Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes.

Recent efforts have shown that structural variations (SVs) can disrupt three-dimensional genome organization and induce enhancer hijacking, yet no computational tools exist to identify such events from chromatin interaction data. Here, we develop NeoLoopFinder, a computational framework to identify the chromatin interactions induced by SVs, including interchromosomal translocations, large deletions and inversions. Our framework can automatically resolve complex SVs, reconstruct local Hi-C maps surrounding the breakpoints, normalize copy number variation and allele effects and predict chromatin loops induced by SVs. We applied NeoLoopFinder in Hi-C data from 50 cancer cell lines and primary tumors and identified tens of recurrent genes associated with enhancer hijacking. To experimentally validate NeoLoopFinder, we deleted the hijacked enhancers in prostate adenocarcinoma cells using CRISPR-Cas9, which significantly reduced expression of the target oncogene. In summary, NeoLoopFinder enables identification of critical oncogenic regulatory elements that can potentially reveal therapeutic targets.

Placental trophoblast-specific overexpression of chemerin induces preeclampsia-like symptoms.

Maternal circulating levels of the adipokine chemerin are elevated in preeclampsia, but its origin and contribution to preeclampsia remain unknown. We therefore studied (1) placental chemerin expression and release in human pregnancy, and (2) the consequences of chemerin overexpression via lentivirus-mediated trophoblast-specific gene manipulation in both mice and immortalized human trophoblasts. Placental chemerin expression and release were increased in women with preeclampsia, and their circulating chemerin levels correlated positively with the soluble Fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio, a well-known biomarker of preeclampsia severity. Placental trophoblast chemerin overexpression in mice induced a preeclampsia-like syndrome, involving hypertension, proteinuria, and endotheliosis, combined with diminished trophoblast invasion, a disorganized labyrinth layer, and up-regulation of sFlt-1 and the inflammation markers nuclear factor-κB (NFκB), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. It also led to embryo resorption, while maternal serum chemerin levels correlated negatively with fetal weight in mice. Chemerin overexpression in human trophoblasts up-regulated sFlt-1, reduced vascular endothelial factor-A, and inhibited migration and invasion, as well as tube formation during co-culture with human umbilical vein endothelial cells (HUVECs). The chemokine-like receptor 1 (CMKLR1) antagonist α-NETA prevented the latter phenomenon, although it did not reverse the chemerin-induced down-regulation of the phosphoinositide 3-kinase/Akt pathway. In conclusion, up-regulation of placental chemerin synthesis disturbs normal placental development via its CMKLR1 receptor, thereby contributing to fetal growth restriction/resorption and the development of preeclampsia. Chemerin might be a novel biomarker of preeclampsia, and inhibition of the chemerin/CMKLR1 pathway is a promising novel therapeutic strategy to treat preeclampsia.

Nanoengineered CAR-T Biohybrids for Solid Tumor Immunotherapy with Microenvironment Photothermal-Remodeling Strategy.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable clinical success in eradicating hematologic malignancies. However, hostile microenvironment in solid tumors severely prevents CAR-T cells migrating, infiltrating, and killing. Herein, a nanoengineered CAR-T strategy is reported for enhancing solid tumor therapy through bioorthogonal conjugation with a nano-photosensitizer (indocyanine green nanoparticles, INPs) as a microenvironment modulator. INPs engineered CAR-T biohybrids (CT-INPs) not only retain the original activities and functions of CAR-T cells, but it is further armed with fluorescent tracing and microenvironment remodeling abilities. Irradiated with laser, CT-INPs demonstrate that mild photothermal intervention destroys the extracellular matrix, expanded blood vessels, loosened compact tissue, and stimulated chemokine secretion without damping CAR-T cell activities. Those regulations induce an immune-favorable tumor microenvironment for recruitment and infiltration of CT-INPs. CT-INPs triggered photothermal effects collapse the physical and immunological barriers of solid tumor, and robustly boosted CAR-T immunotherapy. Therefore, CAR-T biohybrids provide reliable treatment strategy for solid tumor immunotherapy via microenvironment reconstruction.

Contribution of placental 11ß-HSD2 to the pathogenesis of preeclampsia.

