Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

BACKGROUND: Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. METHODS: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. RESULTS: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. CONCLUSIONS: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

The Population-level Effect of Adjuvant Therapies on Breast Cancer Recurrence: Application of the Trend-in-Trend Design.

PURPOSE: Breast cancer has an average 10-year relative survival reaching 84%. This favorable survival is due, in part, to the introduction of biomarker-guided therapies. We estimated the population-level effect of the introduction of two adjuvant therapies-tamoxifen and trastuzumab-on recurrence using the trend-in-trend pharmacoepidemiologic study design. METHODS: We ascertained data on women diagnosed with nonmetastatic breast cancer who were registered in the Danish Breast Cancer Group clinical database. We used the trend-in-trend design to estimate the population-level effect of the introduction of (1) tamoxifen for postmenopausal women with estrogen receptor (ER)-positive breast cancer in 1982, (2) tamoxifen for premenopausal women diagnosed with ER-positive breast cancer in 1999, and (3) trastuzumab for women <60 years diagnosed with human epidermal growth factor receptor 2-positive breast cancer in 2007. RESULTS: For the population-level effect of the introduction of tamoxifen among premenopausal women diagnosed with ER-positive breast cancer in 1999, the risk of recurrence decreased by nearly one-half (OR = 0.52), consistent with evidence from clinical trials; however, the estimate was imprecise (95% confidence interval [CI] = 0.25, 1.85). We observed an imprecise association between tamoxifen use and recurrence from the time it was introduced in 1982 (OR = 1.24 95% CI = 0.46, 5.11), inconsistent with prior knowledge from clinical trials. For the introduction of trastuzumab in 2007, the estimate was also consistent with trial evidence, though imprecise (OR = 0.51; 95% CI = 0.21, 22.4). CONCLUSIONS: We demonstrated how novel pharmacoepidemiologic analytic designs can be used to evaluate the routine clinical care and effectiveness of therapeutic advancements in a population-based setting while considering some limitations of the approach.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Phyllosphere bacterial strains Rhizobium b1 and Bacillus subtilis b2 control tomato leaf diseases caused by Pseudomonas syringae pv. tomato and Alternaria solani.

AIMS: Show that tomato leaf phyllosphere bacteria are candidates for biocontrol of tomato leaf diseases. METHODS AND RESULTS: Seven bacterial isolates from surface-sterilized Moneymaker tomato plants were tested for growth inhibition of 14 tomato pathogens on potato dextrose agar. Biocontrol assays were conducted with tomato leaf pathogens, Pseudomonas syringae pv. tomato (Pto) and Alternaria solani (A. solani). Two potential isolates showing the greatest inhibition were identified by 16S rDNA sequencing as Rhizobium sp. (isolate b1) and Bacillus subtilis (isolate b2), both produce protease and isolate b2 cellulase. Both reduced tomato leaf infections by Pto and A. solani in detached leaf bioassays. Both bacteria b1 and b2 reduced pathogen development in a tomato growth trial. Bacteria b2 also induced the tomato plant salicylic acid (SA) immune response pathway. Disease suppression in biocontrol assays with b1 and b2 varied between five commercial tomato varieties. CONCLUSIONS: Tomato phyllosphere bacteria when used as phyllosphere inoculants, inhibited tomato diseases caused by Pto and A. solani.

Amplified Ca2+ dynamics and accelerated cell proliferation in breast cancer tissue during purinergic stimulation.

Intracellular Ca2+ dynamics shape malignant behaviors of cancer cells. Whereas previous studies focused on cultured cancer cells, we here used breast organoids and colonic crypts freshly isolated from human and murine surgical biopsies. We performed fluorescence microscopy to evaluate intracellular Ca2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco-/endoplasmic reticulum Ca2+ ATPase with cyclopiazonic acid elicited larger Ca2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca2+ levels and acetylcholine-induced increases in intracellular Ca2+ concentrations were unaffected and ATP- and UTP-induced Ca2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca2+ responses, ATP and UTP substantially increased proliferative activity-evaluated by bromodeoxyuridine incorporation-in breast cancer tissue, whereas the effect was minimal in normal breast tissue. ATP caused cell death-identified with ethidium homodimer-1 staining-in breast cancer tissue only at concentrations above the expected pathophysiological range. We conclude that intracellular Ca2+ responses are amplified in breast cancer tissue, but not in colon cancer tissue, and that nucleotide signaling stimulates breast cancer cell proliferation within the extracellular concentration range typical for solid cancer tissue.

