Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

The "Beacon" Structural Model of Protein Folding: Application for Trp-Cage in Water.

Protein folding is a process in which a polypeptide must undergo folding process to obtain its three-dimensional structure. Thermodynamically, it is a process of enthalpy to overcome the loss of conformational entropy in folding. Folding is primarily related to hydrophobic interactions and intramolecular hydrogen bondings. During folding, hydrophobic interactions are regarded to be the driving forces, especially in the initial structural collapse of a protein. Additionally, folding is guided by the strong interactions within proteins, such as intramolecular hydrogen bondings related to the α-helices and ß-sheets of proteins. Therefore, a protein is divided into the folding key (FK) regions related to intramolecular hydrogen bondings and the non-folding key (non-FK) regions. Various conformations are expected for FK and non-FK regions. Different from non-FK regions, it is necessary for FK regions to form the specific conformations in folding, which are regarded as the necessary folding pathways (or "beacons"). Additionally, sequential folding is expected for the FK regions, and the intermediate state is found during folding. They are reflected on the local basins in the free energy landscape (FEL) of folding. To demonstrate the structural model, molecular dynamics (MD) simulations are conducted on the folding pathway of the TRP-cage in water.

Pal Affects the Proliferation in Macrophages and Virulence of Brucella, and as Mucosal Adjuvants, Provides an Effective Protection to Mice Against Salmonella Enteritidis.

The purpose of this study was to elucidate the roles of peptidoglycan-associated lipoprotein (Pal protein) in the proliferation of Brucella in macrophage and bacterial virulence, and to evaluate the immune effect of Pal protein to Salmonella enteritidis. Murine macrophage-like cell line Raw264.7 was stimulated by recombinant Pal protein, and the expression of TNF-α and IFN-γ were up-regulated, but not it of IL-1ß and IL-6. The macrophages infection and in vitro simulated stress assays showed that deletion of pal gene reduced the proliferation of Brucella in macrophages, the survival in acidic, oxidative and polymyxin B-contained environment. The mice infection assay showed that mice challenged with the pal mutant strain were found to have more severe splenomegaly, but less bacterial load. After oral immunization of mice, Pal protein induced a higher titer of mucosal and humoral antibody (IgA and IgG) against heat-killed Salmonella enteritidis, and a stronger Th1 cellular immune response. The challengte experiments showed Pal protein elevated the survival rate and reduced the bacterial load of spleens in immunized mice. In conclusion, our results revealed the important roles of pal gene in Brucella virulence, and Pal protein was a potentially valuable adjuvant against mucosal pathogens, such as Salmonella enteritidis.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Population structure of Han population in China revealed by 41 STR loci.

Background: Currently, the Han population in China may be comprised of different genetic groups due to geographic, cultural and economic factors. Understanding population structure is very important for forensic purposes. However, knowledge of the genetic substructure within the whole Han population in China is still limited.Aim: This study is designed to ascertain the genetic structure of the Han population in China through genetic data from autosomal short tandem repeats (STRs).Subjects and methods: A set of 41 STR markers were analysed in 8725 unrelated Han Chinese males from the seven geographic regions of Northeast, North, East, Central, South, Southwest and Northwest in mainland China. Allele frequencies and F-statistics were estimated. Principal coordinate analysis (PCoA), phylogenetic analyses, analysis of molecular variance (AMOVA) and discriminant analysis of principal components (DAPC) were performed to explore the population structure.Results: Rare alleles that have not been observed in previous samples were detected. The small overall Fst values (0.0008), AMOVA and DAPC indicated that there is no population structure in Han Chinese. However, the PCoA and phylogenetic tree disclose a genetic differentiation pattern from north to south.Conclusions: There is no apparent population substructure in the Han population in China. However, genetic distances among the Han populations correlate with geographic locations.

Robust, synergistic regulation of human gene expression using TALE activators.

Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

CRISPR RNA-guided activation of endogenous human genes.

Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish.

