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The biological functions of nitric oxide (NO) depend on its concentration, and excessive levels of NO induce various harmful situations known as nitrosative stress. Therefore, organisms possess many kinds of strategies to regulate the intracellular NO concentration and/or to detoxify excess NO. Here, we used genetic screening to identify a novel nitrosative stress tolerance gene, RIB1, encoding GTP cyclohydrolase II (GTPCH2), which catalyzes the first step in riboflavin biosynthesis. Our further analyses demonstrated that the GTPCH2 enzymatic activity of Rib1 is essential for RIB1-dependent nitrosative stress tolerance, but that riboflavin itself is not required for this tolerance. Furthermore, the reaction mixture of a recombinant purified Rib1 was shown to quench NO or its derivatives, even though formate or pyrophosphate, which are byproducts of the Rib1 reaction, did not, suggesting that the reaction product of Rib1, 2,5-diamino-6-(5-phospo-d-ribosylamino)-pyrimidin-4(3 H)-one (DARP), scavenges NO or its derivatives. Finally, it was revealed that 2,4,5-triamino-1H-pyrimidin-6-one, which is identical to a pyrimidine moiety of DARP, scavenged NO or its derivatives, suggesting that DARP reacts with N2O3 generated via its pyrimidine moiety.

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

In this work, the cytotoxicity of black TiO2 nanotubes in comparison to their white counterparts is exploited for the first time. To provide a comprehensive overview, the toxicity is also assessed in comparison to multi-walled carbon nanotubes (MWCNTs) using two different cell lines. The TiO2 nanotube layers were prepared by anodic oxidation of Ti foils and investigated in the as-prepared white form, and in the black form, achieved by reduction via comproportionation reaction of Ti and TiO2. Additionally, a part of the black TiO2 nanotube layers was coated with a thin TiO2 coating with a nominal thickness of similar to 0.275 nm by Atomic Layer Deposition (ALD). Pulmonary A549 cells and neural SH-SY5Y cells were exposed to a wide range of TiO2 nanotube concentrations (1-500 mu g/mL) fo(r) 24 and 48 h. Then, the cytotoxicity was evaluated using cell viability and glutathione assessments. Essentially, no reduction in cell viability was observed after incubating the cells with TiO2 nanotubes up to 100 mu g/mL, while MWCNTs decreased the cell viability at a similar concentration significantly. In addition, ALD TiO2 coating on the black TiO2 nanotubes caused disappearance of mild toxic effects of white and black TiO2 nanotubes due to the shading of carbon and fluorine species incorporated within the TiO2 nanotube walls.

In this work, the cytotoxicity of black TiO2 nanotubes in comparison to their white counterparts is exploited for the first time. To provide a comprehensive overview, the toxicity is also assessed in comparison to multi-walled carbon nanotubes (MWCNTs) using two different cell lines. The TiO2 nanotube layers were prepared by anodic oxidation of Ti foils and investigated in the as-prepared white form, and in the black form, achieved by reduction via comproportionation reaction of Ti and TiO2. Additionally, a part of the black TiO2 nanotube layers was coated with a thin TiO2 coating with a nominal thickness of similar to 0.275 nm by Atomic Layer Deposition (ALD). Pulmonary A549 cells and neural SH-SY5Y cells were exposed to a wide range of TiO2 nanotube concentrations (1-500 mu g/mL) fo(r) 24 and 48 h. Then, the cytotoxicity was evaluated using cell viability and glutathione assessments. Essentially, no reduction in cell viability was observed after incubating the cells with TiO2 nanotubes up to 100 mu g/mL, while MWCNTs decreased the cell viability at a similar concentration significantly. In addition, ALD TiO2 coating on the black TiO2 nanotubes caused disappearance of mild toxic effects of white and black TiO2 nanotubes due to the shading of carbon and fluorine species incorporated within the TiO2 nanotube walls.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Severe defects in cell size are a nearly universal feature of cancer cells. Yet, the mechanisms that drive size defects in cancer cells remain unknown, and it is unclear whether they are a direct consequence of primary oncogenic drivers or a secondary consequence of mutations that accumulate during evolution of cancer cells. Hyperactive mutants of Ras are amongst the most prevalent oncogenic drivers. In budding yeast, previous studies have suggested that hyperactive versions of the Ras homolog (ras2G19V) cause defects in cell size. However, the mechanisms by which ras2G19V drives cell size defects are unknown, and it is unclear whether the size defects are due to accumulation of suppressor mutations. Here, I developed a system for inducible expression of ras2G19V that allows for investigation into the immediate consequences of ras2G19V expression. I found that ras2G19V causes a delay in G1 phase, increased cell size, and aberrant expression of G1 cyclin proteins. Furthermore, ras2G19V appears to inhibit a key step in cell cycle entry, in which an early G1 cyclin induces transcription of late G1 cyclins. The data further suggest that Ras does not influence cell size solely via effects in G1 phase. Finally, we found that expression of oncogenic Ras alone is sufficient to cause cell size defects in NIH 3T3 cells, which suggests that defects in cell size in cancer cells could be a direct consequence of primary oncogenic signals. Together, the data suggest that hyperactive forms of Ras influence cell size in both yeast and mammals. Further analysis will determine whether Ras influences cell size via conserved mechanisms.

