG) stimulation. Results suggested that high levels intracellular calcium interfered with TG compromised oocyte survival (84.4 % vs. 93.4 %, p < 0.01) and blastocyst formation in fresh and cryopreservation oocytes (78.1 % vs. 86.4 %, and 60.5 % vs. 72.5 %, p < 0.05) compared with that of 2-APB pretreated oocytes in which Ca2+ was stabilized even though no differences in fertilization and cleavage was detected (p > 0.05). Examination by transmission electron microscopy indicated that the microvilli decreased and shortened, cortical granules considerably decreased in the cortex area, mitochondrial vesicles and vacuoles increased, and the proportion of vacuole mitochondria increased after oocytes were exposed to cryoprotectants. The cryopreservation-warming process deteriorated the negative effects on organelles of survival oocytes. By contrast, a low level of intracellular calcium mediated with 2-APB was supposed to contribute to the protection of organelles. These findings suggested oocyte injuries induced by cryoprotectants and low temperatures can be alleviated. More studies are necessary to confirm the relationship among Ca2+ concentration of the cytoplasm, ultrastructural injuries, and disrupted developmental potential in oocytes subjected to cryopreservation and warming.

HIF-1α Contributes to Hypoxia-induced VSMC Proliferation and Migration by Regulating Autophagy in Type A Aortic Dissection.

Type A aortic dissection (AD) is a catastrophic cardiovascular disease. Hypoxia-inducible factor-1α (HIF-1α) and autophagy are reported to be upregulated in the AD specimens. However, the interaction between HIF-1α and autophagy in the pathogenesis of AD remains to be explored. HIF-1α and LC3 levels are evaluated in 10 AD and 10 normal aortic specimens. MDC staining, autophagic vacuoles, and autophagic flux are detected in human aortic smooth muscle cells (HASMCs) under hypoxia treatment. CCK-8, transwell, and wound healing assay are used to identify proliferation and migration under hypoxia treatment. Furthermore, 3-MA is used to inhibit autophagy in hypoxia-treated HASMCs. This study reveals that AD tissues highly express HIF-1α and the LC3. Autophagy is induced under hypoxia in a time-dependent manner, and autophagy is positively related to HIF-1α in HASMCs. Moreover, the proliferation and migration of HASMCs are enhanced by hypoxia, whereas the knockdown of HIF-1α attenuates this effect. Additionally, inhibiting autophagy with 3-MA ameliorates hypoxia-induced proliferation and migration of HASMCs. In summary, the above results indicate that HIF-1α facilitates HASMC proliferation and migration by upregulating autophagy in a hypoxic microenvironment. Thus, inhibition of autophagy may be a novel therapeutic target for the prevention and treatment of AD.

Prediction of pancreatic fibrosis by dual-energy CT-derived extracellular volume fraction: Comparison with MRI.

OBJECTIVES: To investigate the correlation between dual-energy CT (DECT) and MRI measurements of the extracellular volume fraction (ECV) and to assess the accuracy of both methods in predicting pancreatic fibrosis (PF). METHODS: We retrospectively analyzed 43 patients who underwent pancreatectomy and preoperative pancreatic DECT and MRI between November 2018 and May 2022. The ECV was calculated using the T1 relaxation time (for MR-ECV) or absolute enhancement (for DECT-ECV) at equilibrium phase (180 s after contrast injection in our study). Pearson coefficient and Bland-Altman analysis were used to compare the correlation between the two ECVs, Spearman correlations were used to investigate the association between imaging parameters and PF, Receiver operating characteristic (ROC) curves were used to assess the diagnostic performance of the ECVs for advanced fibrosis (F2-F3), and multivariate logistic regression analysis was used to examine the relationship between PF and imaging parameters. RESULTS: There was a strong correlation between DECT- and MR-derived ECVs (r = 0.948; p < 0.001). The two ECVs were positively correlated with PF (DECT: r = 0.647, p < 0.001; MR: r = 0.614, p < 0.001), and the mean values were 0.34 ± 0.08 (range: 0.22-0.62) and 0.35 ± 0.09 (range: 0.24-0.66), respectively. The area under the operating characteristic curve (AUC) for subjects with advanced fibrosis diagnosed by ECV was 0.86 for DECT-ECV and 0.87 for MR-ECV. Multivariate logistic regression analysis showed that the DECT-ECV was an independent predictor of PF. CONCLUSIONS: The ECV could be an effective predictor of histological fibrosis, and DECT is equivalent to MRI for characterizing pancreatic ECV changes.

