Enzyme Secretion Research Articles

Cumulative and potential synergistic effects of seven different bisphenols on human granulosa cells in vitro?

Bisphenol (BP) structural analogues of BPA are widely used. Previous studies showed similar effects of BPA and BPS on reproduction in several species including human. We hypothesised that the similar effects of several bisphenols (BPs) could accumulate in granulosa cells (GCs) and affects steroidogenesis. This study investigated the effects of seven BP analogues and their equimolar cocktail on human granulosa cells (hGC) and assessed BPA, BPS, BPF and BPAF level exposures in the follicular fluid of 277 women undergoing Assisted Reproductive Technology. The hGCs were recovered after women oocyte punctures and treated with the seven BP analogues (BPS, BPA, BPAF, BPF, BPAP, BPE and BPB) or their equimolar cocktail of 7 × 1.43 or 7 × 7.14 μM for each of the seven BPs, the sum of BPs reaching 10 (”∑BPs 10 μM”), or 50 μM (”∑BPs 50 μM”), respectively. Oestradiol and progesterone secretion, cell proliferation, viability and expression of steroidogenic enzymes were investigated. Progesterone secretion was decreased by 6 BPs 10 μM and the cocktail “∑BPs 10 μM”, (−17.8 to −41.3%) and by all seven BPs 50 μM and “∑BPs 50 μM” (−21.8 to −84.2%). Oestradiol secretion was decreased only by 50 μM BPAF and BPAP (−37.8% and −44%, respectively), with corresponding decreases in CYP17A1 and CYP19A1 gene expression. Cellular proliferation was decreased after treatment with 50 μM BPAF (−32.2%), BPAP (−29%), BPB (−24%) and the equimolar cocktail “∑BPs 50 μM” (−33.1%). BPB (50 μM) and the cocktail “∑BPs 50 μM” increased HSD3B2 mRNA expression. At least one BP was detected in 64 of 277 (23.1%) women follicular fluids.Similar effects of the seven BPs or their cocktail were observed on progesterone secretion and/or on cell proliferation, suggesting cumulative effects of BPs. Our results highlight the urge to consider all BPs simultaneously and to further investigate the potential additive or synergistic effects of several BPs.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

Candida albicans in oral cavity

Candida albicans is a fungus that often colonizes the human body as a commensal without causing any symptoms. Several C. albicans virulence factors have been described in the literature, of which the best known are adhesion to host mucosa cells and secretion of hydrolytic enzymes. The transition from colonization to infection depends not only on the virulence of the fungus, but also on the response of the host to the invasion. This literature review focuses on the different forms of oral candidiasis, and discusses the symptoms, clinical picture and treatment of this disease.

Elucidating the immunological landscape of acute myocardial infarction using an integrated single-cell enzyme and antibody quantification (iSEAQ) assay

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Background Characterizing enzyme secretion with surface antigen expression of circulating leukocytes provides deep immunological insights that may benefit diagnostics and therapeutics of acute myocardial infarction (AMI). Current methods are limited by laborious sample preparation, lengthy assay time and limited analytical throughput. Purpose We aimed to develop a point-of-care platform for real-time, granular immune profiling of AMI during index hospitalization. Methods We developed a system for a 40-min assay termed integrated single-cell enzyme and antigen quantification (iSEAQ), consisting of an integrated microfluidic device for a streamlined process to recover and isolate single leukocytes from whole blood, and a customized 5-channel high-speed droplet fluorescence cytometer for sensitive quantification of key protein activity resembling live leukocyte activity in-vivo (Figure 1). Using a panel of 2 secreted enzymes (granzyme B (GzB) and neutrophil elastase (NE)) and 3 surface antigens (cluster of differentiation (CD) 66b, CD3 and CD31), we tracked the longitudinal shift of immune functionality of 9 patients admitted for acute ST-segment elevation myocardial infarction (STEMI) patients (median age 52 years, 100% males) at 3 timepoints during the index hospitalization: 1) on admission prior to coronary revascularization, 2) within 24 hours of revascularization, and 3) pre-discharge. The 5-channel fluorescence data were imported using a python script and pre-processed, before piping into Numpy, Pandas, Scipy and UMAP library for data preparation and classification. Seaborn and Matplotlib were used for all data visualization and plots. Data was analyzed using student two-tailed t-tests, with a significance level of <5%. Results Leveraging on its ability to dissect enzyme secretion by phenotype class, iSEAQ unraveled distinct contributions of CD31, NE and GzB from peripheral granulocytes, T lymphocytes and other cells (Figure 2). The majority immune response is expressed in CD66b+ granulocytes showing increased NE+ secretion and CD31 surface antigen expression denoted in Class 0 in Figure 2. Compared to samples from healthy donors and STEMI patients on admission, the NE+ and CD31+ expressions were significantly lower in pre-discharge samples of STEMI patients (p<0.05). iSEAQ also distinguished anterior from inferior STEMI, as evident from a significantly elevated NE+CD31-secretion for CD66b+ granulocytes in anterior STEMI (365±70 vs 83±35 fg, p < 0.001), showing preliminary promise in the ability of iSEAQ to risk stratify. Conclusion iSEAQ provided a comprehensive granular immune atlas of the longitudinal immune responses to STEMI during hospital admission. With its open design for assay panel using commercial biosensors, iSEAQ is a potentially powerful tool for taking functionality snapshots of immune system in many diseases.