Cell cycle progression is dependent upon cell growth. Cells must, therefore, translate growth into a proportional signal that can be used to determine when sufficient growth has occurred for cell cycle progression. In budding yeast, a protein kinase called Gin4 is required for normal control of cell growth and undergoes gradual hyperphosphorylation that is dependent upon and proportional to added membrane growth in metaphase within the mitotic phase of the cell cycle. These observation suggest that growth-dependent phosphorylation of Gin4 could play a role in mechanisms that measure cell growth in mitosis. However, the molecular mechanisms that drive growth-dependent hyperphosphorylation of Gin4 are poorly understood. Here, we utilized a combination of biochemical reconstitution and genetic analysis to define the molecular mechanisms that drive growth dependent phosphorylation of Gin4. Our working model is that lipid vesicles that drive plasma membrane growth drive also deliver key signaling molecules that drive Gin4 hyperphosphorylation, which would suggest a simple mechanistic explanation for how growth-dependent Gin4 phosphorylation signals are generated. Gin4 has a KA1 domain that binds phosphatidylserine lipid and is required for growth-dependent phosphorylation of Gin4. Our work rules out a simple model in which phosphatidylserine delivered to sites of membrane growth binds and activates Gin4 to undergo autophosphorylation. Rather, the data suggest that phosphatidylserine recruits Gin4 to sites of membrane growth, where it is phosphorylated and activated by Elm1 in a Yck1-dependent manner. In this manner, Gin4 phosphorylation signals are generated to drive inhibition of the mitotic inhibitor Swe1 to promote the metaphase to anaphase transition, allowing cells to complete division.

The adult human body is made up of trillions of cells and yet began as just one. This could not be possible without careful coordination of cell growth and cell division. The molecular mechanisms that drive and control cell growth as well as its coordination with the cell cycle remain poorly understood. Cell size is the outcome of cell growth. Thus, a decoupling of growth and division would result in cells of increasingly heterogenous sizes. Interestingly, the severity of a cancer is often correlated with increasingly heterogenous size of cells within a tissue. Furthermore, genes that are thought to play important roles in cell growth are often dysregulated in a variety of cancers. This suggests that the misregulation of mechanisms that control cell growth and cell size plays a role in cancer cell development. How cell growth influences cell cycle progression to control cell size is perhaps best understood in budding yeast where key cell cycle transitions are delayed until sufficient growth has occurred. It had been long believed that the predominant cell size checkpoint in budding yeast occurred at cell cycle entry. Recent work, however, suggests that bud growth strongly influences cell cycle progression in mitosis. In this thesis, I present evidence to support a model in which cells influence the extent and duration of bud growth by controlling mitotic exit, a cell cycle transition that seizes bud growth and finalizes daughter cell size.

In this work, the cytotoxicity of black TiO2 nanotubes in comparison to their white counterparts is exploited for the first time. To provide a comprehensive overview, the toxicity is also assessed in comparison to multi-walled carbon nanotubes (MWCNTs) using two different cell lines. The TiO2 nanotube layers were prepared by anodic oxidation of Ti foils and investigated in the as-prepared white form, and in the black form, achieved by reduction via comproportionation reaction of Ti and TiO2. Additionally, a part of the black TiO2 nanotube layers was coated with a thin TiO2 coating with a nominal thickness of similar to 0.275 nm by Atomic Layer Deposition (ALD). Pulmonary A549 cells and neural SH-SY5Y cells were exposed to a wide range of TiO2 nanotube concentrations (1-500 mu g/mL) fo(r) 24 and 48 h. Then, the cytotoxicity was evaluated using cell viability and glutathione assessments. Essentially, no reduction in cell viability was observed after incubating the cells with TiO2 nanotubes up to 100 mu g/mL, while MWCNTs decreased the cell viability at a similar concentration significantly. In addition, ALD TiO2 coating on the black TiO2 nanotubes caused disappearance of mild toxic effects of white and black TiO2 nanotubes due to the shading of carbon and fluorine species incorporated within the TiO2 nanotube walls.

Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth–death regulation., Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth–death regulation.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Cells, regardless of whether they are in an organ in the human body or a component of a bacterial colony, can sense the chemical composition of the environment, the presence of neighboring cells, and even the types of their neighboring cells. Depending on the identity of a cell and the information it receives from its environment, it can grow (increase in size), proliferate (make more cells), become quiescent (stop growing and dividing), differentiate (make different types of cells), or die. How cells achieve the astonishing feat of appropriately sensing and responding to their environment has been a major question in biology. In this course we will read and critically discuss the primary scientific literature with the goal of highlighting the basic principles of cell growth, adaptation, and differentiation. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

This course deals with the biology of cells of higher organisms: The structure, function, and biosynthesis of cellular membranes and organelles; cell growth and oncogenic transformation; transport, receptors, and cell signaling; the cytoskeleton, the extracellular matrix, and cell movements; chromatin structure and RNA synthesis.

Cells, regardless of whether they are in an organ in the human body or a component of a bacterial colony, can sense the chemical composition of the environment, the presence of neighboring cells, and even the types of their neighboring cells. Depending on the identity of a cell and the information it receives from its environment, it can grow (increase in size), proliferate (make more cells), become quiescent (stop growing and dividing), differentiate (make different types of cells), or die. How cells achieve the astonishing feat of appropriately sensing and responding to their environment has been a major question in biology. In this course we will read and critically discuss the primary scientific literature with the goal of highlighting the basic principles of cell growth, adaptation, and differentiation. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

The highly conserved TOR signaling pathway is crucial for coordinating cellular growth with the cell cycle machinery in eukaryotes. One of the two TOR complexes in budding yeast, TORC1, integrates environmental cues and promotes cell growth. While cells grow, they need to copy their chromosomes, segregate them in mitosis, divide all their components during cytokinesis, and finally physically separate mother and daughter cells to start a new cell cycle apart from each other. To maintain cell size homeostasis and chromosome stability, it is crucial that mechanisms that control growth are connected and coordinated with the cell cycle. Successive periods of high and low TORC1 activity would participate in the adequate cell cycle progression. Here, we review the known molecular mechanisms through which TORC1 regulates the cell cycle in the budding yeast Saccharomyces cerevisiae that have been extensively used as a model organism to understand the role of its mammalian ortholog, mTORC1.

Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth–death regulation., Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth–death regulation.

Skeletal muscle harbors a pool of stem cells called muscle satellite cells (MuSCs) that are mainly responsible for its robust regenerative capacities. Adult satellite cells are mitotically quiescent in uninjured muscles under homeostasis, but they exit quiescence upon injury to re‐enter the cell cycle to proliferate. While most of the expanded satellites cells differentiate and fuse to form new myofibers, some undergo self‐renewal to replenish the stem cell pool. Specifically, quiescence exit describes the initial transition of MuSCs from quiescence to the first cell cycle, which takes much longer than the time required for subsequent cell cycles and involves drastic changes in cell size, epigenetic and transcriptomic profiles, and metabolic status. It is, therefore, an essential period indispensable for the success of muscle regeneration. Diverse mechanisms exist in MuSCs to regulate quiescence exit. In this review, we summarize key events that occur during quiescence exit in MuSCs and discuss the molecular regulation of this process with an emphasis on multiple levels of intrinsic regulatory mechanisms. A comprehensive understanding of how quiescence exit is regulated will facilitate satellite cell‐based muscle regenerative therapies and advance their applications in various disease and aging conditions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Skeletal muscle harbors a pool of stem cells called muscle satellite cells (MuSCs) that are mainly responsible for its robust regenerative capacities. Adult satellite cells are mitotically quiescent in uninjured muscles under homeostasis, but they exit quiescence upon injury to re‐enter the cell cycle to proliferate. While most of the expanded satellites cells differentiate and fuse to form new myofibers, some undergo self‐renewal to replenish the stem cell pool. Specifically, quiescence exit describes the initial transition of MuSCs from quiescence to the first cell cycle, which takes much longer than the time required for subsequent cell cycles and involves drastic changes in cell size, epigenetic and transcriptomic profiles, and metabolic status. It is, therefore, an essential period indispensable for the success of muscle regeneration. Diverse mechanisms exist in MuSCs to regulate quiescence exit. In this review, we summarize key events that occur during quiescence exit in MuSCs and discuss the molecular regulation of this process with an emphasis on multiple levels of intrinsic regulatory mechanisms. A comprehensive understanding of how quiescence exit is regulated will facilitate satellite cell‐based muscle regenerative therapies and advance their applications in various disease and aging conditions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

This thesis analyzes data on E. coli cell growth in a bioreactor to investigate the possibility of finding predictable correlations between the environmental parameters (sensor data) and the growth using machine learning. Discovering these correlations could be a first step toward optimizing the growth of cells to be used for cell therapy: an effective but very expensive treatment method for cancer. This could ultimately lead to decreased manufacturing costs and larger treatment availability. The data first underwent a thorough preprocessing to obtain useful features that were divided into batches. In addition, a few separate further processing methods were applied to the data for further analysis. Thereafter several different machine learning methods were implemented and evaluated on the data. All possible sensor combinations were then fed into the best-performing network and the mean absolute error was calculated for each combination. The results showed that the implemented machine learning models did not find predictable patterns between sensor inputs and growth, as the predictions did not follow the growth variations and the models mainly predicted the average yield. However, the possibility that the used approach would benefit from additional data should not be discarded., Detta examensarbete analyserar data som beskriver celltillväxt av E. coli i en bioreaktor för att undersöka möjligheten att hitta samband mellan inputparametrar och tillväxt med hjälp av maskininlärning. Att upptäcka dessa samband kan vara ett första steg mot att optimera tillväxten av celler som används för cellterapi: en effektiv men väldigt dyr behandlingsmetod för cancer. Detta kan i slutändan leda till minskade tillverkningskostnader och en större tillgänglighet av behandlingen. All data genomgick först en ingående förberedande bearbetning för att erhålla användbara features som var uppdelade i batcher. Ett antal separata vidarebearbetningsmetoder tillämpades också för vidare analys. Därefter implementerades och evaluerades ett flertal olika maskininlärningsmetoder. Den bäst presterande modellen blev tränad på alla möjliga sensorkombinationer och medelabsolutfelet beräknades. Resul- taten visade att de implementerade maskininlärningsmodellerna inte hittade förutsägbara mönster mellan sensorinput och celltillväxt, då förutsägelserna inte följde tillväxtvariationerna och modellerna främst förutspådde den genomsnittliga celltillväxten. Trots resultatet bör möjligheten att ytterligare data kan gynna det använda tillvägagångssättet inte förkastas.

