Fractionation Experiments Research Articles

Synchronizing the release rates of topotecan and paclitaxel from a self-eroding crosslinked chitosan – PLGA platform

This study is a continuation of a previous study in which two model drugs, sodium salicylate (highly water-soluble) and indomethacin (low water-soluble) were loaded into an erodible hydrogel, made of ionically crosslinked chitosan (x-Ct). The erosion rate of the x-Ct matrix was controlled by its immersion in calcium chloride solutions (de-crosslinker) of different concentrations, leading to synchronization of the release rates of the two drugs over 2 h. In the present study, a modified platform was developed in order to (a) synchronize the release rates of the two cytotoxic drugs, topotecan (TT, highly water soluble) and paclitaxel (PTX, poorly water soluble); (b) prolong the erosion duration and the derived concomitant release of the two drugs to several days. TT was loaded into a PLGA sphere, which was co-loaded with calcium chloride (CaCl2). The sphere was then placed in an aqueous solution of chitosan (Ct) in which PTX was dispersed. A PLGA core-containing hydrogel was then produced by ionically crosslinking the Ct. The formulation screening section of the study includes a statistically designed Fractional Factorial experiment. It was comprised of the following five experimental factors: (a) the type of Ct and (b) its relative amount in the formulation, (c) the type of ionic crosslinker (citric acid or oxalic acid), (d) the concentration of the ionic crosslinker and (e) the co-loaded amounts of CaCl2 (the constitutional de-crosslinking agent). The difference factor, f1, and the similarity factor, f2, of the TT and PTX release profiles into water, were used as the experimental responses. The computerized prediction models were employed to assess the collective effects of the pre-determined experimental factors on the difference factor, f1, and the similarity factor, f2 (the response factors), by employing a fractional factorial design and multifactorial analysis, without the need to account for the exact mechanisms of the release processes involved. The final composite platform was capable of releasing TT and PTX, at similar (concomitant) rates, over a period of 7 days, a finding which suggests that the novel polymeric platform may serve as a multi-drug implant. An attractive medical application for such a device would be post-operative local treatment that could benefit from localized combination chemotherapy after the removal of malignant tissues, in the surgical treatment of breast cancer, ovarian cancer, glioma and peritoneal carcinomatosis.

Characterization of nitrogen isotope fractionation during nitrification based on a coastal time series

AbstractIsotopic enrichment factors are key to using stable isotope signatures in biogeochemical studies. However, these are typically determined in laboratory experiments and their applicability to environmental conditions is difficult to test. Here, we analyzed nitrogen stable isotope changes associated with nitrification in a coastal basin using weekly time‐series measurements of δ15N in particulate nitrogen, ammonium, nitrite, and nitrate. Two year‐long time series were selected as contrasting natural experiments in the ammonium‐rich, aphotic bottom water of Bedford Basin, Nova Scotia, Canada. In 2014, ammonia oxidation (AO) was associated with Thaumarchaeota and nitrite concentrations remained low (< 0.5 μmol kg−1). In contrast, transient nitrite accumulation (~ 8 μmol kg−1) and a more rapid δ15NNH4 increase in the fall of 2017 were likely caused by ammonia‐oxidizing bacteria, associated with higher AO rates and, possibly, stronger nitrogen‐isotope enrichment (15εAO). Estimates of 15εAO (21.8 ± 2.2‰, 24.1 ± 1.1‰) were derived empirically using Rayleigh models applied to field data from restricted periods during which the bottom waters approximated a closed system and influence on 15εAO from other processes was demonstrably insignificant. Using a numerical reactive‐transport model, we found that the best fit for the δ15N data was obtained with 15εAO values (18.9‰, 25.1‰) close to those determined by the Rayleigh models. The time series also revealed substantial (~ 7‰) 15N‐enrichment of the particulate nitrogen due to light‐independent assimilation of partially nitrified ammonium. Consistent with previous studies, these field‐based nitrogen isotope fractionation experiments suggest that the range of 15εAO values relevant for marine systems may be narrower than determined in laboratory studies.

