Reconstructing Signaling Networks Using Biosensor Barcoding.

Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.

STAT1-Deficient HPV E6/E7-Associated Cancers Maintain Host Immunocompetency against Therapeutic Intervention.

Human papillomavirus (HPV) remains a global health concern because it contributes to the initiation of various HPV-associated cancers such as anal, cervical, oropharyngeal, penile, vaginal, and vulvar cancer. In HPV-associated cancers, oncogenesis begins with an HPV infection, which is linked to the activation of the Janus protein tyrosine kinase (JAK)/STAT signaling pathway. Various STAT signaling pathways, such as STAT3 activation, have been well documented for their tumorigenic role, yet the role of STAT1 in tumor formation remains unclear. In the current study, STAT1-/- mice were used to investigate the role of STAT1 in the tumorigenesis of a spontaneous HPV E6/E7-expressing oral tumor model. Subsequently, our candidate HPV DNA vaccine CRT/E7 was administered to determine whether the STAT1-/- host preserves a therapeutic-responsive tumor microenvironment. The results indicated that STAT1-/- induces robust tumorigenesis, yet a controlled tumor response was attained upon CRT/E7 vaccination. Characterizing this treatment effect, immunological analysis found a higher percentage of circulating CD4+ and CD8+ T cells and tumor-specific cytotoxic T cells. In addition, a reduction in exhaustive lymphocyte activity was observed. Further analysis of a whole-cell tumor challenge affirmed these findings, as spontaneous tumor growth was more rapid in STAT1-/- mice. In conclusion, STAT1 deletion accelerates tumorigenesis, but STAT1-/- mice maintains immunocompetency in CRT/E7 treatments.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Salmonella immunotherapy engineered with highly efficient tumor antigen coating establishes antigen-specific CD8+ T cell immunity and increases in antitumor efficacy with type I interferon combination therapy.

Bacteria-based cancer therapy employs various strategies to combat tumors, one of which is delivering tumor-associated antigen (TAA) to generate specific immunity. Here, we utilized a poly-arginine extended HPV E7 antigen (9RE7) for attachment on Salmonella SL7207 outer membrane to synthesize the bacterial vaccine Salmonella-9RE7 (Sal-9RE7), which yielded a significant improvement in the amount of antigen presentation compared to the previous lysine-extended antigen coating strategy. In TC-1 tumor mouse models, Sal-9RE7 monotherapy decreased tumor growth by inducing E7 antigen-specific immunity. In addition, pairing Sal-9RE7 with adjuvant Albumin-IFNß (Alb-IFNß), a protein cytokine fusion, the combination significantly increased the antitumor efficacy and enhanced immunogenicity in the tumor microenvironment (TME). Our study made a significant contribution to personalized bacterial immunotherapy via TAA delivery and demonstrated the advantage of combination therapy.

Targeting Ras signaling excitability in cancer cells through combined inhibition of FAK and PI3K.

The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.

Localization of Salmonella and albumin-IL-2 to the tumor microenvironment augments anticancer T cell immunity.

BACKGROUND: For centuries, microbial-based agents have been investigated as a therapeutic modality for the treatment of cancer. In theory, these methods would be cheap to produce, broadly applicable in a wide array of cancer types, and could synergize with other cancer treatment strategies. We aimed to assess the efficacy of combining microbial-based therapy using Salmonella SL7207 with interleukin-2 (IL-2), a potent immunostimulatory agent, in the treatment of murine colon carcinoma. METHODS: Female BALB/c mice were implanted subcutaneously with CT26 tumors, a model of colon carcinoma. Mice bearing tumors were selected and administered Albumin-IL-2 (Alb-IL2), a fusion protein, for further analysis of anticancer effect. RESULTS: We demonstrated that Salmonella SL7207, a genetically modified strain of Salmonella enterica serovar Typhimurium, preferentially accumulates in the tumor microenvironment, potentiating it to stimulate localized innate immunity. We delivered IL-2 as a fusion protein, Alb-IL2, which we demonstrate to have preferential accumulation properties, bringing it to the tumor and secondary lymphoid organs. Treatment of tumor-bearing mice with Salmonella + Alb-IL2 leads to superior tumor control and enhanced overall survival compared to controls. When assessing immunological factors contributing to our observed tumor control, significantly enhanced T cell population with superior effector function was observed in mice treated with Salmonella + Alb-IL2. We confirmed that these T cells were indispensable to the observed tumor control through antibody-mediated T cell depletion experiments. CONCLUSIONS: These findings highlight the ability of Salmonella + Alb-IL2 to serve as a novel therapeutic approach to induce T cell-mediated antitumor immunity and exert long-term tumor control in a murine model of cancer.