Protein kinases are central regulators of numerous cellular processes including growth, cell cycle progression, apoptosis and metabolism by phosphorylation of a plethora of protein substrates. Mitochondria are cell organelles which take the center stage in energy production and are involved in signalling and promoting cell death, among other functions. Deregulated kinase and mitochondrial activities have been shown to participate in the progression of several diseases including cancer. In recent years, kinases have become key targets for therapeutic intervention which is underlined by currently more than 60 FDA-approved kinase inhibitor drugs. Kinases are subject of intensive research and inhibitors are among the most effective drugs in cancer therapy. Interdependencies of kinase functions and mitochondrial activities have been described leading to the question of how perturbation of their activities affects mitochondria allocated functions. In this thesis, the effect of several small molecules targeting kinase functions was analysed using the Oroboros O2ks, the gold-standard for high-resolution respirometry. First, melanoma drugs of key MAPK pathway kinase inhibitors (BRAF: Vemurafenib, PLX8394; MEK: Trametinib; BRAF and MEK Combination: Dabrafenib and Trametinib) were analysed to describe their effects on mitochondrial respiration in the melanoma cell line A375 in a time and dose-dependent manner. Second, mitochondria and kinase profiles of the small bioactive metabolic regulator Spermidine were determined using the colon cancer cell line SW620 and non-cancer human embryonic kidney cell line HEK293T. Third, protein conformation reporters were generated to track dynamic changes of key kinases involved in central signalling cascades. Overall, no strong direct impact of these small molecules on mitochondrial function was revealed. Kinase profiling of Spermidine regarding key cellular signalling kinases (MEK, ERK, CREB, AMPK and Akt) showed no major impact on their ph, Masterarbeit Universität Innsbruck 2022

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

This thesis analyzes data on E. coli cell growth in a bioreactor to investigate the possibility of finding predictable correlations between the environmental parameters (sensor data) and the growth using machine learning. Discovering these correlations could be a first step toward optimizing the growth of cells to be used for cell therapy: an effective but very expensive treatment method for cancer. This could ultimately lead to decreased manufacturing costs and larger treatment availability. The data first underwent a thorough preprocessing to obtain useful features that were divided into batches. In addition, a few separate further processing methods were applied to the data for further analysis. Thereafter several different machine learning methods were implemented and evaluated on the data. All possible sensor combinations were then fed into the best-performing network and the mean absolute error was calculated for each combination. The results showed that the implemented machine learning models did not find predictable patterns between sensor inputs and growth, as the predictions did not follow the growth variations and the models mainly predicted the average yield. However, the possibility that the used approach would benefit from additional data should not be discarded., Detta examensarbete analyserar data som beskriver celltillväxt av E. coli i en bioreaktor för att undersöka möjligheten att hitta samband mellan inputparametrar och tillväxt med hjälp av maskininlärning. Att upptäcka dessa samband kan vara ett första steg mot att optimera tillväxten av celler som används för cellterapi: en effektiv men väldigt dyr behandlingsmetod för cancer. Detta kan i slutändan leda till minskade tillverkningskostnader och en större tillgänglighet av behandlingen. All data genomgick först en ingående förberedande bearbetning för att erhålla användbara features som var uppdelade i batcher. Ett antal separata vidarebearbetningsmetoder tillämpades också för vidare analys. Därefter implementerades och evaluerades ett flertal olika maskininlärningsmetoder. Den bäst presterande modellen blev tränad på alla möjliga sensorkombinationer och medelabsolutfelet beräknades. Resul- taten visade att de implementerade maskininlärningsmodellerna inte hittade förutsägbara mönster mellan sensorinput och celltillväxt, då förutsägelserna inte följde tillväxtvariationerna och modellerna främst förutspådde den genomsnittliga celltillväxten. Trots resultatet bör möjligheten att ytterligare data kan gynna det använda tillvägagångssättet inte förkastas.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Pioneering work carried out over 60 years ago discovered that bacterial cell size is proportional to the growth rate set by nutrient availability. This relationship is traditionally referred to as the growth law. Subsequent studies revealed the growth law to hold across all orders of life, a remarkable degree of conservation. However, recent work suggests the relationship between growth rate, nutrients, and cell size is far more complicated and less deterministic than originally thought. Focusing on bacteria and yeast, here we review efforts to understand the molecular mechanisms underlying the relationship between growth rate and cell size.

Protein kinases are central regulators of numerous cellular processes including growth, cell cycle progression, apoptosis and metabolism by phosphorylation of a plethora of protein substrates. Mitochondria are cell organelles which take the center stage in energy production and are involved in signalling and promoting cell death, among other functions. Deregulated kinase and mitochondrial activities have been shown to participate in the progression of several diseases including cancer. In recent years, kinases have become key targets for therapeutic intervention which is underlined by currently more than 60 FDA-approved kinase inhibitor drugs. Kinases are subject of intensive research and inhibitors are among the most effective drugs in cancer therapy. Interdependencies of kinase functions and mitochondrial activities have been described leading to the question of how perturbation of their activities affects mitochondria allocated functions. In this thesis, the effect of several small molecules targeting kinase functions was analysed using the Oroboros O2ks, the gold-standard for high-resolution respirometry. First, melanoma drugs of key MAPK pathway kinase inhibitors (BRAF: Vemurafenib, PLX8394; MEK: Trametinib; BRAF and MEK Combination: Dabrafenib and Trametinib) were analysed to describe their effects on mitochondrial respiration in the melanoma cell line A375 in a time and dose-dependent manner. Second, mitochondria and kinase profiles of the small bioactive metabolic regulator Spermidine were determined using the colon cancer cell line SW620 and non-cancer human embryonic kidney cell line HEK293T. Third, protein conformation reporters were generated to track dynamic changes of key kinases involved in central signalling cascades. Overall, no strong direct impact of these small molecules on mitochondrial function was revealed. Kinase profiling of Spermidine regarding key cellular signalling kinases (MEK, ERK, CREB, AMPK and Akt) showed no major impact on their ph, submitted by Fabian Eichner, B.Sc., Masterarbeit University of Innsbruck 2022