Preeclampsia, a major human pregnancy-specific disorder, leads to maternal and fetal morbidity and mortality. Here we reported that 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), an enzyme that degrades active glucocorticoids, is one of the key factors that contributes to preeclampsia development. In the pregnant rat model, we firstly confirmed that administration of 11ß-HSD2 inhibitor carbenoxolone (CBX) subcutaneously or by placenta-targeted delivery system could lead to a decrease in placental 11ß-HSD2 expression and activity and an increase in corticosterone level in placenta and maternal circulation. Then, we showed that subcutaneous administration and placenta-targeted delivery of CBX resulted in the hallmark of preeclampsia-like features including hypertension, proteinuria, renal damages as well as elevated circulatory soluble fms-like tyrosine kinase 1 (sFlt1) and increased sFlt1/placental growth factor (PlGF) ratio in pregnant rats. These animals displayed decreased trophoblast invasion in uterus, impaired spiral artery remodeling, and reduced placental blood flow. Preeclampsia-like features could also be induced by administration of dexamethasone in pregnant rats. In the cultured human trophoblast models, we found that cortisol only inhibited migration and invasion of the extravillous trophoblasts with 11ß-HSD2 knockdown, and promoted sFlt1 release in the cultured syncytiotrophoblasts with 11ß-HSD2 knockdown. Furthermore, we elucidated that cortisol stimulated a disintegrin and metalloprotease (ADAM)17 expression in placentas, thereby promoting sFlt1 release in placenta. Collectively, our study provided the evidence that placental 11ß-HSD2 dysfunction plays a key role in the development of preeclampsia and immediately identified innovative target to counteract preeclampsia.

Stent placement to prevent strictures after esophageal endoscopic submucosal dissection: a systematic review and meta-analysis.

Endoscopic submucosal dissection (ESD) is an important method for the treatment of early esophageal cancer. However, post-procedure stenosis is one of the most common long-term complications. This meta-analysis aimed to investigate whether stent placement is effective in the stenosis prevention, and which type of stent would be more effective. A systematic and electronic search of clinical trials and observational studies conducted before March 2020 on the efficacy of stent placement in preventing esophageal stricture after ESD was performed. Search terms included "ESD," "esophageal stenosis," "esophageal stricture," and "stents." We conducted a bias risk assessment of the eligible reports and a meta-analysis of the data using Revman 5.3 software. We included two randomized controlled trials (RCTs) and a prospective cohort study involving 163 patients with esophageal mucosal defects encompassing at least three-quarters of the esophagus circumference after ESD. The meta-analysis results showed that post-ESD stenosis rates (RR, 0.37; 95% CI, 0.22-0.64; P = 0.0003) and the number of endoscopic balloon dilations (EBDs) (MD, -1.74; 95% CI, -2.46 to -1.01; P < 0.00001) were reduced in the pooled analysis of three studies, indicating that stent placement was effective for stenosis prevention, especially a polyglycolic acid (PGA) sheet combined with stent placement can prevent stenosis (RR, 0.41; 95% CI, 0.23-0.74; P = 0.003) and reduce the number of EBDs (MD, -1.65; 95% CI, -2.40 to -0.90; P < 0.0001) significantly. Stent placement can reduce the rate of esophageal stenosis after ESD, especially when stents are covered with PGA sheets. However, more high-quality, low-bias RCTs with a sufficient sample size are needed to demonstrate its effectiveness.

Screening and surveillance of multiple solid tumours using plasma placental-like chondroitin sulfate A (pl-CSA).