Strain-Induced Defect Evolution for the Construction of Porous Cu2-xSe with Enhanced Thermoelectric Performance.

Superionic Cu2-xSe, with disordered and even liquid-like Cu ions, has been extensively studied as a high efficiency thermoelectric material. However, the relationship between lattice stability and microstructure evolution in Cu2-xSe under strain, which is crucial for its application, has seldom been explored in previous research. In this study, we investigate the impacts of hydrostatic compression strain on the microstructural evolution and, consequently, its implications for thermoelectric performance. Molecular dynamics (MD) simulations show that high hydrostatic compression strain could induce local diffusion of Cu ions and Se twin evolution, resulting in the breaking and reforming of Cu-Se dynamic bonds and the unstable Se sublattice. The subsequent annealing process of the destabilized structure promoted Se evaporation from the sublattice and resulted in lotus-seedpod-like pores. The reduced sound velocity and intensified phonon scattering, due to pores, lead to a reduction in the lattice thermal conductivity from 0.44 W m-1 K-1 to 0.24 W m-1 K-1 at 800 K, a decrease of approximately 45%, in the porous Cu1.92Se sample. These findings reveal the relationship between stability and defect evolution in Cu2-xSe under high hydrostatic compression, offering a straightforward strategy of defect engineering for designing unique microstructures by leveraging the instability in superionic conductor materials.

CeRNA Network Reveals the Circular RNA Characterization in Goat Ear Fibroblasts Reprogramming into Mammary Epithelial Cells.

Circular RNAs (circRNAs) are a type of non-coding RNA that play a crucial role in the development and lactation of mammary glands in mammals. A total of 107 differentially expressed circRNAs (DE circRNAs) were found, of which 52 were up-regulated and 55 were down-regulated. We also found that DE circRNA host genes were mainly involved in GO terms related to the development process of mammary epithelial cells and KEGG pathways were mostly related to mammary epithelial cells, lactation, and gland development. Protein network analysis found that DE circRNAs can competitively bind to miRNAs as key circRNAs by constructing a circRNA-miRNA-mRNA network. CircRNAs competitively bind to miRNAs (miR-10b-3p, miR-671-5p, chi-miR-200c, chi-miR-378-3p, and chi-miR-30e-5p) involved in goat mammary gland development, mammary epithelial cells, and lactation, affecting the expression of core genes (CDH2, MAPK1, ITGB1, CAMSAP2, and MAPKAPK5). Here, we generated CiMECs and systematically explored the differences in the transcription profile for the first time using whole-transcriptome sequencing. We also analyzed the interaction among mRNA, miRNA, and cirRNA and predicted that circRNA plays an important role in the maintenance of mammary epithelial cells.

Long-chain noncoding RNA sequencing analysis reveals the molecular profiles of chemically induced mammary epithelial cells.