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

How gene activities and biomechanics together direct organ shapes is poorly understood. Plant leaf and floral organs develop from highly similar initial structures and share similar gene expression patterns, yet they gain drastically different shapes later-flat and bilateral leaf primordia and radially symmetric floral primordia, respectively. We analyzed cellular growth patterns and gene expression in young leaves and flowers of Arabidopsis thaliana and found significant differences in cell growth rates, which correlate with convergence sites of phytohormone auxin that require polar auxin transport. In leaf primordia, the PRESSED-FLOWER-expressing middle domain grows faster than adjacent adaxial domain and coincides with auxin convergence. In contrast, in floral primordia, the LEAFY-expressing domain shows accelerated growth rates and pronounced auxin convergence. This distinct cell growth dynamics between leaf and flower requires changes in levels of cell-wall pectin de-methyl-esterification and mechanical properties of the cell wall. Data-driven computer model simulations at organ and cellular levels demonstrate that growth differences are central to obtaining distinct organ shape, corroborating in planta observations. Together, our study provides a mechanistic basis for the establishment of early aerial organ symmetries through local modulation of differential growth patterns with auxin and biomechanics.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Organ-on-chip (OoC) technology is one of the most promising in vitro tools to replace the traditional animal experiment-based paradigms of risk assessment. However, the use of OoC in drug discovery and toxicity studies remain still limited by the low capacity for high-throughput production and the incompatibility with standard laboratory equipment. Moreover, polydimethylsiloxanes, the material of choice for OoC, has several drawbacks, particularly the high absorption of drugs and chemicals. In this work, we report the development of a microfluidic device, using a process adapted for mass production, to culture liver cell line in dynamic conditions. The device, made of cyclic olefin copolymers, was manufactured by injection moulding and integrates Luer lock connectors compatible with standard medical and laboratory instruments. Then, the COC device was used for culturing HepG2/C3a cells. The functionality and behaviour of cultures were assessed by albumin secretion, cell proliferation, viability and actin cytoskeleton development. The cells in COC device proliferated well and remained functional for 9 days of culture. Furthermore, HepG2/C3a cells in the COC biochips showed similar behaviour to cells in PDMS biochips. The present study provides a proof-of-concept for the use of COC biochip in liver cells culture and illustrate their potential to develop OoC.

Skeletal muscle harbors a pool of stem cells called muscle satellite cells (MuSCs) that are mainly responsible for its robust regenerative capacities. Adult satellite cells are mitotically quiescent in uninjured muscles under homeostasis, but they exit quiescence upon injury to re‐enter the cell cycle to proliferate. While most of the expanded satellites cells differentiate and fuse to form new myofibers, some undergo self‐renewal to replenish the stem cell pool. Specifically, quiescence exit describes the initial transition of MuSCs from quiescence to the first cell cycle, which takes much longer than the time required for subsequent cell cycles and involves drastic changes in cell size, epigenetic and transcriptomic profiles, and metabolic status. It is, therefore, an essential period indispensable for the success of muscle regeneration. Diverse mechanisms exist in MuSCs to regulate quiescence exit. In this review, we summarize key events that occur during quiescence exit in MuSCs and discuss the molecular regulation of this process with an emphasis on multiple levels of intrinsic regulatory mechanisms. A comprehensive understanding of how quiescence exit is regulated will facilitate satellite cell‐based muscle regenerative therapies and advance their applications in various disease and aging conditions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

Comprehensive maps of genetic interactions in mammalian cells are daunting to construct because of the large number of potential interactions, ~ 2 × 108 for protein coding genes. We previously used co-inheritance of distant genes from published radiation hybrid (RH) datasets to identify genetic interactions. However, it was necessary to combine six legacy datasets from four species to obtain adequate statistical power. Mapping resolution was also limited by the low density PCR genotyping. Here, we employ shallow sequencing of nascent human RH clones as an economical approach to constructing interaction maps. In this initial study, 15 clones were analyzed, enabling construction of a network with 225 genes and 2,359 interactions (FDR < 0.05). Despite its small size, the network showed significant overlap with the previous RH network and with a protein-protein interaction network. Consumables were ≲$50 per clone, showing that affordable, high quality genetic interaction maps are feasible in mammalian cells.

Although neo-Darwinian (and less often Lamarckian) dynamics are regularly invoked to interpret cancer’s multifarious molecular profiles, they shine little light on how tumorigenesis unfolds and often fail to fully capture the frequency and breadth of resistance mechanisms. This uncertainty frames one of the most problematic gaps between science and practice in modern times. Here, we offer a theory of adaptive cancer evolution, which builds on a molecular mechanism that lies outside neo-Darwinian and Lamarckian schemes. This mechanism coherently integrates non-genetic and genetic changes, ecological and evolutionary time scales, and shifts the spotlight away from positive selection towards purifying selection, genetic drift, and the creative-disruptive power of environmental change. The surprisingly simple use-it or lose-it rationale of the proposed theory can help predict molecular dynamics during tumorigenesis. It also provides simple rules of thumb that should help improve therapeutic approaches in cancer

