Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus.

A circadian clock coordinates physiology and behavior in diverse groups of living organisms. Another major cyclic cellular event, the cell cycle, is regulated by the circadian clock in the few cases where linkage of these cycles has been studied. In the cyanobacterium Synechococcus elongatus, the circadian clock gates cell division by an unknown mechanism. Using timelapse microscopy, we confirm the gating of cell division in the wild-type and demonstrate the regulation of cytokinesis by key clock components. Specifically, a state of the oscillator protein KaiC that is associated with elevated ATPase activity closes the gate by acting through a known clock output pathway to inhibit FtsZ ring formation at the division site. An activity that stimulates KaiC phosphorylation independently of the KaiA protein was also uncovered. We propose a model that separates the functions of KaiC ATPase and phosphorylation in cell division gating and other circadian behaviors.

Flow-NMR as a Process-Monitoring Tool for mRNA IVT Reaction.

Messenger RNA (mRNA) based vaccines were instrumental in accelerating the end of the SARS-CoV-2 pandemic and are being aggressively developed as prophylaxes for a range of viral diseases. The swift adoption of mRNA-based therapeutics has also left open vast areas of opportunity for improving the development of mRNA-based drugs. One such area with immense potential focuses on the mRNA drug substance production, where mRNA is generated by a cell-free reaction called in vitro transcription (IVT). Process analytical technologies (PAT) are integral to the pharmaceutical industry and are necessary to facilitate agile process optimization and enhance process quality, control, and understanding. Due to the complexity and novelty inherent to the IVT reaction, there is a need for effective PAT that would provide in-depth, real-time insight into the reaction process to allow delivery of novel mRNA vaccines to patients faster in a more cost-effective way. Herein, we showcase the development of flow-nuclear magnetic resonance (flow-NMR) as a highly effective process-analytical tool for monitoring mRNA IVT reactions to support process development, optimization, and production.

CircPKM2 aggravates the progression of non-small cell lung cancer by regulating MTDH via miR-1298-5p.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with high morbidity and mortality. The role of dysregulated circular RNAs (circRNAs) in human diseases are receiving more and more attention. In this study, we focused on the role and mechanism of circPKM2 in the progression of NSCLC. METHODS: The expression levels of circPKM2, microRNA-1298-5p (miR-1298-5p) and metadherin (MTDH) in NSCLC were measured by real-time quantitative PCR (qRT-PCR) or Western blot. Cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, transwell and tube formation assays were conducted to evaluate the effects of circPKM2 on malignant phenotypes of NSCLC. Western blot was used to measure related marker protein levels. RESULTS: CircPKM2 and MTDH were highly expressed in NSCLC tissues and cells, while miR-1298-5p was downregulated. CircPKM2 knockdown effectively suppressed cell proliferation, migration, invasion and tube formation whereas induced apoptosis in vitro. CircPKM2 had a potential targeting site with miR-1298-5p and negatively regulated the expression of miR-1298-5p. MiR-1298-5p inhibitor reversed the effect of circPKM2 knockdown on the progression of NSCLC. CircPKM2 induced MTDH expression via sponging miR-1298-5p to promote the progression of NSCLC. MiR-1298-5p directly targeted MTDH, and the addition of MTDH partially attenuated the inhibition of miR-1298-5p on the progression of NSCLC. In addition, the downregulation of circPKM2 significantly slowed down the growth of xenograft tumors in vivo. CONCLUSION: Our findings demonstrated that circPKM2 mediated NSCLC progression via regulating miR-1298-5p/MTDH axis, providing a novel therapeutic target for NSCLC.

Increased homocysteine regulated by androgen activates autophagy by suppressing the mammalian target of rapamycin pathway in the granulosa cells of polycystic ovary syndrome mice.