StomachSim: An in-silico simulator of gastric biomechanics with application to pyloroplasty

Aim: We have developed StomachSim, an in-silico simulator that is designed to simulate the biomechanics and fluid mechanics of a wide range of gastric conditions as well as gastric surgical procedures. Here we demonstrate its use in quantifying the effects of motility disorders and pyloroplasty on the hydrolysis and emptying of a liquid meal inside the stomach. Methods: A physiologically realistic model of the stomach, including the antral motility, is synthesized based on in-vivo imaging, and the mixing and gastric emptying are simulated using computational fluid dynamics (CFD). The enzymatic biochemistry for protein hydrolysis is also implemented by incorporating the secretion of a proteolytic enzyme from the proximal stomach walls and the subsequent enzymatic reaction with the protein dissolved in the liquid meal. Using this model, we measured the effect of stomach motility and the diameter of the pyloric orifice on the retropulsive and pyloric jets, the rate of hydrolysis in different regions, and the emptying of gastric contents. Results: This study presents a proof-of-concept for the application of StomachSim in gastric disorders and surgeries. The emptying rate of our model for the healthy case agreed with the in-vivo measurements of similar caloric density liquid meals. A stomach with a 50% lower motility amplitude had a 35% lower net emptying rate and a 41% lower emptying rate of the hydrolyzed protein. Furthermore, the emptying rate showed a non-linear variation with respect to the motility of the stomach. For a simulated pyloroplasty procedure where the pyloric orifice diameter is doubled and the orifice stays open permanently, the emptying rate of the lower motility case doubled over its original value. Conclusion: StomachSim offers a novel and potentially effective modality for examining the effect of gastric conditions and gastric surgical procedures on gastric function. Our simulations show that gastroparesis and pyloroplasty not only affect the emptying rate but also alter the mixing of contents thereby changing the rate of hydrolysis. The model helped quantify the changes in the flow of contents in the antro-duodenal region for varying degrees of gastroparesis and pyloroplasty. The authors acknowledge support from NSF award CBET 2019405 and NIH award 1R21GM139073-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The endoplasmic reticulum acetyl-CoA transporter AT-1 plays a role in the protective unfolded protein response in the exocrine pancreas

Pancreatic acinar cells are responsible for the synthesis and secretion of digestive enzymes. In order to sustain high rates of secretory protein production, acinar cells rely on adaptive mechanisms to mitigate endoplasmic reticulum (ER) stress and maintain secretory homeostasis. The adaptive unfolded protein response (UPR), mediated by the IRE1-XBP1s axis, is essential for acinar function and protects against cellular injury. Recent work has demonstrated that the ER acetyl-CoA transporter, AT-1, is necessary for nascent secretory protein acetylation and export out of the ER and is regulated by IRE1-XBP1s. Furthermore, AT-1 is rapidly downregulated during experimental pancreatitis. To investigate the role of AT-1 in exocrine pancreas function, we generated inducible, acinar-specific AT-1 knockout mice (Ela-Cre AT-1-/-). Loss of AT-1 produced a chronic pancreatitis phenotype characterized by inflammation, immune cell infiltration, tissue fibrosis, and aberrant intracellular activation of the proteolytic digestive enzyme trypsin. Despite the histological and biochemical dysfunction observed, Ela-Cre AT-1-/- mice grow normally, have typical lifespans, and do not lose pancreatic mass. Interestingly, XBP1s expression is significantly upregulated in Ela-Cre AT-1-/- and may confer a protective effect with loss of AT-1. Our data suggests a role for AT-1 in the IRE1-XBP1s axis and highlights the previously undescribed role of protein acetylation in exocrine pancreas physiology. Given the numerous potential downstream effects of dysregulated acetyl-CoA transport, this model will advance our understanding of pancreatic function and disease. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial Specific Deletion of Autotaxin Improves Stroke Outcomes