As per the latest Canadian Cancer Statistics, pancreatic cancer is the 4th leading cause of cancer-related deaths in Canada, with a net 5 year survival of 8%. Pancreatic cancer mostly refers to pancreatic ductal adenocarcinoma (PDAC) that accounts for almost 85% of the cases diagnosed. Currently, gemcitabine is the first line of treatment for PDAC. Gemcitabine is known to cause lethal DNA damage by inducing DNA double strand breaks (DSBs). DSB can be repaired by the non-homologous end joining repair (NHEJ) pathway, of which DNA-PK plays a key role. It was shown that PDAC tissues display elevated levels of DNA-PKcs. Besides supporting growth, DNA-PK was proposed to offer chemoprotection to the PDAC cells. Still, the mechanisms involved behind DNA-PK mediated PDAC growth sustenance remains poorly investigated. It is now accepted that a vital cellular process, autophagy, plays critical role in PDAC growth. A number of reports have suggested that autophagy could be regulated by DNA-PK in certain forms of cancer. As nothing is known about the regulation of autophagy by DNA-PK in the context of PDAC, we hypothesized that DNA-PK sustains autophagy and pancreatic cancer cell growth. The PDAC cell lines MIA PaCa-2 and PANC1 were used as study models. DNA-PK activity was targeted through a pharmacological strategy using the specific DNA-PK inhibitor NU7441 to analyse the impact of DNA-PK inhibition on PDAC cell growth and autophagy. We observed drastic reduction in PDAC cell growth upon NU7441 treatment, in both dose- and time dependent manner. DNA-PK inhibition also abrogated colony forming ability of PDAC cells. NU7441 treatment caused cleavage of PARP and caspase-7, suggesting an apoptotic response upon DNA-PK inhibition. Furthermore, DNA-PK inhibition via NU7441 led to an accumulation of autophagy markers LC3B II and p62. Measurement of autophagic flux confirmed that this accumulation in the levels of the autophagy markers is due to blockade of autophagy. Overa, Selon les récentes statistiques, le cancer pancréatique est la quatrième cause de décès par cancer au Canada avec une survie à 5 ans de 8%. Le cancer du pancréas réfère à l'adénocarcinome canalaire pancréatique (PDAC) qui représente près de 85% des cas diagnostiqués. Actuellement, la gemcitabine demeure la première ligne de traitement du PDAC malgré une efficacité limitée. Il est connu que la gemcitabine provoque des dommages à l'ADN en provoquant des cassures double-brin. Ces cassures peuvent être réparées par la voie de réparation de jonction d'extrémités non-homologues, la DNA-PK y jouant un rôle clé. Il a été observé que les tissus PDAC présentent des niveaux d’expression élevés de la DNA-PKcs. De plus, il a été suggéré que la DNA-PK soutient la croissance des cellules PDAC. Les mécanismes impliqués demeurent toutefois mal étudiés. Récemment, il a été démontré que l'autophagie joue un rôle important dans la croissance des cellules PDAC. Dans certains types de cancer, la DNA-PK a été proposée comme une régulatrice de l’autophagie. Ainsi, nous avons entrepris cette étude en émettant l'hypothèse que la DNA-PK soutient l'autophagie et la croissance des cellules PDAC. Les lignées cellulaires PDAC MIA PaCa-2 et PANC1 ont été utilisées comme modèles d'étude. L'inhibiteur spécifique de la DNA-PK, le NU7441, a été exploitée pour explorer le rôle de la DNA-PK dans la croissance et l’autophagie des cellules PDAC. Une réduction, dépendante de la dose et du temps, de la croissance des cellules PDAC a été observée lors du traitement au NU7441. L'inhibition de la DNA-PK a également restreint la capacité des cellules PDAC à former des colonies lors d’essais de clonogénicité. Le traitement au NU7441 a provoqué un clivage de PARP et de la caspase-7 suggérant que l’inhibition de la DNA-PK induit l'apoptose. Le traitement au NU7441 a mené à une accumulation des niveaux de marqueurs d'autophagie soient LC3B II et p62. La mesure du flux autophagique a confirmé que cette accumulation

Cells, the building blocks of life, are vastly complex. This complexity confers to every living organism the ability to maintain oneself, reproduce oneself, and evolve. Creating a minimal system from nonliving components that is capable of self-maintenance, self-reproduction, and evolvability, will greatly increase our understanding of life. Essential features of every cell, synthetic or otherwise, are the compartment, a form of information transfer, and the ability to proliferate. In chapter 1, I argue that autonomy, that is self-governance, is another key characteristic of life. Therefore, to create a synthetic cell, aforementioned features should be recapitulated in an autonomous manner. This informs the synthetic cell design that is adhered to in this thesis. Phospholipid vesicles, so called liposomes, serve as a compartment. Reconstitution of the central dogma of molecular biology by cell-free gene expression with the PURE system enables the use of a genetic program encoded on DNA. By encoding its working instructions on its own DNA, the cell will be autonomous. Proliferation is carried out by a set of modules encoded on the DNA. These modules will serve to replicate the DNA itself, to replenish the gene expression machinery, and to stimulate growth and trigger division of the compartment. The goal of this thesis is to reconstitute compartment growth by cell-free gene expression of DNA encoding for phospholipid synthesis machinery., BN/Christophe Danelon Lab

