Chlamydomonas CHT7 Is Required for an Effective Quiescent State by Regulating Nutrient-Responsive Cell Cycle Gene Expression.

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.

Chloroplast Sec14-like 1 (CPSFL1) is essential for normal chloroplast development and affects carotenoid accumulation in Chlamydomonas.

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of Chlamydomonas reinhardtii and isolated cpsfl1 The cpsfl1 mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The CPSFL1 gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The cpsfl1 mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and ß-carotene when expressed in Escherichia coli and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

Modulation of CHT7 Complexes during Light/Dark- and Nitrogen-Mediated Life Cycle Transitions of Chlamydomonas.

The Chlamydomonas reinhardtii Compromised Hydrolysis of Triacylglycerols7 (CHT7) protein has been previously implicated in the regulation of DNA metabolism and cell-cycle-related gene expression during nitrogen (N) deprivation, and its predicted protein interaction domains are necessary for function. Here, we examined impacts of the cht7 mutation during the cell division cycle under nutrient deficiency in light-dark synchronized cultures. We explored the potential mechanisms affecting CHT7 complex activities during the cell cycle and N starvation, with a focus on the possible interaction between CHT7 and the C. reinhardtii retinoblastoma tumor suppressor (RB) protein homolog MAT3. Notably, the absence of CHT7 did not negatively impact the synchrony of cell division and cell cycle progression during diel growth. Although the majority of CHT7 and MAT3/RB proteins were observed in separate complexes by blue native-PAGE, the two proteins coimmunoprecipitated both during synchronized growth and following N deprivation, suggesting the presence of low abundance subcomplexes containing CHT7 and MAT3/RB. Furthermore, we observed several phosphorylated isoforms of CHT7 under these conditions. To test the potential role of phosphorylation on the structure and function of CHT7, we performed site-directed mutagenesis of previously identified phosphorylated amino acids within CHT7. These phosphorylated residues were dispensable for CHT7 function, but phosphorylated variants of CHT7 persisted, indicating that yet-unidentified residues within CHT7 are also likely phosphorylated. Based on the interaction of CHT7 and MAT3/RB, we postulate the presence of a low-abundance or transient regulatory complex in C. reinhardtii that may be similar to DREAM-like complexes in other organisms.

In vivo lipid 'tag and track' approach shows acyl editing of plastid lipids and chloroplast import of phosphatidylglycerol precursors in Arabidopsis thaliana.

In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexity in vivo, we developed an in vivo lipid 'tag and track' method. Essentially, we probed the lipid metabolism in Arabidopsis thaliana by expressing a coding sequence for a fatty acid desaturase from Physcomitrella patens (Δ6D). This enzyme places a double bond after the 6th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at its sn-2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to 'tag and track' the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence of ER-derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this 'tag and track' method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions.

The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas.

Microalgae are prolific photosynthetic organisms that have the potential to sustainably produce high-value chemical feedstocks. However, an industry based on microalgal biomass still is faced with challenges. For example, microalgae tend to accumulate valuable compounds, such as triacylglycerols, only under stress conditions that limit growth. To investigate the fundamental mechanisms at the base of this conundrum--the inverse relationship between biomass production and storage compound accumulation-we applied a combination of cell biological and genetic approaches. Conceptually, nutrient deprivation, which commonly is used to induce the accumulation of triacylglycerol in microalgae, leads to a state of cellular quiescence defined by a halt of cell divisions that is reversible upon nutrient resupply. To identify factors that govern cellular quiescence, we screened for mutants of the model alga Chlamydomonas reinhardtii that, in contrast to wild-type cells placed under conditions of nitrogen deprivation, were unable to degrade triacylglycerols following nitrogen resupply. One of the mutants described here in detail, compromised hydrolysis of triacylglycerols 7 (cht7), was severely impaired in regrowth following removal of different conditions inducing cellular quiescence. The mutant carries a deletion affecting four genes, only one of which rescued the quiescence phenotype when reintroduced. It encodes a protein with similarity to mammalian and plant DNA binding proteins. Comparison of transcriptomes indicated a partial derepression of quiescence-related transcriptional programs in the mutant under conditions favorable to growth. Thus, CHT7 likely is a repressor of cellular quiescence and provides a possible target for the engineering of high-biomass/high-triacylglycerol microalgae.

Direct regulation of abiotic responses by the Arabidopsis circadian clock component PRR7.

Up to 30% of the plant transcriptome is circadian clock-regulated in different species; however, we still lack a good understanding of the mechanisms involved in these genome-wide oscillations in gene expression. Here, we show that PSEUDO-RESPONSE REGULATOR 7 (PRR7), a central component of the Arabidopsis clock, is directly involved in the repression of master regulators of plant growth, light signaling and stress responses. The expression levels of most PRR7 target genes peak around dawn, in an antiphasic manner to PRR7 protein levels, and were repressed by PRR7. These findings indicate that PRR7 is important for cyclic gene expression by repressing the transcription of morning-expressed genes. In particular we found an enrichment of the genes involved in abiotic stress responses, and in accordance we observed that PRR7 is involved in the oxidative stress response and the regulation of stomata conductance.

