A Transformable Specific-Responsive Peptide for One-Step Synergistic Therapy of Bladder Cancer.

Synergistic therapy has shown greater advantages compared with monotherapy. However, the complex multiple-administration plan and potential side effects limit its clinical application. A transformable specific-responsive peptide (TSRP) is utilized to one-step achieve synergistic therapy integrating anti-tumor, anti-angiogenesis and immune response. The TSRP is composed of: i) Recognition unit could specifically target and inhibit the biological function of FGFR-1; ii) Transformable unit could self-assembly and trigger nanofibers formation; iii) Reactive unit could specifically cleaved by MMP-2/9 in tumor micro-environment; iv) Immune unit, stimulate the release of immune cells when LTX-315 (Immune-associated oncolytic peptide) exposed. Once its binding to FGFR-1, the TSRP could cleaved by MMP-2/9 to form the nanofibers on the cell membrane, with a retention time of up to 12 h. Through suppressing the phosphorylation levels of ERK 1/2 and PI3K/AKT signaling pathways downstream of FGFR-1, the TSRP significant inhibit the growth of tumor cells and the formation of angioginesis. Furthermore, LTX-315 is exposed after TSRP cleavage, resulting in Calreticulin activation and CD8+ T cells infiltration. All above processes together contribute to the increasing survival rate of tumor-bearing mice by nearly 4-folds. This work presented a unique design for the biological application of one-step synergistic therapy of bladder cancer.

A novel prognostic risk-scoring system based on m5C methylation regulator-mediated patterns for glioma patients.

N5-methylcytosine (m5C) methylation modification plays a crucial role in the epigenetic mechanisms underlying tumorigenesis, aggressiveness, and malignancy in diffuse glioma. Our study aimed to develop a novel prognostic risk-scoring system to assess the impact of m5C modification in glioma patients. Initially, we identified two distinct m5C clusters based on the expression level of m5C regulators in The Cancer Genome Atlas glioblastoma (TCGA-GBM) dataset. Differentially expressed genes (DEGs) between the two m5C cluster groups were determined. Utilizing these m5C regulation-related DEGs, we classified glioma patients into three gene cluster groups: A, B, and C. Subsequently, an m5C scoring system was developed through a univariate Cox regression model, quantifying the m5C modification patterns utilizing six DEGs associated with disease prognosis. The resulting scoring system allowed us to categorize patients into high- or low-risk groups based on their m5C scores. In test (TCGA-GBM) and validation (Chinese Glioma Genome Atlas [CGGA]-1018 and CGGA-301) datasets, glioma patients with a higher m5C score consistently exhibited shorter survival durations, fewer isocitrate dehydrogenase (IDH) mutations, less 1p/19q codeletion and higher World Health Organization (WHO) grades. Additionally, distinct immune cell infiltration characteristics were observed among different m5C cluster groups and risk groups. Our study developed a novel prognostic scoring system based on m5C modification patterns for glioma patients, complementing existing molecular classifications and providing valuable insights into prognosis for glioma patients.

LRP1 in GABAergic neurons is a key link between obesity and memory function.

OBJECTIVE: Low-density lipoprotein receptor-related protein-1 (LRP1) regulates energy homeostasis, blood-brain barrier integrity, and metabolic signaling in the brain. Deficiency of LRP1 in inhibitory gamma-aminobutyric acid (GABA)ergic neurons causes severe obesity in mice. However, the impact of LRP1 in inhibitory neurons on memory function and cognition in the context of obesity is poorly understood. METHODS: Mice lacking LRP1 in GABAergic neurons (Vgat-Cre; LRP1loxP/loxP) underwent behavioral tests for locomotor activity and motor coordination, short/long-term and spatial memory, and fear learning/memory. This study evaluated the relationships between behavior and metabolic risk factors and followed the mice at 16 and 32 weeks of age. RESULTS: Deletion of LRP1 in GABAergic neurons caused a significant impairment in memory function in 32-week-old mice. In the spatial Y-maze test, Vgat-Cre; LRP1loxP/loxP mice exhibited decreased travel distance and duration in the novel arm compared with controls (LRP1loxP/loxP mice). In addition, GABAergic neuron-specific LRP1-deficient mice showed a diminished capacity for performing learning and memory tasks during the water T-maze test. Moreover, reduced freezing time was observed in these mice during the contextual and cued fear conditioning tests. These effects were accompanied by increased neuronal necrosis and satellitosis in the hippocampus. Importantly, the distance and duration in the novel arm, as well as the performance of the reversal water T-maze test, negatively correlated with metabolic risk parameters, including body weight, serum leptin, insulin, and apolipoprotein J. However, in 16-week-old Vgat-Cre; LRP1loxP/loxP mice, there were no differences in the behavioral tests or correlations between metabolic parameters and cognition. CONCLUSIONS: Our findings demonstrate that LRP1 from GABAergic neurons is important in regulating normal learning and memory. Metabolically, obesity caused by GABAergic LRP1 deletion negatively regulates memory and cognitive function in an age-dependent manner. Thus, LRP1 in GABAergic neurons may play a crucial role in maintaining normal excitatory/inhibitory balance, impacting memory function, and reinforcing the potential importance of LRP1 in neural system integrity.

PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway.

ABSTRACT: Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Visualization and quantification of cadmium accumulation, chelation and antioxidation during the process of vacuolar compartmentalization in the hyperaccumulator plant Solanum nigrum L.

Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the proportion of vacuolar Cd in protoplasts showed its ultimate storage capacity (82.24 %-83.40 %), and the Cd concentration stored in the protoplast maintained at a certain level (73.81-77.46 mg L-1). Besides, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2 = 0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.

Label-Free and Highly-Sensitive Detection of Ochratoxin A Using One-Pot Synthesized Reduced Graphene Oxide/Gold Nanoparticles-Based Impedimetric Aptasensor.

Ochratoxin A (OTA) primarily obtained by the genera aspergillus and penicillium, is one of the toxic substances for different organs and systems of the human body such as the kidney, liver, neurons and the immune system. Moreover, it is considered to cause tumors and fetal malformation even at a very low concentration. Fast and sensitive assay for detection of OTA at ultralow levels in foods and agricultural products has been an increasing demand. In this study, a new label-free electrochemical biosensor based on three-dimensional reduced graphene oxide/gold nanoparticles/aptamer for OTA detection was constructed. The 3D-rGO/Au NPs nanocomposites were firstly synthesized using a one-pot hydrothermal process under optimized experimental conditions. The 3D-rGO/Au NPs with considerable particular surface area and outstanding electrical conductivity was then coated on a glass carbon electrode to provide tremendous binding sites for -SH modified aptamer via the distinctive Au-S linkage. The presence of OTA was specifically captured by aptamer and resulted in electrochemical impedance spectroscopy (EIS) signal response accordingly. The constructed impedimetric aptasensor obtained a broad linear response from 1 pg/mL to 10 ng/mL with an LOD of 0.34 pg/mL toward OTA detection, highlighting the excellent sensitivity. Satisfactory reproducibility was also achieved with the relative standard deviation (RSD) of 1.393%. Moreover, the proposed aptasensor obtained a good recovery of OTA detection in red wine samples within the range of 93.14 to 112.75% along with a low LOD of 0.023 ng/mL, indicating its applicability for OTA detection in real samples along with economical, specific, susceptible, fast, easy, and transportable merits.

Identification and characterization of caspases genes in rainbow trout (Oncorhynchus mykiss) and their expression profiles after Aeromonas salmonicida and Vibrio anguillarum infection.