Rhizosphere bacteria induce programmed cell death defence genes and signalling in chilli pepper.

AIM: To understand how beneficial bacteria assist chilli plants (Capsicum annuum) in defence against biotrophic or hemibiotrophic pathogens. METHOD AND RESULTS: We quantified marker genes of plant defence pathways in Phytophthora capsici-infected chilli pepper treated with anti-oomycete plant growth-promoting rhizobacteria, Bacillus amyloliquefaciens, Bacillus velezensis and Acinetobacter sp. Plants displayed strong resistance, and the pathogen load in the roots was significantly lower in infected plants treated with bacterial biocontrol agents at all time points tested (1, 2 and 7 days after pathogen inoculation, p < 0.05). Gene expression profiling revealed that P. capsici infection in the absence of beneficial bacteria led to the upregulation of a wide array of defence genes. The addition of biocontrol bacteria modulated defence by further enhancing genes involved in programmed cell death, such as CaLOX1, CaPAL1, CaChitIV and CaPTI1, while suppressing others CaLRR1, a negative regulator of cell death. CONCLUSIONS: Our results suggest that the bacteria exerted a combined effect by directly antagonizing the pathogen and enhancing the expression of key plant defence genes, including those involved in cell death, causing resistance at early stages of infection by this hemibiotrophic pathogen.

Hypoxia-inducible factor-1α expression and breast cancer recurrence in a Danish population-based case control study.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that facilitates the adaptation of cancer cells to hypoxic conditions and may be prognostic of breast cancer recurrence. We evaluated the association of HIF-1α expression with breast cancer recurrence, and its association with timing of breast cancer recurrence. METHODS: In this population-based case-control study, we included women diagnosed with stage I-III breast cancer between 1985 and 2001, aged 35-69 years, registered in the Danish Breast Cancer Group. We identified 541 cases of breast cancer recurrence among women with estrogen receptor (ER)-positive disease who were treated with tamoxifen for at least 1 year (ER+ TAM+). We also enrolled 300 breast cancer recurrence cases among women with ER-negative disease, not treated with tamoxifen, who survived at least 1 year (ER-/TAM-). Controls were recurrence-free breast cancer patients at the time of case diagnosis, matched to recurrence cases on ER/TAM status, date of surgery, menopausal status, cancer stage, and county of residence. Expression of HIF-1α was measured by immunohistochemistry on tissue microarrays. We fitted logistic regression models to compute odds ratios (ORs) and 95% confidence intervals (CIs) associating HIF-1α expression with recurrence, and with timing of recurrence. RESULTS: HIF-1α expression was observed in 23% of cases and 20% of controls in the ER+/TAM+ stratum, and in 47% of cases and 48% of controls in the ER-/TAM- stratum. We observed a near-null association between HIF-1α expression in both ER/TAM groups (ER+/TAM+ OR = 1.21, 95%CI 0.88, 1.67 and ER-/TAM- OR = 0.97, 95%CI 0.68, 1.39). HIF-1α expression was not associated with time to recurrence among women in the ER+/TAM+ stratum, but was associated with early recurrence among women in the ER-/TAM- stratum. CONCLUSION: In this study, HIF-1α expression was not associated with breast cancer recurrence overall but may be associated with early recurrence among women diagnosed with ER- breast cancer.

Evidence for the plant recruitment of beneficial microbes to suppress soil-borne pathogens.