Autotaxin (ATX) is an extracellular secretory enzyme (lysophospholipase D) that catalyzes the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA). The ATX-LPA axis is a well-known pathological mediator of liver fibrosis, metastasis in cancer, pulmonary fibrosis, atherosclerosis, and neurodegenerative diseases. Also, it is believed that LPA may cause vascular permeability. In ischemic stroke, vascular permeability leading to hemorrhagic transformation is a major limitation for therapies and an obstacle to stroke management. So, in this study, we generated an endothelial-specific ATX deletion in mice (ERT2 ATX-/-) to observe stroke outcomes in a mouse stroke model to analyze the role of endothelial ATX. The AR2 probe and Evans Blue staining were used to perform the ATX activity and vascular permeability assays, respectively. Laser speckle imaging was used to observe the cerebral blood flow following stroke. In this study, we observed that stroke outcomes were alleviated with endothelial deletion of ATX. Permeability and infarct volume was reduced in ERT2 ATX-/- mice compared to ischemia-reperfusion (I/R) only mice. In addition, cerebral blood flow was retained in ERT2 ATX-/- compared to I/R mice. The outcomes in the stroke model are alleviated due to limited LPA concentration, reduced ATX concentration, and ATX activity in ERT2 ATX-/- mice. In this study, we observed that ATX produced by the endothelium could be a major factor responsible for increases in permeability and infarcts, and adverse effects on the cerebral blood flow during cerebral ischemic reperfusion. LPA produced specifically by endothelial cells in the vasculature can disrupt the BBB leading ultimately to neuronal loss and glial activation. With the deletion of ATX from the endothelium, the microvasculature might be more resilient due to reduced LPA. We found evidence that the functional outcomes of ischemic stroke were alleviated with ATX deletion. Therefore, endothelial ATX can be targeted to make better and more effective therapies for stroke management. his research was supported by Louisiana State University Health Sciences-Shreveport Intramural Grant 110101074A to SM; National Institutes of Health grants HL141998 and HL141998-01S1 to SM; grants AA025744, AA026708, and AA025744-02S1 to MP; grants HL122354 and HL145753 to MSB; P20GM12130 to Dr. Christopher Kevil. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Wheat Class III Peroxidase TaPOD70 Is a Potential Susceptibility Factor Negatively Regulating Wheat Resistance to Blumeria graminis f. sp. tritici.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important diseases on wheat worldwide and can lead to a large reduction in wheat production. Class III peroxidases (PODs), a kind of secretory enzyme and members of a multigene family in higher plants, have been linked to various plant physiological functions and defensive responses. However, the role of PODs in wheat resistance to Bgt remains unclear. TaPOD70, a class III POD gene, was identified from the proteomics sequencing of the incompatible interaction between wheat (Triticum aestivum) cultivar Xingmin 318 and Bgt isolate E09. After transient expression of the TaPOD70-GFP fusion protein in Nicotiana benthamiana leaves, TaPOD70 was located in the membrane region. Yeast secretion assay showed that TaPOD70 was a secretory protein. Furthermore, Bax-induced programmed cell death was inhibited by transient expression of TaPOD70 in N. benthamiana. The transcript expression level of TaPOD70 was significantly upregulated in the wheat-Bgt compatible interaction. More crucially, knocking down TaPOD70 using virus-induced gene silencing increased wheat resistance to Bgt compared with the control plants. In response to Bgt, histological analyses indicated that hyphal development of Bgt was significantly reduced, whereas H2O2 production was enhanced in TaPOD70-silenced leaves. These findings imply that TaPOD70 may act as a susceptibility factor, adversely regulating wheat resistance to Bgt.