Long noncoding RNAs (lncRNAs) were important regulators affecting the cellular reprogramming process. Previous studies from our group have demonstrated that small molecule compounds can induce goat ear fibroblasts to reprogram into mammary epithelial cells with lactation function. In this study, we used lncRNA-Sequencing (lncRNA-seq) to analyze the lncRNA expression profile of cells before and after reprogramming (CK vs. 5i8 d). The results showed that a total of 3,970 candidate differential lncRNAs were detected, 1,170 annotated and 2,800 new lncRNAs. Compared to 0 d cells, 738 lncRNAs were significantly upregulated and 550 were significantly downregulated in 8 d cells. Heat maps of lncrnas and target genes with significant differences showed that the fate of cell lineages changed. Functional enrichment analysis revealed that these differently expressed (DE) lncRNAs target genes were mainly involved in signaling pathways related to reprogramming and mammary gland development, such as the Wnt signaling pathway, PI3K-Akt signaling pathway, arginine and proline metabolism, ECM-receptor interaction, and MAPK signaling pathway. The accuracy of sequencing was verified by real-time fluorescence quantification (RT-qPCR) of lncRNAs and key candidate genes, and it was also demonstrated that the phenotype and genes of the cells were changed. Therefore, this study offers a foundation for explaining the molecular mechanisms of lncRNAs in chemically induced mammary epithelial cells.

Dissecting the molecular trajectory of fibroblast reprogramming to chemically induced mammary epithelial cells.

Introduction: The plasticity of cell identity allows cellular reprogramming that manipulates the lineage of cells to generate the target cell types, bringing new avenues for disease modeling and autologous tailored cell therapy. Previously, we had already successfully established a technical platform for inducing fibroblast reprogramming to chemically induced mammary epithelial cells (CiMECs) by small-molecule compounds. However, exactly how the molecular mechanism driving the lineage conversion remains unknown. Methods: We employ the RNA-sequencing technology to investigate the transcriptome event during the reprogramming process and reveal the molecular mechanisms for the fate acquisition of mammary lineage. Results: The multi-step reprogramming process first overcomes multiple barriers, including the inhibition of mesenchymal characteristics, pro-inflammatory and cell death signals, and then enters an intermediate plastic state. Subsequently, the hormone and mammary development genes were rapidly activated, leading to the acquisition of the mammary program together with upregulation of the milk protein synthesis signal. Moreover, the gene network analyses reveal the potential relationship between the TGF-ß signaling pathway to mammary lineage activation, and the changes in the expression of these genes may play important roles in coordinating the reprogramming process. Conclusion: Together, these findings provide critical insights into the molecular route and mechanism triggered by small-molecule compounds that induce fibroblast reprogramming into the fate of mammary epithelial cells, and they also laid a foundation for the subsequent research on the development and differentiation of mammary epithelial cells and lactation.

Revealing the reinforcing effect of a nanorod network on a polymer matrix through molecular dynamics simulations.

Controlling the spatial morphology of the nanorods (NRs) in a polymer matrix and understanding the structure-property relationship are crucial for fabricating high-performance polymer nanocomposites (PNCs). By employing molecular dynamics simulations, we systematically studied the structural and mechanical properties of NR filled PNCs. The simulated results showed that the NRs gradually self-assembled into a three-dimensional (3D) network upon increasing the NR-NR interaction strength. The generated 3D NR network transferred loads along the NR backbone, differing from the well dispersed system which transfers loads between NRs and nearby polymer chains. Increase of the nanorod diameter or NR content further enhanced the PNCs by improving the NR network integrity. These findings provide insights into the reinforcement mechanism of NRs toward polymer matrices and provide guidance for designing PNCs with excellent mechanical performance.

Effect of Cathodal Transcranial Direct Current Stimulation for Lower Limb Subacute Stroke Rehabilitation.