An emerging experimental framework suggests that plants under biotic stress may actively seek help from soil microbes, but empirical evidence underlying such a 'cry for help' strategy is limited. We used integrated microbial community profiling, pathogen and plant transcriptive gene quantification and culture-based methods to systematically investigate a three-way interaction between the wheat plant, wheat-associated microbiomes and Fusarium pseudograminearum (Fp). A clear enrichment of a dominant bacterium, Stenotrophomonas rhizophila (SR80), was observed in both the rhizosphere and root endosphere of Fp-infected wheat. SR80 reached 3.7 × 107 cells g-1 in the rhizosphere and accounted for up to 11.4% of the microbes in the root endosphere. Its abundance had a positive linear correlation with the pathogen load at base stems and expression of multiple defence genes in top leaves. Upon re-introduction in soils, SR80 enhanced plant growth, both the below-ground and above-ground, and induced strong disease resistance by boosting plant defence in the above-ground plant parts, but only when the pathogen was present. Together, the bacterium SR80 seems to have acted as an early warning system for plant defence. This work provides novel evidence for the potential protection of plants against pathogens by an enriched beneficial microbe via modulation of the plant immune system.

Development of High-Level Omega-3 Eicosapentaenoic Acid (EPA) Production from Phaeodactylum tricornutum.

Phaeodactylum tricornutum is a lipid-rich marine diatom that contains a high level of omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA). In an effort to reduce costs for large-scale cultivation of this microalga, this study first established a New BBM medium (0.3 x strength BBM with only 3% of the initial phosphate level) to replace the traditional F/2 medium. Phaeodactylum tricornutum could grow in extremely low phosphate concentrations (25 µM), without compromising the EPA content. In the presence of sea salts, silicate addition was not necessary for high rate growth, high EPA content, or lipid accumulation in this species. Using urea as the sole nitrogen source tended to increase EPA contents per dry biomass (by 24.7%) while not affecting growth performance. The use of sea salts, rather than just sodium chloride, led to significantly improved biomass yields (20% increase) and EPA contents of total fatty acid (46-52% increase), most likely because it supplied sufficient essential elements such as magnesium. A salinity level of 35 led to significantly higher biomass yields compared with 20, but salinity had no significant influence on EPA content. EPA became the dominant fatty acid with average levels of 51.8% of total fatty acids during the exponential growth phase at 20 ppt in New BBM medium with sea salts.

Impact of the intersection of anaesthesia and gender on burnout and mental health, illustrated by the COVID-19 pandemic.

Physician burnout and poor mental health are prevalent and often stigmatised. Anaesthetists may be at particular risk and this is further increased for women anaesthetists due to biases and inequities within the specialty. However, gender-related risk factors for and experiences of burnout and poor mental health remain under-researched and under-reported. This negatively impacts individual practitioners, the anaesthesia workforce and patients and carries significant financial implications. We discuss the impact of anaesthesia and gender on burnout and mental health using the COVID-19 pandemic as an example illustrating how women and men differentially experience stressors and burnout. COVID-19 has further accentuated the gendered effects of burnout and poor mental health on anaesthetists and brought further urgency to the need to address these issues. While both personal and organisational factors contribute to burnout and poor mental health, organisational changes that recognise and acknowledge inequities are pivotal to bolster physician mental health.

Predictive pharmacogenetic biomarkers for breast cancer recurrence prevention by simvastatin.