On the interactions involving serine proteases obtained from Monacrosporium thaumasium (Ascomycota: Orbiliomycetes) and deoxyribonucleic acid (DNA): biological macromolecules in action

The use of force spectroscopy approaches performed with optical tweezers can be very useful in determining the binding modes and the physical chemistry of DNA interactions with ligands, from small drugs to proteins. Helminthophagous fungi, on the other hand, have important enzyme secretion mechanisms for various purposes, and the interactions between such enzymes and nucleic acids are very poorly studied. Therefore, the main goal of the present work was to investigate, at the molecular level, the mechanisms of interaction between fungal serine proteases and the double-stranded (ds) DNA molecule. Experimental assays performed with this single molecule technique consist in exposing different concentrations of the protease of this fungus to dsDNA until saturation while monitoring the changes on the mechanical properties of the macromolecular complexes formed, from where the physical chemistry of the interaction can be deduced. It was found that the protease binds strongly to the double-helix, forming aggregates and changing the persistence length of the DNA molecule. The present work thus allowed us to infer information at the molecular level on the pathogenicity of these proteins, an important class of biological macromolecules, when applied to a target specimen.

Extracellular enzymes secreted in the mycelial block of Lentinula edodes during hyphal growth

Lentinula edodes (shiitake mushroom) is one of the most widely cultivated edible mushrooms and is primarily cultivated using sawdust medium. While there have been improvements in the cultivation technology, the mechanism of mycelial block cultivation, such as mycelial growth and enzymatic sawdust degradation, has not been clarified. In this study, the mycelium was elongated longitudinally in the bottle sawdust culture for 27 days, and the cultivated sawdust medium was divided into three sections (top, middle, and bottom parts). To determine spatial heterogeneity in the enzyme secretion, the enzymatic activities of each part were analyzed. Lignocellulose degradation enzymes, such as endoglucanase, xylanase, and manganese peroxidase were highly secreted in the top part of the medium. On the other hand, amylase, pectinase, fungal cell wall degradation enzyme (β-1,3-glucanase, β-1,6-glucanase, and chitinase), and laccase activities were higher in the bottom part. The results indicate that the principal sawdust degradation occurs after mycelial colonization. Proteins with the laccase activity were purified from the bottom part of the medium, and three laccases, Lcc5, Lcc6 and Lcc13, were identified. In particular, the expression of Lcc13 gene was higher in the bottom part compared with the level in the top part, suggesting Lcc13 is mainly produced from the tip region and have important roles for mycelial spread and nutrient uptake during early stage of cultivation.

Compounds Interacting with Cholecystokinin as Potential Drugs Against Excessive Weight Gain and Obesity

Cholecystokinin (CCK) is a peptide family that functions pancreatic enzyme secretion, gallbladder contraction, intestinal motility, satiety, and stomach acid secretion. The CCK peptides are derived from pro-CCK with a specific amino acid sequence (Asp, Tyr, Met, Gly, Trp, Met, Asp, and Phe) at their C-terminus, where the Tyr residue is sulfated. The CCK peptides are ligands of the CCK receptors 1 (CCK1) and 2 (CCK2). In this study, we propose an approach for treating overweight and obese patients, preventing or reducing CCK1 activation using compounds that specifically interact with CCK peptides to regulate their interactions with CCK1. The compounds were selected by molecular docking using a chemical library with the selected residues (Asp, Tyr, Met, Gly, Trp, Met, Asp, and Phe) in CCK. Their effects were evaluated in a diet-induced obese mice model. We show that compounds K2 and K5 decreased and maintained the body weight of the diet-induced obese mice model, respectively, likely by preventing/hindering interactions between CCK peptides and CCK1, modifying its activity. This study demonstrates another approach to treating overweight and obesity by regulating CCK1 functions. The K2 and K5 compounds have properties supporting their continued development as new drugs against these risk factors.

Cofilin activation in pancreatic acinar cells plays a pivotal convergent role for mediating CCK-stimulated enzyme secretion and growth.