Caspases are highly conserved cysteine-dependent aspartyl-specific proteases that play an important role in regulating cell death and inflammation. However, the caspase genes have not been systematically studied in rainbow trout (Oncorhynchus mykiss). Rainbow trout experienced 4 rounds (4R) of genome duplication in the evolutionary history. Thereby an increased numbers of paralogs are observed in trout, probably with more complicated gene functions. We identified 18 caspase genes in rainbow trout, including two inflammatory caspases (casp1a, casp1b), six apoptosis executioner caspases (casp3, casp3a1, casp3a2, casp3b, casp6, and casp7), nine apoptosis initiator caspases (casp2a, casp2b, casp8, casp9a, casp9b, casp10a, casp10b, casp20a, and casp20b) and one uncategorized caspase gene (casp17). To investigate the potentially physiological functions of caspase genes, we challenged the rainbow trout with Aeromonas salmonicida (A. salmonicida) and Vibrio anguillarum (V. anguillarum). Results showed that the CASP3-regulated intrinsic apoptosis was activated after A. salmonicida infection, while the CASP8 and CASP6-regulated extrinsic apoptosis exerted the greatest effect on trout challenged with V. anguillarum. In response to V. anguillarum infection, the data of RNA-Seq further showed the casp8 was tightly integrated with the significantly enriched Gene Ontology terms and functional pathways, including apoptosis regulation, pathogen detection and immunomodulation. Our study provides a foundation for the physiological functions and regulatory network of the caspase genes in teleosts.

Evidence for the evolution, clonal expansion and global dissemination of water treatment-resistant naturalized strains of Escherichia coli in wastewater.

We previously demonstrated the existence of naturalized strains of E. coli in wastewater and herein perform an in-depth comparative whole genome analysis of these strains (n = 17). Fourteen of the Canadian E. coli strains, isolated from geographically separated wastewater treatment plants, were virtually identical at the core genome and were ≥96% similar at the whole genome level, suggesting clonal-relatedness among these isolates. Remarkably, these strains were shown to be extremely similar to the genome of an E. coli isolated from wastewater in Switzerland, suggesting a global distribution of these strains. The genomes of three other Canadian wastewater strains were more diverse but very similar to the genomes of E. coli isolates collected from U.S. wastewater samples. Based on maximum likelihood phylogenetic analysis, wastewater strains from Canada, the U.S. and Switzerland formed a clade separate from other known enteric phylogroups (i.e., A, B1, B2, D, E) and the cryptic clades. All Canadian, Swiss and U.S. wastewater strains possessed a common SNP biomarker pattern across their genomes, and a sub-population (i.e., 14 Canadian and 1 Swiss strain) also possessed a previously identified wastewater-specific marker known as uspC-IS30-flhDC element. Biochemical heat mapping of 518 categories of genes recapitulated phylogeny, with wastewater strains phenotypically clustering separately from enteric and cryptic clades. Wastewater strains were enriched for stress-response genes (i.e., nutrient acquisition/deprivation, DNA repair, oxidative stress, and UV resistance) - elements reflective of their environmental survival challenges. Wastewater strains were shown to carry a plethora of known antibiotic resistance (AR) genes, the patterns of which were remarkably similar among all Canadian, U.S. and Swiss wastewater strains. Virulence gene composition was also similar among all the wastewater strains, with an abundant representation of virulence genes commonly associated with urinary pathogenic E. coli (UPEC) as well as enterohemorrhagic (EHEC) E. coli. The remarkable degree of similarity between all wastewater strains from Canada, Switzerland and the U.S. suggests the evolution and global-dissemination of water treatment-resistant clone of E. coli. These finding, along with others, raise some important concerns about the potential for emergence of E. coli pathotypes resistant to water-treatment.

Mutations in GPR101 as a potential cause of X-linked acrogigantism and acromegaly.

X-linked acrogigantism (XLAG) is a recently described early-onset gigantism due to GPR101 duplication that induces growth hormone (GH) oversecretion. GPR101, which belongs to Family A rhodopsin-like family of G protein-coupled receptors, is predominantly expressed in hypothalamus and pituitary, suggesting that GPR101 might be important in regulating diverse functions such as energy balance and reproduction. Most mammalian GPR101s have extremely long third intracellular loops (ICL3); however, zebrafish GPR101 has a much shorter ICL3, but a longer C-terminus. GnRH-(1-5), a GnRH metabolite, can modulate the hypothalamus-pituitary-gonad axis and cancer cell migration via activating GPR101. GPR101 couples to both Gαs and Gαi proteins. GPR101 duplication has a causative role in XLAG, while GPR101 variants, especially c.924G>C (E308D), located at ICL3, are attributed to acromegaly. Some GPR101 mutations that are associated with a small proportion of pituitary tumors without GH oversecretion have also been identified recently. This chapter will summarize studies on GPR101, including its molecular cloning and tissue distribution, physiology, pharmacology, and pathophysiology.