Background: Statins treat hyperlipidemia and prevent cardiovascular morbidity and mortality. Evidence suggests that they also have anti-neoplastic activity. Several studies show a reduced rate of breast cancer recurrence among lipophilic statin users (e.g., simvastatin), motivating calls for clinical trials of statins in breast cancer patients. We measured the impact of genetic variation in statin-metabolizing enzymes and drug transporters on the recurrence rate in simvastatin-treated breast cancer patients.Methods: We conducted a nested case-control study among Danish women diagnosed with non-metastatic, invasive breast cancer between 2004-2010 who had filled ≥1 prescription for simvastatin after diagnosis. Cases were all breast cancer recurrences from the source population; one control was matched to each case on cancer stage, estrogen receptor and hormone therapy status, calendar period of diagnosis, and duration of simvastatin exposure. We genotyped variants in simvastatin-metabolizing enzymes (CYP3A4/rs35599367 and CYP3A5/rs776746) and drug transporters (ABCB1/rs2032582 and SLCO1B1/rs4149056), and estimated their association with recurrence with logistic regression models.Results: We observed protective (though imprecisely-measured) associations between variants in genes encoding drug transporters (ABCB1 and SLCO1B1) and simvastatin-metabolizing enzymes (CYP3A4 and CYP3A5) and breast cancer recurrence in simvastatin-treated women. For example, carrying two variant alleles in ABCB1 was associated with a 31% lower rate of recurrence (multivariable OR = 0.69, 95% CI: 0.31, 1.5).Conclusion: Our study provides weak evidence to support the use of genetic variation in ABCB1, SLCO1B1, CYP3A4, and CYP3A5 as biomarkers of breast tumor response to simvastatin. Validation of these findings within adjuvant clinical trials is encouraged.

Evaluation of microalgae and cyanobacteria as potential sources of antimicrobial compounds.

In recent decades, marine microorganisms have become known for their ability to produce a wide variety of secondary bioactive metabolites. Several compounds have been isolated from marine microorganisms for the development of novel bioactives for the food and pharmaceutical industries. In this study, a number of microalgae were evaluated for their antimicrobial activity against gram-positive and gram-negative bacteria, including food and plant pathogens, using various extraction techniques and antimicrobial assays. Disc diffusion and spot-on-lawn assays were conducted to confirm the antimicrobial activity. To measure the potency of the extracts, minimum inhibition concentrations (MIultCs) were measured. Three microalgae, namely Isochrysis galbana, Scenedesmus sp. NT8c, and Chlorella sp. FN1, showed strong inhibitory activity preferentially against gram-positive bacteria. These microalgal species were then selected for further purification and analysis, leading to compound identification. By using a mixture of different chromatography techniques gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), we were able to separate and identify the dominant compounds that are responsible for the inhibitory activity. Additionally, nuclear magnetic resonance (NMR) was used to confirm the presence of these compounds. The dominant compounds that were identified and purified in the extracts are linoleic acid, oleic acid, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These compounds are the potential candidates that inhibit the growth of gram-positive bacteria. This indicates the potential use of microalgae and their antimicrobial compounds as biocontrol agents against food and plant pathogens.

The AtHSP17.4C1 Gene Expression Is Mediated by Diverse Signals that Link Biotic and Abiotic Stress Factors with ROS and Can Be a Useful Molecular Marker for Oxidative Stress.

Reactive oxygen species (ROS) are highly controlled signaling species that are involved in regulating gene expression in response to different environmental cues. The production of heat shock proteins (HSPs) is a key strategy that plants use to defend themselves against diverse stresses, including oxidative stress. In this study, expression patterns of the Arabidopsis HSP17.4CI gene, a cytosolic class I small HSP, were systematically profiled under different abiotic, biotic and oxidative stresses. Our data show that HSP17.4CI was early and highly induced by heat, cold, salt, drought and high-light. HSP17.4CI also showed high expression levels in Arabidopsis plants infected with the biotrophic pathogen Pseudomonas syringae, but not in response to the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum. Oxidative stress treatments including H2O2 and the herbicide methyl viologen led to induction of HSP17.4CI. The plant hormones abscisic acid (ABA) and salicylic acid (SA) induced the expression of HSP17.4CI, whereas methyl jasmonate (MJ) did not affect the expression level of this gene. Furthermore, we found enhanced expression of HSP17.4CI in catalase mutant plants, which are deficient in catalase 2 activity and accumulate intracellular H2O2. Taken together, data presented here suggest that HSP17.4CI expression is regulated by various signals that connect biotic and abiotic stresses with ROS and can be used as a molecular marker for oxidative stress.

Heavy metal bioremediation of coal-fired flue gas using microalgae under different CO2 concentrations.

