ARTICLE | doi:10.20944/preprints202109.0256.v3
Subject: Biology And Life Sciences, Plant Sciences Keywords: germination; Leucadendron; seed storage; seed viability estimation; viability loss
Online: 17 November 2021 (13:15:02 CET)
Seed viability is routinely measured on seeds that fail to germinate at the end of an experiment. Together with the number of germinants, this is used to estimate viability of the seeds at start of the experiment (i.e., initial viability) and provides the comparative basis on which germination success is determined. Perusal of the literature shows that sometimes (perhaps often, as the problem has yet to be recognized or reported) prolonged duration in the treatment, especially the control where little germination occurs, can lead to loss of viability. This results in underestimation of initial viability if that treatment is used. I caution against the routine use of end-of-trial germination and viability of ungerminated seeds as an estimate of initial viability in determining germination success of various treatments. I explore ways to deal with the problem but the preference is for estimates of initial viability to be undertaken on a separate sample of seeds concurrently with the experiment as this avoids the risk of seed death during the trial.
ARTICLE | doi:10.20944/preprints202309.1776.v1
Subject: Biology And Life Sciences, Toxicology Keywords: lung cancer; dehydroepiandrosterone; proliferation; viability; migration; autophagy
Online: 26 September 2023 (11:59:48 CEST)
Lung cancer is a prevalent and aggressive neoplasm worldwide, contributing to significant mortality rates. Dehydroepiandrosterone (DHEA) constitutes the bulk of the steroid hormone in human plasma, has a robust antiproliferative effect, and induces cell death in various tumor cells. However, its role in lung cancer cells remains unexplored. This study aimed to investigate the influence of DHEA on the proliferation, viability, autophagy, and migration of several lung cancer cell lines, including A549, HCC827, and NCI-H2347. Cell proliferation was assessed through crystal violet staining; cell number and viability were evaluated using trypan blue staining; viability was confirmed by MTT reduction, a method that is also an indicator of metabolic activity; migration was assessed via a wound healing assay. Autophagy was evaluated using a specific kit, while cell death was determined by annexin-V-FITC/propidium iodide staining and caspase-3/7 activity assay. The results indicate that DHEA significantly reduced proliferation, cell number, metabolic activity, and migration in all examined lung tumor cells. These effects correlate with an increased autophagy induced by DHEA. No signs of apoptosis or necrosis were observed across the range of DHEA concentrations used. Although these findings are preliminary, they suggest that DHEA could hold promise as an alternative treatment option for various subtypes of lung cancer.
ARTICLE | doi:10.20944/preprints202209.0333.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: rosé wine; probiotic yeast; fermentation; distillation; viability
Online: 22 September 2022 (05:44:56 CEST)
This paper reports for the first time on the production of probiotic alcoholic and non-alcoholic rosé wines with enhanced health benefits made with Saccharomyces cerevisiae var. boulardii probiotic yeast. The alcohol, sugar, volatile acidity lactic and malic acid contents were assessed for S. cerevisiae var. boulardii before and after fermentation and distillation and compared with a conventional Saccharomyces cerevisiae (ex-bayanus) yeast. The free amino nitrogen and gluconic acid concentrations in the musts were determined. Yeast viability was evaluated after fermentation and distillation as a function of time (0, 15 days, 3 months and 6 months) both at room temperature (25±0.5ºC) and refrigerator temperature (4±0.5ºC). The results obtained showed that the probiotic rosé wine produced with S. cerevisiae var. boulardii possesses the typical values and sensory attributes of other commercial wines produced with S. cerevisiae (ex-bayanus). The probiotic S. cerevisiae var. boulardii yeast survives the high alcohol content produced during fermentation and vacuum distillation. The study also showed that this probiotic rosé wine stored either at room temperature or in a refrigerator keeps its probiotic viability for at least six months, which makes it a promising for large-scale production, in which long storage times are required by both producers and consumers.
ARTICLE | doi:10.20944/preprints202111.0403.v1
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: anticancer agent; cytotoxicity; cell viability test; QSAR
Online: 22 November 2021 (14:18:14 CET)
2-(Morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine having QSAR-predicted anti-tumor activity was tested for the cytotoxicity using MTT and LDH cell viability tests. The experiments were conducted using human fibroblasts, peripheral blood mononuclear cells and breast cancer cells and allowed to identify effective cytotoxic concentration ant therapeutic range of this compound. The data obtained suggest the feasibility of the further studies of the test compound as a potential anti-cancer agent.
