The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

A universal trade-off between growth and lag in fluctuating environments.

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.

Evolution and stability of complex microbial communities driven by trade-offs.

Microbial communities are ubiquitous in nature and play an important role in ecology and human health. Cross-feeding is thought to be core to microbial communities, though it remains unclear precisely why it emerges. Why have multi-species microbial communities evolved in many contexts and what protects microbial consortia from invasion? Here, we review recent insights into the emergence and stability of coexistence in microbial communities. A particular focus is the long-term evolutionary stability of coexistence, as observed for microbial communities that spontaneously evolved in the E. coli long-term evolution experiment (LTEE). We analyze these findings in the context of recent work on trade-offs between competing microbial objectives, which can constitute a mechanistic basis for the emergence of coexistence. Coexisting communities, rather than monocultures of the 'fittest' single strain, can form stable endpoints of evolutionary trajectories. Hence, the emergence of coexistence might be an obligatory outcome in the evolution of microbial communities. This implies that rather than embodying fragile metastable configurations, some microbial communities can constitute formidable ecosystems that are difficult to disrupt.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy.

Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.

Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells.

Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87MG cells significantly increased ciliation. Those PCs were significantly longer than the control cells. Such increased ciliation possibly inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC disassembly and cilia length.

Glycolysis/gluconeogenesis specialization in microbes is driven by biochemical constraints of flux sensing.

Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic reorganization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis, and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. We combined growth dynamics and dynamic changes in intracellular metabolite levels with a coarse-grained model that integrates fluxes, regulation, protein synthesis, and growth and uncovered fundamental limitations of the regulatory network: After nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade-offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.

Biopolymer-based nanocomposite films and coatings: recent advances in shelf-life improvement of fruits and vegetables.

Consumers increasingly prefer healthy and nutritious diet worldwide, and demands for fresh fruits and vegetables are rapidly growing. Fresh produce are perishable commodities, and physical damage, moisture loss, biochemical changes, and postharvest microbial decay are primary causes of quality loss and reduced shelf-life. Packaging, including plastic films and coatings is an effective strategy to improve postharvest-life of whole and cut fruits and vegetables. However, plastic packaging is a significant environmental concern globally. Biopolymer based films and/or coatings are environment-friendly alternative packaging for food. But, these biopolymers, derived from plant, animal and microbial sources, lack some of the primary physico-chemical and mechanical properties compared to conventional plastic packaging. Reinforcement of biopolymer with nanomaterials addresses these shortcomings, and adds functional properties such as antimicrobial and/or antioxidant activities to the nanocomposites. Organic (e.g. nanocellulose fibrils), and inorganic (e.g. montmorillonite, zinc oxide, silver) nanomaterials are effective in achieving these improvements in biopolymer based nanocomposite. Plant-extracts and compounds derived from plant (e.g. essential oil) are also effective in imparting antimicrobial and antioxidant properties to biopolymer based nanocomposites. This is an extensive review of research works on effectiveness of biopolymer based nanocomposite films and coatings used for packaging of whole and cut fruits and vegetables to extend their shelf-life. Numerous reports have demonstrated effectiveness of biopolymer based nanocomposites in improvement in shelf-life of packaged and/or coated whole and cut fruits and vegetables by at least 4-5 days to as much as a few months.HighlightsFresh produce are perishable commodities requiring package or coating.Conventional plastics and waxes are major environmental and health concerns.Biopolymer based nanocomposites are environment-friendly alternatives.These nanocomposite films and coatings are effective in enhancing shelf-life.

New quantitative approach reveals heterogeneity in mitochondrial structure-function relations in tumor-initiating cells.

Steady-state mitochondrial structure or morphology is primarily maintained by a balance of opposing fission and fusion events between individual mitochondria, which is collectively referred to as mitochondrial dynamics. The details of the bidirectional relationship between the status of mitochondrial dynamics (structure) and energetics (function) require methods to integrate these mitochondrial aspects. To study the quantitative relationship between the status of mitochondrial dynamics (fission, fusion, matrix continuity and diameter) and energetics (ATP and redox), we have developed an analytical approach called mito-SinCe2 After validating and providing proof of principle, we applied mito-SinCe2 on ovarian tumor-initiating cells (ovTICs). Mito-SinCe2 analyses led to the hypothesis that mitochondria-dependent ovTICs interconvert between three states, that have distinct relationships between mitochondrial energetics and dynamics. Interestingly, fusion and ATP increase linearly with each other only once a certain level of fusion is attained. Moreover, mitochondrial dynamics status changes linearly with ATP or with redox, but not simultaneously with both. Furthermore, mito-SinCe2 analyses can potentially predict new quantitative features of the opposing fission versus fusion relationship and classify cells into functional classes based on their mito-SinCe2 states.This article has an associated First Person interview with the first author of the paper.