Rationale: Placental-like chondroitin sulfate A (pl-CSA) is known to be exclusively synthesized in multiple cancer tissues and associated with disease severity. Here, we aimed to assess whether pl-CSA is released into bio-fluids and can serve as a cancer biomarker. Methods: A novel ELISA was developed to analyse pl-CSA content in bio-fluids using pl-CSA binding protein and an anti-pl-CSA antibody. Immunohistochemical staining of tissue chips was used as the gold standard control. Results: The developed ELISA method was specific and sensitive (1.22 µg/ml). The pl-CSA content was significantly higher in lysates and supernatants of cancer cell lines than in those of normal cell lines, in plasma from mouse cancer models than in that from control mice, and in plasma from patients with oesophageal, cervical, ovarian, or lung cancer than in that from healthy controls. Similar to the tissue chip analysis, which showed a significant difference in pl-CSA positivity between cancer tissues and normal adjacent tissues, the plasma pl-CSA analysis had 100% sensitivity and specificity for differentiating oesophageal and lung cancer patients from healthy controls. Importantly, in oesophageal and lung cancer patients, the pl-CSA content was significantly higher in late-stage disease than in early-stage disease, and it dramatically decreased after surgical resection of the tumour. Conclusion: These data indicate a direct link between plasma pl-CSA content and tumour presence, indicating that plasma pl-CSA may be a non-invasive biomarker with clinical applicability for the screening and surveillance of patients with multiple types of solid tumours.

Surface-Functionalized Nanoparticles as Efficient Tools in Targeted Therapy of Pregnancy Complications.

Minimizing exposure of the fetus to medication and reducing adverse off-target effects in the mother are the primary challenges in developing novel drugs to treat pregnancy complications. Nanomedicine has introduced opportunities for the development of novel platforms enabling targeted delivery of drugs in pregnancy. This review sets out to discuss the advances and potential of surface-functionalized nanoparticles in the targeted therapy of pregnancy complications. We first describe the human placental anatomy, which is fundamental for developing placenta-targeted therapy, and then we review current knowledge of nanoparticle transplacental transport mechanisms. Meanwhile, recent surface-functionalized nanoparticles for targeting the uterus and placenta are examined. Indeed, surface-functionalized nanoparticles could help prevent transplacental passage and promote placental-specific drug delivery, thereby enhancing efficacy and improving safety. We have achieved promising results in targeting the placenta via placental chondroitin sulfate A (plCSA), which is exclusively expressed in the placenta, using plCSA binding peptide (plCSA-BP)-decorated nanoparticles. Others have also focused on using placenta- and uterus-enriched molecules as targets to deliver therapeutics via surface-functionalized nanoparticles. Additionally, we propose that placenta-specific exosomes and surface-modified exosomes might be potential tools in the targeted therapy of pregnancy complications. Altogether, surface-functionalized nanoparticles have great potential value as clinical tools in the targeted therapy of pregnancy complications.

Targeted Delivery Prodigiosin to Choriocarcinoma by Peptide-Guided Dendrigraft Poly-l-lysines Nanoparticles.

The available and effective therapeutic means to treat choriocarcinoma is seriously lacking, mainly due to the toxic effects caused by chemotherapy and radiotherapy. Accordingly, we developed a method for targeting delivery of chemotherapeutical drugs only to cancer cells, not normal cells, in vivo, by using a synthetic placental chondroitin sulfate (CSA)-binding peptide (plCSA-BP) derived from malarial protein VAR2CSA. A 28 amino acids placental CSA-binding peptide (plCSA-BP) from the VAR2CSA was synthesized as a guiding peptide for tumor-targeting delivery, dendrigraft poly-L-lysines (DGL) was modified with plCSA-BP and served as a novel targeted delivery carrier. Choriocarcinoma was selected to test the effect of targeted delivery carrier, and prodigiosin isolated from Serratia marcescens subsp. lawsoniana was selected as a chemotherapeutical drug and encapsulated in the DGL modified by the plCSA-BP nanoparticles (DGL/CSA-PNPs). DGL/CSA-PNPs had a sustained slow-release feature at pH 7.4, which could specifically bind to the JEG3 cells and exhibited better anticancer activity than that of the controls. The DGL/CSA-PNPs induced the apoptosis of JEG3 cells through caspase-3 and the P53 signaling pathway. DGL/CSA-PNPs can be used as an excellent targeted delivery carrier for anticancer drugs, and the prodigiosin could be an alternative chemotherapeutical drug for choriocarcinoma.

Interaction Effects between Doxorubicin and Hernandezine on the Pharmacokinetics by Liquid Chromatography Coupled with Mass Spectrometry.

