Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

Wnt signaling during cochlear development

Wnt signaling is a hallmark of all embryonic development with multiple roles at multiple developmental time points. Wnt signaling is also important in the development of several organs, one of which is the inner ear, where it participates in otic specification, the formation of vestibular structures, and the development of the cochlea. In particular, we focus on Wnt signaling in the auditory organ, the cochlea. Attempting to dissect the multiple Wnt signaling pathways in the mammalian cochlea is a challenging task due to limited expression data, particularly at proliferating stages. To offer predictions about Wnt activity, we compare cochlear development with that of other biological systems such as Xenopus retina, brain, cancer cells and osteoblasts. Wnts are likely to regulate development through crosstalk with other signaling pathways, particularly Notch and FGF, leading to changes in the expression of Sox2 and proneural (pro-hair cell) genes. In this review we have consolidated the known signaling pathways in the cochlea with known developmental roles of Wnts from other systems to generate a potential timeline of cochlear development.     

Disialoganglioside GD3-synthase over expression inhibits survival and angiogenesis of pancreatic cancer cells through cell cycle arrest at S-phase and disruption of integrin-β1-mediated anchorage

Gangliosides play important roles in the development, differentiation and proliferation of mammalian cells. They bind to other cell membrane components through their terminal sialic acids. Different gangliosides influence cellular functions based on the positions and linkages of sialic acids. Expression of gangliosides mainly depends on the status of sialic acid-modulatory enzymes, such as different types of sialyltransferases and sialidases. One such sialyltransferase, disialoganglioside GD3 synthase, is specifically responsible for the production of GD3. Pancreatic ductal adenocarcinoma, making up more than 90% of pancreatic cancers, is a fatal malignancy with poor prognosis. Despite higher sialylation status, the disialoganglioside GD3 level is very low in this cancer. However, the exact status and function of this disialoganglioside is still unknown. Here, we intended to study the intracellular mechanism of disialoganglioside GD3-induced apoptosis and its correlation with the adhesion and angiogenic pathways in pancreatic cancer. We demonstrated that disialoganglioside GD3 synthase-transfected cells showed enhanced apoptosis and it caused the arrest of these cells in the S-phase of the cell cycle. Integrins, a family of transmembrane proteins play important role in cell–cell recognition, invasion, adhesion and migration. disialoganglioside GD3 co-localised with integrin-β1 and thereby inhibited it's downstream signalling in transfected cells. Transfected cells exhibited inhibition of cell adhesion with extracellular matrix proteins. Enhanced GD3 expression down regulated angiogenesis-regulatory proteins and inhibited epidermal growth factor/vascular endothelial growth factor-driven angiogenic cell growth in these cells. Taken together, our study provides support for the GD3-induced cell cycle arrest, disruption of integrin-β1-mediated anchorage, inhibition of angiogenesis and thereby induced apoptosis in pancreatic cancer cells.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2 Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.     

Evaluation of blood-brain barrier penetration and examination of binding to human serum albumin of 7-O-arylpiperazinylcoumarins as potential antipsychotic agents

The delivery of drugs to the brain is complicated by the multiple factors including low blood–brain barrier (BBB) passive permeability, active BBB efflux systems, and plasma protein binding. Thus, a detailed understanding of the transport of the new potent substances through the membranes is vitally important and their physico-chemical characteristics should be analyzed at first. This work presents an evaluation of drug likeness of eight 7-O-arylpiperazinylcoumarin derivatives with high affinity towards serotoninergic receptors 5-HT_1A and 5-HT_2A with particular analysis of the requirements for the CNS chemotherapeutics. The binding constants to human serum albumin (HSA) were determined at physiological pH using fluorescence spectroscopy, and then their mode of action was explained by analysis of theoretical HSA complexes. Dynamic simulation of systems allowed for reliable evaluation of the interaction strength. The analyzed coumarins were able to pass BBB, and they present good drug likeness properties. They showed high affinities to HSA (log K_Q = 5.3–6.0 which corresponds to −8.12 to −7.15 kcalmol⁻¹ of Gibbs free energy). The changes of the emission intensity upon binding to HSA were scrutinized showing the different mode of action for 4-phenylpiperazinylcoumarins. The values of computed Gibbs free energy and determined on the basis of experimentally obtained binding constants log K_Q coincide suggesting a good quality of the theoretical model. Overall the 8-acetyl-7-O-arylpiperazinyl-4-methylcoumarin derivatives represent valuable lead compounds to be further tested in various preclinical assays as a possible chemotherapeutics against CNS diseases. Studied coumarins can be metabolized by cytochrome P450 to aldehydes and hydroxy derivatives. The existence of other binding sites inside HSA than Sudlow’s site 1 was postulated. The longer aliphatic linker between coumarin and piperazine moieties favored binding to HSA in other than Sudlow site 1 pocket.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

Nicotinamide adenine dinucleotide, NAD⁺, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD⁺ and NADH). NAD⁺ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD⁺ as a substrate. In this review, we highlight the significance of NAD⁺ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.     