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

Exploring the potential of jujube seed powder in polysaccharide based functional film: Characterization, properties and application in fruit preservation.

In this study, we fabricated a novel biodegradable functional film using natural polysaccharides by adding jujube seed powder as an active ingredient. Scanning electron microscopy analysis showed agglomerate formation in the film with increasing concentration of seed powder. Fourier transform-infrared spectroscopy study demonstrated an electrostatic interaction between pectin and chitosan. The water solubility and swelling degree significantly decreased from 55.5 to 47.7 % and 66.0 to 41.9 %, respectively, depicting the film's water resistance properties. Higher opacity and lower transmittance value of the film indicated its protective effect towards light-induced oxidation of food. It was observed that the fabricated active film biodegraded to 82.33 % in 6 days. The DPPH radical scavenging activity of 98.02 % was observed for the functional film. The film showed antifungal activity against B. cinerea and P. chrysogenum. The highest zone of inhibition was obtained against food spoiling bacteria B. subtilis followed by S. aureus, P. aeruginosa and E. coli. Genotoxicity studies with the fabricated film showed a mitotic index of 8 % compared to 3 % in the control film. We used the fabricated film to preserve grapefruits, and the result showed that it could preserve grapes for ten days with an increase in antioxidant activity and polyphenolic content.

Homeostasis of cytoplasmic crowding by cell wall fluidization and ribosomal counterions.

In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained3, although recent works have revealed that these processes are indeed coupled4,5. Here, we report a striking increase of turgor pressure with growth rate in E. coli, suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics6.

Chitosan-based composite films containing eugenol nanoemulsion, ZnO nanoparticles and Aloe vera gel for active food packaging.

Biopolymer-based food packaging films are gaining increasing popularity, as consumers' demands for sustainable alternatives and environmental concerns associated with synthetic plastic packaging grow. In this research work, chitosan-based active antimicrobial films reinforced with eugenol nanoemulsion (EuNE), Aloe vera gel, and zinc oxide nanoparticles (ZnONPs) were fabricated and characterized for their solubility, microstructure, optical properties, antimicrobial and antioxidant activities. The rate of release of EuNE from the fabricated films was also evaluated to determine active nature of the films. The EuNE droplet size was about 200 nm, and they were uniformly distributed throughout the film matrices. Incorporation of EuNE in chitosan drastically improved UV-light barrier property of the fabricated composite film by 3 to 6 folds, while maintaining their transparency. The XRD spectra of the fabricated films showed good compatibility between the chitosan and the incorporated active agents. The incorporation of ZnONPs significantly improved their antibacterial properties against foodborne bacteria and tensile strength about 2-folds, whereas incorporation of EuNE and AVG improved DPPH scavenging activities of the chitosan film up to 95 %, respectively.

Long noncoding RNAs are substrates for cytoplasmic capping enzyme.

Cytoplasmic capping returns a cap to specific mRNAs, thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long noncoding RNAs (lncRNAs) are substrates of the cytoplasmic capping enzyme (cCE). The subcellular localisation of 14 lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5' rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5'-RACE cloned sequences, suggesting that these uncapped lncRNAs are targets of cytoplasmic capping.

Coexisting ecotypes in long-term evolution emerged from interacting trade-offs.

Evolution of complex communities of coexisting microbes remains poorly understood. The long-term evolution experiment on Escherichia coli (LTEE) revealed the spontaneous emergence of stable coexistence of multiple ecotypes, which persisted for more than 14,000 generations of continuous evolution. Here, using a combination of experiments and computer simulations, we show that the emergence and persistence of this phenomenon can be explained by the combination of two interacting trade-offs, rooted in biochemical constraints: First, faster growth is enabled by higher fermentation and obligate acetate excretion. Second, faster growth results in longer lag times when utilizing acetate after glucose is depleted. This combination creates an ecological niche for a slower-growing ecotype, specialized in switching to acetate. These findings demonstrate that trade-offs can give rise to surprisingly complex communities with evolutionarily stable coexistence of multiple variants in even the simplest environments.

Current progress in valorization of food processing waste and by-products for pectin extraction.

