CPT1A loss disrupts BCAA metabolism to confer therapeutic vulnerability in TP53-mutated liver cancer.

Driver genomic mutations in tumors define specific molecular subtypes that display distinct malignancy competence, therapeutic resistance and clinical outcome. Although TP53 mutation has been identified as the most common mutation in hepatocellular carcinoma (HCC), current understanding on the biological traits and therapeutic strategies of this subtype has been largely unknown. Here, we reveal that fatty acid ß oxidation (FAO) is remarkable repressed in TP53 mutant HCC and which links to poor prognosis in HCC patients. We further demonstrate that carnitine palmitoyltransferase 1 (CPT1A), the rate-limiting enzyme of FAO, is universally downregulated in liver tumor tissues, and which correlates with poor prognosis in HCC and promotes HCC progression in the de novo liver tumor and xenograft tumor models. Mechanically, hepatic Cpt1a loss disrupts lipid metabolism and acetyl-CoA production. Such reduction in acetyl-CoA reduced histone acetylation and epigenetically reprograms branched-chain amino acids (BCAA) catabolism, and leads to the accumulation of cellular BCAAs and hyperactivation of mTOR signaling. Importantly, we reveal that genetic ablation of CPT1A renders TP53 mutant liver cancer mTOR-addicted and sensitivity to mTOR inhibitor AZD-8055 treatment. Consistently, Cpt1a loss in HCC directs tumor cell therapeutic response to AZD-8055. CONCLUSION: Our results show genetic evidence for CPT1A as a metabolic tumor suppressor in HCC and provide a therapeutic approach for TP53 mutant HCC patients.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Discovery of novel cholesteryl ester transfer protein (CETP) inhibitors by a multi-stage virtual screening.

Cholesteryl ester transfer protein (CETP) is a promising therapeutic target for cardiovascular diseases. It effectively lowers the low-density lipoprotein cholesterol levels and increases the high-density lipoprotein cholesterol levels in the human plasma. This study identified novel and highly potent CETP inhibitors using virtual screening techniques. Molecular docking and molecular dynamics (MD) simulations revealed the binding patterns of these inhibitors, with the top 50 compounds selected according to their predicted binding affinity. Protein-ligand interaction analyses were performed, leading to the selection of 26 compounds for further evaluation. A CETP inhibition assay confirmed the inhibitory activities of the selected compounds. The results of the MD simulations revealed the structural stability of the protein-ligand complexes, with the binding site remaining significantly unchanged, indicating that the five compounds (AK-968/40709303, AG-690/11820117, AO-081/41378586, AK-968/12713193, and AN-465/14952302) identified have the potential as active CETP inhibitors and are promising leads for drug development.

Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch.

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmid-based multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the "BacAmp", a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis, which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis.

Protein of yak milk residue: Structure, functionality, and the effects on the quality of non-fat yogurt.

The purpose of this study was to compare the structural and functional of protein from yak milk residue, which collected from different elevations (MRP1 and MRP2) in Tibet, as well as their potential for enhancing the quality of non-fat yogurt. The results showed that MRP1 exhibited higher levels of ß-sheet, turbidity, particle size, and gel properties. MRP2 had better flexibility, emulsification, foaming, water/oil absorption capacity. The addition of MRP1 (3%) could improve texture and sensory properties of yogurt. Although MRP2 yogurt had higher hardness, gumminess, chewiness and water holding capacity, poor mouthfeel. Rheological test showed that MRPs yogurt exhibited typical gel-like and shear-thinning behavior. Moreover, the fortification of non-fat yogurts with MRP1 brought the formation of larger protein clusters with a more tightly knit network of smaller pores. These results indicate that MRP1 can be used as a fat substitute to improve the quality of non-fat yogurt.

Genome-wide characterization of Remorin gene family and their responsive expression to abiotic stresses and plant hormone in Brassica napus.

KEY MESSAGE: Remorin proteins could be positively related to salt and osmotic stress resistance in rapeseed. Remorins (REMs) play a crucial role in adaptations to adverse environments. However, their roles in abiotic stress and phytohormone responses in oil crops are still largely unknown. In this study, we identified 47 BnaREM genes in the B.napus genome. Phylogenetic relationship and synteny analysis revealed that they were categorized into 5 distinct groups and have gone through 55 segmental duplication events under purifying selection. Gene structure and conserved domains analysis demonstrated that they were highly conserved and all BnaREMs contained a conserved Remorin_C domain, with a variable N-terminal region. Promoter sequence analysis showed that BnaREM gene promoters contained various hormones and stress-related cis-acting elements. Transcriptome data from BrassicaEDB database exhibited that all BnaREMs were ubiquitously expressed in buds, stamens, inflorescences, young leaves, mature leaves, roots, stems, seeds, silique pericarps, embryos and seed coats. The qRT-PCR analysis indicated that most of them were responsive to ABA, salt and osmotic treatments. Further mutant complementary experiments revealed that the expression of BnaREM1.3-4C-1 in the Arabidopsis rem1.3 mutant restored the retarded growth phenotype and the ability to resistance to salt and osmotic stresses. Our findings provide fundamental information on the structure and evolutionary relationship of the BnaREM family genes in rapeseed, and reveal the potential function of BnaREM1.3-4C-1 in stress and hormone response.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Teneligliptin mitigates diabetic cardiomyopathy by inhibiting activation of the NLRP3 inflammasome.

