The Study of Antistaphylococcal Potential of Omiganan and Retro-Omiganan Under Flow Conditions.

Staphylococcus aureus is considered one of the leading pathogens responsible for infections in humans and animals. The heterogeneous nature of diseases caused by these bacteria is due to the occurrence of multiple strains, differentiated by several mechanisms of antibiotic resistance and virulence factors. One of these is the ability to form biofilm. Biofilm-associated bacteria exhibit a different phenotype that protects them from external factors such as the activity of immune system or antimicrobial substances. Moreover, it has been shown that the majority of persistent and recurrent infections are associated with the presence of the biofilm. Omiganan, an analog of indolicidin - antimicrobial peptide (AMP) derived from bovine neutrophil granules, was found to exhibit high antistaphylococcal and antibiofilm potential. Furthermore, its analog with a reversed sequence (retro-omiganan) was found to display enhanced activity against a variety of pathogens. Based on experience of our group, we found out that counterion exchange can improve the antistaphylococcal activity of AMPs. The aim of this study was to investigate the activity of both compounds against S. aureus biofilm under flow conditions. The advantage of this approach was that it offered the opportunity to form and characterize the biofilm under more controlled conditions. To do this, unique flow cells made of polydimethylsiloxane (PDMS) were developed. The activity against pre-formed biofilm as well as AMPs-treated bacteria was measured. Also, the incorporation of omiganan and retro-omiganan into the channels was conducted to learn whether or not it would inhibit the development of biofilm. The results of the microbiological tests ultimately confirmed the high potential of the omiganan and its retro-analog as well as the importance of counterion exchange in terms of antimicrobial examination. We found out that retro-omiganan trifluoroacetate had the highest biofilm inhibitory properties, however, acetates of both compounds exhibited the highest activity against planktonic and biofilm cultures. Moreover, the developed methodology of investigation under flow conditions allows the implementation of the studies under flow conditions to other compounds.

IRE1-mediated degradation of pre-miR-301a promotes apoptosis through upregulation of GADD45A.

The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here, we determine IRE1's role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A's 3' UTR with a target protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these results indicate that IRE1's endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis. Video Abstract.

Dual RNase activity of IRE1 as a target for anticancer therapies.

The unfolded protein response (UPR) is a cellular mechanism that protects cells during stress conditions in which there is an accumulation of misfolded proteins in the endoplasmic reticulum (ER). UPR activates three signaling pathways that function to alleviate stress conditions and promote cellular homeostasis and cell survival. During unmitigated stress conditions, however, UPR activation signaling changes to promote cell death through apoptosis. Interestingly, cancer cells take advantage of this pathway to facilitate survival and avoid apoptosis even during prolonged cell stress conditions. Here, we discuss different signaling pathways associated with UPR and focus specifically on one of the ER signaling pathways activated during UPR, inositol-requiring enzyme 1α (IRE1). The rationale is that the IRE1 pathway is associated with cell fate decisions and recognized as a promising target for cancer therapeutics. Here we discuss IRE1 inhibitors and how they might prove to be an effective cancer therapeutic.

HIF-1-Induced hsa-miR-429: Understanding Its Direct Targets as the Key to Developing Cancer Diagnostics and Therapies.

MicroRNAs (miRNAs) play a critical role in the regulation of mRNA stability and translation. In spite of our present knowledge on the mechanisms of mRNA regulation by miRNAs, the utilization and translation of these ncRNAs into clinical applications have been problematic. Using hsa-miR-429 as an example, we discuss the limitations encountered in the development of efficient miRNA-related therapies and diagnostic approaches. The miR-200 family members, which include hsa-miR-429, have been shown to be dysregulated in different types of cancer. Although these miR-200 family members have been shown to function in suppressing epithelial-to-mesenchymal transition, tumor metastasis, and chemoresistance, the experimental results have often been contradictory. These complications involve not only the complex networks involving these noncoding RNAs, but also the problem of identifying false positives. To overcome these limitations, a more comprehensive research strategy is needed to increase our understanding of the mechanisms underlying their biological role in mRNA regulation. Here, we provide a literature analysis of the verified hsa-miR-429 targets in various human research models. A meta-analysis of this work is presented to provide better insights into the role of hsa-miR-429 in cancer diagnosis and any potential therapeutic approach.

