Humanized mouse models: A valuable platform for preclinical evaluation of human cancer.

Animal models are routinely employed to assess the treatments for human cancer. However, due to significant differences in genetic backgrounds, traditional animal models are unable to meet bioresearch needs. To overcome this restriction, researchers have generated and optimized immunodeficient mice, and then engrafted human genes, cells, tissues, or organs in mice so that the responses in the model mice could provide a more reliable reference for treatments. As a bridge connecting clinical application and basic research, humanized mice are increasingly used in the preclinical evaluation of cancer treatments, particularly after gene interleukin 2 receptor gamma mutant mice were generated. Human cancer models established in humanized mice support exploration of the mechanism of cancer occurrence and provide an efficient platform for drug screening. However, it is undeniable that the further application of humanized mice still faces multiple challenges. This review summarizes the construction approaches for humanized mice and their existing limitations. We also report the latest applications of humanized mice in preclinical evaluation for the treatment of cancer and point out directions for future optimization of these models.

The morphology of osseous structure in subtalar joint with chronic ankle instability.

BACKGROUND: Osseous structures have been demonstrated as risk factors for chronic ankle instability (CAI). Previously, the researchers only focused on the osseous structures of ankle, but ignored the osseous structures of subtalar joint(STJ). Accordingly, the aim of our study was to investigate the morphological characteristics of STJ osseous structures in CAI. METHODS: 52 patients with CAI and 52 sex- and age- matched control subjects were enrolled from The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University. The lateral radiographs of ankle in weight-bearing were used to compare the diversity of the two groups. Specifically, The Length of calcaneus, Calcaneal facet height and Absolute foot height, Böhler's angle, Gissane's angle, Calcaneal inclination angle, Talocalcaneal angle, Tibiotalar angle, Tibiocalcaneal angle, Talar-horizontal angle, talar declination angle, facet inclination angle were gauged in the two groups. RESULTS: The Böhler's angle, Calcaneal inclination, Talocalcaneal angle, Tibiotalar angle, Talar-horizontal angle, Talar declination angle, Facet inclination angle and Absolute foot height of CAI group were significantly higher than normal control group (P < 0.05). There were no significant differences in Gissane's angle, Tibiocalcaneal angle, Length of calcaneus and Calcaneal facet height between patients with CAI and normal controls (P > 0.05). CONCLUSIONS: The osseous structures of STJ in CAI patients are different from normal people in morphology. Therefore, we should pay more attention to the changes of STJ anatomical parameters in the diagnosis and prevention of CAI. LEVEL OF EVIDENCE: Ⅲ.

Arctigenin impairs UBC12 enzyme activity and cullin neddylation to attenuate cancer cells.

Neddylation is a type of posttranslational protein modification that has been observed to be overactivated in various cancers. UBC12 is one of two key E2 enzymes in the neddylation pathway. Reports indicate that UBC12 deficiency may suppress lung cancer cells, such that UBC12 could play an important role in tumor progression. However, systematic studies regarding the expression profile of UBC12 in cancers and its relationship to cancer prognosis are lacking. In this study, we comprehensively analyzed UBC12 expression in diverse cancer types and found that UBC12 is markedly overexpressed in most cancers (17/21), a symptom that negatively correlates with the survival rates of cancer patients, including gastric cancer. These results demonstrate the suitability of UBC12 as a potential target for cancer treatment. Currently, no effective inhibitor targeting UBC12 has been discovered. We screened a natural product library and found, for the first time, that arctigenin has been shown to significantly inhibit UBC12 enzyme activity and cullin neddylation. The inhibition of UBC12 enzyme activity was newly found to contribute to the effects of arctigenin on suppressing the malignant phenotypes of cancer cells. Furthermore, we performed proteomics analysis and found that arctigenin intervened with cullin downstream signaling pathways and substrates, such as the tumor suppressor PDCD4. In summary, these results demonstrate the importance of UBC12 as a potential therapeutic target for cancer treatment, and, for the first time, the suitability of arctigenin as a potential compound targeting UBC12 enzyme activity. Thus, these findings provide a new strategy for inhibiting neddylation-overactivated cancers.