Hemangioblast, including primitive hematopoietic progenitor cells, play an important role in hematopoietic development, however, the underlying mechanism for the propagation of hematopoietic progenitor cells remains elusive. A variety of regulatory molecules activated in early embryonic development play a critical role in the maintenance of function of hematopoietic progenitor cells. Homeobox transcription factors are an important class of early embryonic developmental regulators determining hematopoietic development. However, the effect of homeobox protein Hox-B4 (HOXB4) ectopic expression on the development of hemangioblasts has not been fully addressed. This study aimed to investigate the role of Hoxb4a, an ortholog gene of HOXB4 in zebrafish, in the hematopoietic development in zebrafish. A transgenic zebrafish line was established with Cre-loxP system that stably overexpressed enhanced green fluorescent protein (EGFP)-tagged Hoxb4a protein under the control of hemangioblast-specific lmo2 promoter. Overexpression of Hoxb4a in the development of hemangioblasts resulted in a considerable increase in the number of stem cell leukemia (scl) and lmo2-positive primitive hematopoietic progenitor cells occurring in the posterior intermediate cell mass (ICM). Interestingly, Hoxb4a overexpression also disrupted the development of myelomonocytes in the anterior yolk sac and the posterior ICM, without affecting erythropoiesis in the posterior ICM. Taken together, these results indicate that Hoxb4a favours the development of hematopoietic progenitor cells originated from hemangioblasts in vivo.

Cross shard leader accountability protocol based on two phase atomic commit.

Sharding blockchain is a technology designed to improve the performance and scalability of traditional blockchain systems. However, due to its design, communication between shards depends on shard leaders for transmitting information, while shard members are unable to detect communication activities between shards. Consequently, Byzantine nodes can act as shard leaders, engaging in malicious behaviors to disrupt message transmission. To address these issues, we propose the Cross shard leader accountability protocol (CSLAP), which is based on the two-phase atomic commit protocol (2PC). CSLAP employs byzantine broadcast/byzantine agreement (BB/BA) for Byzantine fault tolerance to generate cross-shard leader re-election certificates, thereby reducing the impact of shard leaders on inter-shard communication. It also uses Round-robin mechanism to facilitate leader re-election. Moreover, we demonstrate that CSLAP maintains the security and liveness of sharding transactions while providing lower communication latency. Finally, we conducted an experimental comparison between CSLAP and other cross-shard protocols. The results indicate that CSLAP exhibits superior performance in reducing communication latency.

Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear. AIM OF THE STUDY: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl4)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis. MATERIALS AND METHODS: In this study, CCl4-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting. RESULTS: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl4 and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis. CONCLUSION: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.

Spatiotemporal variation and influencing factors of air pollution in Anhui Province.

Anhui Province locates in the Yangtze River Delta region. The spatial difference between the north and the south is significant, and the air quality is improved over time. Studying the spatial and temporal changes of air pollution and its influencing factors for the coordinated control of air pollutants in the Yangtze River Delta region is significant. This study used the annual and monthly average data of six pollutants, PM2.5, PM10, O3, NO2, SO2, and CO, in Anhui Province and various cities from 2015 to 2021 and analyzed the spatiotemporal change characteristics using Excel and GIS software. Meanwhile, this paper used the SPSS correlation analysis method to analyze the correlation between pollutants and meteorological factors and analyzed the impact of economic development and environmental protection policies. The results are shown below. (1) The concentrations of SO2, NO2, and CO showed an overall downward trend at an interannual level. Meanwhile, the concentrations of PM10 and PM2.5 first increased slowly before 2017 and then declined, while the concentrations of O3 increased significantly before 2018 and then declined slowly. On a monthly scale, O3 presented an M-shaped change, while the other five pollutants basically presented a U-shaped change mode. The top monthly pollutants in each city followed the order of PM2.5, O3, PM10, and NO2. (2) PM2.5 and PM10 showed apparent characteristics of high concentrations in the north and low concentrations in the south in space. There were no significant differences in NO2, SO2, and CO pollution between the north and the south in space, and the differences in spatial pollution among cities were reduced significantly. (3) Five pollutants (SO2, NO2, PM10, PM2.5, and CO) except O3 were positively correlated, and the degree of correlation was correlated, strongly correlated, and above. However, five pollutants were negatively correlated with O3. The temperature had the most significant impact of negative correlation on five pollutants except for O3. The sunshine duration had the most significant impact on O3. (4) Economic growth and environmental protection policies in Anhui Province have positively affected environmental governance.

Sodium cholate ameliorates nonalcoholic steatohepatitis by activation of FXR signaling.