Glycosylated Lipopeptides-Synthesis and Evaluation of Antimicrobial Activity and Cytotoxicity.

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that may be used to combat pathogens such as bacteria and fungi. USCLs consist of a few basic amino acid residues and at least one lipid moiety, usually a fatty acid chain. Generally, USCLs are potent antimicrobials but their major shortcoming is a relatively high cytotoxicity and hemolytic activity. Glycopeptide antibiotics (e.g. vancomycin) are essential in combating bacterial infections and are popular in medicinal practice. However, literature concerning the effect of glycosylation of peptides on their antimicrobial activity is rather scarce. For the first time, this study highlights the effect of USCLs glycosylation on in vitro biological activity. The aim of this study was to evaluate the impact of glycosylation of a series of USCLs on antimicrobial activity, cytotoxicity and hemolytic activity. Straight-chain fatty acids (C14, C16, C18) were attached to the N-terminal amino group of tripeptides-SRR-NH2, RSR-NH2 and RRS-NH2. Two groups of the lipopeptides were synthetized, the first with unmodified L-serine (USCLs) and the other with L-serine O-glycosylated by N-acetyl-ß-d-glucosamine to produce new class of glycosylated ultrashort cationic lipopeptide (gUSCLs). Both USCLs and gUSCLs were tested against planktonic and biofilm cultures of ESKAPE strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Candida glabrata, and hemolytic activity on human erythrocytes and cytotoxicity against the HaCaT cell line was examined. Generally, USCLs and gUSCLs proved to be active against all the tested strains. The highest activity displayed was by lipopeptides containing the C18 fatty acid. Antimicrobial, hemolytic and cytotoxic activities were mainly correlated with amino acid sequence (position of serine/glycosylated serine) and hydrophobicity of molecule and were found to be highly strain-dependent. In general, glycosylation did not guarantee an increased antimicrobial activity or a decreased hemolytic and cytotoxic activities. However, in some cases, gUSCLs proved to be superior to their USCLs analogs. The most pronounced differences were found for peptides with C18 fatty acid and serine at the first and second position against both planktonic cells and biofilm of C. glabrata, as well as the second and third position against S. aureus. It is noteworthy that gUSCLs were also more active against biofilm than were USCLs.

The transition from HIF-1 to HIF-2 during prolonged hypoxia results from reactivation of PHDs and HIF1A mRNA instability.

The hypoxia-inducible factors (HIF) are transcription factors that activate the adaptive hypoxic response when oxygen levels are low. The HIF transcriptional program increases oxygen delivery by inducing angiogenesis and by promoting metabolic reprograming that favors glycolysis. The two major HIFs, HIF-1 and HIF-2, mediate this response during prolonged hypoxia in an overlapping and sequential fashion that is referred to as the HIF switch. Both HIF proteins consist of an unstable alpha chain and a stable beta chain. The instability of the alpha chains is mediated by prolyl hydroxylase (PHD) activity during normoxic conditions, which leads to ubiquitination and proteasomal degradation of the alpha chains. During normoxic conditions, very little HIF-1 or HIF-2 alpha-beta dimers are present because of PHD activity. During hypoxia, however, PHD activity is suppressed, and HIF dimers are stable. Here we demonstrate that HIF-1 expression is maximal after 4 h of hypoxia in primary endothelial cells and then is dramatically reduced by 8 h. In contrast, HIF-2 is maximal at 8 h and remains elevated up to 24 h. There are differences in the HIF-1 and HIF-2 transcriptional profiles, and therefore understanding how the transition between them occurs is important and not clearly understood. Here we demonstrate that the HIF-1 to HIF-2 transition during prolonged hypoxia is mediated by two mechanisms: (1) the HIF-1 driven increase in the glycolytic pathways that reactivates PHD activity and (2) the much less stable mRNA levels of HIF-1α (HIF1A) compared to HIF-2α (EPAS1) mRNA. We also demonstrate that the alpha mRNA levels directly correlate to the relative alpha protein levels, and therefore to the more stable HIF-2 expression during prolonged hypoxia.

