Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer.

Although only a fraction of CTCF motifs are bound in any cell type, and few occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique binding properties of each protein, but also on the genomic context of bound sites and enrichment of motifs for expressed TFs abutting these sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.

MethNet: a robust approach to identify regulatory hubs and their distal targets in cancer.

Aberrations in the capacity of DNA/chromatin modifiers and transcription factors to bind non-coding regions can lead to changes in gene regulation and impact disease phenotypes. However, identifying distal regulatory elements and connecting them with their target genes remains challenging. Here, we present MethNet, a pipeline that integrates large-scale DNA methylation and gene expression data across multiple cancers, to uncover novel cis regulatory elements (CREs) in a 1Mb region around every promoter in the genome. MethNet identifies clusters of highly ranked CREs, referred to as 'hubs', which contribute to the regulation of multiple genes and significantly affect patient survival. Promoter-capture Hi-C confirmed that highly ranked associations involve physical interactions between CREs and their gene targets, and CRISPRi based scRNA Perturb-seq validated the functional impact of CREs. Thus, MethNet-identified CREs represent a valuable resource for unraveling complex mechanisms underlying gene expression, and for prioritizing the verification of predicted non-coding disease hotspots.

High-throughput probing macrophage-bacteria interactions at the single cell level with microdroplets.

Pathogenic infections may lead to disruption of homeostasis, thus becoming a serious threat to the human health. Understanding the interactions between bacteria and macrophages is critical for therapeutic development against sepsis or inflammatory bowel disease. Here, we report a technique using droplet biosensors for the detection of nitric oxide (NO) secreted by a single macrophage under inflammatory stimuli. We demonstrated that the limit of detection can be promoted more than two orders of magnitude by our approach, in comparison to the conventional microplate format. The experiments of co-encapsulating single macrophages and different numbers of Escherichia coli (E. coli) enabled fluorescence monitoring of NO secretion by single macrophages over the incubation, and investigation of their interactions inside the isolated droplet for their separate fates. Our approach provides a unique platform to study the bacteria-macrophage interactions at the single cell level.

Folic acid-modified fluorescent dye-protein nanoparticles for the targeted tumor cell imaging.

Serum albumin has a wide range of applications in biochemical experiments and pharmaceutical field. We found that a cyanine dye, dimethylindole red (Dir), could selectively interact with bovine serum albumin (BSA). Dir exhibited very weak red fluorescence, while the fluorescence intensity at 630 nm was enhanced up to 130-fold upon noncovalently interacting with 30 µM BSA. Besides, Dir showed a highly selective response to BSA over human serum albumin (HSA). For the detection of BSA, a limit of detection as low as 23 nM was obtained. Then biocompatible Dir-BSA nanoparticles were prepared by the desolvation technique. The Dir-BSA nanoparticles possess excellent fluorescence properties with a quantum yield of 32%. Furthermore, folic acid as a targeting group was conjugated to Dir-BSA nanoparticles and these nanoparticles were characterized by TEM and laser particle analyzer, etc. Folic acid-modified Dir-BSA nanoparticles were successfully used for tumor cell-targeted imaging.

The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery.

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

Effects of electroacupuncture on the cortical extracellular signal regulated kinase pathway in rats with cerebral ischaemia/reperfusion.

OBJECTIVE: To explore the effects of electroacupuncture (EA) on the phosphorylated extracellular signal regulated kinase (p-ERK) pathway of the cerebral cortex in a rat model of focal cerebral ischaemia/reperfusion (I/R). METHODS: 160 adult Sprague-Dawley rats underwent middle carotid artery occlusion (MCAO) to establish I/R injury and were randomly divided into four groups (n=40 each) that remained untreated (I/R group) or received EA at LU5, LI4, ST36 and SP6 (I/R+EA group), the ERK inhibitor PD98059 (I/R+PD group), or both interventions (I/R+PD+EA groups). An additional 40 rats undergoing sham surgery formed a healthy control group. Eight rats from each group were sacrificed at the following time points: 2 hours, 6 hours, 1 day, 3 days and 1 week. Neurological function was assessed using neurological deficit scores, morphological examination was performed following haematoxylin-eosin staining of cortical tissues, and apoptotic indices were calculated after terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling. Cortical protein and mRNA expression of p-ERK and ERK were measured by immunohistochemistry and real-time quantitative PCR, respectively. RESULTS: Compared with the I/R group, neurological deficit scores and apoptotic indices were lower in the I/R+EA group at 1 and 3 days, whereas mRNA/protein expression of ERK/p-ERK was higher in the EA group at all time points studied. CONCLUSION: Our results suggest that EA can alleviate neurological deficits and reduce cortical apoptosis in rats with I/R injury. These anti-apoptotic effects may be due to upregulation of p-ERK. Moreover, apoptosis appeared to peak at 1 day after I/R injury, which might therefore represent the optimal time point for targeting of EA.