Sustainability assessments have revealed that integration of CO2 from coal-fired flue gas with microalgae cultivation systems could reduce greenhouse gas emissions. The technical goal of this integration is to utilize exhaust from coal power plants to enhance microalgae cultivation processes by capturing and recycling of carbon dioxide from a more toxic to a less toxic form. However, heavy metals are also introduced along with CO2 to the cultivation system which could contaminate biomass and have deleterious effects on products derived from such systems. The present study aimed at shedding some light on capability of microalgae to sustain their diversity and propagate them under different CO2 concentrations from coal-fired flue gas. Mixed microalgal culture was grown in nutrient rich medium and heavy metals (Al, Cu, Fe, Mn and Zn) are expected to be introduced from flue gas. Three concentrations (1%, 3% and 5.5%) of CO2 were evaluated (reference concentrations from flue gas). Comparative studies were carried out by flue gas and control systems in photobioreactors. Under the 3% CO2 (30% flue gas), the highest fraction of B, Mn and Zn were found to be internalized by the cells (46.8 ±9.45 gL-1, 253.66 ± 40.62 gL-1 and 355.5 ±50.69 gL-1 respectively) during their cultivation period into biomass. Hence, microalgae may offer solution to two major challenges: providing potential biofuel feedstock for energy security and reducing heavy metal pollution to the air.

Self-reference, emotion inhibition and somatosensory disturbance: preliminary investigation of network perturbations in conversion disorder.

BACKGROUND AND PURPOSE: Conversion disorder (CD), or functional neurological disorder, is manifested as a neurological disturbance that is not macroscopically visible on clinical structural neuroimaging and is instead ascribed to underlying psychological stress. Known for many years in neuropsychiatry, a comprehensive explanation of the way in which psychological stress leads to a neurological deficit of a structural-like origin is still lacking. METHODS: We applied whole-brain network-based data-driven analyses on resting-state functional magnetic resonance imaging, recorded in seven patients with acute-onset, stroke-like CD with unilateral paresis and hypoesthesia as compared with 15 age-matched healthy controls. We used a clustering analysis to measure functional connectivity (FC) strength within 10 different brain networks, as well as between these networks. Finally, we tested FC of specific brain regions that are known to be involved in CD. RESULTS: We found a significant increase in FC strength only within the default-mode network (DMN), which manages self-referential processing. Examination of inter-connectivity between networks showed a structure of disturbed connectivity, which included decreased connectivity between the DMN and limbic/salience network, increased connectivity between the limbic/salience network and body-related temporo-parieto-occipital junction network, decreased connectivity between the temporo-parieto-occipital junction and memory-related medial temporal lobe, and decreased connectivity between the medial temporal lobe and sensorimotor network. Region-specific FC analysis showed increased connectivity between the hippocampus and DMN. DISCUSSION: These preliminary results of disturbances in brain networks related to memory, emotions and self-referential processing, and networks involved in motor planning and execution, suggest a role of these cognitive functions in the psychopathology of CD.

Use of prescription drugs and risk of postoperative red blood cell transfusion in breast cancer patients: a Danish population-based cohort study.