Non-alcoholic steatohepatitis (NASH) has become a major cause of liver transplantation and liver-associated death. The gut-liver axis is a potential therapy for NASH. Sodium cholate (SC) is a choleretic drug whose main component is bile acids and has anti-inflammatory, antifibrotic, and hepatoprotective effects. This study aimed to investigate whether SC exerts anti-NASH effects by the gut-liver axis. Mice were fed with an high-fat and high-cholesterol (HFHC) diet for 20 weeks to induce NASH. Mice were daily intragastric administrated with SC since the 11th week after initiation of HFHC feeding. The toxic effects of SC on normal hepatocytes were determined by CCK8 assay. The lipid accumulation in hepatocytes was virtualized by Oil Red O staining. The mRNA levels of genes were determined by real-time quantitative PCR assay. SC alleviated hepatic injury, abnormal cholesterol synthesis, and hepatic steatosis and improved serum lipid profile in NASH mice. In addition, SC decreased HFHC-induced hepatic inflammatory cell infiltration and collagen deposition. The target protein-protein interaction network was established through Cytoscape software, and NR1H4 [farnesoid x receptor (FXR)] was identified as a potential target gene for SC treatment in NASH mice. SC-activated hepatic FXR and inhibited CYP7A1 expression to reduce the levels of bile acid. In addition, high-dose SC attenuated the abnormal expression of cancer markers in NASH mouse liver. Finally, SC significantly increased the expression of FXR and FGF15 in NASH mouse intestine. Taken together, SC ameliorates steatosis, inflammation, and fibrosis in NASH mice by activating hepatic and intestinal FXR signaling so as to suppress the levels of bile acid in NASH mouse liver and intestine.

An evolutionarily conserved PTEN-C/EBPalpha-CTNNA1 axis controls myeloid development and transformation.

Loss of function of tumor suppressor genes, such as PTEN, CEBPAlpha, and CTNNA1 (encoding the alpha-catenin protein), has been found to play an essential role in leukemogenesis. However, whether these genes genetically interact remains largely unknown. Here, we show that PTEN-mammalian target of rapamycin signaling acts upstream to dictate the ratio of wild-type p42 C/EBPalpha to its dominant-negative p30 isoform, which critically determines whether p30 C/EBPalpha (lower p42/p30 ratio) or p42 C/EBPalpha (higher p42/p30 ratio) binds to the proximal promoter of the retained CTNNA1 allele. Binding of p30 C/EBPalpha recruits the polycomb repressive complex 2 to suppress CTNNA1 transcription through repressive H3K27me3 modification, whereas binding of p42 C/EBPalpha relieves this repression and promotes CTNNA1 expression through activating H3K4me3 modification. Loss of Pten function in mice and zebrafish induces myelodysplasia with abnormal invasiveness of myeloid progenitors accompanied by significant reductions in both wild-type C/EBPalpha and alpha-catenin protein. Importantly, frame-shift mutations in either PTEN or CEBPA were detected exclusively in the primary LICs with low CTNNA1 expression. This study uncovers a novel molecular pathway, PTEN-C/EBPalpha-CTNNA1, which is evolutionarily conserved and might be therapeutically targeted to eradicate LICs with low CTNNA1 expression.

TCP-WBQ: a backlog-queue-based congestion control mechanism for heterogeneous wireless networks.

In heterogeneous wireless networks, random packet loss and high latency lead to conventional TCP variants performing unsatisfactorily in the case of competing communications. Especially on high-latency wireless links, conventional TCP variants are unable to estimate congestion degrees accurately for fine-grained congestion control because of the effects of random packet loss and delay oscillations. This paper proposes a TCP variant at the sender side to identify congestion degrees, namely TCP-WBQ, which quickly responses to the real congestion and effectively shields against random packet loss and oscillations of latency time. The proposed algorithm of congestion control firstly constructs a backlog-queue model based on the dynamics of the congestion window, and deduces the two bounds of the model which delimit oscillations of the backlog queue for non-congestion and random packet loss respectively. TCP-WBQ detects congestion degrees more accurately and thus implements the corresponding schemes of adjusting the congestion window, maintaining a tradeoff between high throughputs and congestion avoidance. The comprehensive simulations show that TCP-WBQ works efficiently in bandwidth utilization with single and multiple bottleneck scenarios, and achieves high performance and competitive fairness in heterogeneous wireless networks.

Fufang Zhenzhu Tiaozhi Capsule Prevents Intestinal Inflammation and Barrier Disruption in Mice With Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) has become a major cause of liver transplantation and liver-associated death. Targeting the gut-liver axis is a potential therapy for NASH. The Fufang Zhenzhu Tiaozhi (FTZ) capsule, a traditional Chinese medicine commonly used in clinical practice, has recently emerged as a promising drug candidate for metabolic diseases such as NASH. The present study aimed to investigate whether FTZ exerts an anti-NASH effect by targeting the gut-liver axis. Mice were fed with a high-fat diet (HFD) for 20 weeks to induce NASH. HFD-fed mice were daily intragastrically administrated with FTZ at 10 weeks after tbe initiation of HFD feeding. The mRNA levels of genes associated with the intestinal tight junction, lipid metabolism, and inflammation were determined by the q-PCR assay. Hepatic pathology was evaluated by H&E staining. The gut microbiota was analyzed by 16S rRNA gene sequencing. FTZ attenuated HFD-induced obesity, insulin resistance, and hepatic steatosis in mice. FTZ treatment decreased the elevated levels of serum aminotransferases and liver triglyceride in NASH mice. Furthermore, FTZ treatment reduced hepatic inflammatory cell infiltration and fibrosis in mice. In addition, FTZ attenuated the intestinal inflammatory response and improved intestinal barrier function. Mechanistically, FTZ-treated mice showed a different gut microbiota composition compared with that in HFD-fed mice. Finally, we identified eight differential metabolites that may contribute to the improvement of NASH with FTZ treatment. In summary, FTZ ameliorates NASH by inhibiting gut inflammation, improving intestinal barrier function, and modulating intestinal microbiota composition.