Doxorubicin (DOX) is an effective anti-tumor drug widely used in clinics. Hernandezine (HER), isolated from a Chinese medicinal herb, has a selective inhibitory effect on DOX multidrug resistance, making DOX more effective in treating cancer. The aim of this study was to investigate the effect of the interaction of HER and DOX on pharmacokinetics. Male Sparague-Dawley rats were randomly divided into three groups: a single DOX group, a single HER group, and a combination group. Plasma concentrations of DOX and HER were determined by the LC-MS/MS method at specified time points after administration, and the main pharmacokinetic parameters were estimated. The results showed that there were significant differences in the Cmax and AUC0-∞ of DOX in the single drug group and combined drug group, indicating that HER could improve the absorption of DOX. However, DOX in combination, in turn, reduced the free drug concentration of HER, possibly because DOX enhanced the HER drug-protein binding effect. The results could be used as clinical guidance for DOX and HER to avoid adverse reactions.

Clinical and biological significance of a - 73A > C variation in the CDH1 promoter of patients with sporadic gastric carcinoma.

BACKGROUND: CDH1 germline mutations lead to hereditary diffuse gastric carcinomas. However, it is unclear whether genetic variations in the CDH1 promoter affect the progression of sporadic gastric carcinomas (SGCs). METHODS: SGC patients in two independent cohorts with follow-up data were enrolled. The CDH1 genotypes, including the - 73A > C polymorphism (rs28372783), were determined by PCR sequencing. The CDH1 promoter activity was determined using reporter assays. SNAIL bound to CDH1 alleles was determined by chromatin immunoprecipitation primer extension PCR. CDH1 DNA methylation was determined by bisulfite-based PCR analyses. RESULTS: Kaplan-Meier analyses showed that the overall survival (OS) of the - 73C/C patients was significantly longer than that of the - 73A/C or - 73A/A patients in a Chinese cohort [n = 526; hazard ratio 0.68 (95% CI 0.47-1.00)], which was validated in an independent Korea cohort [n = 215; hazard ratio 0.49 (95% CI 0.26-0.94)]. Moreover, the transcription activity of the - 73C alleles was significantly higher than that of the - 73A alleles in vitro and in vivo. The ratio of SNAIL recruited to the promoter regions of the - 73C and - 73A alleles was 1:10, indicating a strong influence of this polymorphism on the recruitment of SNAIL to the flanking E-box. The prevalence of DNA methylation of the CpG island and shore within the promoter of the - 73C allele was much less than that of the - 73A allele in both gastric tissues and cancer cell lines. CONCLUSION: The - 73A > C variation may lead to differences in the overall survival of SGC patients and allele-specific repressions of CDH1.

Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation.

OBJECTIVE: To investigate the relationship between the transcription of ANRIL, P15, P14 and P16 at the same locus and the regulation mechanism of ANRIL. METHODS: Publicly available database of Cancer Cell Line Encyclopedia (CCLE) was used in bioinformatic analyses. Methylation of CpG islands was detected by denaturing high performance liquid chromatography (DHPLC). Gene transcript levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR) assays. An engineered P16-specific transcription factor and DNA methyltransferase were used to induce P16-specific DNA demethylation and methylation. RESULTS: The expression level of ANRIL was positively and significantly correlated with that of P16 but not with that of P15 in the CCLE database. This was confirmed in human cell lines and patient colon tissue samples. In addition, ANRIL was significantly upregulated in colon cancer tissues. Transcription of ANRIL and P16 was observed only in cell lines in which the P16 alleles were unmethylated and not in cell lines with fully methylated P16 alleles. Notably, P16-specific methylation significantly decreased transcription of P16 and ANRIL in BGC823 and GES1 cells. In contrast, P16-specific demethylation re-activated transcription of ANRIL and P16 in H1299 cells (P<0.001). Alteration ofANRIL expression was not induced by P16 expression changes. CONCLUSIONS: ANRIL and P16 are coordinately transcribed in human cells and regulated by the methylation status of the P16 CpG islands around the transcription start site.

Critical evaluation of Cbx7 downregulation in primary colon carcinomas and its clinical significance in Chinese patients.

