Liver organoid culture methods.

Organoids, three-dimensional structures cultured in vitro, can recapitulate the microenvironment, complex architecture, and cellular functions of in vivo organs or tissues. In recent decades, liver organoids have been developed rapidly, and their applications in biomedicine, such as drug screening, disease modeling, and regenerative medicine, have been widely recognized. However, the lack of repeatability and consistency, including the lack of standardized culture conditions, has been a major obstacle to the development and clinical application of liver organoids. It is time-consuming for researchers to identify an appropriate medium component scheme, and the usage of some ingredients remains controversial. In this review, we summarized and compared different methods for liver organoid cultivation that have been published in recent years, focusing on controversial medium components and discussing their advantages and drawbacks. We aimed to provide an effective reference for the development and standardization of liver organoid cultivation.

Advances and Challenges in Cytomegalovirus Detection Methods for Liver Transplant Donors.

Cytomegalovirus (CMV) infection is a highly prevalent opportunistic infection among liver transplant recipients. When the liver donor is infected with CMV, there is a risk of transmission to the recipient, leading to CMV infection. To improve the postoperative outcome of liver transplantation, it is crucial to shift the focus of CMV detection to the donor and achieve early diagnosis, as well as implement effective preventative and therapeutic measures. However, the commonly used CMV detection methods in the past had limitations that prevented their early and accurate diagnosis in liver transplant donors. This review focuses on the latest advancements in CMV detection methods that can potentially be applied to liver transplant donors. The objective is to compare and evaluate their clinical utility, thereby providing guidance and support for rapid and accurate diagnosis of CMV infection in the clinic. The clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR-Cas) system-based assay emerges as a promising method for detecting the virus, offering great prospects for early and expedient CMV infection diagnosis in clinical settings.

Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology.

Hypothermic Oxygenated Machine Perfusion Promotes Mitophagy Flux against Hypoxia-Ischemic Injury in Rat DCD Liver.

Hypothermic oxygenated machine perfusion (HOPE) can enhance organ preservation and protect mitochondria from hypoxia-ischemic injury; however, an understanding of the underlying HOPE mechanism that protects mitochondria is somewhat lacking. We hypothesized that mitophagy may play an important role in HOPE mitochondria protection. Experimental rat liver grafts were exposed to 30 min of in situ warm ischemia. Then, grafts were procured, followed by cold storage for 3 or 4 h to mimic the conventional preservation and transportation time in donation after circulatory death (DCD) in clinical contexts. Next, the grafts underwent hypothermic machine perfusion (HMP) or HOPE for 1 h through portal vein only perfusion. The HOPE-treated group showed a better preservation capacity compared with cold storage and HMP, preventing hepatocyte damage, nuclear injury, and cell death. HOPE can increase mitophagy marker expression, promote mitophagy flux via the PINK1/Parkin pathway to maintain mitochondrial function, and reduce oxygen free radical generation, while the inhibition of autophagy by 3-methyladenine and chloroquine could reverse the protective effect. HOPE-treated DCD liver also demonstrated more changes in the expression of genes responsible for bile metabolism, mitochondrial dynamics, cell survival, and oxidative stress. Overall, HOPE attenuates hypoxia-ischemic injury in DCD liver by promoting mitophagy flux to maintain mitochondrial function and protect hepatocytes. Mitophagy could pave the way for a protective approach against hypoxia-ischemic injury in DCD liver.

Exosome is involved in liver graft protection after remote ischemia reperfusion conditioning.

BACKGROUND: Remote ischemic perconditioning (RIPerC) has been demonstrated to protect grafts from hepatic ischemia-reperfusion injury (IRI). This study investigated the role of exosomes in RIPerC of liver grafts in rats. METHODS: Twenty-five rats (including 10 donors) were randomly divided into five groups (n = 5 each group): five rats were used as sham-operated controls (Sham), ten rats were for orthotopic liver transplantation (OLT, 5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). Liver architecture and function were evaluated. RESULTS: Compared to the OLT group, the OLT + RIPerC group exhibited significantly improved liver graft histopathology and liver function (P < 0.05). Furthermore, the number of exosomes and the level of P-Akt were increased in the OLT + RIPerC group. CONCLUSIONS: RIPerC effectively improves graft architecture and function, and this protective effect may be related to the increased number of exosomes. The upregulation of P-Akt may be involved in underlying mechanisms.

NMI: a potential biomarker for tumor prognosis and immunotherapy.