Contextualization: Currently, interest in the production of microalgae has increased due to the different uses of microalgal biomass in some industrial sectors, in addition to being used in the mitigation of greenhouse gases thanks to its high capacity for CO2 capture. Knowledge gap: Being part of the Paris Agreement in 2015, Colombia is committed to reduce its greenhouse gases emissions by 20%, to contribute to the goal set for 2050, for this reason, it must adopt research and action plans focused on this objective. Purpose: the purpose of this study was to evaluate the effect of CO2 induction at different concentrations on the growth kinetics of Chlorella Vulgaris CV_2714A and Scenedesmus Obliquus SOB_001 in a laboratory scale photobioreactor system in a volume of 3.8 L of culture medium (NPK solution + micronutrients). Methodology: The tests were carried out in photoperiods with 12 hours of light and 12 hours of darkness for 5 consecutive days. The completely randomized experimental design whose sources of variation were microalgae species and CO2 dosage (0.2, 6.5 and 12.8 L. day-1) with artificial lighting by a 4400-lumen fluorescent lamp. The cellular concentration was estimated by means of the numerical approximation in the “Neubauer” chamber with the methodology used by Darki et al. (2017), in addition to growth rate and cell doubling time in days according to Andersen, (2005). The data were submitted to analysis of variance and Tukey's mean tests (p<0.05). Results and conclusions: The results in the different tests showed a higher cell concentration in C. vulgaris compared to S. obliquus. Regarding CO2 supply, the 2 species obtained a significantly greater response (p<0.05) at the dose of 6.5 L. day-1, reaching a cellular concentration of 2.59 x 107 cel.ml-1 and 4.62 x 106 cel.ml-1, respectively. These results allow us to conclude that the cultivation of these microalgae associated with the dose of CO2 can favor their rapid growth and biomass production, whi, Contextualización: actualmente se ha incrementado el interés en la producción de microalgas debido a los diferentes usos de la biomasa microalgal en algunos sectores industriales, además de la posibilidad de emplearse en la mitigación de gases de efecto invernadero gracias a su alta capacidad para la captura de CO2. Vacío de conocimiento: como parte del Acuerdo de Paris en 2015, Colombia se comprometió en reducir un 20% sus emisiones de gases efecto invernadero, con el propósito de contribuir a la meta establecida para 2050; por esta razón, se deben desarrollar investigaciones y planes de acción enfocados a este objetivo. Propósito del estudio: el propósito de este estudio es evaluar del efecto de la inducción de CO2 en diferentes concentraciones sobre la cinética de crecimiento de Chlorella Vulgaris CV_2714A y Scenedesmus Obliquus SOB_001 en un sistema de fotobiorreactores a escala laboratorio, en un volumen de 3,8 L de medio de cultivo (solución NPK + micronutrientes). Metodología: los ensayos se realizaron en fotoperiodos con 12 h de luz y 12 h de oscuridad durante 5 días consecutivos. El diseño experimental aleatorizado cuyas fuentes de variación fueron: especie microalgas y dosificación de CO2 (0, 2, 6,5, y 12,8 L. día-1) con iluminación artificial por lámpara fluorescente de 4400 lúmenes. Se estimó la concentración celular por medio de la aproximación numérica en cámara de “Neubauer” con la metodología usada por Darki et al. (2017), además de tasa de crecimiento y tiempo de duplicación celular en días de acuerdo con Andersen, (2005). Los datos fueron sometidos al análisis de varianza y pruebas de medias de Tukey (p < 0,05). Resultados y conclusiones: los resultados en los diferentes ensayos mostraron mayor concentración celular en C. vulgaris en comparación con S. obliquus. En cuanto al suministro de CO2, las 2 especies obtuvieron respuesta significativamente mayor (p <0,05) a la dosis de 6.5 L. día-1 alcanzando concentración es celulares de 2.59 x 107 cel.m

Plants possess cell wall, a polysaccharide exoskeleton which encompasses all plant cells. Cell wall gives plant cells mechanical support, defines their shape, enables growth and water transport through a plant. It also has important role in communication with the external environment. Regulation of plant cell wall biosynthesis and cell and organ morphogenesis depends on cell’s ability to detect mechanical signals originating both from the external environment and from internal plant tissues. Thanks to the presence of the cell wall, all living plant cells develop constant internal pressure generated by the active water uptake, known as turgor pressure, which enables them to grow. Thus, actively growing cells in the tissue are exerting mechanical stress to each other. In order to properly coordinate cell growth, tissue morphogenesis and maintain cell-to-cell adhesion, plant cell have to detect these mechanical signals. That is performed by a group of still not well enough characterized plant mechanosensitive proteins. Mechanosensors are proteins capable of detecting changes in mechanical stress patterns and translating them into physiological and developmental outputs. One of plant mechanosensitive proteins, DEFECTIVE KERNEL1 (DEK1) has shown to be a very important in proper plant development. DEK1 bears similarity with animal cysteine proteases of Calpain superfamily. DEK1 is very important for plant development since all null alleles are embryo lethal. During the last 20 years of DEK1 studies, this protein has proven to be a very difficult for different molecular and biochemical manipulations. As a consequence, very little is known about its direct target proteins. Wang and co-workers (2003) and Johnson and co-workers (2008) have given a valuable contribution to biochemical understanding of DEK1 by determining that it functions as Cys-protease in similar way as animal calpains. However, a lot of indirect knowledge was gathered about the effects of disruption and mod

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

Comprehensive maps of genetic interactions in mammalian cells are daunting to construct because of the large number of potential interactions, ~ 2 × 108 for protein coding genes. We previously used co-inheritance of distant genes from published radiation hybrid (RH) datasets to identify genetic interactions. However, it was necessary to combine six legacy datasets from four species to obtain adequate statistical power. Mapping resolution was also limited by the low density PCR genotyping. Here, we employ shallow sequencing of nascent human RH clones as an economical approach to constructing interaction maps. In this initial study, 15 clones were analyzed, enabling construction of a network with 225 genes and 2,359 interactions (FDR < 0.05). Despite its small size, the network showed significant overlap with the previous RH network and with a protein-protein interaction network. Consumables were ≲$50 per clone, showing that affordable, high quality genetic interaction maps are feasible in mammalian cells.