BACKGROUND: CBX7 is a Polycomb group protein that shows variable expression changes in various cancers that are often contradictive. A mouse knockout experiment has validated the tumor suppressor role in carcinogenesis. The purpose of this study is to verify the tumor suppressor role of Cbx7 in human colon carcinomas (CC). METHODS: Frozen CC and the surgical margin (SM) tissue samples from patients (n = 97) were obtained from the Peking University Cancer Hospital. All patients had follow-up data for at least three years. The level of Cbx7 mRNA and protein was determined by quantitative RT-PCR, immunohistochemistry and Western blot, respectively. The association between Cbx7 mRNA level and clinicopathological characteristics of CC patients was then statistically analyzed. RESULTS: CBX7 expression changes detected through immunohistochemistry and Western blot in 10 pairs of representative CC samples significantly correlated with their corresponding mRNA levels when Alu, but not GAPDH, was used as the endogenous reference control in quantitative RT-PCR. The Alu-normalized Cbx7 mRNA levels were significantly increased in SM tissues when compared with CC tissues or colon biopsies taken from non-cancer patients (Student's t-test, P < 0.036 or 0.007). Furthermore, decreased levels of Cbx7 mRNA positively correlated with lymph metastasis (P = 0.029). Overall survival (OS) of CC patients classified as Cbx7 expression-low was considerably shorter than those classified as Cbx7 expression-high (Hazard ratio = 2.97, 95% CI [1.68 ~ 5.25]; P <0.001). Multiple variant analyses showed that the Cbx7 expression-low was an independent predictor of short OS (Hazard ratio = 3.16, 95% CI [1.58-6.30]; P < 0.001). CONCLUSION: Cbx7 is downregulated in CCs, and Cbx7 expression-low tumors correlated with lymph metastasis and poor overall survival of CC patients.

Liraglutide reduces the body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients.

AIM: To investigate the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) receptor activator, on body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. METHODS: A total of 328 Chinese overweight and obese type 2 diabetic patients were included in this multi-center, open-labeled and self-controlled clinical study. The patients were subcutaneously injected with liraglutide once daily for 24 weeks as add-on therapy to their previous hypoglycemic treatments. Statistical analyses were performed using SPSS software package version 11.5 for Windows. RESULTS: Liraglutide treatment caused significant reduction of the mean body weight (from 86.61±14.09 to 79.10±13.55 kg) and waist circumference (from 101.81±13.96 to 94.29±14.17 cm), resulting in body weight lose of 5%-10% in 43.67% patients, and body weight loss above 10% in 34.06% patients, who had significant lower plasma creatinine levels. Baseline waist circumference, BMI and HOMA-IR were independently correlated with the body weight loss. Furthermore, liraglutide treatment significantly decreased HbA1c levels (from 8.66%±2.17% to 6.92%±0.95%) with HbA1c<7.0% in 35.37% patients, who had a significantly lower baseline level of HbA1c, but higher baseline levels of C peptide and glucagon. Moreover, liraglutide treatment resulted in greater body weight loss in patients with a long duration of diabetes, and better glycemic control in patients with a short duration of diabetes. CONCLUSION: Liraglutide significantly reduces body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. Patients with apparent visceral obesity, insulin resistance and a long duration of diabetes may have greater body weight loss; whereas patients with high insulin-secreting ability, hyperglucagonemia, and short-duration diabetes may obtain better glycemic control with liraglutide.

Kaiso mainly locates in the nucleus in vivo and binds to methylated, but not hydroxymethylated DNA.

OBJECTIVE: Kaiso is upregulated in many cancers and proposed to bind with both methylated- and unmethylated-DNA in the nucleus as a transcriptional repressor. The objective is to define its subcellular localization in vivo and exact binding DNA sequences in cells. METHODS: Compartmentalization of exogenous Kaiso in cells was tracked with enhanced green fluorescence protein (EGFP) tag. The endogenous Kaiso expression in gastric carcinoma tissue was examined with immunohistochemical staining. Kaiso-DNA binding was tested using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). RESULTS: Kaiso mainly localized in the nucleus of cancer and stromal cells in vivo, but remained in the cytoplasm of cultured cells. Most importantly, nuclear Kaiso can bind with the methylated-CGCG-containing sequence in the CDKN2A promoter, but not with the hydroxymethylated-CGCG sequence in HCT116 cells. CONCLUSIONS: Kaiso locates mainly in the nucleus in vivo where it binds with the methylated-CGCG sequences, but does not bind with the hydroxymethylated-CGCG sequences.