Understanding the Role of Self-Assembly and Interaction with Biological Membranes of Short Cationic Lipopeptides in the Effective Design of New Antibiotics.

This study investigates short cationic antimicrobial lipopeptides composed of 2-4 amino acid residues and C12-C18 fatty acids attached to the N-terminal part of the peptides. The findings were discussed in the context of the relationship among biological activity, self-assembly, stability, and membrane interactions. All the lipopeptides showed the ability to self-assemble in PBS solution. In most cases, the critical aggregation concentration (CAC) much surpassed the minimal inhibitory concentration (MIC) values, suggesting that monomers are the main active form of lipopeptides. The introduction of ß-alanine into the peptide sequence resulted in a compound with a high propensity to fibrillate, which increased the peptide stability and activity against S. epidermidis and C. albicans and reduced the cytotoxicity against human keratinocytes. The results of our study indicated that the target of action of lipopeptides is the bacterial membrane. Interestingly, the type of peptide counterion may affect the degree of penetration of the lipid bilayer. In addition, the binding of the lipopeptide to the membrane of Gram-negative bacteria may lead to the release of calcium ions necessary for stabilization of the lipopolysaccharide layer.

The Role of the Hypoxia-Related Unfolded Protein Response (UPR) in the Tumor Microenvironment.

Despite our understanding of the unfolded protein response (UPR) pathways, the crosstalk between the UPR and the complex signaling networks that different cancers utilize for cell survival remains to be, in most cases, a difficult research barrier. A major problem is the constant variability of different cancer types and the different stages of cancer as well as the complexity of the tumor microenvironments (TME). This complexity often leads to apparently contradictory results. Furthermore, the majority of the studies that have been conducted have utilized two-dimensional in vitro cultures of cancer cells that were exposed to continuous hypoxia, and this approach may not mimic the dynamic and cyclic conditions that are found in solid tumors. Here, we discuss the role of intermittent hypoxia, one of inducers of the UPR in the cellular component of TME, and the way in which intermittent hypoxia induces high levels of reactive oxygen species, the activation of the UPR, and the way in which cancer cells modulate the UPR to aid in their survival. Although the past decade has resulted in defining the complex, novel non-coding RNA-based regulatory networks that modulate the means by which hypoxia influences the UPR, we are now just to beginning to understand some of the connections between hypoxia, the UPR, and the TME.

The hypoxia-induced changes in miRNA-mRNA in RNA-induced silencing complexes and HIF-2 induced miRNAs in human endothelial cells.

The cellular adaptive response to hypoxia relies on the expression of hypoxia-inducible factors (HIFs), HIF-1 and HIF-2. HIFs regulate global gene expression changes during hypoxia that are necessary for restoring oxygen homeostasis and promoting cell survival. In the early stages of hypoxia, HIF-1 is elevated, whereas at the later stages, HIF-2 becomes the predominant form. What governs the transition between the two HIFs (the HIF switch) and the role of miRNAs in this regulation are not completely clear. Genome-wide expression studies on the miRNA content of RNA-induced silencing complexes (RISC) in HUVECs exposed to hypoxia compared to the global miRNA-Seq analysis revealed very specific differences between these two populations. We analyzed the miRNA and mRNA composition of RISC at 2 h (mainly HIF-1 driven), 8 h (HIF-1 and HIF-2 elevated), and 16 h (mainly HIF-2 driven) in a gene ontology context. This allowed for determining the direct impact of the miRNAs in modulating the cellular signaling pathways involved in the hypoxic adaptive response. Our results indicate that the miRNA-mRNA RISC components control the adaptive responses, and this does not always rely on the miRNA transcriptional elevations during hypoxia. Furthermore, we demonstrate that the hypoxic levels of the vast majority of HIF-1-dependent miRNAs (including miR-210-3p) are also HIF-2 dependent and that HIF-2 governs the expression of 11 specific miRNAs. In summary, the switch from HIF-1 to HIF-2 during hypoxia provides an important level of miRNA-driven control in the adaptive pathways in endothelial cells.

