Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters.

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and disease modeling. Here, we present a protocol for establishing edited hPSC cell lines utilizing visualized orthogonal selective reporters. We describe steps for constructing plasmids, carrying out cell culture and electroporation, as well as performing drug-fluorescent dual enrichment, clone screening, and cell line characterization. This protocol facilitates the achievement of single-base homozygous mutations and reporter knockins, offering a reliable approach for precision genome editing.

NTRK3 exhibits a pro-oncogenic function in upper tract urothelial carcinomas.

Neurotrophic receptor tyrosine kinase 3 (NTRK3) has pleiotropic functions: it acts not only as an oncogene in breast and gastric cancers but also as a dependence receptor in tumor suppressor genes in colon cancer and neuroblastomas. However, the role of NTRK3 in upper tract urothelial carcinoma (UTUC) is not well documented. This study investigated the association between NTRK3 expression and outcomes in UTUC patients and validated the results in tests on UTUC cell lines. A total of 118 UTUC cancer tissue samples were examined to evaluate the expression of NTRK3. Survival curves were generated using Kaplan-Meier estimates, and Cox regression models were used for investigating survival outcomes. Higher NTRK3 expression was correlated with worse progression-free survival, cancer-specific survival, and overall survival. Moreover, the results of an Ingenuity Pathway Analysis suggested that NTRK3 may interact with the PI3K-AKT-mTOR signaling pathway to promote cancer. NTRK3 downregulation in BFTC909 cells through shRNA reduced cellular migration, invasion, and activity in the AKT-mTOR pathway. Furthermore, the overexpression of NTRK3 in UM-UC-14 cells promoted AKT-mTOR pathway activity, cellular migration, and cell invasion. From these observations, we concluded that NTRK3 may contribute to aggressive behaviors in UTUC by facilitating cell migration and invasion through its interaction with the AKT-mTOR pathway and the expression of NTRK3 is a potential predictor of clinical outcomes in cases of UTUC.

Welded Carbon Nanotube-Graphene Hybrids with Tunable Strain Sensing Behavior for Wide-Range Bio-Signal Monitoring.

Carbon nanotubes (CNTs) and graphene have commonly been applied as the sensitive layer of strain sensors. However, the buckling deformation of CNTs and the crack generation of graphene usually leads to an unsatisfactory strain sensing performance. In this work, we developed a universal strategy to prepare welded CNT-graphene hybrids with tunable compositions and a tunable bonding strength between components by the in situ reduction of CNT-graphene oxide (GO) hybrid by thermal annealing. The stiffness of the hybrid film could be tailored by both initial CNT/GO dosage and annealing temperature, through which its electromechanical behaviors could also be defined. The strain sensor based on the CNT-graphene hybrid could be applied to collect epidermal bio-signals by both capturing the faint skin deformation from wrist pulse and recording the large deformations from joint bending, which has great potential in health monitoring, motion sensing and human-machine interfacing.

Differences in the association of oral health knowledge, attitudes, and practices with frailty among community-dwelling older people in China.

BACKGROUND: Oral health and frailty are significantly related and should be well examined, especially in late life. Few studies have explored the relationship of oral health knowledge, attitudes, and practices with frailty and examined sociodemographic variations in this association. This study aimed to examine the association between oral health knowledge, attitudes, practices and frailty, with a special focus on comparing differences in their association among the Chinese community-dwelling older population. METHODS: This study included 4218 community-dwelling older adults (aged ≥ 60 years) who participated in a cross-sectional survey. Sociodemographic characteristics, oral health knowledge, attitudes, practices, and frail status (non-frailty, pre-frailty, and frailty) were collected with a face-to-face questionnaire-based interview. Multivariate logistic regression models were used to evaluate the association of oral health knowledge, attitudes, and practices with frailty. RESULTS: Of the 4218 participants, 36.2% (n = 1527) and 18.8% (n = 792) were classified as pre-frailty and frailty. Age, gender and educational attainments differences existed in the association of oral health knowledge with frailty. Urban-rural differences in the association of oral health knowledge and practices with frailty were also found. Specifically, oral health knowledge was significantly related to frailty only among participants aged 70-79 years (adjusted odds ratio [95% confidence interval]) (1.08 [1.02-1.15]), females (1.05 [1.00-1.10]), rural residents (1.06 [1.01-1.12]), and those who were primary school and lower education (1.06 [1.01-1.11]), whereas oral health practices were related to frailty only among urban participants (0.96 [0.92-1.00]). CONCLUSION: This study confirmed the different associations of oral health knowledge and practices with frailty among community-dwelling older people in China. Further research is needed to better understand the abovementioned differences and public health strategies are required to improve oral health literacy and thereby contain the development of frailty in later life.

