Ubiquitin C-Terminal Hydrolase L1 Is Expressed in Mouse Pituitary Gonadotropes In Vivo and Gonadotrope Cell Lines In Vitro

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating variousbiological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinatingenzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly andrestrictedly expressed in neural and reproductive tissues and plays significant roles inthese organs. Although the expression of UCH-L1 in the anterior pituitary gland has beenreported, the detailed localization and the role of UCH-L1 remain obscure. In the presentstudy, we detected UCH-L1 protein exclusively in hormone-producing cells, but notnon-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition,the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes andmammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed acomparative analysis using genetically UCH-L1-deficient gad mice.Significant decreases in the numbers of gonadotropes and mammotropes were observed ingad mice, suggesting a close involvement of UCH-L1 in these cells.Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Takentogether, this is the first report to definitely demonstrate the presence of UCH-L1 inmouse anterior pituitary gland, and our results might provide a novel insight for betterunderstanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in thereproduction.     

UBE2C triggers HIF‐1α‐glycolytic flux in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy in Taiwan. Therefore, refining the diagnostic sensitivity of biomarkers for early‐stage tumours and identifying therapeutic targets are critical for improving the survival rate of HNSCC patients. Metabolic reprogramming contributes to cancer development and progression. Metabolic pathways, specifically, play a crucial role in these diverse biological and pathological processes, which include cell proliferation, differentiation, apoptosis and carcinogenesis. Here, we investigated the role and potential prognostic value of the ubiquitin‐conjugating enzyme E2 (UBE2) family in HNSCC. Gene expression database analysis followed by tumour comparison with non‐tumour tissue showed that UBE2C was upregulated in tumours and was associated with lymph node metastasis in HNSCC patients. Knockdown of UBE2C significantly reduced the invasion/migration abilities of SAS and CAL27 cells. UBE2C modulates glycolysis pathway activation and HIF‐1α expression in SAS and CAL27 cells. CoCl₂ (HIF‐1α inducer) treatment restored the expression of glycolytic enzymes and the migration/invasion abilities of UBE2C knockdown cells. Based on our findings, UBE2C expression mediates HIF‐1α activation, increasing glycolysis pathway activation and the invasion/migration abilities of cancer cells. UBE2C may be an independent prognostic factor and a therapeutic target in HNSCC.     

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response‐related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan‐induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)‐γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN‐γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN‐γ doses (0.2 μg/kg) either alone or in combination with the ß‐glucan‐binding CD5 protein (0.7 mg/kg) leading to improved post zymosan‐induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10⁴ CFU/gr) were ameliorated by low‐dose IFN‐γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF‐β1/Smad pathway

Epithelial‐mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N‐α‐acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF‐β1/Smad and EMT‐related molecules. The Transwell migration, invasion, qRT‐PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF‐β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF‐β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα‐induced change in the migration, invasion and EMT‐related molecules in OSCC cells after TGF‐β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF‐β1/Smad, a novel pathway for preventing OSCC.     

Pancreatic cancer-derived exosomal microRNA-19a induces β-cell dysfunction by targeting ADCY1 and EPAC2

New-onset diabetes mellitus has a rough correlation with pancreatic cancer (PaC), but the underlying mechanism remains unclear. This study aimed to explore the exosomal microRNAs and their potential role in PaC-induced β-cell dysfunction. The pancreatic β cells were treated with isolated exosomes from PaC cell lines, SW1990 and BxPC-3, before measuring the glucose-stimulated insulin secretion (GSIS), validating that SW1990 and BxPC-3 might disrupt GSIS of both β cell line MIN6 and primary mouse pancreatic islets. The difference in expression profiles between exosomes and exosome-free medium of PaC cell lines was further defined, revealing that miR-19a secreted by PaC cells might be an important signaling molecule in this process. Furthermore, adenylyl cyclase 1 (Adcy1) and exchange protein directly activated by cAMP 2 (Epac2) were verified as the direct targets of exogenous miR-19a, which was involved in insulin secretion. These results indicated that exosomes might be an important mediator in the pathogenesis of PaC-DM, and miR-19a might be the effector molecule. The findings shed light on the pathogenesis of PaC-DM.     

SDF‐1 inhibits the dedifferentiation of islet β cells in hyperglycaemia by up‐regulating FoxO1 via binding to CXCR4

Islet β cell dedifferentiation is one of the most important mechanisms in the occurrence and development of diabetes. We studied the possible effects of chemokine stromal cell‐derived factor‐1 (SDF‐1) in the dedifferentiation of islet β cells. It was noted that the number of dedifferentiated islet β cells and the expression of SDF‐1 in pancreatic tissues significantly increased with diabetes. In islet β cell experiments, inhibition of SDF‐1 expression resulted in an increase in the number of dedifferentiated cells, while overexpression of SDF‐1 resulted in a decrease. This seemed to be contradicted by the effect of diabetes on the expression of SDF‐1 in pancreatic tissue, but it was concluded that this may be related to the loss of SDF‐1 activity. SDF‐1 binds to CXCR4 to form a complex, which activates and phosphorylates AKT, subsequently increases the expression of forkhead box O1 (FOXO1), and inhibits the dedifferentiation of islet β cells. This suggests that SDF‐1 may be a novel target in the treatment of diabetes.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.     

The Relationship Between the Tumor Cell Expression of Hypoxic Markers and Survival in Patients With ER-positive Invasive Ductal Breast Cancer

The prognostic significance of hypoxia markers, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), and carbonic anhydrase IX (CAIX), was investigated in estrogen receptor (ER)-positive breast cancer patients. Immunohistochemistry determined the expression of makers in two independent ductal ER-positive cohorts (Training set, n=373 and Validation set, n=285) and was related to clinicopathological parameters and disease-free survival (DFS). In the training cohort, nuclear HIF-1α (1) was independently associated with poorer DFS in luminal A tumors [hazard ratio (HR) = 0.53 95% confidence interval (CI): 0.30–0.94, p=0.030]. In the validation cohort, both HIF-1α (1) and CAIX were independently associated with decreased DFS in the entire cohort (HR = 1.85 95% CI: 1.10–3.11, p=0.019; HR = 1.74 95% CI: 1.08–2.82, p=0.023), in luminal A disease (HR = 1.98 95% CI: 1.02–3.83, p=0.042), and in luminal B disease (HR = 2.75 95% CI: 1.66–4.55, p<0.001), respectively. Taken together, elevated cytoplasmic HIF-1α (1) expression was an independent prognostic factor in luminal A disease, whereas CAIX was an independent prognostic factor in luminal B disease. Further work in large tissue cohorts is required.     

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3^H]-thymidine incorporation, protein abundance, and mRNA expression.     

Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F₁ mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F₁ mice and R26GRR /Ins1-Cre F₁ mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).     

CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3′ UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.