Abstract 3307: Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition

Abstract Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR inhibition as a strategy for radiosensitization in AR+ TNBC. Despite its role in radioresistance in AR+ TNBC, the mechanistic role of AR and specifically its role in mediating DNA damage repair in response to radiation therapy (RT) remains unknown. Methods: Nuclear fractionation experiments were performed to assess cellular localization of AR protein in AR+ TNBC cell lines (ACC-422, MDA-MB-453). Cells were cultured in media containing hormones (FBS) with treatment of enzalutamide (ENZA), apalutamide (APA), or darolutamide (DARO). Cells were alternatively cultured in media containing charcoal stripped serum (CSS) without hormones with R1881 stimulation. RNA-sequencing was performed to compare AR+ TNBC cells treated with CSS or R1881 stimulation alone or in combination with ionizing radiation. Reverse phase protein arrays were performed in cells treated with ENZA, RT, or combination treatment. Results: While stimulation with R1881 was sufficient to induce nuclear translocation of AR in MDA-MB-453 cells, AR inhibition with ENZA, APA, or DARO blocked AR nuclear translocation under CSS or FBS growth conditions. When cells were treated with R1881+RT, AR nuclear translocation was induced at similar or greater levels compared to R1881 alone in MDA-MB-453 and ACC-422 cells. Combination treatment of RT with ENZA in the presence of hormones reduced AR nuclear localization (39% reduction in MDA-MB-453 cells and 32% reduction in ACC-422 cells) compared to RT alone. These results suggest that decreased promoter region binding, and gene expression upregulation may be a mechanism of radiosensitization with AR inhibition. In addition, transcriptomic analyses demonstrated at least 979 genes differentially expressed in multiple models. Pathway analyses in these models showed common affected pathways included ECM-receptor interaction, PPAR-gamma activation, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway. Proteomic analysis in the same cell lines identified apoptosis, DNA damage, and cell cycle pathway changes after RT when AR-signaling was blocked. Common affected pathways in combined analyses identified PI3K-Akt and MAPK/ERK signaling pathway changes that may be responsible for this radiosensitizing phenotype. Conclusions: Our data suggest that AR inhibition in AR+ TNBC is sufficient to inhibit AR nuclear translocation suggesting that AR may play a nuclear role in response to RT to promote DNA repair and radioresistance. We identify potential pathways, including ECM-receptor interaction, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway that may be regulated by AR in response to RT and therefore may be responsible for radioresistance. Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Leah Moubadder, Stephanie The, Breanna McBean, Caleb Cheng, Lori J. Pierce, Corey W. Speers. Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3307.

Focal Adhesion Kinase (FAK)-Hippo/YAP transduction signaling mediates the stimulatory effects exerted by S100A8/A9-RAGE system in triple-negative breast cancer (TNBC)

BackgroundUnderstanding the intricate signaling network involved in triple-negative breast cancer (TNBC) represents a challenge for developing novel therapeutic approaches. Here, we aim to provide novel mechanistic insights on the function of the S100A8/A9-RAGE system in TNBC.MethodsTNM plot analyzer, Kaplan-Meier plotter, Meta-analysis, GEPIA2 and GOBO publicly available datasets were used to evaluate the clinical significance of S100A8/A9 and expression levels of S100A8/A9, RAGE and Filamin family members in breast cancer (BC) subtypes. METABRIC database and Cox proportional hazard model defined the clinical impact of high RAGE expression in BC patients. Multiple bioinformatics programs identified the main enriched pathways within high RAGE expression BC cohorts. By lentiviral system, TNBC cells were engineered to overexpress RAGE. Western blotting, immunofluorescence, nucleus/cytoplasm fractionation, qRT-PCR, gene silencing and luciferase experiments were performed to identify signal transduction mediators engaged by RAGE upon stimulation with S100A8/A9 in TNBC cells. Proliferation, colony formation and transwell migration assays were carried out to evaluate the growth and migratory capacity of TNBC cells. Statistical analysis was performed by ANOVA and independent t-tests.ResultsWe found a remarkable high expression of S100A8 and S100A9 in BC, particularly in HER2-positive and TNBC, with the latter associated to worst clinical outcomes. In addition, high RAGE expression correlated with a poor overall survival in BC. Next, we determined that the S100A8/A9-RAGE system triggers FAK activation by engaging a cytoskeleton mechanosensing complex in TNBC cells. Through bioinformatics analysis, we identified the Hippo pathway as the most enriched in BC patients expressing high RAGE levels. In accordance with these data, we demonstrated the involvement of S100A8/A9-RAGE-FAK signaling in the control of Hippo/YAP activities, and we established the crucial contribution of RAGE-FAK-YAP circuitry in the growth and migratory effects initiated by S100A8/A9 in TNBC cells.ConclusionsThe present study provides novel mechanistic insights on RAGE actions in TNBC. Moreover, our findings suggest that RAGE-FAK-YAP transduction pathway could be exploited as a druggable system halting the aggressive TNBC subtype.

Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level.

O-GlcNAcylation is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. Only two enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase), control the attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells and RAW 264.7 cells, as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a Bioluminescence Resonance Energy Transfer (BRET)-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we show that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA has no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.

Interventions to optimise the outputs of national clinical audits to improve the quality of health care: a multi-method study including RCT

Background National clinical audit programmes aim to improve patient care by reviewing performance against explicit standards and directing action towards areas not meeting those standards. Their impact can be improved by (1) optimising feedback content and format, (2) strengthening audit cycles and (3) embedding randomised trials evaluating different ways of delivering feedback. Objectives The objectives were to (1) develop and evaluate the effects of modifications to feedback on recipient responses, (2) identify ways of strengthening feedback cycles for two national audits and (3) explore opportunities, costs and benefits of national audit participation in a programme of trials. Design An online fractional factorial screening experiment (objective 1) and qualitative interviews (objectives 2 and 3). Setting and participants Participants were clinicians and managers involved in five national clinical audits – the National Comparative Audit of Blood Transfusions, the Paediatric Intensive Care Audit Network, the Myocardial Ischaemia National Audit Project, the Trauma Audit & Research Network and the National Diabetes Audit – (objective 1); and clinicians, members of the public and researchers (objectives 2 and 3). Interventions We selected and developed six online feedback modifications through three rounds of user testing. We randomised participants to one of 32 combinations of the following recommended specific actions: comparators reinforcing desired behaviour change; multimodal feedback; minimised extraneous cognitive load for feedback recipients; short, actionable messages followed by optional detail; and incorporating ‘the patient voice’ (objective 1). Main outcome measures The outcomes were intended actions, including enactment of audit standards (primary outcome), comprehension, user experience and engagement (objective 1). Results For objective 1, the primary analysis included 638 randomised participants, of whom 566 completed the outcome questionnaire. No modification independently increased intended enactment of audit standards. Minimised cognitive load improved comprehension (+0.1; p = 0.014) and plans to bring audit findings to colleagues’ attention (+0.13, on a –3 to +3 scale; p = 0.016). We observed important cumulative synergistic and antagonistic interactions between modifications, participant role and national audit. The analysis in objective 2 included 19 interviews assessing the Trauma Audit Research Network and the National Diabetes Audit. The identified ways of strengthening audit cycles included making performance data easier to understand and guiding action planning. The analysis in objective 3 identified four conditions for effective collaboration from 31 interviews: compromise – recognising capacity and constraints; logistics – enabling data sharing, audit quality and funding; leadership – engaging local stakeholders; and relationships – agreeing shared priorities and needs. The perceived benefits of collaboration outweighed the risks. Limitations The online experiment assessed intended enactment as a predictor of actual clinical behaviour. Interviews and surveys were subject to social desirability bias. Conclusions National audit impacts may be enhanced by strengthening all aspects of feedback cycles, particularly effective feedback, and considering how different ways of reinforcing feedback act together. Future work Embedded randomised trials evaluating different ways of delivering feedback within national clinical audits are acceptable and may offer efficient, evidence-based and cumulative improvements in outcomes. Trial registration This trial is registered as ISRCTN41584028. Funding details This project was funded by the National Institute for Health and Care Research (NIHR) Health and Social Care Delivery Research programme and will be published in full in Health and Social Care Delivery Research; Vol. 10, No. 15. See the NIHR Journals Library website for further project information.

The area under the effect curve as an efficacy determinant for anti-infectives.