Mir-144 selectively regulates embryonic alpha-hemoglobin synthesis during primitive erythropoiesis.

Precise transcriptional control of developmental stage-specific expression and switching of alpha- and beta-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating beta-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling alpha-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic alpha-globin, but not embryonic beta-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both alpha-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic alpha-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.

Long non-coding RNA TCONS_00814106 regulates porcine granulosa cell proliferation and apoptosis by sponging miR-1343.

Recent evidence shows that long non-coding RNAs (lncRNAs), a class of non-coding RNAs, are involved in the regulation of reproductive processes. In this study, we identified a lncRNA, TCONS_00814106, that was upregulated in high-fecundity sow ovarian tissues and influenced by reproductive hormones. Bioinformatics analyses and luciferase reporter assays showed that TCONS_00814106 is a miR-1343 target. Cell counting kit (CCK)-8 and apoptosis assays showed that TCONS_00814106 promotes proliferation and inhibits apoptosis in porcine granulosa cells (GCs), and that this could be reversed by miR-1343. Also, we observed that transforming growth factor-ß receptor type I (TGFBR1) is a functional target of miR-1343 in GCs. TCONS_00814106 serves as a competing endogenous RNA to regulate TGFBR1 expression by sponging miR-1343, thereby exerting regulatory functions in GCs. Overall, these results provide new insights into the biological function of the lncRNA TCONS_00814106.

In vivo interstitial migration of primitive macrophages mediated by JNK-matrix metalloproteinase 13 signaling in response to acute injury.

Interstitial cell migration through extracellular matrix is a hallmark of the inflammation response, tumor invasion, and metastasis. We have established a stable zebrafish transgenic line expressing enhanced GFP under the lysozyme C promoter for visualizing and measuring primitive macrophage migration in vivo. We show that tissue-resident primitive macrophages migrate rapidly through extracellular matrix to the site of acute injury induced by tail transection. Mechanistically, the specific inhibition of JNK, but not p38 and ERK, dramatically abolished the chemotactic migration in a dose-dependent manner, suppressing the trauma-induced recruitment of phosphorylated C-Jun transcription factor to proximal AP-1 sites in the promoter of matrix metalloproteinase 13 (mmp13), a gene specifically expressed in primitive macrophages during embryogenesis and required for the interstitial migration. Furthermore, dexamethasone suppressed the trauma-induced JNK phosphorylation and macrophage migration accompanied by simultaneous up-regulation of mkp-1, a well-known phosphatase capable of inactivating phosphorylated JNK. The results indicate that the JNK-Mmp13 signaling pathway plays an essential role in regulating the innate immune cell migration in response to severe injury in vivo.

Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis.

MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. Despite increasing number of genes has been predicted to be miRNA targets by bioinformatics analysis and luciferase-based reporter assay in vitro (RA-In Vitro), few of them have been experimentally validated in physiological context. Using transient reporter assay in vivo (TRA-In Vivo), we identified hydroxymethylbilane synthase b (hmbsb) and Krüppel-like transcription factor d (klfd) as potential target gene for miR-451 and miR-144, respectively. Although hmbsb, miR-451, klfd and miR-144 are all co-expressed in the developing erythroid progenitors during zebrafish erythropoiesis, only klfd can be validated as a bona fide physiological target of miR-144 using a transgene-based physiological reporter assay in vivo (PRA-In Vivo). Thus, failure to verify hmbsb as miR-451 target in physiological context raises a crucial question as to how to determine bona fide target of miRNA. The results address the importance of using multiple approaches combined with Western blot analysis to validate the physiological target of a given miRNA.

Radiation induces apoptosis primarily through the intrinsic pathway in mammalian cells.

Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in "initiator" caspases, "effector" caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both "initiator" and "effector" caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.