Methods: A pilot double-blind and randomized clinical trial. Ninety-one subjects with subacute stroke were treated with cathodal/sham stimulation tDCS based on CGR (physiotherapy 40 min/d and occupational therapy 20 min/d) once daily for 20 consecutive working days. Computer-based stratified randomization (1 : 1) was employed by considering age and sex, with concealed assignments in opaque envelopes to ensure no allocation errors after disclosure at the study's end. Patients were evaluated at T0 before treatment, T1 immediately after the posttreatment assessment, and T2 assessment one month after the end of the treatment. The primary outcome index was assessed: lower limb Fugl-Meyer motor score (FMA-LE); secondary endpoints were other gait assessment and relevant stroke scale assessment. Results: Patients in the trial group performed significantly better than the control group in all primary outcome indicators assessed posttreatment T1 and at follow-up T2: FMA-LE outcome indicators between the two groups in T1 (P = 0.032; effect size 1.00, 95% CI: 0.00 to 2.00) and FMA-LE outcome indicators between the two groups in T2 (P = 0.010; effect size 2.00, 95% CI: 1.00 to 3.00). Conclusion: In the current pilot study, ctDCS plus CGR was an effective treatment modality to improve lower limb motor function with subacute stroke. The effectiveness of cathodal tDCS in poststroke lower limb motor dysfunction is inconclusive. Therefore, a large randomized controlled trial is needed to verify its effectiveness.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Rapid direct conversion of bovine non-adipogenic fibroblasts into adipocyte-like cells by a small-molecule cocktail.

Introduction: The molecular regulation mechanism of fat deposition in bovine and its improvement on beef quality are important research directions in the livestock industry. The research of molecular mechanisms that govern the regulation and differentiation of adipocytes may conduct to understand the mechanism of obesity, lipid disorders, and fat deposition. In the recent decade, small-molecule compounds have been widely used in reprogramming and transdifferentiation fields, which can promote the induction efficiency, replace exogenous genes, or even induce cell fate conversion alone. Furthermore, small-molecule compound induction is expected to be a novel approach to generate new cell types from somatic cells in vitro and in vivo. Methods: In this study, we established rapid chemically induced platform for transdifferentiation of bovine ear fibroblasts into adipocyte-like cells using a small-molecule cocktail (Repsox, VPA, TTNPB). The chemically induced adipocytes (CiADCs) were characterized by lipid staining, qRT-PCR and WB. Bovine natural adipocytes were used as positive control, and the expression of adipocyte-related marker genes in CiADCs were analyzed. Moreover, RNA-Seq explore the mechanism of RVB in the regulation of Bovine adipocyte transdifferentiation. Results: In this study, the chemically induced adipocytes (CiADCs) could be identified as early as day 6. The CiADCs appeared to be circular and rich of lipid droplets. The adipocyte-specific genes of LPL, PPARγ, IGF1, GPD1, C/EBPδ, ADIPOQ, PCK2, FAS, C/EBPß, PPARGC1A, C/EBPα, and CFD were detected to be significantly upregulated in both CiADCs and natural adipocytes. Western blot analysis also confirmed the increase C/EBPα and PPARγ protein level in induced adipocytes (CiADCs-6d) treated with RVB. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. Discussion: In the present study, a combination of small-molecule compounds RVB was used to transdifferentiate bovine ear fibroblasts into the chemically-induced adipocyte cells (CiADCs) that have a large number of lipid droplets. Importantly, the small-molecule cocktail significantly shortened the reprogramming turnaround time. The morphology of CiADCs is close to the "ring type" of natural differentiated adipocytes on sixth day. And, the CiADCs showed similar adipocyte-specific gene expression patterns to natural adipocytes. Furthermore, RVB increased protein expression of PPARγ and C/EBPα in the chemically-induced adipocytes (CiADCs-6d). Our findings reveal that the signaling pathways of C/EBPα and PPARγ play pivotal roles in this transdifferentiation process. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. In general, this study provides valuable evidence to deepen our understanding of the molecular mechanism of small molecule cocktails in regulating adipogenesis.

A HD-ZIP transcription factor specifies fates of multicellular trichomes via dosage-dependent mechanisms in tomato.

Hair-like structures are shared by most living organisms. The hairs on plant surfaces, commonly referred to as trichomes, form diverse types to sense and protect against various stresses. However, it is unclear how trichomes differentiate into highly variable forms. Here, we show that a homeodomain leucine zipper (HD-ZIP) transcription factor named Woolly controls the fates of distinct trichomes in tomato via a dosage-dependent mechanism. The autocatalytic reinforcement of Woolly is counteracted by an autoregulatory negative feedback loop, creating a circuit with a high or low Woolly level. This biases the transcriptional activation of separate antagonistic cascades that lead to different trichome types. Our results identify the developmental switch of trichome formation and provide mechanistic insights into the progressive fate specification in plants, as well as a path to enhancing plant stress resistance and the production of beneficial chemicals.