Pharmacokinetic/pharmacodynamic (PK/PD) indices making use of area under the curve, maximum concentration, and the duration that in vivo drug concentration is maintained above a critical level are commonly applied to clinical dose prediction from animal efficacy experiments in the infectious disease arena. These indices make suboptimal use of the nonclinical data, and the prediction depends on the shape of the PK profiles in the animals, determined by the species‐specific absorption, distribution, metabolism, and elimination properties, and the dosing regimen used in the efficacy experiments. Motivated by the principle that efficacy is driven by pharmacology, we conducted simulations using a generalized pathogen dynamic model, to assess the properties of an alternative efficacy predictor: the area under the effect curve (AUEC), computed using in vitro PD and in vivo PK. Across a wide range of hypothetical scenarios, the AUEC consistently showed regimen‐independent strong correlation (R 2 0.76–0.98) with in vivo efficacy, superior to all other indices. These findings serve as proof of concept that AUEC should be considered in practice as a translation tool for cross‐species dose prediction. Using AUEC for clinical dose prediction could also potentially cut down animal use by reducing or avoiding dose fractionation experiments.

A randomised fractional factorial screening experiment to predict effective features of audit and feedback

BackgroundAudit and feedback aims to improve patient care by comparing healthcare performance against explicit standards. It is used to monitor and improve patient care, including through National Clinical Audit (NCA) programmes in the UK. Variability in effectiveness of audit and feedback is attributed to intervention design; separate randomised trials to address multiple questions about how to optimise effectiveness would be inefficient. We evaluated different feedback modifications to identify leading candidates for further “real-world” evaluation.MethodsUsing an online fractional factorial screening experiment, we randomised recipients of feedback from five UK NCAs to different combinations of six feedback modifications applied within an audit report excerpt: use effective comparators, provide multimodal feedback, recommend specific actions, provide optional detail, incorporate the patient voice, and minimise cognitive load. Outcomes, assessed immediately after exposure to the online modifications, included intention to enact audit standards (primary outcome, ranked on a scale of −3 to +3, tailored to the NCA), comprehension, user experience, and engagement.ResultsWe randomised 1241 participants (clinicians, managers, and audit staff) between April and October 2019. Inappropriate repeated participant completion occurred; we conservatively excluded participant entries during the relevant period, leaving a primary analysis population of 638 (51.4%) participants.None of the six feedback modifications had an independent effect on intention across the five NCAs. We observed both synergistic and antagonistic effects across outcomes when modifications were combined; the specific NCA and whether recipients had a clinical role had dominant influences on outcome, and there was an antagonistic interaction between multimodal feedback and optional detail. Among clinical participants, predicted intention ranged from 1.22 (95% confidence interval 0.72, 1.72) for the least effective combination in which multimodal feedback, optional detail, and reduced cognitive load were applied within the audit report, up to 2.40 (95% CI 1.88, 2.93) for the most effective combination including multimodal feedback, specific actions, patient voice, and reduced cognitive load.ConclusionPotentially important synergistic and antagonistic effects were identified across combinations of feedback modifications, audit programmes, and recipients, suggesting that feedback designers must explicitly consider how different features of feedback may interact to achieve (or undermine) the desired effects.Trial registrationInternational Standard Randomised Controlled Trial Number: ISRCTN41584028

Application of experiment planning in the development of a nutrient medium in the production cycle of cultivation of <i>G.Mellonella</i>

Relevance. The study aimed to develop the composition of an artificial nutrient medium for the rearing of Gallery mellonella larvae. In the process of the research, the relative contributions of the feed component to the biological growth parameters of larvae and their morphological indicators were determined.Methods. In the experiment the plan 27-4 was used, that is, fractional factor experiments were varied in which seven factors — feed ingredients. The matrix of the complete factor experiment 23 was taken as a basis, and the coefficients in the interaction of three or more factors were assumed to be insignificant and replaced by additional factors. Morphophysiological parameters of larvae were used as the main indicators: mass, length, width of the head capsule.Results To obtain biomass in the production cycle, the mass of larvae should be at least 0.15 g, length 20-22 mm, width of the head capsule 1.9-2.3 mm (VI-early VII age), survival rate of at least 85%. In the regression equation for larval masses, wheat flour (X1), yeast (X3) and honey (X5) turned out to be significant ingredients. In the regression equation for the length of the larvae, the ingredients that make the maximum contribution are again yeast (X3) and honey (X5). Yeast (X3) was significant for the width of the head capsule. The obtained regression equations enable mathematical modeling within the framework of a linear model represented by regression equations in the studied range of morphological parameters of larvae, depending on the composition of the feed.