Introduction: The actin regulatory protein, cofilin plays a key signaling role in many cells for numerous cellular responses including in proliferation, development, motility, migration, secretion and growth. In the pancreas it is important in islet insulin secretion, growth of pancreatic cancer cells and in pancreatitis. However, there are no studies on its role or activation in pancreatic acinar cells. Methods: To address this question, we studied the ability of CCK to activate cofilin in pancreatic acinar cells, AR42J cells and CCK1-R transfected Panc-1 cells, the signaling cascades involved and its effect on enzyme secretion and MAPK activation, a key mediator of pancreatic growth. Results: CCK (0.3 and 100nM), TPA, carbachol, Bombesin, secretin and VIP decreased phospho-cofilin (i.e., activate cofilin) and both phospho-kinetic and inhibitor studies of cofilin, LIM kinase (LIMK) and Slingshot Protein Phosphatase (SSH1) demonstrated these conventional activators of cofilin were not involved. Serine phosphatases inhibitors (calyculin A and okadaic acid), however inhibited CCK/TPA-cofilin activation. Studies of various CCK-activated signaling cascades showed activation of PKC/PKD, Src, PAK4, JNK, ROCK mediated cofilin activation, but not PI3K, p38, or MEK. Furthermore, using both siRNA and cofilin inhibitors, cofilin activation was shown to be essential for CCK-mediated enzyme secretion and MAPK activation. Conclusion: These results support the conclusion that cofilin activation plays a pivotal convergent role for various cell signaling cascades in CCK mediated growth/enzyme secretion in pancreatic acini.

Remediation of petroleum contaminated soil by persulfate oxidation coupled with microbial degradation

This study was conducted on soil polluted with high concentrations of crude oil (12,835 ± 572.76 mg/kg) with different dosages of persulfate (PS) coupled with hydrocarbon-degrading mixed bacteria (including Enterobacteriaceae, Stenotrophomonas, Pseudomonas, Acinetobacter, and Achromobacter) obtained from long-term oil-contaminated soil. The interaction among total petroleum hydrocarbons (TPH), soil parameters, and microbial communities during a 187-d combined remediation process was evaluated. The result showed that the TPH removal of 1% PS oxidation combined with bioremediation was 80.05%; this was 4.88% and 20.94% higher than that of bioremediation and 1% PS oxidation combined with natural attenuation. This is a result of 1%PS stimulated enzyme secretion and dissolved organic carbon content, especially fulvic acid, improving TPH mobility and bioavailability in soil. Furthermore, introducing mixed bacteria after oxidation further increased enzyme activities and TPH-degrading ability, thus promoting carbon substrate conversion and biodegradation rate. Conversely, the excessive oxidation of 3% and 5% PS had a negative impact on the soil environment and microbial diversity resulting in lower TPH degradation (36%-57%). High-throughput sequencing revealed that the abundance and diversity of soil bacteria decreased in all remediation groups compared to those in the pristine soil. Redundancy analysis showed that the main factors affecting 1% PS oxidation combined with bioremediation during subsequent microbial remediation were pH, cation exchange capacity, and lipase activity, which may indirectly influence TPH biodegradation by increasing the abundance of genus Immundisolibacteraceae, Comamonadaceae, and Acinetobacter. This study provides data on in situ chemical oxidation integrated with the bioremediation of petroleum hydrocarbon-contaminated soils.

Research Progress of Polyphenols in the Prevention and Treatment of AD by Regulating Intestinal Flora

The human gut microbiota is known as the "second brain". Intestinal flora is a large number of microorganisms in the human gut, which is a highly diverse bacterial homeostasis system. If this homeostasis is out of balance, the intestinal flora will be disturbed, which will affect the physical health. A growing body of research has shown that intestinal flora participates in two-way communication between gut and brain through gut-brain axis (GBA) and is strongly associated with Alzheimer's disease (AD). At present, there is still a lack of effective treatment for AD. Recent studies have shown that polyphenols play a neuroprotective role by exerting their anti-inflammatory and antioxidant effects. This topic refers to a large number of recent literatures in the same field and makes a comprehensive review of them. It was found that polyphenols with complex structure could not be directly digested and absorbed by human organism, and their bioavailability was low, which affected the effective play of their health effects. The secretory enzymes in the intestinal flora convert them into small molecular metabolites that are biologically active and easy for the body to absorb. At the same time, polyphenols can the composition and function of intestinal flora by acting on its growth or metabolism, and further act on immune, endocrine and intestinal neural pathways in GBA, forming a complex network of relationships. By exerting its neuroprotective effect, it is expected to achieve the purpose of preventing and treating AD.

Recent Trends on Mitigative Effect of Probiotics on Oxidative-Stress-Induced Gut Dysfunction in Broilers under Necrotic Enteritis Challenge: A Review.