Uridylation: a vital way for cellular RNA surveillance.

RNA uridylation is an efficient posttranscriptional regulator of gene expression conserved in almost all eukaryotes. Terminal uridylyltransferase (TUTase) are responsible for monouridylation and oligouridylation of various RNA substrates, including snRNA, miRNA, mRNA and other ncRNAs. Studies have demonstrated that monouridylation on ncRNA intermediates alters their ultimate products and processing rates, whereas oligouridylation is often employed to degrade particular RNAs with spatio-temporal specificity and responsible for clearance of the aberrant RNAs and viral RNAs. Uridylation regulates gene expression by these two ways, therefore affects several important biological processes including organismal reproduction and early development, apoptosis, tumorigenesis, as well as virus infection. In this review, we provide the summarization of current researches on uridylation, introduce several techniques widely used for RNA 3' terminus detection, put more emphases on describing the mechanisms of how uridylation controls gene expression, and summarize the key roles of uridylation in RNA surveillance and several biological processes. Furthermore, we discuss other unsolved issues and crucial aspects of future research as well, with the aim of providing new ideas for anti-tumor and anti-virus therapies.

Association between subtalar articular surface typing and flat foot deformity: which type is more likely to cause flat foot deformity.

BACKGROUND: Previous studies have shown a wide range of anatomical classifications of the subtalar joint (STJ) in the population and this is related to the different force line structures of the foot. Different subtalar articular surface morphology may affect the occurrence and development of flat foot deformity, and there are fewer studies in this area. The main objective of our study was to determine the association of different subtalar articular surface with the occurrence and severity of flat foot deformity. METHODS: We analyzed the imaging data of 289 cases of STJ. The articular surface area, Gissane's angle and Bohler's angle of subtalar articular surface of different types were counted. The occurrence and severity of flat foot deformity in different subtalar articular surface were judged by measuring the Meary angle of foot. RESULTS: We classified 289 cases of subtalar articular surface into five types according to the morphology. According to Meary angle, the flat foot deformity of Type I and Type IV are significantly severer than Type II (P < 0.05). Type II (7.65 ± 1.38 cm2) was significantly smaller than Type I (8.40 ± 1.79 cm2) in the total joint facet area(P < 0.05). Type III (9.15 ± 1.92 cm2) was smaller than Type I (8.40 ± 1.79 cm2), II (7.65 ± 1.38 cm2) and IV (7.81 ± 1.74 cm2) (P < 0.05). Type II (28.81 ± 7.44∘) was significantly smaller than Type I (30.80 ± 4.61 degrees), and IV (32.25 ± 5.02 degrees) in the Bohler's angle (P < 0.05). Type II (128.49 ± 6.74 degrees) was smaller than Type I (131.58 ± 7.32 degrees), and IV (131.94 ± 5.80 degrees) in the Gissane's angle (P < 0.05). CONCLUSIONS: After being compared and analyzed the measurement of morphological parameters, joint facet area and fusion of subtalar articular surface were closely related to the severity of flat foot deformity and Type I and IV were more likely to develop severer flat foot deformity. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Celastrol inhibits cancer metastasis by suppressing M2-like polarization of macrophages.

In recent years, a large amount of clinical and experimental data has shown that M2-like polarized tumor-associated macrophages (TAMs) play an important role in cancer metastasis. Therefore, TAMs, especially M2-like TAMs is a promising target for anti-tumor metastasis therapy. Here, we found that celastrol dose-dependently suppressed IL-13 induced CD206 expression both in RAW264.7 and in primary macrophages. Consistently, celastrol also inhibited the expression of M2-like specific genes, including MRC1, Arg1, Fizz1, Mgl2 and CD11c. Further, by the employment of 4T1 breast cancer model, we found that celastrol significantly prevented cancer metastasis in vivo. Mechanistically, celastrol completely ameliorated STAT6 phosphorylation, which is the key signal molecule responsible for M2 polarization. Our research puts forward a new application of celastrol in anti-cancer metastasis, by intervening M2-like polarization through inhibiting STAT6.

