Effects of copper mineralogy and methanobactin on cell growth and sMMO activity in <i>Methylosinus trichosporium</i> OB3b

Controls on in situ methanotroph activity are not well understood. One potentially important parameter is copper (Cu) because it is the metal-centre of particulate methane monooxygenase (pMMO), the most active enzyme for oxidizing methane to methanol. Further, Cu-to-cell ratios influence the relative expression of pMMO versus the alternate soluble MMO (sMMO) in some species. However, most methanotroph studies only have assessed readily soluble forms of Cu (e.g. CuCl₂) and there is a dearth of Cu-related activity data for Cu sources more common in the environment. Here we quantified sMMO activity (as a practical indicator of Cu availability) and growth kinetics in <i>Methylosinus trichosporium</i> OB3b, an organism that expresses both pMMO and sMMO, when grown on Cu-minerals with differing dissolution equilibria to assess how mineral type and methanobactin (mb) might influence in situ methanotroph activity. Mb is a molecule produced by <i>M. trichosporium</i> OB3b that has a high affinity for Cu, reduces Cu toxicity, and may influence Cu availability in terrestrial systems. CuCO₃.Cu(OH)₂ and CuO were chosen for study based on modelling data, reflecting more and less soluble minerals, respectively, and were found to affect <i>M. trichosporium</i> OB3b activity differently. Cells grew without growth lag and with active pMMO on CuCO₃.Cu(OH)₂, regardless of the amount of mineral supplied (<500 μmoles Cu-total l^&minus;1). The organism also grew well on CuO; however, significant sMMO activity was retained up to 50 μmoles Cu-total l^&minus;1, although sMMO activity was suppressed by supplemental mb and-or direct cell-mineral contact. Mb addition increased growth rates (<i>p</i> < 0.05) with both minerals. Results show mb broadly stimulates growth, but Cu mineralogy and mb dictate whether sMMO or pMMO is active in the cells. This explains why sMMO activity has been seen in soils with high Cu and also has implications for predicting dominant MMO activity in terrestrial bioremediation applications

Optimising Nature-Based Treatment Systems for Management of Mine Water

\ua9 2025 by the authors.Deployment of nature-based systems for mine water treatment is constrained by system size, and the evidence suggests decreasing hydraulic conductivity (Ksat) of organic substrates over time compromises performance. In lab-scale continuous-flow reactors, we investigated (1) the geochemical and hydraulic performance of organic substrates used in nature-based systems for metals removal (via bacterial sulfate reduction) from mine water, and then (2) the potential to operate systems modestly contaminated with Zn (0.5 mg/L) at reduced hydraulic residence times (HRTs). Bioreactors containing limestone, straw, and wood chips, with and without compost and/or sewage sludge all achieved 88%–90% Zn removal, but those without compost/sludge had higher Ksat (929–1546 m/d). Using a high Ksat substrate, decreasing the HRT from 15 to 9 h had no impact on Zn removal (92.5% to 97.5%). Although the sulfate reduction rate decreased at a shorter HRT, microbial analysis showed high relative abundance (2%–7%) of sulfate reducing bacteria, and geochemical modelling pointed to ZnS(s) precipitation as the main attenuation mechanism (mean ZnS saturation index = 3.91–4.23). High permeability organic substrate treatment systems operated at a short HRT may offer potential for wider deployment of such systems, but pilot-scale testing under ambient environmental conditions is advisable

Experimental procedures for studying microbial reactions under high hydrogen gas saturations in microcosms

\ua9 2025. This methodology is proposed to investigate the response of microbial communities through analysis of headspace composition under high saturations of hydrogen. Changes in headspace composition will be related to specific communities and environmental conditions that will influence their response and result in changes in gases produced or potential changes in the liquid phase of microcosms pertaining to the hydrogen consumption rate through microbial metabolic processes. A step-by-step procedure is documented here. • Methodology includes an easy setup utilising common laboratory equipment. • The method showed minor appreciable loss of hydrogen from the microcosm setup/storage and the use of exetainers for gas measurements. • Actively studied microbial hydrogen consumption across 18 days at 30 \ub0C and 50 \ub0C This method is useful for the first instances in scientific studies towards understanding species or microbial communities found in environments with high percentages of hydrogen: underground hydrogen storage sites, hydrogen pipelines, and hydrogen leakage into subsurface soils

Does pre-enrichment of anodes with acetate to select for <em>Geobacter</em> spp. enhance performance of microbial fuel cells when switched to more complex substrates?