Trophoblast-Targeted Liposomes for Placenta-Specific Drug Delivery.

A major challenge in developing potential treatments for pregnancy complications is minimizing adverse effects to the fetus and mother. Placenta-targeted drug delivery could reduce the risks of drug treatments in pregnancy by targeting tissue where most pregnancy complications originate and decreasing dosages. We previously developed a tool for the targeted delivery of drug-carrying nanoparticles to the placenta using a synthetic placental chondroitin sulfate A-binding peptide (plCSA-BP) derived from the malarial protein VAR2CSA, which binds a distinct type of chondroitin sulfate A (CSA) exclusively expressed by placental trophoblasts. Liposomes are a type of nanoparticle already approved for use in humans by the Food and Drug Administration (FDA) and used successfully for the treatment of a wide range of diseases. Here, we present a detailed method to create plCSA-BP-decorated liposomes that can be used to deliver drugs specifically to placental trophoblasts. Liposomes are first generated by the standard film method and then conjugated to plCSA-BPs using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS) bioconjugate technique. This protocol may facilitate bench-to-bedside translation of drug discovery for the treatment of pregnancy disorders by reducing risks of side effects, and enabling rapid and scalable production.

T cell cascade regulation initiates systemic antitumor immunity through living drug factory of anti-PD-1/IL-12 engineered probiotics.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but only works in a subset of patients due to the insufficient infiltration, persistent exhaustion, and inactivation of T cells within a tumor. Herein, we develop an engineered probiotic (interleukin [IL]-12 nanoparticle Escherichia coli Nissle 1917 [INP-EcN]) acting as a living drug factory to biosynthesize anti-PD-1 and release IL-12 for initiating systemic antitumor immunity through T cell cascade regulation. Mechanistically, INP-EcN not only continuously biosynthesizes anti-PD-1 for relieving immunosuppression but also effectively cascade promote T cell activation, proliferation, and infiltration via responsive release of IL-12, thus reaching a sufficient activation threshold to ICB. Tumor targeting and colonization of INP-EcNs dramatically increase local drug accumulations, significantly inhibiting tumor growth and metastasis compared to commercial inhibitors. Furthermore, immune profiling reveals that anti-PD-1/IL-12 efficiently cascade promote antitumor effects in a CD8+ T cell-dependent manner, clarifying the immune interaction of ICB and cytokine activation. Ultimately, such engineered probiotics achieve a potential paradigm shift from T cell exhaustion to activation and show considerable promise for antitumor bio-immunotherapy.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION: P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

BACKGROUND: It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions. RESULTS: In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability. CONCLUSIONS: Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

Biocompatible smart micro/nanorobots for active gastrointestinal tract drug delivery.

INTRODUCTION: Oral delivery is the most commonly used route of drug administration owing to good patient compliance. However, the gastrointestinal (GI) tract contains multiple physiological barriers that limit the absorption efficiency of conventional passive delivery systems resulting in a low drug concentration reaching the diseased sites. Micro/nanorobots can convert energy to self-propulsive force, providing a novel platform to actively overcome GI tract barriers for noninvasive drug delivery and treatment. AREAS COVERED: In this review, we first describe the microenvironments and barriers in the different compartments of the GI tract. Afterward, the applications of micro/nanorobots to overcome GI tract barriers for active drug delivery are highlighted and discussed. Finally, we summarize and discuss the challenges and future prospects of micro/nanorobots for further clinical applications. EXPERT OPINION: Micro/nanorobots with the ability to autonomously propel themselves and to load, transport, and release payloads on demand are ideal carriers for active oral drug delivery. Although there are many challenges to be addressed, micro/nanorobots have great potential to introduce a new era of drug delivery for precision therapy.