BACKGROUND: Diabetic cardiomyopathy (DCM), which is a complication of diabetes, poses a great threat to public health. Recent studies have confirmed the role of NLRP3 (NOD-like receptor protein 3) activation in DCM development through the inflammatory response. Teneligliptin is an oral hypoglycemic dipeptidyl peptidase-IV inhibitor used to treat diabetes. Teneligliptin has recently been reported to have anti-inflammatory and protective effects on myocardial cells. AIM: To examine the therapeutic effects of teneligliptin on DCM in diabetic mice. METHODS: Streptozotocin was administered to induce diabetes in mice, followed by treatment with 30 mg/kg teneligliptin. RESULTS: Marked increases in cardiomyocyte area and cardiac hypertrophy indicator heart weight/tibia length reductions in fractional shortening, ejection fraction, and heart rate; increases in creatine kinase-MB (CK-MB), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels; and upregulated NADPH oxidase 4 were observed in diabetic mice, all of which were significantly reversed by teneligliptin. Moreover, NLRP3 inflammasome activation and increased release of interleukin-1ß in diabetic mice were inhibited by teneligliptin. Primary mouse cardiomyocytes were treated with high glucose (30 mmol/L) with or without teneligliptin (2.5 or 5 µM) for 24 h. NLRP3 inflammasome activation. Increases in CK-MB, AST, and LDH levels in glucose-stimulated cardiomyocytes were markedly inhibited by teneligliptin, and AMP (p-adenosine 5'-monophosphate)-p-AMPK (activated protein kinase) levels were increased. Furthermore, the beneficial effects of teneligliptin on hyperglycaemia-induced cardiomyocytes were abolished by the AMPK signaling inhibitor compound C. CONCLUSION: Overall, teneligliptin mitigated DCM by mitigating activation of the NLRP3 inflammasome.

Effectiveness of biological drying for citric acid dewatered sludge: Evaluating the impact of energy-efficient ventilation strategies.

The effectiveness of dehydration and utilization processes for citric acid dewatered sludge is hampered by its high concentrations of polysaccharides, proteins, and water-binding properties of microbial extracellular polymers (EPS). This research explores the efficacy and mechanisms involved in extracting water from this type of sludge using biological drying technology, with varying rates of ventilation. Especially pertinent was the use of low ventilation rates as control variables. Our results suggest that a scheduled intermittent ventilation at lower rates allows for the most efficient removal of water, achieving a rate of 41.71 % within eight days, according to the zero-order kinetic model. Remarkably, the peak temperature registered was 60 °C, reaching this threshold in just 0.1 days and maintaining high temperatures for approximately 5.9 days. Component analysis of organic matter illustrated a preferential degradation process for lipids under these ventilation conditions which is pivotal for releasing and transforming bound water for efficient extraction, as well as facilitating the breakdown of easily hydrolysable materials. Further, polysaccharide/protein (EPS) decomposition contributed to water removal, though less significantly. The periodic ventilation strategy allowed for the maximum cumulative temperature to be sustained, demonstrating superior efficiency in harnessing bio-generated heat (82.77 % for water evaporation), resulting in dry sludge suitable for self-sustained combustion at relatively low cost ($26.61/t). Highlighted by this study is the considerable potential of energy-efficient ventilation methods in the biological drying treatment of citric acid fermented sludge and similar industrial waste materials.

GLI1 polymorphisms influence remission rate and prognosis of young de novo acute myeloid leukemia patients treated with cytarabine-based chemotherapy.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy. Cytarabine (Ara-C)-based chemotherapy is the primary treatment for AML, but currently known prognostic risk stratification factors cannot fully explain the individual differences in outcome of patients. In this article, we reported that patients with homozygous GLI1 rs2228224 mutation (AA genotype) had a significantly lower complete remission rate than those with GG wild type (54.17% vs.76.02%, OR = 1.993, 95% CI: 1.062-3.504, P = 0.031). GLI1 rs2229300 T allele carriers had remarkably shorter overall survival (513 vs. 645 days, P = 0.004) and disease-free survival (342 vs. 456 days, P = 0.033) than rs2229300 GG carriers. Rs2229300 G > T variation increased the transcriptional activity of GLI1. CCND1, CD44 and PROM1 were potential target genes differentially regulated by GLI1 rs2229300. Our results demonstrated for the first time that GLI1 polymorphisms influence chemosensitivity and prognosis of young de novo AML patients treated with Ara-C.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

PD-1 antibody in combination with chemotherapy for the treatment of SMARCA4-deficient advanced undifferentiated carcinoma of the duodenum: Two case reports.

BACKGROUND: SMARCA4 is a component of chromatin remodeling of SWItch/sucrose-nonfermenting (SWI/SNF) complexes and plays an essential role in oncogenesis. SMARCA4-deficient malignancies arising from the gastrointestinal tract are rare and have a poor prognosis. There is no standard treatment for advanced and undifferentiated SMARCA4-deficient duodenal malignancies. Programmed death 1 (PD-1) antibodies, known as immune checkpoint inhibitor antibodies, potentially play a role in treating gastrointestinal tract malignancies. CASE SUMMARY: We present two patients with SMARCA4 deficiency and TP53 gene mutation in advanced undifferentiated carcinomas of the duodenum. For both patients, SMARCA4 deficiency was confirmed by immunohistochemical staining for the BRG1 protein, while TP53 gene mutations were observed via next-generation sequencing. Both patients were administered chemotherapy in combination with an anti-PD-1 antibody. The two patients exhibited completely different responses to treatment and had different prognoses. Case 1 experienced rapid progression after PD-1 infusion and chemotherapy, case 2 experienced a remarkable response after treatment, and the progression-free survival was more than 6 months. CONCLUSION: This study described our clinical and pathological observations of SMARCA4-deficient advanced undifferentiated carcinoma of the duodenum. PD-1 combined with chemotherapy showed a certain efficacy in select patients, providing options for treating these highly malignant tumors. Patients with liver metastases had a worse prognosis than did those with only lymph node metastasis.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Application of additively manufactured bone scaffold: a systematic review.

The application of additive manufacturing (AM) technology plays a significant role in various fields, incorporating a wide range of cutting-edge technologies such as aerospace, medical treatment, electronic information, and materials. It is currently widely adopted for medical services, national defense, and industrial manufacturing. In recent years, AM has also been extensively employed to produce bone scaffolds and implant materials. Through AM, products can be manufactured without being constrained by complex internal structures. AM is particularly advantageous in the production of macroscopically irregular and microscopically porous biomimetic bone scaffolds, with short production cycles required. In this paper, AM commonly used to produce bone scaffolds and orthopedic implants is overviewed to analyze the different materials and structures adopted for AM. The applications of antibacterial bone scaffolds and bone scaffolds in biologically relevant animal models are discussed. Also, the influence on the comprehensive performance of product mechanics, mass transfer, and biology is explored. By identifying the reasons for the limited application of existing AM in the biomedical field, the solutions are proposed. This study provides an important reference for the future development of AM in the field of orthopedic healthcare. In conclusion, various AM technologies, the requirements of bone scaffolds and the important role of AM in building bridges between biomaterials, additives, and bone tissue engineering scaffolds are described and highlighted. Nevertheless, more caution should be exercised when designing bone scaffolds and conducting in vivo trials, due to the lack of standardized processes, which prevents the accuracy of results and reduces the reliability of information.

Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a "Design-Build-Test-Learn" Framework.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (kcat /Km ) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol-1 reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in kcat-GlcNAc6P /Km-GlcNAc6P and a 59% decrease in kcat-Glc6P /Km-Glc6P is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L-1 with a yield of 0.597 g (g glucose)-1 in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

Erratum: Author correction to "Immune-awakening Saccharomyces-inspired nanocarrier for oral target delivery to lymph and tumors" [Acta Pharmaceutica Sinica B 12 (2022) 4501-4518].

[This corrects the article DOI: 10.1016/j.apsb.2022.04.018.].

Clinicopathological, immunohistochemical and molecular features of acinic cell carcinoma of the breast.

Breast acinic cell carcinoma (ACC) is a rare subtype of breast cancer. Accurate diagnosis of ACC using core needle biopsy (CNB) is pivotal for the use of effective treatments and patient prognosis. In the present study, a detailed analysis of the morphological, immunohistochemical and gene mutation features of 2 cases of ACC was performed. CNB was performed prior to surgical excision. The breast ACC in the present cases exhibited overt burrowing labyrinthine networks or 'hand-holding-hand' features. The tumor cells in both of the present cases expressed cytokeratin (CK)7, S100 and CK5/6, but were negative for p63, estrogen receptor and progesterone receptor. GATA binding protein 3 was positive in case 1 but negative in case 2. Fluorescence in situ hybridization indicated no ETS variant transcription factor 6 break-apart probe detection. Next-generation sequencing results revealed the same mutation and a similar abundance in exon 27 (NM_005120.2; c.3817G>T; p.A1273S) of the mediator of RNA polymerase II transcription, subunit 12 homolog (MED12) gene in both patients. To conclude, the findings of the present study suggested that recognition of this rare 'hand-holding-hand' structure could potentially be beneficial for avoiding patient misdiagnosis. In addition, it could be suggested that a mutation in the MED12 exon 27 was associated with the formation of a burrowing labyrinthine network or 'hand-holding-hand' feature.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia.

BACKGROUND: The E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. OBJECTIVES: We aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. METHODS: CD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5' untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). RESULTS: CD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase-protein kinase B (AKT) signaling, decreased CBLB expression, and 5' UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. CONCLUSION: These results indicate that the 5' UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.