Copyright \ua9 2023 Christgen, Spurr, Milner, Izadi, McCann, Yu, Curtis, Scott and Head. Many factors affect the performance of microbial fuel cells (MFCs). Considerable attention has been given to the impact of cell configuration and materials on MFC performance. Much less work has been done on the impact of the anode microbiota, particularly in the context of using complex substrates as fuel. One strategy to improve MFC performance on complex substrates such as wastewater, is to pre-enrich the anode with known, efficient electrogens, such as Geobacter spp. The implication of this strategy is that the electrogens are the limiting factor in MFCs fed complex substrates and the organisms feeding the electrogens through hydrolysis and fermentation are not limiting. We conducted a systematic test of this strategy and the assumptions associated with it. Microbial fuel cells were enriched using three different substrates (acetate, synthetic wastewater and real domestic wastewater) and three different inocula (Activated Sludge, Tyne River sediment, effluent from an MFC). Reactors were either enriched on complex substrates from the start or were initially fed acetate to enrich for Geobacter spp. before switching to synthetic or real wastewater. Pre-enrichment on acetate increased the relative abundance of Geobacter spp. in MFCs that were switched to complex substrates compared to MFCs that had been fed the complex substrates from the beginning of the experiment (wastewater-fed MFCs - 21.9 \ub1 1.7% Geobacter spp.; acetate-enriched MFCs, fed wastewater - 34.9 \ub1 6.7% Geobacter spp.; Synthetic wastewater fed MFCs – 42.5 \ub1 3.7% Geobacter spp.; acetate-enriched synthetic wastewater-fed MFCs - 47.3 \ub1 3.9% Geobacter spp.). However, acetate pre-enrichment did not translate into significant improvements in cell voltage, maximum current density, maximum power density or substrate removal efficiency. Nevertheless, coulombic efficiency (CE) was higher in MFCs pre-enriched on acetate when complex substrates were fed following acetate enrichment (wastewater-fed MFCs – CE = 22.0 \ub1 6.2%; acetate-enriched MFCs, fed wastewater – CE =58.5 \ub1 3.5%; Synthetic wastewater fed MFCs – CE = 22.0 \ub1 3.2%; acetate-enriched synthetic wastewater-fed MFCs – 28.7 \ub1 4.2%.) The relative abundance of Geobacter ssp. and CE represents the average of the nine replicate reactors inoculated with three different inocula for each substrate. Efforts to improve the performance of anodic microbial communities in MFCs utilizing complex organic substrates should therefore focus on enhancing the activity of organisms driving hydrolysis and fermentation rather the terminal-oxidizing electrogens

Identification of differentially expressed microRNAs in human male breast cancer

<p>Abstract</p> <p>Background</p> <p>The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.</p> <p>Methods</p> <p>The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.</p> <p>Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.</p> <p>Results</p> <p>Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.</p> <p>Conclusions</p> <p>Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.</p

Erbb2 mrna expression and response to ado-trastuzumab emtansine (T-dm1) in her2-positive breast cancer