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Probiotics; food matrices; cell viability; model digestion
Online: 1 December 2020 (11:36:11 CET)
The aim of this work was to evaluate the influence of various food and beverages on the viability of probiotic bacteria during passing through artificial digestion. As a model food, solutions with various concentrations of alcohol, sugar, salt, protein and acid were prepared. Different types of real foods and beverages were used as well. Viability in presence of food matrices was tested on monocultures of Lactobacillus acidophilus CCM4833 and Bifidobacterium breve CCM7825T and on mixed commercial culture with 9 different strains of probiotic microorganisms( Lactobacillus, Bifidobacterium, Streptococcus). The concentration and viability of probiotic bacteria was tested by cultivation assay and flow cytometry. In model foods, the best growth of was determined in the presence of 10% albumin and 10% ethanol. Survival of the probiotics delivered in different food matrices through a simulated gastrointestinal tract was quantitatively different. As the best food environment for probiotics complex food matrices such as pasta with cream sauce, chocolate spread and homemade beef broth were selected, followed by mixed vegetables, potato salad, salted chips, fruits and yoghurt. Among beverages the best option was milk, followed by black tea, coffee and Coca Cola. Probiotic microorganisms are more viable when consumed with meals than with beverages only. In general, the highest viability of probiotic cells has been observed in presence of foods containing high concentration of sugar and fat or their suitable combination. The increase of cell viability observed in such foods during model digestion may further contribute to the positive effect of probiotics on human health.
ARTICLE | doi:10.20944/preprints202309.1479.v1
Subject: Chemistry And Materials Science, Food Chemistry Keywords: antidiabetic; antioxidative activity; Bacillus coagulans spores; microencapsulation; viability
Online: 21 September 2023 (11:33:31 CEST)
This research explores a novel encapsulating agent for establishing a synbiotic relationship with Bacillus (B.) coagulans by combining different ratios of wall materials, including skim milk powder (S), maltodextrin (M), and cellulose acetate phthalate (C). The ratio options are represented as SMC1 (1:2:1), SMC3 (3:2:1), SMC5 (5:2:1), and SMC7 (7:2:1). These wall materials are incorporated with 5% inulin (a prebiotic). The results show that these agents effectively protect B. coagulans spores during spray-drying, resulting in spore vitality exceeding 6 log cfu/g; SMC5 and SMC7 yielded the highest spore viability values. Furthermore, SMC5 demonstrated the highest levels of antioxidant activity, encompassing the scavenging of DPPH, hydroxy radicals, and superoxide radicals. Additionally, it displayed notable antidiabetic effects, including inhibitions of α-amylase and α-glucosidase. In comparison, SMC7 exhibited the most potent antidiabetic activity by inhibiting DPP-IV. Subsequently, after simulated gastrointestinal digestion, SMC5 and SMC7 showed a slight decrease in spore viability during the 6-hour simulation. Therefore, the most suitable condition for synbiotic production was found to be SMC5, which can potentially protect B. coagulans spores and exhibit bioactive activities after microencapsulation and passage through gastrointestinal digestion. This innovative product can be utilized as an advanced food delivery system and as a functional ingredient in functional food products.
ARTICLE | doi:10.20944/preprints202209.0408.v1
Subject: Engineering, Automotive Engineering Keywords: Public transportation; Automated vehicles; economic viability; business model
Online: 27 September 2022 (03:37:23 CEST)
During the past few years many projects and initiatives were undertaken deploying and testing automated vehicles for public transportation and logistics. However in spite of their ambition, all of these deployments stayed on the level of elaborated experimentation deploying no more than 4 maximum 5 AVs in rather small sites (few Kms of roads) and never really reached the level of large scale “commercial” deployment of transport services. The reasons for this are many, but the most important being the lack of economically viability and commercially realistic models, the lack of scalability of the business and operating models, and the lack of inclusive citizen/user centric services required for the large end-user acceptation and adoption of the solutions. In this paper, based on the experience gained in the H2020 AVENUE project, we present the missing pieces of the puzzle, ad which will be addressed in the Horizon Europe project ULTIMO.
ARTICLE | doi:10.20944/preprints202209.0356.v1
Subject: Chemistry And Materials Science, Biomaterials Keywords: PEG; viability; osteoblasts; fibroblasts; pH; polyurethane; polyethylene glycol
Online: 23 September 2022 (03:52:52 CEST)
Grafting polyethylene glycol (PEG) on polymers surface is widely used to improve biocompatibility by reducing protein and cell adhesion. Although PEG is considered to be bioinert, its incorporation to biomaterials has shown to improve cell viability depending on the amount and molecular weight (MW) used. This phenomenon was studied here by grafting PEG of three MW onto polyurethane (PU) substrata at three molar concentrations to assess their effect on PU surface properties and on the viability of osteoblasts and fibroblasts. PEG formed a covering on the substrata which increased the hydrophilicity and surface energy of PUs. Among the results it was observed that osteoblast viability increased for all MW and grafting densities of PEG employed compared with unmodified PU. However, fibroblast viability only increased at certain combinations of MW and grafting densities of PEG, suggesting an optimal level of these parameters. PEG grafting also promoted a more spread cell morphology than that exhibited by unmodified PU; nevertheless, cells became apoptotic-like as PEG MW and grafting density were increased. These effects on cells could be due to PEG affecting culture medium pH, which became more alkaline at higher MW and concentrations of PEG. Results support the hypothesis that surface energy of PU substrates can be tuned by controlling the MW and grafting density of PEG, but these parameters should be optimized to promote cell viability without inducing apoptotic-like behavior.