Gabrb3 is required for the functional integration of pyramidal neuron subtypes in the somatosensory cortex.

Dysfunction of gamma-aminobutyric acid (GABA)ergic circuits is strongly associated with neurodevelopmental disorders. However, it is unclear how genetic predispositions impact circuit assembly. Using in vivo two-photon and widefield calcium imaging in developing mice, we show that Gabrb3, a gene strongly associated with autism spectrum disorder (ASD) and Angelman syndrome (AS), is enriched in contralaterally projecting pyramidal neurons and is required for inhibitory function. We report that Gabrb3 ablation leads to a developmental decrease in GABAergic synapses, increased local network synchrony, and long-lasting enhancement in functional connectivity of contralateral-but not ipsilateral-pyramidal neuron subtypes. In addition, Gabrb3 deletion leads to increased cortical response to tactile stimulation at neonatal stages. Using human transcriptomics and neuroimaging datasets from ASD subjects, we show that the spatial distribution of GABRB3 expression correlates with atypical connectivity in these subjects. Our studies reveal a requirement for Gabrb3 during the emergence of interhemispheric circuits for sensory processing.

Serum IgG N-glycans enable early detection and early relapse prediction of colorectal cancer.

Colorectal cancer (CRC) develops mainly from colorectal advanced adenomas (AA), which are considered precancerous lesions. Novel early diagnostic biomarkers are urgently needed to distinguish CRC and AA from healthy control (HC). Alternative glycosylation of serum IgG has been shown to be closely associated with CRC. We aimed to explore the potential of IgG N-glycan as biomarkers in the early differential diagnosis of CRC. The study population was strictly matched to the exclusion criteria process. Serum IgG N-glycan profiles were analyzed by a robust and reliable relative quantitative method based on ultra-performance liquid chromatography (UPLC). Relative quantification and classification performance of IgG N-glycans were evaluated by Mann-Whitney U tests and ROC curve based on directly detected and derived glycan traits, respectively. Six and 14 directly detected glycan traits were significantly changed in AA and CRC, respectively, compared with HC. GP1 and GP3 were able to accurately distinguish AA from HC for early precancerous lesions screening. GP4 and GP14 provided a high value in discriminating CRC from HC. A novel combined index named GlycoF, including GP1, GP3, GP4, GP14 and CEA was developed to provide a potential early diagnostic biomarker in discriminating simultaneously AA (AUC = 0.847) and CRC (AUC = 0.844) from HC. GlycoF also demonstrated a superior CRC detection rate across CRC all stages and conspicuous prediction ability of risk of relapse. Serum IgG N-glycans analysis provided powerful early screening biomarkers that can efficiently differentiate CRC and AA from HC.

Role of metabolic equivalent between calcium intake and vertebral fractures: a cross-sectional study of NHANES 2013-2014.