An RNA-RNA crosstalk network involving HMGB1 and RICTOR facilitates hepatocellular carcinoma tumorigenesis by promoting glutamine metabolism and impedes immunotherapy by PD-L1+ exosomes activity.

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA-RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA-RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3'untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

MiR-552-3p promotes malignant progression of gallbladder carcinoma by reactivating the Akt/ß-catenin signaling pathway due to inhibition of the tumor suppressor gene RGMA.

BACKGROUND: Gallbladder carcinoma (GBC) remains a highly lethal disease worldwide. MiR-552 family members promote the malignant progression of a variety of digestive system tumors, but the role of miR-552-3p in GBC has not been elucidated. miR-552-3p was predicted to target the 3'-untranslated region (3'UTR) of the mRNA for the tumor suppressor gene "repulsive guidance molecule BMP co-receptor a" (RGMA). The aim of the present study was to clarify the roles and mechanisms of miR-552-3p targeting RGMA in the malignant progression of GBC. METHODS: In vitro: expression of miR-552-3p was detected by real-time quantitative PCR (qRT-PCR) in tumor and non-tumor adjacent tissues (NATs). Lentivirus-miR-552-3p was employed to knockdown this miRNA in GBC cell lines. Stem cell-related transcription factors and markers were assessed by qRT-PCR. Cell Counting Kit-8 (CCK-8), sphere formation and transwell assays were used to determine the malignant phenotypes of GBC cells. Targeting the 3'UTR of RGMA by miR-552-3p was verified by integrated analysis including bioinformatics prediction, luciferase assays, measures of changes of gene expression and rescue experiments. In vivo: mouse models of subcutaneous tumors and lung metastases were established to observe the effect of miR-552-3p on tumorigenesis and organ metastasis, respectively. RESULTS: MiR-552-3p was abnormally highly expressed in GBC tissues and cancer stem cells. Interference with miR-552-3p in SGC-996 and GBC-SD cells significantly inhibited GBC stem cell expansion. Reciprocally, miR-552-3p promoted GBC cell proliferation, migration and invasion both in vitro and in vivo; hence, interference with this miRNA impeded the malignant progression of GBC. Furthermore, the important tumor suppressor gene RGMA was identified as a target of miR-552-3p. The effects of miR-552-3p on cell proliferation and metastasis were abrogated or enhanced by gain or loss of RGMA function, respectively. Mechanistically, miR-552-3p promoted GBC progression by reactivating the Akt/ß-catenin pathway and epithelial-mesenchymal transformation (EMT). Clinically, miR-552-3p correlated with multi-malignant characteristics of GBC and acted as a prognostic marker for GBC outcome. CONCLUSIONS: MiR-552-3p promotes the malignant progression of GBC by inhibiting the mRNA of the tumor suppressor gene RGMA, resulting in reactivation of the Akt/ß-catenin signaling pathway.

Targeted next-generation sequencing identifies a novel frameshift EYA1 variant causing branchio-otic syndrome in a Chinese family.

OBJECTIVE: To evaluate the genotype-phenotype correlation of branchio-otic syndrome (BOS) in a Chinese family. METHODS: The proband in this study was an 18-month-old boy with hearing loss, preauricular pit, and branchial fistula without a renal anomaly. We collected blood samples from 6 family members, including 4 who were affected by the syndrome. Targeted next-generation sequencing and Sanger sequencing were performed to identify pathogenic mutations in this family. RESULTS: Pedigree analysis indicated that the mode of inheritance in the family was consistent with the autosomal dominant pattern. Hearing loss was the most common manifestation, occurring in 4 patients. Other findings included preauricular pits (n=2), cervical fistulas (n=3) and abnormal pinnae (n=4). None of the patients had renal anomalies. Evaluation by pure-tone audiometry and temporal bone imaging demonstrated bilateral mixed hearing loss, as well as middle ear and inner ear deformities, in two patients. Mutational analysis of candidate genes in the selected patients led to the identification of a novel frameshift variant NM_000503.4: c.1075_1077delinsAT (p.Gly359Ilefs*7) in the EYA1 gene. CONCLUSIONS: The EYA1 c.1075_1077delinsAT mutation is the causative variant in the Chinese family with BOS, although the penetrance is variable within patients.