BACKGROUND: Several frequently used prescription drugs may affect bleeding risk. We investigated use of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), selective serotonin reuptake inhibitors (SSRIs), and statins and risk of postoperative red blood cell transfusion in breast cancer patients. METHODS: Using Danish population-based registries, we identified a cohort of women who underwent surgery for primary breast cancer (n = 22,238) during 2005-2012 and ascertained their use of aspirin, NSAIDs, SSRIs, and statins. For each drug, patients were categorized as users if they filled ≥1 prescription in the 60 days prior to surgery. We calculated the 14-day risk of red blood cell transfusion and relative risks (RRs) with 95% confidence intervals (CIs), comparing users with nonusers for each drug and adjusting for age, cancer stage, and Charlson Comorbidity Index score. RESULTS: In our cohort, 1385 (6.2%) women were aspirin users, 1794 (8.0%) were NSAID users, 1110 (4.9%) were SSRI users, and 2053 (9.1%) were statin users. The overall risk of red blood cell transfusion was 1.3%. The 14-day risk of RBC transfusion was 3.5% among aspirin users versus 1.1% among aspirin nonusers (adjusted RR = 1.9, 95% CI: 1.4-2.7), and 1.8% among SSRI users versus 1.2% among SSRI nonusers (adjusted RR = 1.2, 95% CI: 0.7-1.9). Red blood cell transfusion risk was increased among NSAID users, but not in a sensitivity analysis with a 30-day exposure window. Red blood cell transfusion risk was not increased among SSRI and statin users. CONCLUSIONS: Primary breast cancer surgery confers a low risk of RBC transfusion. Still, use of aspirin and possibly NSAIDs, but not SSRIs and statins, is associated with increased red blood cell transfusion. This increased risk is not explained by differences in age, stage, or comorbidity level.

Impact of age, intrinsic subtype and local treatment on long-term local-regional recurrence and breast cancer mortality among low-risk breast cancer patients.

AIM: To evaluate the long-term prognostic impact of age, local treatment and intrinsic subtypes on the risk of local-regional recurrence (LRR) and breast cancer mortality among low-risk patients. MATERIAL AND METHODS: Cohort study with prospectively collected data, balanced five-year age groups, including 514 Danish lymph node negative breast cancer patients diagnosed between 1989 and 1998, treated with mastectomy (N = 320) or breast-conserving therapy (BCT) (N = 194) and without systemic treatment. Intrinsic subtype approximation was performed by combining information on estrogen-, progesterone-, HER2 receptor and Ki67. RESULTS: The majority of the tumors had a luminal subtype: 70% Luminal-A (LumA), 16% Luminal-B (LumB), and 10% Luminal-HER2 + (Lum-HER2+). The distribution of intrinsic subtypes between younger (≤45 years) and older (>45 years) patients was similar. Intrinsic subtypes had no prognostic impact on the 20-year LRR risk, regardless of age. A distinct 20-year mortality pattern was observed among the younger patients: 11% of patients with LumB tumor died of breast cancer within the first five years after primary surgery, 23% of patients with Lum-HER2+ tumor died within a 5-10-year period, whereas patients with LumA tumor died with a constant low rate throughout the 20-year period. After 20 years of follow-up, patients with LumA tumor had breast cancer mortality comparable to that of patients with LumB tumor (20%) and lower than Lum-HER2+ tumor (39%). Among the older patients, no distinct mortality pattern was observed, and the 20-year breast cancer mortality was not associated with intrinsic subtypes. CONCLUSION: Among low-risk patients, 96% of the tumors were Luminal and the distribution of intrinsic subtypes between younger (≤45 years) and older (>45 years) patients was similar. The observed higher frequency of LRR among younger low-risk BCT patients was not associated intrinsic subtype. The 20-year breast cancer mortality was non-significant for LumA tumors among the older patients, whereas among the younger patients, LumA tumors had a comparable mortality with LumB, but lower than for Lum-HER2 + tumors.

Validity of Danish Breast Cancer Group (DBCG) registry data used in the predictors of breast cancer recurrence (ProBeCaRe) premenopausal breast cancer cohort study.