Gut health includes normal intestinal physiology, complete intestinal epithelial barrier, efficient immune response, sustained inflammatory balance, healthy microbiota, high nutrient absorption efficiency, nutrient metabolism, and energy balance. One of the diseases that causes severe economic losses to farmers is necrotic enteritis, which occurs primarily in the gut and is associated with high mortality rate. Necrotic enteritis (NE) primarily damages the intestinal mucosa, thereby inducing intestinal inflammation and high immune response which diverts nutrients and energy needed for growth to response mediated effects. In the era of antibiotic ban, dietary interventions like microbial therapy (probiotics) to reduce inflammation, paracellular permeability, and promote gut homeostasis may be the best way to reduce broiler production losses. The current review highlights the severity effects of NE; intestinal inflammation, gut lesions, alteration of gut microbiota balance, cell apoptosis, reduced growth performance, and death. These negative effects are consequences of; disrupted intestinal barrier function and villi development, altered expression of tight junction proteins and protein structure, increased translocation of endotoxins and excessive stimulation of proinflammatory cytokines. We further explored the mechanisms by which probiotics mitigate NE challenge and restore the gut integrity of birds under disease stress; synthesis of metabolites and bacteriocins, competitive exclusion of pathogens, upregulation of tight junction proteins and adhesion molecules, increased secretion of intestinal secretory immunoglobulins and enzymes, reduction in pro-inflammatory cytokines and immune response and the increased production of anti-inflammatory cytokines and immune boost via the modulation of the TLR/NF-ĸ pathway. Furthermore, increased beneficial microbes in the gut microbiome improve nutrient utilization, host immunity, and energy metabolism. Probiotics along with biosecurity measures could mitigate the adverse effects of NE in broiler production.

Abstract 3645: OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression

Abstract Ductal carcinoma is the most commonly diagnosed breast cancer in women, the presence of metastases drops patient five-year survival from 95% to an abysmal 27%. This makes studying the mechanisms that promote metastasis of critical importance. Research shows that the extracellular matrix (ECM) of the tumor microenvironment (TME) plays an important role in invasive ductal carcinoma (IDC) progression and metastasis. Specifically; the density, stiffness, and orientation of collagen I fibers in the stroma all impact IDC motility, invasion, and metastasis. Lysyl oxidase-like 2 (LOXL2) is a secreted enzymatic protein that is implicit in ECM remodeling. By catalyzing an oxidative reaction in peptidyl lysine/hydroxylysine present on collagen I fibers, LOXL2 initiates crosslinking of the fibers. Our previous publication demonstrates that interleukin-6-related pro-inflammatory cytokine signaling, specifically oncostatin M (OSM), led to the overexpression and secretion of the LOXL2 enzyme. This increased the metastatic potential of IDC cells by promoting the crosslinking and alignment of collagen I fibers, which in turn led to increased invasion in 3D collagen I matrices. Thus, we hypothesize that OSM-induced LOXL2 expression, secretion, and subsequent collagen I fiber crosslinking and alignment, leads to an increase in IDC metastasis. In vivo studies utilizing nude athymic mice injected with either MCF7-Luc-EV control or MCF7-Luc-OSM overexpressing cells were performed in conjunction with LOXL2 inhibition using specially designed chow containing either LOXL2 SMI or control. Further analysis of OSM signaling pathways was performed in MCF7 and MDA-MB-468 IDC cells to determine mechanism for OSM induction of LOXL2. Our studies demonstrate that canonical OSM signaling pathways (pERK, pSTAT3, and pAKT) all contribute to LOXL2 expression in MCF7 cells suggesting a possible mediator protein we identified as c-Myc. Furthermore, OSM-induced LOXL2 played a significant role in OSM promoted tumor growth and metastasis as LOXL2 inhibition led to decreased tumor size and far less metastatic lesions. We also analyzed and confirmed a synergistic interaction between OSM and interleukin-1beta (IL-1β) in LOXL2 expression. These results confirm the significance of LOXL2 expression in OSM promoted metastasis and suggests LOXL2 would be an important target for future breast cancer therapies, specifically in patients with elevated levels of OSM. Furthermore, the synergistic interaction between OSM and IL-1β in LOXL2 expression suggests that patients with high levels of both cytokines are at even greater risk of metastasis, making these great markers for determining at risk IDC patients. Citation Format: Simion Dinca, Cody Wolf, Laura Bond, Cheryl Jorcyk. OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3645.