Home environment and cord blood levels of lead,arsenic, and zinc on neurodevelopment of 24 months children living in Chitwan Valley,Nepal

In a birth cohort living in Chitwan Valley, lowland Nepal, we have previously reported inverse associations between in utero levels of lead (Pb), arsenic (As) and neurodevelopment at birth measured by the Brazelton Neonatal Behavioral Assessment Scale, third edition (NBAS III). In the present paper, a follow-up of the same cohort was made on 24-month-old infants regarding the neurodevelopmental effects of these metals, taking the postnatal environment into account. In total, the same100 mother-infant pairs as the previous study, whose Pb, As, and Zn concentrations in cord blood were known, were recruited. Postnatal raising environment was evaluated using the Home Observation for Measurement of Environment (HOME) scale. Neurodevelopment of children at 24 months of age (n = 74) was assessed using the Bayley Scale of Infant Development, Second Edition (BSID II). Multivariable regression adjusting for covariates was performed to determine the associations of in utero levels of toxic and essential elements and the home environment with neurodevelopment scores. Unlike the NBAS III conducted for newborns, none of the BSID II cluster scores in 24-month-old infants were associated with cord blood levels of Pb, As, and Zn. The total HOME score was positively associated with the mental development scale (MDI) score (coefficient = 0.67, at 95% CI = 0.03 to 1.31). In this cohort, a detrimental effect of in utero Pb and As on neurodevelopmental indicators observed at birth disappeared at 24 months, while an association between neurodevelopment and home environment continued.     

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Inhibition of vascular smooth muscle cell calcification by vasorin through interference with TGFβ1 signaling

Elevated transforming growth factor β1 (TGFβ1) levels are frequently observed in chronic kidney disease (CKD) patients. TGFβ1 contributes to development of medial vascular calcification during hyperphosphatemia, a pathological process promoted by osteo−/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Vasorin is a transmembrane glycoprotein highly expressed in VSMCs, which is able to bind TGFβ to inhibit TGFβ signaling. Thus, the present study explored the effects of vasorin on osteo−/chondrogenic transdifferentiation and calcification of VSMCs. Primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human TGFβ1 or β-glycerophosphate without or with recombinant human vasorin or vasorin gene silencing by siRNA. As a result, TGFβ1 down-regulated vasorin mRNA expression in HAoSMCs. Vasorin supplementation inhibited TGFβ1-induced pathway activation, SMAD2 phosphorylation and downstream target genes expression in HAoSMCs. Furthermore, treatment with exogenous vasorin blunted, while vasorin knockdown augmented TGFβ1-induced osteo−/chondrogenic transdifferentiation of HAoSMCs. In addition, phosphate down-regulated vasorin mRNA expression in HAoSMCs. Phosphate-induced TGFβ1 expression was not affected by addition of exogenous vasorin. Nonetheless, the phosphate-induced TGFβ1 signaling, osteo−/chondrogenic transdifferentiation and calcification of HAoSMCs were all blunted by vasorin. Conversely, silencing of vasorin aggravated osteoinduction in HAoSMCs during high phosphate conditions. Aortic vasorin expression was reduced in the hyperphosphatemic klotho-hypomorphic mouse model of CKD-related vascular calcification. In conclusion, vasorin, which suppresses TGFβ1 signaling and protects against osteo−/chondrogenic transdifferentiation and calcification of VSMCs, is reduced by pro-calcifying conditions. Thus, vasorin is a novel key regulator of VSMC calcification and may represent a potential therapeutic target for vascular calcification during CKD.     

Organelle communication: Signaling crossroads between homeostasis and disease

Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca²⁺ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies.     

Energy metabolism disorders in rare and common diseases. Toward bioenergetic modulation therapy and the training of a new generation of European scientists

Energy metabolism alterations are found in a large number of rare and common diseases of genetic or environmental origin. The number of patients that could benefit from bioenergetic modulation therapy (BIOMET) is therefore very important and includes individuals with pathologies as diverse as mitochondrial diseases, acute coronary syndrome, chronic kidney disease, asthma or even cancer. Although, the alteration of energy metabolism is disease specific and sometimes patient specific, the strategies for BIOMET could be common and target a series of bioenergetic regulatory mechanisms discussed in this article. An excellent training of scientists in the field of energy metabolism, related human diseases and drug discovery is also crucial to form a young generation of MDs, PHDs and Pharma or CRO-group leaders who will discover novel personalized bioenergetic medicines, through pharmacology, genetics, nutrition or adapted exercise training. The Mitochondrial European Educational Training (MEET) consortium was created to pursue this goal, and we dedicated here a special issue of Organelle in Focus (OiF) to highlight their objectives. A total of 10 OiFs articles constitute this Directed Issue on Mitochondrial Medicine. As part of this editorial article, we asked timely questions to the PR. Jan W. Smeitink, professor of Mitochondrial Medicine and CEO of Khondrion, a mitochondrial medicine company. He shared with us his objectives and strategies for the study of mitochondrial diseases and the identification of future treatments.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.