Bioengineered skin organoids: from development to applications.

Significant advancements have been made in recent years in the development of highly sophisticated skin organoids. Serving as three-dimensional models that mimic human skin, these organoids have evolved into complex structures and are increasingly recognized as effective alternatives to traditional culture models and human skin due to their ability to overcome the limitations of two-dimensional systems and ethical concerns. The inherent plasticity of skin organoids allows for their construction into physiological and pathological models, enabling the study of skin development and dynamic changes. This review provides an overview of the pivotal work in the progression from 3D layered epidermis to cyst-like skin organoids with appendages. Furthermore, it highlights the latest advancements in organoid construction facilitated by state-of-the-art engineering techniques, such as 3D printing and microfluidic devices. The review also summarizes and discusses the diverse applications of skin organoids in developmental biology, disease modelling, regenerative medicine, and personalized medicine, while considering their prospects and limitations.

Vitamin D and Diabetic Kidney Disease.

Vitamin D is a hormone involved in many physiological processes. Its active form, 1,25(OH)2D3, modulates serum calcium-phosphate homeostasis and skeletal homeostasis. A growing body of evidence has demonstrated the renoprotective effects of vitamin D. Vitamin D modulates endothelial function, is associated with podocyte preservation, regulates the renin-angiotensin-aldosterone system, and has anti-inflammatory effects. Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease worldwide. There are numerous studies supporting vitamin D as a renoprotector, potentially delaying the onset of DKD. This review summarizes the findings of current research on vitamin D and its role in DKD.

Learning CT projection denoising from adjacent views.

BACKGROUND: Many learning-based low-dose (LD) computed tomography (CT) imaging methods require large paired full- and low-dose datasets for training, which are usually unavailable in clinic. Whereas models trained on simulated data often face the generalization problem on real clinical data. PURPOSE: To develop an unsupervised learning technique to acquire clean CT projection from its adjacent LD projections. METHODS: Given a sequential LD projection set, the method extracts out the middle projection as the target and treats the rest ones as the input. The model is trained with the mean absolute error with proposed inter-view gradient constraint term, which helps to suppress outliers and preserve edges in the denoised projection. The simulated low-dose CT grand challenge dataset and a real physical torso phantom dataset were employed for experiment. The peak signal to noise ratio (PSNR) and structural similarity index measure (SSIM) were calculated for quantitative evaluation. RESULTS: In experiments with both the simulated and real datasets, visual comparisons reveal that the proposed method obtained images superior to unsupervised and supervised methods working in both image and projection domain. For numerical comparison, our method obtains larger SSIMs than other unsupervised methods at quarter and eighth dose levels. As for PSNR, our method obtains larger value at eighth dose whereas smaller value at quarter dose. The supervised models obtain better numerical results than all unsupervised models on simulated datasets. CONCLUSION: The proposed method can reduce the noise in CT projections effectively, making it an attractive tool for practical LDCT pre-processing.

Angiorganoid: vitalizing the organoid with blood vessels.

The emergence of the organoid simulates the native organs and this mini organ offers an excellent platform for probing multicellular interaction, disease modeling and drug discovery. Blood vessels constitute the instructive vascular niche which is indispensable for organ development, function and regeneration. Therefore, it is expected that the introduction of infiltrated blood vessels into the organoid might further pump vitality and credibility into the system. While the field is emerging and growing with new concepts and methodologies, this review aims at presenting various sources of vascular ingredients for constructing vascularized organoids and the paired methodology including de- and recellularization, bioprinting and microfluidics. Representative vascular organoids corresponding to specific tissues are also summarized and discussed to elaborate on the next generation of organoid development.