Food processing waste and by-products such as peel of citrus fruit, melon, mango, pineapple, etc. and fruit pomace can be utilized for manufacturing of several high-value products. Valorization of these waste and by-products for extraction of pectin, can help offset growing environmental concerns, facilitate value-addition of by-products and their sustainable uses. Pectin has many applications in food industries such as gelling, thickening, stabilizing, and emulsifying agent, and as a dietary fibre. This review elaborates on various conventional and advanced, sustainable pectin extraction techniques, and paints a comparative picture between them considering extraction efficiency, quality, and functionality of the pectin. Conventional acid, alkali, and chelating agents-assisted extraction have been profusely used for pectin extraction, but advanced extraction technologies e.g., enzyme, microwave, supercritical water, ultrasonication, pulse electric field and high-pressure extraction are preferred due to less energy consumption, better quality product, higher yield, and minimal or no generation of harmful effluent.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Recent progress in pectin extraction and their applications in developing films and coatings for sustainable food packaging: A review.

Perishable foods like fruits and vegetables, meat, fish, and dairy products have short shelf-life that causes significant postharvest losses, which poses a major challenge for food supply chains. Biopolymers have been extensively studied as sustainable alternatives to synthetic plastics, and pectin is one such biopolymer that has been used for packaging and preservation of foods. Pectin is obtained from abundantly available low-cost sources such as agricultural or food processing wastes and by products. This review is a complete account of pectin extraction from agro-wastes, development of pectin-based composite films and coatings, their characterizations, and their applications in food packaging and preservation. Compared to conventional chemical extraction, supercritical water, ultrasound, and microwave assisted extractions are a few examples of modern and more efficient pectin extraction processes that generate almost no hazardous effluents, and thus, such extraction techniques are more environment friendly. Pectin-based films and coatings can be functionalized with natural active agents such as essential oils and other phytochemicals to improve their moisture barrier, antimicrobial and antioxidant properties. Application of pectin-based active films and coatings effectively improved shelf-life of fresh cut-fruits, vegetables, meat, fish, poultry, milk, and other food perishable products.

Long-term history dependence of growth rates of E. coli after nutrient shifts.

According to a widely accepted paradigm of microbiology, steady-state growth rates are determined solely by current growth conditions1-3 and adaptations between growth states are rapid, as recently recapitulated by simple resource allocation models4. However, even in microbes overlapping regulatory networks can yield multi-stability or long-term cellular memory. Species like Listeria monocytogenes5 and Bacillus subtilis "distinguish" distinct histories for the commitment to sporulation6, but it is unclear if these states can persist over many generations. Remarkably, studying carbon co-utilization of Escherichia coli, we found that growth rates on combinations of carbon sources can depend critically on the previous growth condition. Growing in identical conditions, we observed differences in growth rates of up to 25% and we did not observe convergence of growth rates over 15 generations. We observed this phenomenon occurs across combinations of different phosphotransferase (PTS) substrates with various gluconeogenic carbon sources and found it to depend on the transcription factor Mlc.

Treatment Outcome with High versus low-to-moderate Dosing of Corticosteroids in Early vis-a-vis Late-onset Hypoxic Cases of COVID-19: A Multicentric Retrospective Cohort Study.

Background: Corticosteroid dosing in COVID-19 cases associated with early-onset and late-onset hypoxia have not been separately explored. Methods: In this retrospective cohort study, we divided hypoxic COVID-19 cases into groups based on timing of initiation of corticosteroids relative to onset of symptoms; Group A (≤6th day), Group B (7th-9th day) and Group C (≥10th day), each group being sub-grouped into high and low-to-moderate dose corticosteroid recipients. Cox regression with propensity scoring was used to compare 28-day mortality between high and low-to-moderate dose recipients separately in Group A, Group B, Group C. Results: Among 505 patients included, propensity score matched Cox regression showed greater risk of all-cause mortality among high dose recipients in Group A [HR= 7.35, 95%CI 3.36-16.11, p-value<0·01, N=114] and Group B [HR=3.17, 95%CI 1.65-6.07, p-value<0·01, N=251]. In Group C, mortality was lowest [12.8% (18/140)] with no significant difference between sub-groups [HR=2.52, 95%CI 0.22-29.15, p-value=0.459, N=140]. Kruskal-Wallis Test between Group A, Group B and Group C for six pre-defined exposure variables showed significant differences for Neutrophil:Lymphocyte Ratio (NLR). Conclusion: When steroids were initiated early (owing to an earlier onset of hypoxic symptoms), a high dose of corticosteroid was associated with greater overall 28-day mortality compared to a low-to-moderate dose. NLR, a marker for individual immune response, varied between treatment groups.