Trastuzumab emtansine (T-DM1) is approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive (HER2+) metastatic breast cancer (BC) and for residual disease after neoadjuvant therapy; however, not all patients benefit. Here, we hypothesized that the heterogeneity in the response seen in patients is partly explained by the levels of human epidermal growth factor receptor 2 gene (ERBB2) mRNA. We analyzed ERBB2 expression using a clinically applicable assay in formalin-fixed paraffin-embedded (FFPE) tumors (primary or metastatic) from a retrospective series of 77 patients with advanced HER2+ BC treated with T-DM1. The association of ERBB2 levels and response was further validated in 161 baseline tumors from the West German Study (WGS) Group ADAPT phase II trial exploring neoadjuvant T-DM1 and 9 in vitro BC cell lines. Finally, ERBB2 expression was explored in 392 BCs from an in-house dataset, 368 primary BCs from The Cancer Genome Atlas (TCGA) dataset and 10,071 tumors representing 33 cancer types from the PanCancer TCGA dataset. High ERBB2 mRNA was found associated with better response and progression-free survival in the metastatic setting and higher rates of pathological complete response in the neoadjuvant setting. ERBB2 expression also correlated with in vitro response to T-DM1. Finally, our assay identified 0.20–8.41% of tumors across 15 cancer types as ERBB2-high, including gastric and esophagus adenocarcinomas, urothelial carcinoma, cervical squamous carcinoma and pancreatic cancer. In particular, we identified high ERBB2 mRNA in a patient with HER2+ advanced gastric cancer who achieved a long-lasting partial response to T-DM1. Our study demonstrates that the heterogeneity in response to T-DM1 is partly explained by ERBB2 levels and provides a clinically applicable assay to be tested in future clinical trials of breast cancer and other cancer types

TNBC-DX genomic test in early-stage triple-negative breast cancer treated with neoadjuvant taxane-based therapy

Background: Identification of biomarkers to optimize treatment strategies for early-stage triple-negative breast cancer (TNBC) is crucial. This study presents the development and validation of TNBC-DX, a novel test aimed at predicting both short- and long-term outcomes in early-stage TNBC. The objective of this study was to evaluate the association between TNBC-DX and efficacy outcomes [pathologic complete response (pCR), distant disease-free survival (DDFS) or event-free survival (EFS), and overall survival (OS)] in the validation cohorts. Methods: Information from 1259 patients with early-stage TNBC (SCAN-B, CALGB-40603, and BrighTNess) was used to establish the TNBC-DX scores. Independent validation of TNBC-DX was carried out in three studies: (i) WSG-ADAPT-TN; (ii) MMJ-CAR-2014-01; and (iii) NeoPACT, including 527 patients with stage I-III TNBC undergoing neoadjuvant chemotherapy. In WSG-ADAPT-TN, patients were randomized to receive nab-paclitaxel plus gemcitabine or carboplatin. In MMJ-CAR-2014-01, patients received carboplatin plus docetaxel. In NeoPACT, patients received carboplatin plus docetaxel and pembrolizumab. Results: TNBC-DX test was created incorporating the 10-gene Core Immune Gene module, the 4-gene tumor cell proliferation signature, tumor size, and nodal staging. In the two independent validation cohorts without pembrolizumab, the TNBC-DX pCR score was significantly associated with pCR after adjustment for clinicopathological variables and treatment regimen [odds ratio per 10-unit increment 1.34, 95% confidence interval (CI) 1.20-1.52, P < 0.001]. pCR rates for the TNBC-DX pCR-high, pCR-medium, and pCR-low categories were 56.3%, 53.6%, and 22.5% respectively (odds ratio for pCR-high versus pCR-low 3.48, 95% CI 1.72-7.15, P < 0.001). In addition, the TNBC-DX risk score was significantly associated with DDFS [hazard ratio (HR) high-risk versus low-risk 0.24, 95% CI 0.15-0.41, P < 0.001] and OS (HR 0.19, 95% CI 0.11-0.35, P < 0.001). In the validation cohort with pembrolizumab, the TNBC-DX scores were significantly associated with pCR, EFS, and OS. Conclusions: TNBC-DX predicts pCR to neoadjuvant taxane–carboplatin in stage I-III TNBC and helps to forecast the patient's long-term survival in the absence of neoadjuvant anthracycline–cyclophosphamide, and independent of pembrolizumab use

Nuclear Kaiso Expression Is Associated with High Grade and Triple-Negative Invasive Breast Cancer

Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC