Hexokinase 3 dysfunction promotes tumorigenesis and immune escape by upregulating monocyte/macrophage infiltration into the clear cell renal cell carcinoma microenvironment.

Purpose: This study aimed to identify the potential prognostic role of HK3 and provide clues about glycolysis and the microenvironmental characteristics of ccRCC. Methods: Based on the Cancer Genome Atlas (TCGA, n = 533) and Gene expression omnibus (GEO) (n = 127) databases, real-world (n = 377) ccRCC cohorts, and approximately 15,000 cancer samples, the prognostic value and immune implications of HK3 were identified. The functional effects of HK3 in ccRCC were analyzed in silico and in vitro. Results: The large-scale findings suggested a significantly higher HK3 expression in ccRCC tissues and the predictive efficacy of HK3 for tumor progression and a poor prognosis. Next, the subgroup survival and Cox regression analyses showed that HK3 serves as a promising and independent predictive marker for the prognosis and survival of patients with ccRCC from bioinformatic databases and real-world cohorts. Subsequently, we found that HK3 could be used to modulate glycolysis and the malignant behaviors of ccRCC cells. The comprehensive results suggested that HK3 is highly correlated with the abundance of immune cells, and specifically stimulates the infiltration of monocytes/macrophages presenting surface markers, regulates the immune checkpoint molecules PD-1 and CTLA-4 of exhaustive T cells, restrains the immune escape of tumor cells, and prompts the immune-rejection microenvironment of ccRCC. Conclusion: In conclusion, the large-scale data first revealed that HK3 could affect glycolysis, promote malignant biologic processes, and predict the aggressive progression of ccRCC. HK3 may stimulate the abundance of infiltrating monocytes/macrophages presenting surface markers and regulate the key molecular subgroups of immune checkpoint molecules of exhaustive T cells, thus inducing the microenvironmental characteristics of active anti-tumor immune responses.

Integrated Omics Reveals Tollip as an Regulator and Therapeutic Target for Hepatic Ischemia-Reperfusion Injury in Mice.

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/µL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

CRISPR disruption of TCTP gene impaired normal development in the silkworm Bombyx mori.

The translationally controlled tumor protein (TCTP) is a highly conserved and multifunctional protein with activities ranging from cytoskeletal regulation to transcription regulation in numerous organisms. In insects, TCTP is essential for cell growth and proliferation. Recently, TCTP has been reported to affect the innate intestinal immune pathway in the Bombyx mori silkworm, a lepidopteran model insect. However, the comprehensive physiological roles of TCTP in the silkworm remain poorly understood. Here, we performed functional analysis of BmTCTP by using a binary transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/RNA-guided CRISPER-associated protein 9 nucleases) system. Disruption of BmTCTP led to developmental arrestment and subsequent lethality in third instar larvae. Histological analysis revealed that growth impairment originated from decreased cell size, and the proliferation and differentiation of intestinal epithelial cells were also affected. RNA-seq analysis revealed that genes involved in carbohydrate metabolism, lipid metabolism and digestive system pathways were significantly affected by BmTCTP depletion. Together, the results demonstrated that BmTCTP plays a key role in controlling larval growth and development.

[Variations of the amount of sialic acids on hepatocellular carcinoma cell membrane].

OBJECTIVE: To observe the change in the amount of sialic acids on hepatocellular carcinoma (HCC) cell membrane. METHODS: Surgical specimens of HCC and liver cirrhosis tissues were obtained from 28 patients to prepare carcinoma cell and hepatocyte suspensions by collagenase digestion. For assay of α2, 3 and α2, 6-sialic acids, the cells were suspended in the staining buffer containing either fluorescein isothiocyanate-Maackia amurensis lectin (FITC-MAL) or fluorescein isothiocyanate-Sambucus nigra bark lectin (FITC-SNA) and incubated for 1 h, respectively. Flow cytometric analysis was carried out to measure the mean fluorescence intensity (MFI) on the cell surface. RESULTS: In both FITC-MAL- and FITC-SNA-incubated HCC cells, the MFI on the cell surface was greater than that of the hepatocytes. CONCLUSION: Both of α2, 3 and α2, 6- sialic acids increases significantly on the hepatocyte membrane after the carcinomatous change.

[Intrahepatic transplantation of in vitro induced autologous bone marrow-derived liver stem cells in patients with posthepatitic cirrhosis].

OBJECTIVE: To evaluate the therapeutic effect of in vitro induced autologous bone marrow-derived liver stem cell transplantation for posthepatitic cirrhosis. METHODS: Between Jun 2008 and Mar 2009, 12 patients with posthepatitic cirrhosis and portal hypertensive underwent azygousportal disconnection and splenectomy in our department. The patients were then divided into two groups to receive autologous bone marrow-deprived liver stem cell infusion via the hepatic artery after in vitro induction for 7 days (n=6) or saline (n=6). The therapeutic effects of the operations on the liver functions and liver fibrosis index were evaluated. RESULTS: All the patients recovered uneventfully and no side effect of the operation was found. After the operation, the patients receiving bone marrow-deprived liver stem cell infusion showed better hepatic function improvement than those receiving saline infusion (P<0.05). CONCLUSION: Transplantation of in vitro induced autologous bone marrow-derived liver stem cell via the hepatic artery is safe and effective for treatment of posthepatitic cirrhosis.

[Protocols for cloning human bone marrow-derived hepatic stem cells in vitro].

OBJECTIVE: To explore practical protocols for cloning bone marrow-derived hepatic stem cells in vitro. METHODS: The cell fraction rich in CD117(+) cells and CD184(+) cells was separated from fresh bone marrow by density gradient centrifugation and cultured for 0, 7 and 14 days in high-glucose DMEM supplemented with or without 10% autologous serum or in serum-free high-glucose DMEM. All the media were supplemented with different concentrations of hepatocyte growth promoting factors (HGPF), thrombopoietin (TPO) and interleukin-3 (IL-3). The quantitative changes of CD117(+) cells and CD184(+) cells were measured by flow cytometry. RESULTS: The optimal effect for cell cloning was achieved with high-glucose DMEM with 10% autologous serum group supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3. At day 7 of cell culture in this media, the quantity of CD117(+) cells and CD184(+) cells increased by 6.55 and 6.20 folds, and by 11.62 and 20.57 folds at day 14, respectively. CONCLUSION: It is practical for cloning bone marrow-derived hepatic stem cells in high-glucose DMEM with 10% autologous serum supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3.

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.

We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.

A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines.

The anticancer activity of eight crude extracts of Smilax china L. rhizome (SCR) against HeLa cells was assessed by MTT assay and clonogenic assay, the fraction rich in flavonoids had show good activity against HeLa cells. A bioassay-guided separation on this extract lead to the detection of kaempferol-7-O-beta-D-glucoside (KG), which belongs to flavonoid glycoside, displayed marked anticancer activity. We evaluated its in vitro cytotoxicity and antiproliferative effect in a panel of established cancer cell lines by MTT assay and clonogenic assay. KG induces A375 and HL60 cells apoptosis, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis. Fluorescent staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the A375 and HL60 cells. Flow cytometric analysis shown that A375 and HL60 cells treated with KG resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Quantitation of the hypodiploid cells shows a dose-dependent response to KG, and this result is in good accordance with that of the DNA fragmentation assay by agarose gel electrophoresis. Our results suggested that cell cycle arrest at G(1) phase and induce apoptosis as a mechanism by which KG exerts an antiproliferative effect.

Microbial communities in the larval midgut of laboratory and field populations of cotton bollworm (Helicoverpa armigera).

We compared the bacterial communities in the larval midgut of field and laboratory populations of a polyphagous pest, the cotton bollworm (Helicoverpa armigera), using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences and 16S library sequence analysis. DGGE profiles and 16S rDNA library sequence analysis indicated similar patterns of midgut microbial community structure and diversity: specific bacterial types existed in both populations, and a more diverse microbial community was observed in caterpillars obtained from the field. The laboratory population harbored a rather simple gut microflora consisting mostly of phylotypes belonging to Enterococcus (84%). For the field population, phylotypes belonging to Enterococcus (28%) and Lactococcus (11%), as well as Flavobacterium (10%), Acinetobacter (19%), and Stenotrophomonas (10%) were dominant members. These results provided the first comprehensive description of the microbial diversity of the midgut of the important pest cotton bollworm and suggested that the environment and food supply might influence the diversity of the gut bacterial community.

Preliminary studies on differential defense responses induced during plant communication.

We compared the expression patterns of three representative genes in undamaged tomato and tobacco plants in response to exposure to either tomato or tobacco fed on by Helicoverpa armigera (cotton bollworm). When tomato and tobacco, two species of one family, were incubated in the chambers with the tomato plants damaged by the cotton bollworm, the expression of the PR1, BGL2, and PAL genes was up-regulated in leaves of both plants. However, the levels of gene expression were significantly higher in the tomato than that in the tobacco. In addition, the activities of enzymes, peroxidase, polyphenol oxidase, and lipoxygenase were found to be higher in the tomato than those in the tobacco. Similar results were obtained when the damaged plants were replaced by the tobacco.

The binding of BmK abT, a unique neurotoxin, to mammal brain and insect Na(+) channels using biosensor.

The binding properties of BmK abT (a novel neurotoxic polypeptide abT from Chinese scorpion Buthus martensi Karsch), a unique neurotoxin from Chinese scorpion, on mammal brain and insect sodium channels were investigated using the BIAcore assay. Results showed that BmK abT could bind to rat brain synaptosomes with an association rate constant of about 2.49 x 10(6) M(-1) s(-1) and a dissociation rate constant of about 1.57 x 10(-4) s(-1), and to Heliothis nerve cord synaptosomes with an association rate constant of about 1.21 x 10(7) M(-1) s(-1) and a dissociation rate constant of about 0.99 x 10(-3) s(-1). The binding of BmK abT to rat brain synaptosomes could be partially inhibited by increasing the membrane potential, but not by BmK AS (a novel active polypeptide AS from B. martensi Karsch), BmK IT2 (a depressant insect-selective toxin IT2 from B. martensi Karsch), and BmK I (an alpha-like anti-mammal toxin I from B. martensi Karsch). Binding was not modulated by veratridine. In addition, the binding of BmK abT to Heliothis nerve cord synaptosomes was significantly enhanced by increasing the membrane potential and veratridine concentration and was inhibited by BmK IT2, but not by BmK AS or BmK I. The results suggest that BmK abT binds to a distinct receptor site on mammal brain Na(+) channels and associates with a related site for depressant insect-selective toxins on insect sodium channels.