Tough synthetic spider-silk fibers obtained by titanium dioxide incorporation and formaldehyde cross-linking in a simple wet-spinning process.

Due to its unique mechanical properties, spider silk shows great promise as a strong super-thin fiber in many fields. Although progress has been made in the field of synthesizing spider-silk fiber from recombinant spidroin (spider silk protein) in the last few decades, methods to obtain synthetic spider-silk fibers as tough as natural silk from small-sized recombinant protein with a simple spinning process have eluded scientists. In this paper, a recombinant spidroin (MW: 93.4 kDa) was used to spin tough synthetic spider-silk fibers with a simple wet-spinning process. Titanium oxide incorporation and formaldehyde cross-linking were used to improve the mechanical properties of synthetic spider-silk fibers. Fibers treated with incorporation or/and cross-linking varied in microstructure, strength and extensibility while all exhibited enhanced strength and toughness. In particular, one fiber possessed a toughness of 249 ± 22 MJ/m3. This paper presents a new method to successfully spin tough spider-silk fibers in a simple way.

MRI features and differential diagnoses of congenital vaginal atresia.

Objective: To investigate the MRI manifestations of congenital vaginal atresia, analyze its imaging features, and improve the understanding of the disease. Methods: MRI findings and clinical data of 12 patients with congenital vaginal atresia confirmed by hysteroscopy and laparoscopic surgery were retrospectively analyzed. Vaginal atresia was classified according to vaginal dysplasia in AFS female genital malformation classification system. Results: In this study, 12 cases of congenital vaginal atresia were diagnosed by combined preoperative MRI with operative diagnosis. Among them, 10 patients all had type-I congenital vaginal atresia, and their uterus and cervix were normal (1 patient had ectopic renal malformation combined with left ovarian endometriosis cyst and 1 patient with uterine empyema). The other two cases were diagnosed congenital vaginal atresia type II (1 case merged with residual uterus, 1 case with cervical dysplasia). MRI mainly manifested as dilatation and hemorrhage in the uterine cavity, cervical canal and vaginal upper segment. T1WI showed high signal, T2WI showed slightly lower and slightly higher signal. The dilated vagina was above the perineal level. Conclusion: MRI features of congenital vaginal atresia have certain characteristics. MRI cannot only accurately assess the type of vaginal dysplasia and its associated complications, but also make objective evaluation and diagnosis, so it can be used as the best effective preoperative image evaluation.

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels.

AIM: To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K(ATP)) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs). METHODS: Whole-cell KATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips. RESULTS: Application of iptakalim (10 and 100 µmol/L) significantly increased the whole-cell K(ATP) currents, which were prevented by the specific K(ATP) blocker glibenclamide (1.0 µmol/L). The opening of K(ATP) channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K(ATP) channels when the intracellular ATP or NDPs were at 100 or 1000 µmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 µmol/L) was infused into the cells. In contrast, the K(ATP) opener pinacidil activated K(ATP) channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 µmol/L, and even ATPγS (1000 µmol/L) was infused into the cells. CONCLUSION: Iptakalim activates K(ATP) channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.

Comparison of complications of the suprameatal approach and mastoidectomy with posterior tympanotomy approach in cochlear implantation: a meta-analysis.

BACKGROUND: Cochlear implantation (CI) is a popular procedure to preserve hearing in patients with severe-to-profound hearing loss. Evidence shows that the suprameatal approach (SMA) may help reducing the risk of the incidence of complications and shortening the surgery time, but there is still dispute. OBJECTIVES: The aim of this study was to compare the incidence of complications of SMA and the mastoidectomy with posterior tympanotomy approach (MPTA), and to find whether SMA yields better outcomes than MPTA. METHODS: We searched PubMed, the Cochrane Library, the Web of Science and Chinese Biomedical Literature databases, Chinese National Knowledge Infrastructure, the Chinese Science and Technology Journal Full-Text database, and Wangfang database. The latest data was accessed in March 2013. Review Manager 5.1 software was used for comprehensive quantification data analysis. RESULTS: Three studies were included in the meta-analysis, composed of 799 participants and reporting major and minor complications. The meta-analysis indicated no statistically significant difference in major and minor complications between the two approaches, except for facial nerve and chorda tympani injuries (OR = 0.13; 95% CI: 0.02, 0.67; p = 0.02; I(2) = 0%). CONCLUSIONS: Current evidence suggests that SMA may be clearly a good alternative to the classical surgery technique for CI in terms of reducing the incidence of facial nerve injury and chorda tympani sacrifice.