Co-expression network analysis of the transcriptomes of rice roots exposed to various cadmium stresses reveals universal cadmium-responsive genes.

BACKGROUND: The migration of cadmium (Cd) from contaminated soil to rice is a cause for concern. However, the molecular mechanism underlying the response of rice roots to various Cd stresses remains to be clarified from the viewpoint of the co-expression network at a system-wide scale. RESULTS: We employed a comparative RNAseq-based approach to identify early Cd-responsive differentially expressed genes (DEGs) in rice 'Nipponbare' seedling roots after 1 h of high-Cd treatment. A multiplicity of the identified 1772 DEGs were implicated in hormone signaling and transcriptional regulation, particularly NACs and WRKYs were all upregulated under Cd stress. All of the 6 Cd-upregulated ABC transporters were pleiotropic drug resistance proteins (PDRs), whereas all of the 6 ZRT/IRT-like proteins (ZIPs) were consistently downregulated by Cd treatment. To further confirm our results of this early transcriptomic response to Cd exposure, we then conducted weighted gene co-expression network analysis (WGCNA) to re-analyze our RNAseq data in combination with other 11 previously published RNAseq datasets for rice roots exposed to diverse concentrations of Cd for extended treatment periods. This integrative approach identified 271 transcripts as universal Cd-regulated DEGs that are key components of the Cd treatment coupled co-expression module. A global view of the 164 transcripts with annotated functions in pathway networks revealed several Cd-upregulated key functional genes, including transporter ABCG36/OsPDR9, 12-oxo-phytodienoic acid reductases (OPRs) for JA synthesis, and ZIM domain proteins JAZs in JA signaling, as well as OsWRKY10, NAC, and ZFP transcription factors. More importantly, 104 of these, including ABCG36/OsPDR9, OsNAC3, as well as several orthologs in group metalloendoproteinase, plastocyanin-like domain containing proteins and pectin methylesterase inhibitor, may respond specifically to various Cd pressures, after subtracting the 60 general stress-responsive genes reported to be commonly upregulated following multiple stresses. CONCLUSION: An integrative approach was implemented to identify DEGs and co-expression network modules in response to various Cd pressures, and 104 of the 164 annotatable universal Cd-responsive DEGs may specifically respond to various Cd pressures. These results provide insight into the universal molecular mechanisms beneath the Cd response in rice roots, and suggest many promising targets for improving the rice acclimation process against Cd toxicity.

In Different Gender Groups, What Is the Impact of the Fibular Notch on the Severity of High Ankle Sprain: A Retrospective Study of 360 Cases.