Delta- T noise for weak tunneling in one-dimensional systems: Interactions versus quantum statistics

We study delta-T noise -- excess charge noise at zero voltage but finite temperature bias -- for weak tunneling in 1D interacting systems. We show that the sign of the delta-T noise is generically determined by the nature of the dominating tunneling process (more specifically, its scaling dimension). We clarify the relation between the sign of delta-T noise and the quantum exchange statistics of tunneling quasiparticles. We find that, for infinite systems hosting chiral channels with local interactions (e.g., quantum Hall or quantum spin Hall edges), when the delta-T noise is negative, the tunneling particles are boson-like, revealing their tendency towards bunching. However, the opposite is not true: Boson-like particles do not necessarily produce negative delta-T noise. Importantly, the bosonic nature of particles generating the negative delta-T is not necessarily intrinsic, but can be induced by the interactions. This particularly implies that negative delta-T noise for tunneling between the edge states cannot serve as a smoking gun for detecting "intrinsic anyons". We also establish a connection between the delta-T noise and the temperature derivative of the thermal noise in interacting systems, both governed by the same scaling dimensions. As a demonstration of the above statements, we study tunneling between two interacting quantum spin Hall edges. With bosonization and renormalization-group techniques, we find that many-body interactions can generate negative delta-T noise for both direct tunneling through a point contact and in Kondo exchange tunneling via a localized spin. In both setups, we show that the noise can become negative at sufficiently low temperatures, when interactions renormalize the tunneling to favor boson-like pair-tunneling of electrons. Our findings show that delta-T noise can probe the nature of collective excitations in interacting 1D systems.

A Method to Estimate the Distribution of Proteins across Multiple Compartments Using Data from Quantitative Proteomics Subcellular Fractionation Experiments.

Knowledge of cellular location is key to understanding the biological function of proteins. One commonly used large-scale method to assign cellular locations is subcellular fractionation, followed by quantitative mass spectrometry to identify proteins and estimate their relative distribution among centrifugation fractions. In most of such subcellular proteomics studies, each protein is assigned to a single cellular location by comparing its distribution to those of a set of single-compartment reference proteins. However, in many cases, proteins reside in multiple compartments. To accurately determine the localization of such proteins, we previously introduced constrained proportional assignment (CPA), a method that assigns each protein a fractional residence over all reference compartments (Jadot Mol. Cell Proteomics 2017, 16(2), 194-212. 10.1074/mcp.M116.064527). In this Article, we describe the principles underlying CPA, as well as data transformations to improve accuracy of assignment of proteins and protein isoforms, and a suite of R-based programs to implement CPA and related procedures for analysis of subcellular proteomics data. We include a demonstration data set that used isobaric-labeling mass spectrometry to analyze rat liver fractions. In addition, we describe how these programs can be readily modified by users to accommodate a wide variety of experimental designs and methods for protein quantitation.

Delineating the mechanistic pathway and therapeutic potential of Siglec-6 in Chronic Lymphocytic Leukemia

Abstract Sialic acid-binding immunoglobulin-like lectins (Siglec) regulate immune function via glycan recognition. Here, we show for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in chronic lymphocytic leukemia (CLL). Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ CLL− bone marrow stromal cells. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a Siglec-6+ CLL mouse model (Siglec-6 x TCL1). Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating in vivo efficacy of Siglec-6/CD3 targeted bispecific antibody. Natarajan Muthusamy: NIH-R21 Grant and Pelotonia Idea grant Jessica Nunes: Pelotonia Graduate Fellowship