N-Myc and STAT Interactor protein (NMI) is an interferon inducible protein participating in various cellular activities, and is widely involved in the process of tumorigenesis and progression. Studies have shown that the loss of NMI expression in breast cancer can promote its progression by inducing epithelial-mesenchymal transition (EMT). However, the expression level of NMI in other tumors and its impact on immune cell infiltration, patient prognosis, and drug treatment are still unclear. Here, we analyzed the role of NMI in pan-cancer through multiple omics data. We found that NMI was abnormally expressed in a variety of tumor tissues. The expression of NMI was closely related to the unique molecular and immunotyping, diagnosis and prognosis of various tumor tissues. In addition, we identified the main proteins that interact with NMI, and focused on the relationship between the clinical parameters of lower grade glioma (LGG) and NMI expression. Subsequently, we found that the expression of NMI was correlated with the infiltration of multiple immune cells and the expression of immune checkpoints. Finally, we also found that the expression of NMI was correlated with the sensitivity to multiple antitumor drugs. In conclusion, our comprehensive pan-cancer analysis of NMI revealed that it is a potential molecular marker for tumor diagnosis and treatment, plays an important role in tumor immunity, and is a promising molecular target for cancer treatment.

Gαi3 nuclear translocation causes irradiation resistance in human glioma cells.

We have previously shown that Gαi3 is elevated in human glioma, mediating Akt activation and cancer cell proliferation. Here, we imply that Gαi3 could also be important for irradiation resistance. In A172 human glioma cells, Gαi3 knockdown (by targeted shRNAs) or dominant-negative mutation significantly potentiated irradiation-induced cell apoptosis. Reversely, forced over-expression of wild-type or constitutively-active Gαi3 inhibited irradiation-induced A172 cell apoptosis. Irradiation in A172 cells induced Gαi3 translocation to cell nuclei and association with local protein DNA-dependent protein kinase (DNA-PK) catalytic subunit. This association was important for DNA damage repair. Gαi3 knockdown, depletion (using Gαi3 knockout MEFs) or dominant-negative mutation potentiated irradiation-induced DNA damages. On the other hand, expression of the constitutively-active Gαi3 in A172 cells inhibited DNA damage by irradiation. Together, these results indicate a novel function of Gαi3 in irradiation-resistance in human glioma cells.

Preclinical study of cinobufagin as a promising anti-colorectal cancer agent.

Here, we assessed the anti-colorectal cancer (CRC) cell activity of cinobufagin (CBG). We found that CBG exerted potent cytotoxic and anti-proliferative activity against CRC lines (HCT-116 and HT-29) and primary human CRC cells. Meanwhile, it activated apoptosis, and disrupted cell-cycle progression in the cells. At the signaling level, CBG treatment in CRC cells provoked endoplasmic reticulum stress (ER stress), the latter was evidenced by caspase-12 activation, CHOP expression, as well as PERK and IRE1 phosphorylations. Contrarily, the ER stress inhibitor salubrinal, the caspase-12 inhibitor and CHOP shRNA remarkably attenuated CBG-induced CRC cell death and apoptosis. Further, CBG in-activated mammalian target or rapamycin complex 1 (mTORC1), which appeared responsible for proliferation inhibition in CRC cells. Introduction of a constitutively-active S6K1 ("ca-S6K1") restored proliferation of CBG-treated CRC cells. Finally, CBG intraperitoneal injection suppressed HCT-116 xenograft tumor growth in the nude mice. CHOP upregulation and mTORC1 in-activation were also noticed in CBG-treated HCT-116 tumors. The results of this preclinical study suggest that CBG could be tested as promising anti-CRC agent.

SC79 rescues osteoblasts from dexamethasone though activating Akt-Nrf2 signaling.

Dexamethasone (Dex) causes osteoblast cell injuries. In the present research, we tested the potential effect of SC79, a novel and specific Akt activator, against Dex in osteoblasts. In primary murine osteoblasts and osteoblastic MC3T3-E1 cells, pretreatment with SC79 significantly attenuated Dex-induced cell death. Further, Dex-induced mitochondrial permeability transition pore (mPTP) opening, cytochrome C release and apoptosis activation were dramatically alleviated with SC79 pretreatment in above cells. At the molecular level, SC79 activated Akt, which was indispensable for subsequent osteoblast protection against Dex. Akt inhibitors (LY294002, perifosine and MK-2206) blocked SC79-induced Akt activation and abolished its anti-Dex actions in osteoblasts. Further, SC79 activated Akt downstream Nrf2 (NF-E2-related factor 2) signaling and attenuated Dex-induced oxidative stress in osteoblasts. Nrf2 shRNA knockdown or S40T mutation almost reversed SC79-mediated anti-oxidant and cytoprotective activities in osteoblasts. Together, these results suggest that SC79 activates Akt-Nrf2 signaling to protect osteoblasts from Dex.