BACKGROUND: Validation studies of the Danish Breast Cancer Group (DBCG) registry show good agreement with medical records for adjuvant treatment data, but inconsistent recurrence information. No studies have validated changes in menopausal status or endocrine therapy during follow-up. In a longitudinal study, we validated DBCG data using medical records as the gold standard. MATERIAL AND METHODS: From a cohort of 5959 premenopausal women diagnosed during 2002-2010 with stage I-III breast cancer, we selected 151 patients - 77 estrogen-receptor-positive and 74 estrogen-receptor-negative - from three hospitals. We assessed the validity of DBCG registry data on patient, tumor, and treatment factors, and follow-up information on menopausal transition, changes in endocrine therapy, and recurrence. We computed positive predictive values (PPVs) with 95% confidence intervals (95%CI). RESULTS: Agreement was near perfect for tumor size, lymph node involvement, receptor status, surgery type, and receipt of radiotherapy, chemotherapy, or tamoxifen treatment. The PPV for a change in endocrine therapy in the DBCG was 96% (95%CI = 83, 100). The PPV for menopausal transition was 61% (95%CI = 42, 77). The PPV for DBCG-recorded recurrence was 100%. However, of 19 patients who had a recurrence documented in their medical record, 13 had the recurrence registered in DBCG. CONCLUSIONS: DBCG data are valid for most epidemiological studies of breast cancer treatment. Data on menopausal transition may be less valid, though this interpretation depends on the suitability of medical records for making this assessment. Although recurrence is missing for some, this would not bias most ratio measures of association.

Linking Jasmonic Acid Signaling, Root Exudates, and Rhizosphere Microbiomes.

Jasmonic acid (JA) is an essential hormone in plant development and defense responses in Arabidopsis thaliana. Exogenous treatment with JA has recently been shown to alter root exudate profiles and the composition of root-associated bacterial communities. However, it is currently unknown whether disruptions of the JA in the rhizosphere affect root exudation profiles and the relative abundance of bacteria and archaea in the rhizosphere. In the present study, two Arabidopsis mutants that are disrupted in different branches of the jasmonate pathway, namely myc2 and med25, were cultivated in nutrient solution and soil to profile root exudates and bacterial and archaeal communities, respectively. Compared with the wild type, both mutants showed distinct exudation patterns, including lower amounts of asparagine, ornithine, and tryptophan, as well as distinct bacterial and archaeal community composition, as illustrated by an increased abundance of Streptomyces, Bacillus, and Lysinibacillus taxa in the med25 rhizosphere and an Enterobacteriaceae population in myc2. Alternatively, the Clostridiales population was less abundant in the rhizosphere of both mutants. Similarities between plant genotypes were highly correlated, as determined by operational taxonomic units in the rhizosphere and metabolites in root exudates. This strongly suggests that root exudates play a major role in modulating changes in microbial community composition upon plant defense responses.

Na+,HCO3- -cotransport is functionally upregulated during human breast carcinogenesis and required for the inverted pH gradient across the plasma membrane.

Metabolic and biochemical changes during breast carcinogenesis enhance cellular acid production. Extrusion of the acid load from the cancer cells raises intracellular pH, while it decreases extracellular pH creating an inverted pH gradient across the plasma membrane compared to normal cells and promoting cancer cell metabolism, proliferation, migration, and invasion. We investigated the effects of breast carcinogenesis on the mechanisms of cellular pH control using multicellular epithelial organoids freshly isolated from human primary breast carcinomas and matched normal breast tissue. Intracellular pH was measured by fluorescence microscopy, while protein expression was investigated by immunofluorescence imaging and immunoblotting. We found that cellular net acid extrusion increased during human breast carcinogenesis due to enhanced Na(+),HCO3 (-)-cotransport, which created an alkaline shift (~0.3 units of magnitude) in steady-state intracellular pH of human primary breast carcinomas compared to normal breast tissue. Na(+)/H(+)-exchange activity and steady-state intracellular pH in the absence of CO2/HCO3 (-) were practically unaffected by breast carcinogenesis. These effects were evident under both acidic (pH 6.8, representative of the tumor microenvironment) and physiological (pH 7.4) extracellular conditions. Protein expression of the Na(+),HCO3 (-)-cotransporter NBCn1 (SLC4A7), which has been linked to breast cancer susceptibility in multiple genome-wide association studies, was twofold higher in human breast carcinomas compared to matched normal breast tissue. Protein expression of the Na(+)/H(+)-exchanger NHE1 (SLC9A1) was markedly less affected. We propose that upregulated NBCn1 during human breast carcinogenesis contributes to the characteristic acid distribution within human breast carcinomas and thereby plays a pathophysiological role for breast cancer development and progression.