G9a Responds to KRAS‐Mediated Replication Stress by Increased Origin Licensing

Pancreatic Ductal Adenocarcinoma (PDAC) remains as one of the leading causes of cancer death in the US. KRAS mutations are recognized as the leading oncogenic driver of PDAC growth. Activation of KRAS causes increased proliferation that requires a level of adaptation during processes, such as DNA replication. This rapid duplication of the genome and epigenome generates havoc in the timing and coordination of replication origin firing, triggering a response known as replication stress (RS). The epigenomic regulator G9a is responsible for catalyzing histone H3 lysine 9 mono‐ and di‐methylation (H3K9me1 and H3K9me2). G9a is abundantly present in many forms of cancer, including PDAC, and because of its interaction with replication proteins, such as RPA and PCNA, it is known to have an important role during DNA replication. DNA replication happens at the S phase of the cell cycle, which requires that replication origins are recognized at late G1 phase by the origin recognition complex (ORC) followed by assembly of the pre‐replication complex (pre‐RC), comprised of CDC6, cdt1 and mini chromosome maintenance (MCM) proteins, to allow origin licensing and subsequent firing. We have shown that the G9a complex increases in response to KRAS activation and is critical to the growth‐promoting effects of this oncogene in PDAC. Though it is known that KRAS reduces distances between replication origins, there has been a paucity of knowledge regarding the role of G9a during replication origin licensing and whether it is directly involved with the pre‐RC. In the current study, we aim to better understand whether G9a interacts with the replication origins and the pre‐RC to increase origin licensing under conditions of KRAS‐mediated RS. To simulate the early events in oncogene‐induced RS to study compensatory mechanisms, we generated an inducible KRAS model (iKRAS‐HPNE) in an hTERT‐immortalized human non‐cancerous pancreatic ductal cell line, hTERT‐HPNE E6/E7. We found that KRAS induction for 48h in this iKRAS‐HPNE model activated the ATR RS‐response pathway, indicated by increased levels of P‐T1989‐ATR, P‐S345‐CHK1 and P‐S33‐RPA32 by western blot and immunofluorescence‐based microscopy. Concurrently, members of the G9a epigenomic complex (G9a, GLP and WIZ) significantly increased along with two subunits of the ORC (ORC1 and ORC2) and several other pre‐RC proteins (Cdt1, MCM2, MCM5 and MCM7). Co‐immunoprecipitation of G9a in the iKRAS‐HPNE cells revealed that G9a interacts with ORC2 protein under conditions of oncogene‐induced RS. Pharmacological inhibition of G9a with UNC0642 during KRAS‐induced RS resulted in a decrease of chromatin‐bound ORC1 and ORC2 as well as pre‐RC proteins in subcellular fractionation experiments, suggesting that functional G9a is critical for enhanced origin licensing. In summary, our results demonstrate that G9a directly interacts with replication origins and the pre‐RC during origin licensing under conditions of RS. Our study reveals one potential mechanism by which cancerous cells with KRAS mutation meet the demands for rapid cellular proliferation during RS and reinforces the role of G9a as a promising therapeutic target for PDAC.

Implication of Alkane Carbon and Hydrogen Isotopes for Genesis and Accumulation of Over-Mature Shale Gas: A Case Study of Longmaxi Formation Shale Gas in Upper Yangtze Area

To clarify the implication of alkane carbon and hydrogen isotopes for the genesis and accumulation of over-mature shale gas, we carried out a comparative study on Longmaxi shale gases from eight blocks in the Upper Yangtze area. The results show that the δ13CCH4, δ13CC2H6, and δ13CC3H8 of Longmaxi shale gas are all positively correlated with Ro. According to the distribution model of δ13C with thermal maturity, the Longmaxi shale gas lies in the reversal stage. Shale gas is a mixture of the kerogen cracking gas and secondary cracking gas, and the mixing ratio of the two cracking gas can be estimated by isotopic fractionation experiments of thermogenic gas. The proportion of secondary cracking gas in the shale gas of the Longmaxi Formation ranges from 33 to 72%. The increase of secondary cracking gas with lower δ13C would reduce the carbon isotope of the shale gas. The δ13CC2H6 and δ13CC3H8 have acute sensitivity to the occurrence of secondary cracking gas, hence they can be used as potential indicators of shale gas content. The decline of gas generation capacity, the reduction of micropores, and the destruction of tectonic movement are the considerable factors leading to the decrease of gas content in high-maturity shale.

Anti-β2-GPI Antibodies Induce Endothelial Cell Expression of Tissue Factor by LRP6 Signal Transduction Pathway Involving Lipid Rafts.

In this study we analyzed whether anti-β2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-β2-GPI antibodies. Both LRP6 and β-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-β2-GPI antibodies induced LRP6 phosphorylation with consequent β-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-β-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-β2-GPI antibodies effect, indicating that the anti-β2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between β2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-β2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-β2-GPI binding triggers β-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).

The role of intermolecular interactions on melt memory and thermal fractionation of semicrystalline polymers.

The origin of melt memory effects associated with semicrystalline polymers and the physical parameters involved in this process have been widely studied in the literature. However, a comprehensive understanding of the role of intermolecular interactions on melt memory is still being developed. For this purpose, we have considered aliphatic polyesters and we have incorporated amide and additional ester groups. Inserting these additional functional groups, the strength of the intermolecular interactions increases widening the melt memory effect. Not only the presence of the functional groups but also the position of these groups in the repeating unit plays a role in the melt memory effect as it impacts the strength of the intermolecular interactions in the crystals. The study of the effect of intermolecular interactions has been extended to successive self-nucleation and annealing thermal fractionation experiments to explore for the first time the role of intermolecular forces on the fractionation capacity of linear polymers. We demonstrated that intermolecular interactions act as intrinsic defects interrupting the crystallizable chain length, thus facilitating thermal fractionation. Overall, this work sheds light on the role of intermolecular interactions on the crystallization behavior of a series of aliphatic polyesters.

Molecular characterization of nitrogen-containing organic compounds in fractionated atmospheric humic-like substances (HULIS) and its relationship with optical properties

Diverse nitrogen-containing organics are important components of humic-like substances (HULIS) in the atmosphere. In this study, organic components in particulate matter (PM) samples representing multiple sources were separated by successive solvent fractionation, which were then analyzed by mass spectrometric and optical instruments. The CHON compounds were eluted and clustered into the Low-polar, Medium-polar, and High-polar fractions, and discrepancies of the polar-fractions were particularly reflected by molecular descriptors such as aromaticity, oxygen content and molecular weight. In addition, the results from the light-absorbing parameters (i.e., MAE365 and SUVA254) underscored the importance of the Low-polar and High-polar fractions on optical absorption properties. The Low-polar fraction accounted for 40% of the cumulative SUVA254 values, suggesting significant content of ultraviolet-absorbing organics. The High-polar fraction contributed 52% of the cumulative MAE365 values, indicating abundant light absorption capacity and efficiency. Significant improvements were made on statistical analysis of multidimensional data by a combination of the molecular descriptors and optical parameters. Molecular structures, including condensed aromatic, lignin-like, and aliphatic compounds observed in distinct electrospray ionization modes, were found as main contributors to the light absorption capacity and the abundances of fluorophores in individual polar-fractions. Differential contributions of molecular characteristics on types and abundances of fluorophores were further found among the samples of multiple sources. Conclusions obtained from this successive solvent fractionation experiment could promote development of the pretreatment method for exploring the potential light-absorbing organics, which also provide insights into the emission sources of organics that are related to specific light absorption and fluorescence properties.

CircRNA circ_0023984 promotes the progression of esophageal squamous cell carcinoma via regulating miR-134-5p/cystatin-s axis

ABSTRACT Recent studies have shown that circRNAs can act as oncogenic factors or tumor suppressors by sponging microRNAs (miRNAs). The upregulation of circ_0023984 was reported in esophageal squamous cell carcinoma (ESCC). However, its functional role in ESCC remain unclear. In the present study, circ_0023984 expression in ESCC cells and tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). Subcellular fraction experiment was performed to determine relative nuclear-cytoplasmic localization. The loss-of-function effects of circ_0023984 in ESCC cell lines were investigated by shRNA-mediated knockdown. Functional assays including cell Counting Kit-8 (CCK-8), 5-Ethynyl-2’-deoxyuridine (EDU) incorporation, colony formation and Transwell migration assays were conducted to assess the malignant phenotype. The interaction between the two molecules was analyzed by RNA pull-down, luciferase reporter assay and RNA immunoprecipitation (RIP). The subcutaneous tumor model in nude mice was used to assess the role of circ-0023984 in tumorigenesis. We found that ESCC patients with high circ_0023984 expression was associated with a poor prognosis. The knockdown of circ_0023984 suppressed cell growth, invasion, and migration in ESCC cells. Circ_0023984 interacted with miR-134-5p and inhibited its activity, which promoted the expression of CST4 (Cystatin-S). Circ_0023984 also regulated tumorigenesis in a CST4-dependent manner. Together, our study indicates that the oncogenic role of Circ_0023984 is mediated by miR-134-5p/CST4 Axis in ESCC, which could serve as potential targets for future therapeutic strategies.