Light and the circadian clock mediate time-specific changes in sensitivity to UV-B stress under light/dark cycles.

In Arabidopsis, the circadian clock regulates UV-B-mediated changes in gene expression. Here it is shown that circadian clock components are able to inhibit UV-B-induced gene expression in a gene-by-gene-specific manner and act downstream of the initial UV-B sensing by COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and UVR8 (UV RESISTANCE LOCUS 8). For example, the UV-B induction of ELIP1 (EARLY LIGHT INDUCIBLE PROTEIN 1) and PRR9 (PSEUDO-RESPONSE REGULATOR 9) is directly regulated by LUX (LUX ARRYTHMO), ELF4 (EARLY FLOWERING 4), and ELF3. Moreover, time-dependent changes in plant sensitivity to UV-B damage were observed. Wild-type Arabidopsis plants, but not circadian clock mutants, were more sensitive to UV-B treatment during the night periods than during the light periods under diel cycles. Experiments performed under short cycles of 6h light and 6h darkness showed that the increased stress sensitivity of plants to UV-B in the dark only occurred during the subjective night and not during the subjective day in wild-type seedlings. In contrast, the stress sensitivity of Arabidopsis mutants with a compromised circadian clock was still influenced by the light condition during the subjective day. Taken together, the results show that the clock and light modulate plant sensitivity to UV-B stress at different times of the day.

A qualitative study exploring the epistemology of suffering within a Malaysian Indigenous tribe.

Despite the universal nature of suffering, few studies have examined how Indigenous ethnic minorities in non-western regions understand and respond to adversity. This study explored the epistemology of suffering among the Temiar ethnic group of Peninsular Malaysia using participant observation and semi-structured interviews. Interview transcripts of 43 participants were coded through inductive thematic analysis and a consensual qualitative approach. Three-tier themes were defined and named after subsequent analysis of core ideas and domains in the data. Major adversities reported included a lack of basic needs, lack of land-rights and unjust treatment from authorities, destruction of the forest environment and livelihood, and lack of accessibility and facilities, which were attributed to authorities' negligence of responsibilities, increasing human-animal conflict, environmental threats and imposed lifestyle changes. Faced with adversity, the Temiar endeavoured to survive by working crops and gathering forest resources. They utilized resources from family, fellow villagers, external agencies and spiritual-religious traditions. Theoretical mapping of attribution styles into the Ecological Rationality Framework revealed predominantly external-focused and concrete-perceptual rationalities privileged by strong-ties societies. These findings pointed to the resilience of a strong-ties community while adapting to the systemic suffering and risk factors stemming from a rationality mismatch with modernization and globalization trends. To conclude, we advocate for culture-sensitive mental health and psychiatric practices, as well as sustainable development for the well-being of Indigenous communities locally and globally.

Helicobacter pylori DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium sulphate-induced colitis.

BACKGROUND AND AIMS: Epidemiological data have recently emerged to suggest Helicobacter pylori may protect against certain chronic inflammatory diseases such as inflammatory bowel disease (IBD). However, the mechanism for the observed inverse association between H pylori and IBD has not been described. METHODS: The frequency of immunoregulatory (IRS) to immunostimulatory (ISS) sequences within the genome of various bacteria was calculated using MacVector software. The induction of type I IFN and IL-12 responses by DNA-pulsed murine bone marrow-derived dendritic cells (BMDC) and human plasmacytoid dendritic cells (DC) was analysed by cytokine production. The effect of H pylori DNA on Escherichia coli DNA production of type I IFN and IL-12 was assessed. The in-vivo significance of H pylori DNA suppression was assessed in a dextran sodium sulphate (DSS) model of colitis. The systemic levels of type I IFN were assessed in H pylori-colonised and non-colonised patients. RESULTS: H pylori DNA has a significantly elevated IRS:ISS ratio. In-vitro experiments revealed the inability of H pylori DNA to stimulate type I IFN or IL-12 production from mouse BMDC or human plasmacytoid DC. H pylori DNA was also able to suppress E coli DNA production of type I IFN and IL-12. The administration of H pylori DNA before the induction of DSS colitis significantly ameliorated the severity of colitis compared with E coli DNA or vehicle control in both an acute and chronic model. Finally, the systemic levels of type I IFN were found to be lower in H pylori-colonised patients than non-colonised controls. CONCLUSIONS: This study indicates that H pylori DNA has the ability to downregulate pro-inflammatory responses from DC and this may partly explain the inverse association between H pylori and IBD.

Chlamydomonas CHT7 is involved in repressing DNA replication and mitotic genes during synchronous growth.

In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network.