BACKGROUND: This study was to analyze the association of calcium intake and metabolic equivalent (MET) with vertebral fractures, and to explore the role of MET between calcium intake and vertebral fractures. METHOD: This cross-sectional study used data from the National Health and Nutrition Examination Surveys (NHANES) 2013-2014. The study involved individuals aged ≥ 50 years old with complete information on vertebral fracture, calcium intake, and physical activity. Vertebral fracture assessment is obtained using dual-energy x-ray absorptiometry to perform a lateral scan of the thoracolumbar spine. Calcium intake included total nutrient intake and total dietary supplements. The total MET is the sum of the METs for each activity (Vigorous/ moderate work-related activities, walking or bicycling for transportation and vigorous/ moderate recreational activities). Univariate and multivariate logistic regression analyses were utilized to investigate the effect of calcium intake, MET, and their combined effect on vertebral fracture. RESULTS: A total of 766 participants were included in the analysis, and 54 participants had vertebral fractures. The median calcium intake and MET were 8.43 mcg and 280.00, respectively. Multivariate results showed that neither calcium intake nor MET as continuous or categorical variables was significantly associated with vertebral fractures. MET < 160 and calcium intake ≥ 670 mg group was associated with the decreased risks of vertebral fracture [odds ratio (OR) = 0.47, 95% confidence interval (CI): 0.26-0.83, P = 0.032] after adjusting for age, race, energy, total femur bone mineral density (BMD), and femoral neck BMD. In the group of MET < 160, increased calcium intake was associated with a reduced risk of vertebral fracture, with a decreased OR value. In the group of MET ≥ 160, increased calcium intake was associated with an increased risk of vertebral fracture, with an increased OR value. CONCLUSION: The combination of MET < 160 and calcium intake ≥ 670 mg was associated with decreased risks of vertebral fractures. There may be an interaction between calcium intake and MET on vertebral fracture risk.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Parabolic relationship between SMAD3 expression level and the reprogramming efficiency of goat induced mammary epithelial cells.

Mammary epithelial cells are the only cells of mammary glands with lactation capacity. They are closely related to mammary development and milk yield. Our earlier studies showed that the transformation of goat fibroblasts into induced mammary epithelial cells (iMECs) was closely correlated with SMAD3 overexpression. Therefore, we further explored the role of SMAD3 on iMECs reprogramming in this study. The SMAD3 gene was overexpressed in goat ear fibroblasts using the tetracycline-induced expression method. The outcomes demonstrated that goat ear fibroblasts can be converted into iMECs by overexpressing the SMAD3 gene. In contrast, it was discovered that SMAD3 downregulation by RNA interference significantly decrease the reprogramming efficiency of iMECs. These results show that SMAD3 plays a key regulatory role in the reprogramming of iMECs. Surprisingly, we also found a parabolic relationship between SMAD3 expression level and iMECs reprogramming efficiency, and that the reprogramming efficiency was maximum when the addition of doxycycline concentration was 5 µg/ml. In light of this, our findings may offer new perspectives on the regulatory mechanism governing mammary epithelial cell fate in goats as well as a fresh approach to studying mammary development and differentiation in vitro.

Effective suppression of triple negative breast cancer by paclitaxel nanoparticles conjugated with transmembrane TNF-α monoclonal antibody.

Transmembrane TNF-α (tmTNF), a transmembrane form of TNF-α, was reported overexpressed in approximately 84% of triple-negative breast cancer (TNBC) patients and has emerged as a valid candidate biomarker for targeting TNBC. Paclitaxel is a first-line chemotherapeutic agent for the treatment of triple-negative breast cancer, but suffers from low water solubility, resulting in its low bioavailability. To achieve site-specific delivery of the anticancer chemotherapeutic drug (paclitaxel) on TNBC, we developed tmTNF-α monoclonal antibody (mAb)-conjugated paclitaxel (PTX) nanoparticles (NPs) (tmTNF-α mAb-PTX NPs) as potential nanocarriers. This targeted delivery-therapy nanocarriers was conducted by using an emulsification-evaporation method. tmTNF-α mAb-PTX NPs displayed favorable physicochemical properties. Compared with the control groups, tumor growth in human MDA-MB-231 xenograft mice was suppressed significantly by tmTNF-α mAb-PTX NPs. TmTNF-α mAb-PTX NPs exerts anti-tumor effects via promoting apoptosis and regulating mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) cascade, as well as AMP-activated protein kinase (AMPK) and nuclear factor Kappa-B (NF-κB) pathways. Moreover, tmTNF-α mAb-PTX NPs can inhibit the process of epithelial-mesenchymal transition (EMT) in TNBC to suppress tumor progression and metastasis. Together, the novel tmTNF-α mAb-PTX NPs based targeted drug delivery system is a potentially highly effective approach for treating TNBC.