Hypoxia-inducible factor (HIF)-3a2 serves as an endothelial cell fate executor during chronic hypoxia.

The adaptive response to hypoxia involves the transcriptional induction of three transcription factors called hypoxia inducible factor alpha 1, 2 and 3 (HIF-1α, HIF-2α, and HIF-3α) which dimerize with constitutively expressed beta chains that together form the HIF-1, -2 and -3 transcription factors. During normoxic conditions, the alpha chain is expressed at low levels since its stability is regulated by prolyl-hydroxylation that promotes subsequent ubiquitination and degradation. During hypoxic conditions, however, the prolyl hydroxylases are less active, and the alpha chain accumulates through elevated protein stability and the elevated induction of expression. Two of the three HIFs isoforms present in mammals, HIF-1 and HIF-2, are well characterized and have overlapping functions that promote cell survival, whereas HIF-3's role remains less clear. The HIF-3 response is complicated because the HIF3A gene can utilize different promotors and alternate splicing sites that result in a number of different HIF-3α isoforms. Here, using human umbilical vein endothelial cells (HUVECs), we demonstrate that one of the isoforms of HIF-3α, isoform 2 (HIF-3α2) accumulates at a late stage of hypoxia and induces the expression of DNA damage inducible transcript 3 (DDIT4), a gene known to promote apoptosis. We also demonstrate that caspase 3/7 activity is elevated, supporting that the role of the HIF-3α2 isoform is to promote apoptosis. Furthermore, we provide evidence that HIF-3α2 is also expressed in seven other primary endothelial cell types, suggesting that this may be a common feature of HIF-3α2 in endothelial cells.

Antimicrobial, Cytotoxic and Mutagenic Activity of Gemini QAS Derivatives of 1,4:3,6-Dianhydro-l-iditol.

A series of quaternary diammonium salts derivatives of 1,4:3,6-dianhydro-l-iditol were synthesized, using isommanide (1,4:3,6-dianhydro-d-mannitol) as a starting material. Both aromatic (pyridine, 4-(N,N-dimethylamino)pyridine (DMAP), (3-carboxamide)pyridine; N-methylimidazole) and aliphatic (trimethylamine, N,N-dimethylhexylamine, N,N-dimethyloctylamine, N,N-dimethyldecylamine) amines were used, giving eight gemini quaternary ammonium salts (QAS). All salts were tested for their antimicrobial activity against yeasts, Candida albicans and Candida glabrata, as well as bacterial Staphylococcus aureus and Escherichia coli reference strains. Moreover, antibacterial activity against 20 isolates of S. aureus collected from patients with skin and soft tissue infections (n = 8) and strains derived from subclinical bovine mastitis milk samples (n = 12) were evaluated. Two QAS with octyl and decyl residues exhibited antimicrobial activity, whereas those with two decyl residues proved to be the most active against the tested pathogens, with MIC of 16-32, 32, and 8 µg/mL for yeast, E. coli, and S. aureus reference and clinical strains, respectively. Only QAS with decyl residues proved to be cytotoxic in MTT assay against human keratinocytes (HaCaT), IC50 12.8 ± 1.2 µg/mL. Ames test was used to assess the mutagenic potential of QAS, and none of them showed mutagenic activity in the concentration range 4-2000 µg/plate.

Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor.

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next-generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

Triazoloacridone C-1305 impairs XBP1 splicing by acting as a potential IRE1α endoribonuclease inhibitor.