Intercropping increases soil N-targeting enzyme activities: A meta-analysis

Intercropping rationally utilizes land resources and effectively increases soil biodiversity. The enzyme-mediated biological catabolism of soil organic and mineral components is a crucial step in controlling soil carbon and nutrient circulation and fixation in agroecosystems, and those enzymes involved in nitrogen (N) cycling are particularly important. However, when considering different crop combinations, the effects of intercropping on N-targeting enzyme activities remain uncertain. Given such incomplete knowledge of these belowground processes and interactions, we synthesized results for three kinds of N-targeting enzyme activities in response to intercropping based on 117 peer-reviewed papers, in which 454 observations were included. We found that intercropping significantly increased the activity of N-acetyl-glucosaminidase by 26.1%, protease by 10.2%, and urease by 22.2%. Our results suggest that the intercropping effect is mediated by responses of the soil microbes to the diversity of soil nutrients under different crop combinations. Soil N availability and crop competition for nutrients between plant crops mediate patterns of microbial nutrient acquisition strategies and secretion of N-targeting enzymes. Our comprehensive meta-analysis highlights the importance of intercropping for better soil quality and biodiversity and has implications for the role of intercropping in regulating soil carbon dynamics.

New insight into the mechanisms of autochthonous fungal bioaugmentation of phenanthrene in petroleum contaminated soil by stable isotope probing

Autochthonous fungal bioaugmentation (AFB) is considered a reliable bioremediation approach for polycyclic aromatic hydrocarbon (PAH) contamination, but little is known about its mechanisms in contaminated soils. Here, a microcosm experiment was performed to explore the AFB mechanisms associated with two highly efficient phenanthrene degrading agents of fungi (with laccase-producing Scedosporium aurantiacum GIG-3 and non-laccase-producing Aspergillus fumigatus LJD-29), using stable-isotope-probing (SIP) and high-throughput sequencing. The results showed that each fungus markedly improved phenanthrene removal, and microcosms with both fungi exhibited the best phenanthrene removal performance among all microcosms. Additionally, AFB markedly shifted the composition of the microbial community, particularly the phenanthrene-degrading bacterial taxa. Interestingly, based on SIP results, strains GIG-3 and LJD-29 did not assimilate phenanthrene directly during AFB, but instead played key roles in the preliminary decomposition of phenanthrene though secretion of different extracellular enzymes to oxidize the benzene ring (GIG-3 bioaugmentation with laccase, and LJD-29 bioaugmentation with manganese and lignin peroxidases). In addition, all functional degraders directly involved in phenanthrene assimilation were indigenous bacteria, while native fungi rarely participated in the direct phenanthrene mineralization. Our findings provide a new mechanism of AFB with multiple fungi, and support AFB as a promising strategy for the in situ bioremediation of PAH-contaminated soil.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the β-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.

Possible mechanisms of action of Bacillus wiedmannii AzBw1, a biocontrol agent of the root-knot nematode, Meloidogyne arenaria

BackgroundWith increased environmental concerns and restrictions of chemical control, the importance of other eco-friendly strategies for management of the nematodes is being substantially grown nowadays. One of the most well-known strategies that have attracted the attentions is biological control of these deleterious agents. In our previous study (Moslehi et al. in Egypt J Biol Pest Control 31:1–11, 2021), Bacillus wiedmannii AzBw1 was introduced as a robust antagonistic agent against root-knot nematode Meloidogyne arenaria. Present study addressed the possible mechanisms of action of this strain.ResultsBased on quantitative bioassays it was shown that the strain AzBw1 is able to produce considerable amount of siderophore, protease, and chitinase. In an in vitro assay conducted by bi-plate Petri dishes, it was shown that hatching of the nematode eggs, subjected to bacterial volatile compounds (BVCs) was 34% lower than those of mock-treated control eggs. On the other hand, mortality of BVC-treated juveniles was 33.5% higher than those of mock-treated control juveniles. The secretory proteins from the medium culture of strain AzBw1 were precipitated and fractionated by anion exchange chromatography (AEC). Fractions from AEC were checked for hydrolytic activity and nematicidal effect. It was found that the fractions with the highest protease activity have a strong nematicidal effect. In contrast, significant nematicidal effect in the fraction with Chitinase activity was detected.ConclusionThe results suggested that protease activity played a key role in strain AzBw1 antagonism against root-knot nematode, M. arenaria. Finally, nonvolatile organic compounds were also extracted from the medium culture after removing secretory proteins and enzymes. Obtained results showed that these metabolites had also a strong anti-nematode effect.