Given an increasing focus on environmental sustainability, microbial oils have been suggested as an alternative to petroleum-based products. However, microbial oil production relies on the use of costly sugar-based feedstocks. Substrate limitation, elevated costs, and risk of contamination have sparked the search for alternatives to sugar-based platforms. Volatile fatty acids are generated during anaerobic digestion of organic waste and are considered a promising substrate for microbial oil production. In the present study, two freshwater and one marine microalga along with two thraustochytrids were evaluated for their potential to produce lipids when cultivated on volatile fatty acids generated from food waste via anaerobic digestion using a membrane bioreactor. Freshwater microalgae Auxenochlorella protothecoides and Chlorella sorokiniana synthesized lipids rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). This composition corresponds to that of soybean and jatropha oils, which are used as biodiesel feedstock. Production of added-value polyunsaturated fatty acids (PUFA) mainly omega-3 fatty acids was examined in three different marine strains: Aurantiochytrium sp. T66, Schizochytrium limacinum SR21, and Crypthecodinium cohnii. Only Aurantiochytrium sp. T66 seemed promising, generating 43.19% docosahexaenoic acid (DHA) and 13.56% docosapentaenoic acid (DPA) in total lipids. In summary, we show that A. protothecoides, C. sorokiniana, and Aurantiochytrium sp. T66 can be used for microbial oil production from food waste material.

Given an increasing focus on environmental sustainability, microbial oils have been suggested as an alternative to petroleum-based products. However, microbial oil production relies on the use of costly sugar-based feedstocks. Substrate limitation, elevated costs, and risk of contamination have sparked the search for alternatives to sugar-based platforms. Volatile fatty acids are generated during anaerobic digestion of organic waste and are considered a promising substrate for microbial oil production. In the present study, two freshwater and one marine microalga along with two thraustochytrids were evaluated for their potential to produce lipids when cultivated on volatile fatty acids generated from food waste via anaerobic digestion using a membrane bioreactor. Freshwater microalgae Auxenochlorella protothecoides and Chlorella sorokiniana synthesized lipids rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). This composition corresponds to that of soybean and jatropha oils, which are used as biodiesel feedstock. Production of added-value polyunsaturated fatty acids (PUFA) mainly omega-3 fatty acids was examined in three different marine strains: Aurantiochytrium sp. T66, Schizochytrium limacinum SR21, and Crypthecodinium cohnii. Only Aurantiochytrium sp. T66 seemed promising, generating 43.19% docosahexaenoic acid (DHA) and 13.56% docosapentaenoic acid (DPA) in total lipids. In summary, we show that A. protothecoides, C. sorokiniana, and Aurantiochytrium sp. T66 can be used for microbial oil production from food waste material.

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical. © 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH

Background: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing. © 2021, The Author(s).

High production titers are paramount in lactic fermentation processes aimed towards poly lactic acid (PLA) manufacturing. Optimization of the fermentation stage is therefore essential for the viability of the process. L-lactic acid producing Pediococcus acidilactici strain TY112 was grown in shake flasks and 2L bioreactors with glucose and mannose as carbon sources under different aeration conditions and with an increased concentration of yeast extract. The presence of oxygen in the medium was found to activate undesired oxidative metabolic pathways in the strain through the apparent activation of NADH and pyruvate oxidases. This metabolic shift diverted precursors from lactate synthesis onto acetate synthesis, so the presence of oxygen should therefore be avoided during high-yield fermentation processes. Yeast extract was found to be stimulatory to cell growth, inducing higher sugar consumption and acid production rates without compromising the production rates. Therefore, the presence of yeast extract in the medium leads to an enhanced productivity in the system which is beneficial to large scale processes. However, further research should be conducted to fully identify the specific components in yeast extract responsible for this stimulatory growth., How does our bacterial gym look like, you ask? It’s warm, underwater, woody and full of sugar. Our bacteria love it and they pay us with molecules that we will turn into bio-plastics. Keep reading to know more about our training plan! Ever heard of lactic acid? It’s a molecule found in a lot of fermented foods, like yogurt or sauerkraut, but we can also produce it when we exercise. The best producers, however, are microbes! They can turn basic and complex sugars into lactic acid very efficiently. Now here’s the thing: lactic acid is not only good for food or exercise, there’s also a way to use it to produce bioplastics! However, this technology is not competitive with fossil plastics yet, because the production costs are too high. That’s why there is a lot of research trying to optimize the production process, and a lot of it has to do with how the microbes produce the lactic acid. One line of work is to find or develop strains of microbes that can convert the sugars the most efficiently possible. Another, is to use renewable sugar sources which do not compete with our food, like wood! However, to use the sugars in wood, it first has to be treated so the sugars can escape the wood network and then the microbes can eat them. In this study, we used the bacterial strain Pediococcus acidilactici TY112 to produce lactic acid because it can use the sugars very efficiently, but also because it can survive in the presence of toxic compounds found in wood! We studied how the bacteria behaved when we gave them oxygen to see if it was good as they produce lactic acid. However, we saw that when they have oxygen, they use the sugars to produce acetic acid instead of lactic acid, so of course we don’t want that if our goal is to produce lactic acid! We also studied how they react when we give them a very rich nutrient called yeast extract. Yeast extract is full of nutrients and vitamins, so we thought it would help them grow and produce lactic acid. We grew the cells and gave the