Inositol requiring enzyme 1 alpha (IRE1α) is one of three signaling sensors in the unfolding protein response (UPR) that alleviates endoplasmic reticulum (ER) stress in cells and functions to promote cell survival. During conditions of irrevocable stress, proapoptotic gene expression is induced to promote cell death. One of the three signaling stressors, IRE1α is an serine/threonine-protein kinase/endoribonuclease (RNase) that promotes nonconventional splicing of XBP1 mRNA that is translated to spliced XBP1 (XBP1s), an active prosurvival transcription factor. Interestingly, elevated IRE1α and XBP1s are both associated with poor cancer survival and drug resistance. In this study, we used next-generation sequencing analyses to demonstrate that triazoloacridone C-1305, a microtubule stabilizing agent that also has topoisomerase II inhibitory activity, dramatically decreases XBP1s mRNA levels and protein production during ER stress conditions, suggesting that C-1305 does this by decreasing IRE1α's endonuclease activity.

Utilizing Genome-Wide mRNA Profiling to Identify the Cytotoxic Chemotherapeutic Mechanism of Triazoloacridone C-1305 as Direct Microtubule Stabilization.

Rational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often difficult due to limited information regarding the complete identification of a molecule's biological activities. The increasing affordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound's diverse effects on gene regulation. Here, we used an unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for delineating the antitumor mechanisms of other putative anticancer drug candidates.

Unfolded protein response (UPR) integrated signaling networks determine cell fate during hypoxia.

During hypoxic conditions, cells undergo critical adaptive responses that include the up-regulation of hypoxia-inducible proteins (HIFs) and the induction of the unfolded protein response (UPR). While their induced signaling pathways have many distinct targets, there are some important connections as well. Despite the extensive studies on both of these signaling pathways, the exact mechanisms involved that determine survival versus apoptosis remain largely unexplained and therefore beyond therapeutic control. Here we discuss the complex relationship between the HIF and UPR signaling pathways and the importance of understanding how these pathways differ between normal and cancer cell models.

Lipidated Analogs of the LL-37-Derived Peptide Fragment KR12-Structural Analysis, Surface-Active Properties and Antimicrobial Activity.

An increasing number of multidrug-resistant pathogens is a serious problem of modern medicine and new antibiotics are highly demanded. In this study, different n-alkyl acids (C2-C14) and aromatic acids (benzoic and trans-cinnamic) were conjugated to the N-terminus of KR12 amide. The effect of this modification on antimicrobial activity (ESKAPE bacteria and biofilm of Staphylococcus aureus) and cytotoxicity (human red blood cells and HaCaT cell line) was examined. The effect of lipophilic modifications on helicity was studied by CD spectroscopy, whereas peptide self-assembly was studied by surface tension measurements and NMR spectroscopy. As shown, conjugation of the KR12-NH2 peptide with C4-C14 fatty acid chains enhanced the antimicrobial activity with an optimum demonstrated by C8-KR12-NH2 (MIC 1-4 µg/mL against ESKAPE strains; MBEC of S. aureus 4-16 µg/mL). Correlation between antimicrobial activity and self-assembly behavior of C14-KR12-NH2 and C8-KR12-NH2 has shown that the former self-assembled into larger aggregated structures, which reduced its antimicrobial activity. In conclusion, N-terminal modification can enhance antimicrobial activity of KR12-NH2; however, at the same time, the cytotoxicity increases. It seems that the selectivity against pathogens over human cells can be achieved through conjugation of peptide N-terminus with appropriate n-alkyl fatty and aromatic acids.

Genome-wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress.

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol-requiring protein 1, protein kinase RNA-like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human-immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18-h time course and utilized real-time monitoring of cell survival, next-generation sequencing, and quantitative real-time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT-enhancer-binding protein homologous protein (a.k.a DDIT3), BCL2-binding component 3 (a.k.a. BBC3), and phorbol-12-myristate-13-acetate-induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

Effect of self-assembly on antimicrobial activity of double-chain short cationic lipopeptides.

