Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells.

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells.

The free calcium (Ca2+) levels in pancreatic beta cell organelles have been the subject of many recent investigations. Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The Kds of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 µM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.

Deciphering multi-way interactions in the human genome.

Chromatin architecture, a key regulator of gene expression, can be inferred using chromatin contact data from chromosome conformation capture, or Hi-C. However, classical Hi-C does not preserve multi-way contacts. Here we use long sequencing reads to map genome-wide multi-way contacts and investigate higher order chromatin organization in the human genome. We use hypergraph theory for data representation and analysis, and quantify higher order structures in neonatal fibroblasts, biopsied adult fibroblasts, and B lymphocytes. By integrating multi-way contacts with chromatin accessibility, gene expression, and transcription factor binding, we introduce a data-driven method to identify cell type-specific transcription clusters. We provide transcription factor-mediated functional building blocks for cell identity that serve as a global signature for cell types.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development. METHODS: We performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis. RESULTS: Loss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial-mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata. CONCLUSIONS: Loss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

Retraction notice to "Peptide-induced formation of protein aggregates and amyloid fibrils in human and Guinea pig αA-crystallins under physiological conditions of temperature and pH" [Exp. Eye Res. 179 (2019) 193-205].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al. Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

Antimicrobial and antibiofilm activity of designed and synthesized antimicrobial peptide, KABT-AMP.

Lysine-rich peptide, designated as KABT-AMP, was designed and synthesized to supersede the irrational use of chemical antibiotics as standard therapy. KABT-AMP is a 22-amino acid helical cationic peptide (+10) and amphipathic in nature. The antimicrobial kinetics of the peptide was ascertained in the representative strains of gram-positive, gram-negative, and fungal strains, viz., Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, and Candida albicans MTCC 227, respectively. KABT-AMP was synthesized by solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography which resulted in >95 % purity, and matrix-assisted laser desorption/ionization time of flight revealed the mass of the peptide to be 2.8 kDa. KABT-AMP showed significant broad-spectrum antimicrobial activity against the bacterial and fungal strains analyzed in the present study with survivability of 30.8, 30.6, and 31.7 % in E. coli, S. aureus, and C. albicans, respectively, at 6 h. KABT-AMP also demonstrated antibiofilm activity against the tested biofilm forming clinical isolate, Candida tropicalis. The putative membranolytic activity of the peptide was substantiated by electron microscopic analysis. Results reveal that KABT-AMP will exhibit noteworthy antimicrobial activity against multidrug-resistant bacteria and fungus at micromolar concentrations with minimal cytotoxicity and thus could be conceived for biomedical application.

Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein.

A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.