DNA sequence-dependent fluorescence of doxorubicin for turn-on detection of biothiols in human serum.

Doxorubicin (Dox) is a DNA-targeting anthracycline antibiotic active against a wide spectrum of cancers. The interaction between Dox and double-stranded DNA (dsDNA) was used to load Dox using DNA duplexes as carriers. More importantly, the interesting DNA sequence-dependent fluorescence response of Dox could be exploited in the design of efficient Dox release systems and efficient fluorescence sensors. In this work, we demonstrated that separate introduction of G and C bases into T-rich single-stranded DNA (ssDNA) sequences afforded the best discrimination of Dox binding between dsDNA and ssDNA. For the first time, we successfully utilized this interesting DNA sequence-dependent fluorescence response of Dox as a signal transduction mechanism for the sensitive detection of biothiols in human serum. Cysteine, homocysteine, and glutathione were detected at as low as 26 nM, 37 nM, and 29 nM, respectively. The biosensors exhibited not only good selectivity, stability, and sensitivity in aqueous solutions but also a sensitive response in human serum, demonstrating their potential for diagnosis.

RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence.

Rose (Rosa hybrida) is one of the most important ornamental plants worldwide; however, senescence of its petals terminates the ornamental value of the flower, resulting in major economic loss. It is known that the hormones abscisic acid (ABA) and ethylene promote petal senescence, while gibberellins (GAs) delay the process. However, the molecular mechanisms underlying the antagonistic effects amongst plant hormones during petal senescence are still unclear. Here we isolated RhHB1, a homeodomain-leucine zipper I transcription factor gene, from rose flowers. Quantitative RT-PCR and GUS reporter analyses showed that RhHB1 was strongly expressed in senescing petals, and its expression was induced by ABA or ethylene in petals. ABA or ethylene treatment clearly accelerated rose petal senescence, while application of the gibberellin GA3 delayed the process. However, silencing of RhHB1 delayed the ABA- or ethylene-mediated senescence, and resulted in higher petal anthocyanin levels and lower expression of RhSAG12. Moreover, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. In addition, silencing of RhHB1 blocked the ABA- or ethylene-induced reduction in expression of the GA20 oxidase encoded by RhGA20ox1, a gene in the GA biosynthetic pathway. Furthermore, RhHB1 directly binds to the RhGA20ox1 promoter, and silencing of RhGA20ox1 promoted petal senescence. Eight senescence-related genes showed substantial differences in expression in petals after treatment with GA3 or paclobutrazol. These results suggest that RhHB1 mediates the antagonistic effect of GAs on ABA and ethylene during rose petal senescence, and that the promotion of petal senescence by ABA or ethylene operates through an RhHB1-RhGA20ox1 regulatory checkpoint.

[Association of cytokine gene polymorphisms with cachexia related to sporadic female breast cancer].

OBJECTIVE: To investigate the relation of the frequency of tumor necrosis factor (TNF)-alpha - 308, TNF-beta + 252, IL-1 beta + 3954, and IL-10 - 1082 gene polymorphisms to female breast cancer. METHODS: Peripheral blood samples were collected from 102 breast cancer patients with cachexia and 120 breast cancer patients without cachexia. Biallelic polymorphisms were performed by analyzing the incision enzyme-digested DNA fragment obtained using PCR. RESULTS: The allele frequencies of TNF-beta + 252, IL-1 beta + 3954, and IL-10 -1082 in the patients with cachexia were comparable with those of the patients without cachexia (all P > 0.05). The patients with cachexia showed a significantly higher prevalence of TNF2 than the patients without cachexia (20.6 % vs 10.0 %, P = 0.027). Logistic regression analysis indicated TNF2 as a risk factor for cachexia in breast cancer ( OR = 2.333, 95% CI: 1.085-5.017). CONCLUSION: TNF2 plays an important role in the susceptibility of cachexia in breast cancer.