Short cationic antimicrobial lipopeptides with surfactant-like structure are promising antibiotic candidates that preferentially target microbial membranes. Therefore, we focused our study on double-chain lipopeptides, (C10-16)2Dab-KKK-NH2 and (C10-16)2Dap-KKK-NH2, where Dab and Dap are 2,4-diaminobutyric and 2,3-diaminopropionic acids, respectively. We tried to answer a question how the self-assembly behaviour affects biological activities of the tested compounds. The subject compounds were synthesized by solid-phase method and screened for their antimicrobial and haemolytic activities. Cytotoxicity tests on human keratinocytes were carried out for the most promising lipopeptides. Self-assembly properties were evaluated by both experimental and theoretical methods. Interactions with membrane models were examined using the ITC and FTIR techniques. All the lipopeptides studied showed the tendency to self-assembly in solution, and this behaviour was affected by the length of the hydrocarbon chains. Acyl chain elongation supported the formation of the bilayer structure and deprived the lipopeptides of antimicrobial activity. A multi-step mechanism of interaction with a negatively charged membrane was observed for the short-chain lipopeptides, indicating other processes accompanying the binding process. Short-chain lipopeptides were able to penetrate into the liposome's interior and/or cause the rupture of the liposome, this being compatible with their high antimicrobial activity.

miR-34c-5p modulates X-box-binding protein 1 (XBP1) expression during the adaptive phase of the unfolded protein response.

During endoplasmic reticulum (ER) stress conditions, an adaptive signaling network termed the unfolded protein response (UPR) is activated. The UPR's function is to increase ER protein-folding capacity in order to attenuate ER stress, restore ER homeostasis, and, most importantly, promote cell survival. X-box-binding protein 1 (XBP1) is one component of the UPR and is a proadaptive transcription factor that is subject to transcriptional, post-transcriptional, and post-translational control. In the present study, we identified a post-transcriptional mechanism mediated by miR-34c-5p that governs the expression of both the spliced (active) and unspliced (latent) forms of XBP1 mRNAs. We showed that miR-34c-5p directly attenuates spliced XBP1 (XBP1s) mRNA levels during ER stress and thus regulates the proadaptive component of the UPR that is mediated by XBP1s without interfering with the induction of apoptotic responses.-Bartoszewska, S., Cabaj, A., Dabrowski, M., Collawn, J. F., Bartoszewski, R. miR-34c-5p modulates X-box-binding protein 1 (XBP1) expression during the adaptive phase of the unfolded protein response.

Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

During hypoxia, a cellular adaptive response activates hypoxia-inducible factors (HIFs; HIF-1 and HIF-2) that respond to low tissue-oxygen levels and induce the expression of a number of genes that promote angiogenesis, energy metabolism, and cell survival. HIF-1 and HIF-2 regulate endothelial cell (EC) adaptation by activating gene-signaling cascades that promote endothelial migration, growth, and differentiation. An HIF-1 to HIF-2 transition or switch governs this process from acute to prolonged hypoxia. In the present study, we evaluated the mechanisms governing the HIF switch in 10 different primary human ECs from different vascular beds during the early stages of hypoxia. The studies demonstrate that the switch from HIF-1 to HIF-2 constitutes a universal mechanism of cellular adaptation to hypoxic stress and that HIF1A and HIF2A mRNA stability differences contribute to HIF switch. Furthermore, using 4 genome-wide mRNA expression arrays of HUVECs during normoxia and after 2, 8, and 16 h of hypoxia, we show using bioinformatics analyses that, although a number of genes appeared to be regulated exclusively by HIF-1 or HIF-2, the largest number of genes appeared to be regulated by both.-Bartoszewski, R., Moszynska, A., Serocki, M., Cabaj, A., Polten, A., Ochocka, R., Dell'Italia, L., Bartoszewska, S., Króliczewski, J., Dabrowski, M., Collawn, J. F. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.