The purpose of this study was to explore the potential molecular mechanisms of excess homocysteine in relation to autophagic activity in the ovarian tissue of polycystic ovarian syndrome (PCOS) with hyperandrogenism.A PCOS model was constructed using ICR mice. ELISA was used to detect the Hcy levels in the serum and ovarian tissues of PCOS model. The expression level of key enzymes (Methionine synthase and Betaine-homocysteine methyltransferase, MTR and BHMT) in homocysteine metabolism and autophagy-related proteins were detected in ovarian tissues and mouse granulosa cells (mGCs) that were treated with homocysteine, androgen, autophagy inhibitors or BHMT-expressing plasmid by western blot and immunohistochemistry. Electron microscope experiments were used to evaluate autophagosomes in Hcy-treated mGCs. The prenatally androgenized (PNA) PCOS mouse model showed hyperhomocysteinemia and hyperandrogenism. Homocysteine levels displayed a significant increase, while its metabolic enzymes levels were significantly decreased in ovarian tissues of PCOS mice and dihydrotestosterone (DHT)-stimulated mGCs. The LC3II and Beclin1 expression levels were increased and the P62 and p-mTOR levels were decreased in vivo in ovarian tissue from the PCOS mice. The in vitro data were similarly with the in vivo by stimulation of mGCs with DHT or homocysteine. These effects could be diminished by the autophagy inhibitor (MHY1485), androgen receptor antagonists (ARN509) or BHMT-expressing plasmid. Androgen increases homocysteine concentration by downregulating the key enzymes in homocysteine metabolism. And then Hcy promotes GCs autophagy via the mTOR signal pathway.

The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which, together with ATP, can generate a self-sustained approximately 24 h oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate, whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the "A-loops", are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. A dynamic equilibrium likely exists between the buried and exposed states, which determines the steady-state level of phosphorylation of KaiC. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state, KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation, and we show evidence in support of how this movement may be accomplished.

Telomerase inhibition decreases esophageal squamous carcinoma cell migration and invasion.

Telomerase has been shown to be associated with a variety of cancer types. To elucidate the role of telomerase in esophageal squamous carcinoma (ESCC), tissue samples from 100 patients with ESCC, and paired paracancerous tissues from 75 of these patients, were collected for use in the present study. Using immunohistochemical analysis, the expression of telomerase reverse transcriptase (hTERT) in the cytoplasm of ESCC cells was revealed to be significantly higher compared with that in paracancerous tissues, and no significant difference was observed between hTERT expression in the nucleus of ESCC and paracancerous tissue cells. Combined analysis revealed that the cytoplasmic hTERT-positive rate of patients with ESCC was significantly associated with pathological grade, N stage and Tumor-Node-Metastasis (TNM) stage; these data support the association between hTERT expression and poor patient prognosis. In vitro experiments demonstrated that hTERT knockdown does not inhibit the proliferation of ESCC Kyse410 or Kyse520 cells, but inhibits their migration and invasion abilities. These findings indicate that hTERT expression is associated with ESCC metastasis. Interestingly, decreased colony-formation ability was observed in Kyse410 cells, but not in Kyse520 cells. Collectively, the results of the present study suggest that hTERT may serve as a potential therapeutic target for ESCC.

[Increased expression of transendothelial migration-related molecules CCL4, ICAM-1 and VCAM-1 in lung tissue after surgical removal of mouse tumor-bearing lymph node].

Objective To detect the expression of chemokine and adhesion molecules related to leukocytes' transendothelial migration, meanwhile, to investigate changes of reticular fibers and the expression of vimentin and matrix metalloproteinase-9 (MMP9) in lung tissues after surgical removal of mouse tumor-bearing lymph node, revealing their changes and roles in the formation of pre-metastatic microenvironment in the lung. Methods B16F10 melanoma cells were inoculated into mouse subiliac lymph node (SiLN). Twenty mice were equally divided into groups with or without (as a control group) tumor-bearing SiLN removal. Fifteen days later, tumor-bearing lymph node was surgically removed; 3 days after resection, mouse lung tissues were collected. The change of reticular fibers in lung tissues was observed by silver impregnation staining. The expression of C-C motif chemokine ligand 4 (CCL4), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), vimentin and MMP9 in lung tissues was detected by Western blotting. Results Compared with control group, expression of MMP9 and vimentin increased significantly in the lung tissues of SiLN removal group; reticular fibers were obviously fractured and its per area was reduced. Moreover, expression of CCL4, ICAM-1 and VCAM-1 also significantly increased. Conclusion Expression of CCL4, ICAM-1, VCAM-1, MMP9 and vimentin in mouse lung tissues is promoted after surgical removal of tumor-bearing lymph node, contributing to inflammatory cells' adhesion to and extravasation across vascular endothelium and further resulting in the formation of inflammatory microenvironment.

Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus.

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.

Dynamic localization of the cyanobacterial circadian clock proteins.

BACKGROUND: The cyanobacterial circadian clock system has been extensively studied, and the structures, interactions, and biochemical activities of the central oscillator proteins (KaiA, KaiB, and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell. RESULTS: Here we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC colocalize with each other, as well as with CikA, a key input and output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod-shaped bacterium, suggesting that KaiC localization is not dependent on other clock- or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking delocalization of KaiC. CONCLUSIONS: This work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli.

Gene transfer in Leptolyngbya sp. strain BL0902, a cyanobacterium suitable for production of biomass and bioproducts.

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira ("pirulina" strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a "elper"plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain.

Simplicity and complexity in the cyanobacterial circadian clock mechanism.

The circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 is built on a three-protein central oscillator that can be reconstituted in vitro, a redox-sensitive input for synchronization with the environment, and a bacterial two-component signal transduction pathway for global transcriptional regulation. This review covers the most recent progress in our understanding of the biological and biochemical mechanism of this bacterial clock, such as the discovery of a quinone-binding activity of the oscillator protein KaiA, the molecular mechanism of circadian control of cell division, and the global control of gene expression via modulation of DNA topology.

Circadian gating of the cell cycle revealed in single cyanobacterial cells.

Although major progress has been made in uncovering the machinery that underlies individual biological clocks, much less is known about how multiple clocks coordinate their oscillations. We simultaneously tracked cell division events and circadian phases of individual cells of the cyanobacterium Synechococcus elongatus and fit the data to a model to determine when cell cycle progression slows as a function of circadian and cell cycle phases. We infer that cell cycle progression in cyanobacteria slows during a specific circadian interval but is uniform across cell cycle phases. Our model is applicable to the quantification of the coupling between biological oscillators in other organisms.

How a cyanobacterium tells time.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator composed of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. An input pathway of redox-sensitive proteins uses photosynthetic function to relay light/dark information to the oscillator, and signal transduction proteins of well-known families broadcast temporal information to the genome, where global changes in transcription and a compaction of the chromosome are clock regulated.

The pseudo-receiver domain of CikA regulates the cyanobacterial circadian input pathway.

CikA (circadian input kinase) is a component of the cyanobacterial circadian clock that aids in synchronizing the endogenous oscillator with the external environment. cikA mutants of the prokaryotic circadian model organism Synechococcus elongatus PCC 7942 fail to reset the phase of the circadian rhythm of gene expression after an environmental time cue, and also exhibit reduced amplitude and shortened period of circadian oscillation. CikA has histidine protein kinase (HPK) activity that is modulated in vitro by GAF and pseudo-receiver (PsR) domains. Here we show that the PsR domain negatively regulates HPK activity in vivo and also serves as an interaction module to dock CikA at a specific subcellular location. Phenotypes conferred by alleles that encode CikA variants showed that all domains except the featureless N-terminus are required for CikA function. Overexpression of all alleles that encode the PsR domain, whether or not the HPK is functional, caused a dominant arrhythmic phenotype, whereas overexpressed variants that lack PsR did not. Subcellular localization of intact CikA identified a polar focus whereas a variant without PsR showed uniform distribution in the cell, consistent with a model in which PsR mediates interaction with other input pathway components.