Selectins modify dendritic cells during atherosclerosis.

Dendritic cells (DCs) are professional antigen-presenting cells (APC) that facilitate the development and progression of atherosclerosis. However, DCs also function as novel "switches" between immune activation and immune tolerance and represent a heterogeneous hematopoietic lineage, with cell subsets in different tissues that show a differential morphology, phenotype, and function. Regulatory DCs, depending on their immature state, can be induced by immunosuppressive modulation, which plays an important part in the maintenance of immunologic tolerance via suppression of the immune response. In this review, we describe the current understanding of the generation of regulatory DCs. The novel role of selectins in the modification of DCs in atherosclerosis is also discussed.

Expression of estrogen receptors in female rainbow trout (Oncorhynchus mykiss) during first ovarian development and under dense rearing condition.

To study the expression of four estrogen receptor genes (erα1, erα2, erß1, erß2) of female rainbow trout (Oncorhynchus mykiss) during first ovarian development, trouts were sampled from different ovarian stages. Serum E2 (estradiol) was measured by ELISA and estrogen receptors mRNA expression were examined by qRT-PCR. Our results showed a close association between increased erα1 and vitellogenin mRNA expression during ovarian maturation and increased erα2 mRNA expression in mature ovarian stages. Correlation analysis revealed that a negative relationship between serum E2 and ovarian erß1 (or hepatic erß2), but ovarian erß2 mRNA expression was relatively unchanged during first ovarian development. Trout were also reared in different densities as stocking density 1, 2 and 3 (SD1, 4.6-31.1 kg/m3; SD2, 6.6-40.6 kg/m3; SD3, 8.6-49.3 kg/m3) to elucidate effects of high density on estrogen receptor expression. Histology observation showed ovarian development of trout in higher densities were retard with a relatively early stage and fewer vitellogenin accumulation. Trout in high densities showed significantly decreased serum E2, erα mRNA expression and increasing trends of erß mRNA expression. A noticeable increase of ovarian erß2 mRNA expression was seen in trout when density is approaching to 50 kg/m3. In conclusion, we may hypothesize that increased erß mRNA expression triggered by high density result in decreased erα mRNA expression and vitellogenesis. As a result, ovarian development in higher densities was retard.

Hypothalamus-pituitary-gonad axis of rainbow trout (Oncorhynchus mykiss) during early ovarian development and under dense rearing condition.

The objective of this study was to determine the hypothalamus-pituitary-gonad (HPG) axis of female rainbow trout (Oncorhynchus mykiss) during early ovarian development and under high rearing density. Trouts were sampled from 240 (ovarian stage II) to 540 (ovarian stage IV) days following hatching (DFH) as control group (Ctrl, 4.6-31.1kg/m(3)) to determine HPG axis during early ovarian development. Trouts from the same batch of fertilized eggs were reared in two higher densities during 240-540 DFH as stocking density 1 and 2 (SD1, 6.6-40.6kg/m(3); SD2, 8.6-49.3kg/m(3)) to elucidate effects of high density on reproductive parameters. Dopamine, E2 (estradiol), 17α,20ß-P (17α,20ß-dihydroxy4-pregnen-3-one) and P4 (progesterone) were evaluated by radioimmunoassay or ELISA. mRNA expression of hypothalamic gnrh-1, -2 (gonadotropin-releasing hormone-1, -2), pituitary gonadotropins (fsh/lh, follicle-stimulating hormone/luteinizing hormone) and their cognate receptors (fshr/lhr) in ovaries were examined by qRT-PCR. Our findings demonstrated mRNA expression of hypothalamic sgnrh-1, pituitary fsh and ovarian fshr increased in early ovarian development (360 DFH). Serum 17α,20ß-P and pituitary lh mRNA expression first increased when trouts were in ovarian stage III (420 DFH). Ovaries were at different stages when reared in different densities. Long-term high density treatment (over 31.7kg/m(3)) resulted in decreased hypothalamic sgnrh-1, pituitary fsh, ovarian fshr, serum E2, and increased hypothalamus gnrh-2 and serum dopamine during vitellogenin synthesis, suggesting HPG of rainbow trout might be retarded under dense rearing condition.