OBJECTIVES: The role of the distal tibiofibular ligament in the occurrence of high ankle sprain (HAS) has been widely studied. But previous studies have overlooked the physiological and anatomical differences between males and females and have not further refined gender. Therefore, the impact of the anatomical morphology of fibular notch (FN) on HAS in different genders is still unclear. This study aimed to explore the impact of different types of FN on the severity of HAS and to estimate the prognosis of patients with HAS while excluding anatomical differences caused by gender. METHODS: One hundred and eighty patients with HAS were included in this study as the experimental group (i.e., HAS group). They were further divided into four groups according to gender and FN depth, with deep concave FN ≥ 4 mm and shallow flat FN < 4 mm. Another 180 normal individuals were set as the control group. The FN morphological indicators, tibiofibular distance (TFD), and ankle mortise indexes were measured and compared with those in HAS group. The independent t-test was used to compare continuous variables between groups, the intraclass correlation coefficient (ICC) was used to analyze the reliability of intra-observer measurement, and the Pearson correlation coefficient was used to verify the correlation between FN and the severity of HAS. RESULTS: In males with shallow flat type, the measurements of anterior tibiofibular distance (aTFD), middle tibiofibular distance (mTFD), posterior tibiofibular distance (pTFD), front ankle mortise width (fAMW), middle ankle mortise width (mAMW), posterior ankle mortise width (pAMW), and depth of ankle mortise (DOAM) in HAS group were significantly larger than those in normal group (p < 0.05). In male patients with deep concave type, the measurements of aTFD, mTFD, fAMW, mAMW, and DOAM were significantly larger than those in normal group (p < 0.05). Among female patients with shallow flat type, the measurements of aTFD, mTFD, pTFD, fAMW, mAMW, pAMW, and DOAM were found to be significantly larger than those in normal group (p < 0.05). Among female patients with deep concave type, the measurements of mTFD, pTFD, fAMW, mAMW, and DOAM were found to be significantly larger than those of the normal group (p < 0.05). The depth of FN was negatively correlated with TFD, and the AOFAS score of patients with shallow flat type was significantly lower than that of patients with deep concave type after treatment (p < 0.05). CONCLUSIONS: In different gender groups, compared with the normal controls, the TFD and partial ankle mortise indices were significantly different in HAS patients. Moreover, FN depth was negatively correlated with TFD, and the AOFAS score of shallow flat patients was significantly lower than that of deep concave patients. These suggested that shallow flat FN may be associated with more severe distal tibiofibular ligament injury and ankle mortise widening, leading to poorer prognosis. This should be taken seriously in clinical practice.

The Influence of Sagittal Angle of Posterior Malleolus Fracture on Ankle Joint Stability: A Retrospective Study of 120 Cases.

OBJECTIVE: Due to the different force exerted during the posterior malleolus fracture (PMF), the difference in sagittal angle (SA) between the fracture fragments may affect ankle stability. But this aspect is less well studied and the aim of this study was to investigate the relationship between SA and the stability of PMF. METHODS: The imaging data of 120 patients with PMFs from January 2014 to November 2022 were collected retrospectively and reconstructed. We first measured SA, posterior fragment area (PFA) and fragment area ratio (FAR), reanalyzing the correlation of SA with PFA and FAR, respectively. To better describe the morphological characteristics of the fracture fragments, we further measured the fragment width diameter ratio (FWR), the fragment length ratio (FLR), fragment height (FH), contact area (CA), and finally carried these data into the regression model of SA versus FAR to conduct the intermediary role. RESULTS: SA was negatively correlated with PFA(s) (r = -0.583, P < 0.001), with regression equation s = -0.063SA + 3.066; SA was negatively correlated with FAR (r = -0.204, P < 0.05), with regression equation FAR = -0.002SA + 0.198; A significant correlation was found between FWR, FLR, FH, CA and SA (P < 0.05), as well as between FWR, FLR, FH and FAR (P < 0.05); Further intermediary role analysis showed that FWR, FLR, FH had a partial intermediary role between SA and FAR. CONCLUSIONS: As SA increased, PFA and FAR decreased, so the larger the SA was due to the effect of vertical shear force, reflecting higher ankle stability, meanwhile, FWR, FLR and FH should also be considered on the fixation method of fracture fragments.

Co-infusion of CAR T cells with aAPCs expressing chemokines and costimulatory ligands enhances the anti-tumor efficacy in mice.

Chimeric antigen receptor-modified T (CAR-T) cell therapy has shown curable efficacy for treating hematological malignancies, while in solid tumors, the immunosuppressive microenvironment causes poor activation, expansion and survival of CAR-T cells, accounting mainly for the unsatisfactory efficacy. The artificial antigen-presenting cells (aAPCs) have been used for ex vivo expansion and manufacturing of CAR-T cells. Here, we constructed a K562 cell-based aAPCs expressing human epithelial cell adhesion molecule (EpCAM), chemokines (CCL19 and CCL21) and co-stimulatory molecular ligands (CD80 and 4-1BBL). Our data demonstrated that the novel aAPCs enhanced the expansion, and increased the immune memory phenotype and cytotoxicity of CAR-T cells recognizing EpCAM, in vitro. Of note, co-infusion CAR-T and aAPC enhances the infiltration of CAR-T cells in solid tumors, which has certain potential for the treatment of solid tumors Moreover, IL-2-9-21, a cytokine cocktail, prevents CAR-T cells from entering the state of exhaustion prematurely after continuous antigen engagement and boosts the anti-tumor activity of CAR-T cells co-infused with aAPCs. These data provide a new strategy to enhance the therapeutic potential of CAR-T cell therapy for the treatment of solid tumors.