Circ_SAR1A regulates the malignant behavior of lung cancer cells via the miR-21-5p/TXNIP axis.

BackgroundLung cancer is one of the most common malignancies globally and a significant component of cancer‐related deaths. The lack of early diagnosis accounts for detecting approximately 75% of cancer patients at an intermediate to an advanced stage, with a low 5‐year survival rate. Therefore, a more comprehensive understanding of the molecular mechanisms of lung cancer development is necessary to find reliable and effective therapeutic and diagnostic biomarkers.Methodscirc_SAR1A, miR‐21‐5p, and TXNIP in lung cancer tissues, animal xenografts, and cell lines were validated by qRT‐PCR and western blotting analyses. RNase R digestion and nuclear/cytoplasm fractionation experiments were utilized to determine the stability and localization of circ_SAR1A in lung cancer cells. The binding between miR‐21‐5p and circ_SAR1A or TXNIP was confirmed by luciferase reporter, RNA pull‐down, Spearman's correlation, and rescue assays. CCK‐8, colony formation, flow cytometry, Transwell, and western blotting were utilized to illustrate the malignant behavior of lung cancer cells.Resultscirc_SAR1A and TXNIP were down‐regulated while miR‐21‐5p was up‐regulated in lung cancer samples and cells. circ_SAR1A was located predominantly in the cytoplasm; it inhibited lung cancer growth in vitro and in vivo by sponging to miR‐21‐5p. miR‐21‐5p silencing suppressed lung cancer malignancy by targeting TXNIP.Conclusionscirc_SAR1A is a critical negative regulator of lung carcinogenesis. circ_SAR1A/miR‐21‐5p/TXNIP attenuation inhibited lung cancer progression, presenting an ideal diagnostic and a potential therapeutic target.

Mycobacterium tuberculosis EspK Has Active but Distinct Roles in the Secretion of EsxA and EspB.

The Mycobacterium tuberculosis type-7 protein secretion system ESX-1 is a major driver of its virulence. While the functions of most ESX-1 components are characterized, many others remain poorly defined. In this study, we examined the role of EspK, an ESX-1-associated protein that is thought to be dispensable for ESX-1 activity in members of the Mycobacterium tuberculosis complex. We show that EspK is needed for the timely and optimal secretion of EsxA and absolutely essential for EspB secretion in M. tuberculosis Erdman. We demonstrate that only the EsxA secretion defect can be alleviated in EspK-deficient M. tuberculosis by culturing it in media containing detergents like Tween 80 or tyloxapol. Subcellular fractionation experiments reveal EspK is exported by M. tuberculosis in an ESX-1-independent manner and localized to its cell wall. We also show a conserved W-X-G motif in EspK is important for its interaction with EspB and enabling its secretion. The same motif, however, is not important for EspK localization in the cell wall. Finally, we show EspB in EspK-deficient M. tuberculosis tends to adopt higher-order oligomeric conformations, more so than EspB in wild-type M. tuberculosis. These results suggest EspK interacts with EspB and prevents it from assembling prematurely into macromolecular complexes that are presumably too large to pass through the membrane-spanning ESX-1 translocon assembly. Collectively, our findings indicate M. tuberculosis EspK has a far more active role in ESX-1-mediated secretion than was previously appreciated and underscores the complex nature of this secretion apparatus. IMPORTANCE Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion. Culturing EspK-deficient M. tuberculosis with detergents alleviates EsxA but not EspB secretion defects. We also show that EspK, which is exported by M. tuberculosis in an ESX-1-independent manner to the cell wall, interacts with and prevents EspB from assembling into large structures inside the M. tuberculosis cell that are nonsecretable. Collectively, our observations demonstrate EspK is an active component of the ESX-1 secretion machinery of the tubercle bacillus.