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BACKGROUND: Interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2-depleted metastatic OSCC mouse model. METHODS: Genetically engineered and xenograft tumour models were used to explore the effect of IFIT2-depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C-reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured. RESULTS: IFIT2-depleted metastatic tumours caused marked body weight loss (-18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2-depleted metastatic tumour-bearing mice (quadriceps: -28.7%, gastrocnemius: -29.4%, and tibialis: -24.3%, all P < 0.001). Tumour-derived circulating granulocyte-macrophage colony-stimulating factor (+772.2-fold, P < 0.05), GROα (+1283.7-fold, P < 0.05), IL6 (+245.8-fold, P < 0.001), IL8 (+616.9-fold, P < 0.001), IL18 (+24-fold, P < 0.05), IP10 (+18.8-fold, P < 0.001), CCL2 (+439.2-fold, P < 0.001), CCL22 (+9.1-fold, P < 0.01) and tumour necrosis factor α (+196.8-fold, P < 0.05) were elevated in IFIT2-depleted metastatic tumour-bearing mice. Murine granulocyte colony-stimulating factor (+61.4-fold, P < 0.001) and IL6 (+110.9-fold, P < 0.01) levels were significantly increased in IFIT2-depleted metastatic tumour-bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (-26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (-81%, P < 0.001) and lean tissue (-21.7%, P < 0.01), which was consistent with the reduced food intake (-19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross-sectional area (-23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2-depleted metastatic tumour-bearing mice (i.v.-shIFIT2 group) was associated with elevated IL6 (+2.7-fold, P < 0.05), phospho-p38 (+2.8-fold, P < 0.05), and atrogin-1 levels (+2.3-fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium-induced myotube atrophy (+24.6%, P < 0.01). CONCLUSIONS: Our results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

Learning to Reconstruct CT Images From the VVBP-Tensor.

Deep learning (DL) is bringing a big movement in the field of computed tomography (CT) imaging. In general, DL for CT imaging can be applied by processing the projection or the image data with trained deep neural networks (DNNs), unrolling the iterative reconstruction as a DNN for training, or training a well-designed DNN to directly reconstruct the image from the projection. In all of these applications, the whole or part of the DNNs work in the projection or image domain alone or in combination. In this study, instead of focusing on the projection or image, we train DNNs to reconstruct CT images from the view-by-view backprojection tensor (VVBP-Tensor). The VVBP-Tensor is the 3D data before summation in backprojection. It contains structures of the scanned object after applying a sorting operation. Unlike the image or projection that provides compressed information due to the integration/summation step in forward or back projection, the VVBP-Tensor provides lossless information for processing, allowing the trained DNNs to preserve fine details of the image. We develop a learning strategy by inputting slices of the VVBP-Tensor as feature maps and outputting the image. Such strategy can be viewed as a generalization of the summation step in conventional filtered backprojection reconstruction. Numerous experiments reveal that the proposed VVBP-Tensor domain learning framework obtains significant improvement over the image, projection, and hybrid projection-image domain learning frameworks. We hope the VVBP-Tensor domain learning framework could inspire algorithm development for DL-based CT imaging.

Antitumor Effects of the Novel Quinazolinone Holu-12: Induction of Mitotic Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma CAL27 Cells.

BACKGROUND/AIM: Quinazolinone is a privileged chemical structure employed for targeting various types of cancer. This study aimed to demonstrate the antitumor activity of synthesized 6,7-disubstituted-2-(3-fluorophenyl) quinazolines (HoLu-11 to HoLu-14). MATERIALS AND METHODS: The cytotoxicity was assessed by the sulforhodamine B (SRB) assay. The cell cycle was examined by flow cytometry. The expression levels of cell cycle- and apoptosis-related proteins were estimated by western blotting. A xenograft animal model was used to explore the antitumor effects of HoLu-12. RESULTS: Among four synthetic quinazolinone derivatives, HoLu-12 significantly reduced the viability of oral squamous cell carcinoma (OSCC) cells. HoLu-12 induced G2/M arrest and increased the expression of cyclin B, histone H3 (Ser10) phosphorylation, and cleaved PARP, indicating that HoLu-12 could induce mitotic arrest and then apoptosis. Moreover, the combination of HoLu-12 and 5-fluorouracil (5-FU) displayed synergistic toxic effect on OSCC cells. HoLu-12 significantly inhibited tumor growth in vivo. CONCLUSION: HoLu-12 induces mitotic arrest and leads to apoptosis of OSCC cells. Furthermore, HoLu-12 alone or in combination with 5-FU is a potential therapeutic agent for OSCC.

Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells.

Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.