Cultural adaptation and validation of instruments for measuring the flipped classroom experience.

INTRODUCTION: In 2017, a revamped bachelor of pharmacy program was introduced at Monash University and incorporated a predominantly flipped classroom-based pedagogy. The attitudes and preferences of students towards this program had yet to be assessed using a reliable instrument. Since no instrument was readily available, the objective of this study was to identify, contextualize, and validate a suitable instrument. METHODS: We conducted a literature search to identify and adapt a validated instrument. Cognitive interviews were conducted to examine students' understanding of scales and definitions of items. The instrument was then evaluated by education experts for further refinement. The reliability of the final instrument was assessed in a cohort of students, and unsuitable items were removed. RESULTS: Students had issues understanding the scales and specific terms used in the original instrument, potentially due to differences in terminologies used in the university's context and variance in English proficiency levels and exposure. In the preference domain, wording of the instrument to present exclusively traditional classroom or exclusively flipped classroom statements greatly influenced its reliability. This could be due to exposure of students to a predominantly flipped classroom environment since inception. The final instrument optimized in this study had α = 0.85, 0.86, and 0.9 for the pre-activities, in-class lectures, and in-class workshops attitude domains, respectively, and α = 0.73 for the preference domain. CONCLUSIONS: Our study highlights the necessity of contextualizing instruments to fit the local context in which they are administered and provides key recommendations when conducting such adaptations.

Helicobacter pylori immune escape is mediated by dendritic cell-induced Treg skewing and Th17 suppression in mice.

BACKGROUND & AIMS: Helicobacter pylori infection increases gastric regulatory T cell (Treg) response, which may contribute to H pylori immune escape. We hypothesize that H pylori directs Treg skewing by way of dendritic cells (DCs) and thus inhibits interleukin-17(+) helper T cells (Th17) immunity. METHODS: Two-photon microscopy was used to locate DCs in gastric lamina propria of mice. The induction of Th17 and Treg responses by bacteria-pulsed murine bone marrow-derived DCs was analyzed by cytokine production and stimulation of T-cell proliferation. The effect of VacA, CagA, transforming growth factor-beta (TGF-beta), and IL-10 on Th17/Treg balance was assessed. The in vivo significance of Tregs on the H pylori-specific Th17 response and H pylori density was determined by using anti-CD25 neutralizing antibodies to deplete Tregs in mice. RESULTS: We showed that mucosal CD11c(+) DCs are located near the surface of normal gastric epithelium, and their number increased after H pylori infection. Study of the direct interaction of DCs with H pylori showed a Treg-skewed response. The Treg skewing was independent of H pylori VacA and CagA and dependent on TGF-beta and IL-10. In vivo Treg skewing by adoptive transfer of H pylori-pulsed DCs reduces the ratio of gastric IL-17/Foxp3 mRNA expressions. The depletion of CD25(+) Tregs results in early reduction of H pylori density, which is correlated with enhanced peripheral H pylori-specific Th17, but not Th1, response. CONCLUSIONS: Overall, our study indicates that H pylori alters the DC-polarized Th17/Treg balance toward a Treg-biased response, which suppresses the effective induction of H pylori-specific Th17 immunity.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4.

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.

Nitrogen-dependent coordination of cell cycle, quiescence and TAG accumulation in Chlamydomonas.

Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biopharmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the potential to make algal biofuels more economically viable, a number of challenges continue to hamper algal production systems at all levels. One such hurdle includes the metabolic trade-off often observed between the increased yields of desired products, such as triacylglycerols (TAG), and the growth of an organism. Initial genetic engineering strategies to improve lipid productivity in microalgae, which focused on overproducing the enzymes involved in fatty acid and TAG biosynthesis or inactivating competing carbon (C) metabolism, have seen some successes albeit at the cost of often greatly reduced biomass. Emergent approaches that aim at modifying the dynamics of entire metabolic pathways by engineering of pertinent transcription factors or signaling networks appear to have successfully achieved a balance between growth and neutral lipid accumulation. However, the biological knowledge of key signaling networks and molecular components linking these two processes is still incomplete in photosynthetic eukaryotes, making it difficult to optimize metabolic engineering strategies for microalgae. Here, we focus on nitrogen (N) starvation of the model green microalga, Chlamydomonas reinhardtii, to present the current understanding of the nutrient-dependent switch between proliferation and quiescence, and the drastic reprogramming of metabolism that results in the storage of C compounds following N starvation. We discuss the potential components mediating the transcriptional repression of cell cycle genes and the establishment of quiescence in Chlamydomonas, and highlight the importance of signaling pathways such as those governed by the target of rapamycin (TOR) and sucrose nonfermenting-related (SnRK) kinases in the coordination of metabolic status with cellular growth. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity and of the signaling pathways linking cellular growth to energy and lipid homeostasis, is essential to improve the prospects of biofuels and biomass production in microalgae.

Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779.

Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene ( NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).