Chimeric Antigen Receptor-T Cell Therapy Decreases Distant Metastasis and Inhibits Local Recurrence Post-surgery in Mice.

Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and local recurrence of tumors, respectively. Chimeric antigen receptor (CAR)-modified T cells target residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells infusion. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.

EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Cellular permeation of large molecules mediated by TRPM8 channels.

While most membrane channels are only capable of passing small ions, certain non-selective cation channels have been recently shown to have the capacity to permeate large cations. The mechanisms underlying large molecule permeation are unclear, but this property has been exploited pharmacologically to target molecules, such as nerve conduction blockers, to specific subsets of pain-sensing neurons (nociceptors) expressing the heat-gated transient receptor potential (TRP) channel TRPV1. However, it is not clear if the principal mediator of cold stimuli TRPM8 is capable of mediating the permeation large molecules across cell membranes, suggesting that TRPM8-positive nerves cannot be similarly targeted. Here we show that both heterologous cells and native sensory neurons expressing TRPM8 channels allow the permeation of the large fluorescent cation Po-Pro3. Po-Pro3 influx is blocked by TRPM8-specific antagonism and when channel activity is desensitized. The effects of the potent agonist WS-12 are TRPM8-specific and dye uptake mediated by TRPM8 channels is similar to that observed with TRPV1. Lastly, we find that as with TRPV1, activation of TRPM8 channels can be used as a means to target intracellular uptake of cell-impermeable sodium channel blockers. In a neuronal cell line expressing TRPM8 channels, voltage-gated sodium currents are blocked in the presence of the cell-impermeable, charged lidocaine derivative QX-314 and WS-12. These results show that the ability of somatosensory TRP channels to promote the permeation of large cations also includes TRPM8, thereby suggesting that novel approaches to alter cold pain can also be employed via conduction block in TRPM8-positive sensory neurons.

Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds.

Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα' and 8SGß. The 5'-flanking sequences of 8SGα' has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5'-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding ß-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors.

A Vigna radiata 8S globulin α' promoter drives efficient expression of GUS in Arabidopsis cotyledonary embryos.

Plants are proven effective bioreactors for the production of heterologous proteins including those desired by the biopharmaceutical industry. However, the potential of plants as bioreactors is limited by the availability of characterized plant promoters that can drive target gene expression in relatively distant plant species. Seeds are ideal for protein storage because seed proteins can be kept stably for several months. Hence, a strong promoter that can direct the expression and accumulation of target proteins within seeds represents a powerful tool in plant biotechnology. Toward this end, an effort was made to identify such a promoter from Vigna radiata (mung bean) to drive expression in dicot seeds. A 784-bp 5'-flanking sequence of the gene encoding the 8S globulin α' subunit (8SGα') of the V. radiata seed storage protein was isolated by genome walking. When the 5'-flanking region was analyzed with bioinformatics tools, numerous putative cis-elements were identified. The Green Fluorescent Protein (GFP) regulated by this promoter was observed to be transiently expressed in protoplasts derived from V. radiata cotyledons. Finally, transgenic Arabidopsis plants expressing the ß-glucuronidase (GUS) reporter gene driven from the 8S globulin α' promoter showed strong GUS expression in transgenic embryos in both histochemical and quantitative GUS assays, confirming high expression within seeds. Therefore, the V. radiata 8S α' promoter has shown potential in directing expression in seeds for bioreactor applications.