ARTICLE | doi:10.20944/preprints202105.0591.v1
Subject: Engineering, Industrial And Manufacturing Engineering Keywords: cell viability; device reliability; joule heating; eDEP devices.
Online: 25 May 2021 (08:43:36 CEST)
The application of electrode-based microfluidic devices in biological entity often imposes a problem due to joule heating. The strong applied potentials or micro channels having narrow cross sections generate undesirable temperature inside the microfluidic channels leading to strong thermal distribution inside the micro channel. When intrinsic distribution of temperature, if not fix with threshold value, causes device damage or cell loss. In this work, we investigate the effects of temperature generated due to joules heating effects and we attempt to address the design constraints for minimizing the joule heating effects in the microfluidic device for developing effective microfluidic device. The device reliability was analyzed under different parametric constraints for various types of substrate materials (PDMS, PMMA, Polyimide and glass). We also attempt to investigate the effects of cell reliability due to strong temperature gradients generated through different applied potentials on different cell types. Furthermore, the response of the device performance due to different electrode configuration and different conductivity of the medium was also studied. Our investigation will eventually provide guidelines for microfluidic researchers to fabricate efficient electrode based microfluidic device which will ultimately help to choose a critical channel dimensions, threshold potentials, and conductivity of solutions in order to avoid device damage and cell loss.
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: fire-related death; viability; light microscopy; bronchiolar epithelium
Online: 25 February 2021 (13:12:51 CET)
Fire-related deaths are issues for forensic pathologists particularly in ascertaining if death occurred before or during fire. The authors highlight a unique bronchiolar epithelial cytological clue (nuclear heat-induced elongation) determined by active inhalation of hot gases and fumes, not yet described in the literature at distal small airways level.
ARTICLE | doi:10.20944/preprints201904.0062.v1
Subject: Medicine And Pharmacology, Veterinary Medicine Keywords: BCG; Eudragit, oral vaccine; tuberculosis; in vitro viability
Online: 5 April 2019 (11:59:53 CEST)
Bacillus Calmette-Guérin (BCG) vaccine is the only licensed vaccine against tuberculosis (TB) in humans and animals. It is most commonly administered parenterally but oral delivery is highly advantageous for immunisation of cattle and wildlife hosts of TB in particular. Since BCG is susceptible to inactivation in the gut, vaccine formulations were prepared from suspensions of Eudragit L100 copolymer powder and BCG in PBS, containing Tween 80, with and without the addition of mannitol or trehalose. Samples were frozen at -20oC, freeze-dried and the lyophilised powders were compressed to produce BCG-Eudragit matrices. Production of the dried powders resulted in a reduction in BCG viability. Substantial losses in viability occurred at the initial formulation stage and at the stage of powder compaction. Data indicated that the Eudragit matrix protected BCG against simulated gastric fluid (SGF). The matrices remained intact in SGF and dissolved completely in SIF within three hours. The inclusion of mannitol or trehalose in the matrix provided additional protection to BCG during freeze-drying. Control needs to be exercised over BCG aggregation, freeze-drying and powder compaction conditions to minimise physical damage of the bacterial cell wall and maximise the viability of oral BCG vaccines prepared by dry powder compaction.
ARTICLE | doi:10.20944/preprints202308.0917.v1
Subject: Biology And Life Sciences, Agricultural Science And Agronomy Keywords: seed storage; seed viability; seedling emergence; seed ageing; germination
Online: 11 August 2023 (10:06:48 CEST)
Seed quality is crucial for the success of crop production, as it affects germination capacity, emergence potential, and seedling growth. This study investigated the impact of extended stor-age on the germination capacity, oleoresin and essential oil contents of black cumin seeds from three black cumin varieties in Ethiopia: "Aden", "Dershaye", and "Darbera". Seeds were stored for up to three years, and germination studies were conducted using a Completely Randomized De-sign. Results showed that storage period significantly affected various germination parameters, with a decrease in germination percentage observed with increasing storage period. However, variety and the interaction between storage period and variety had no statistically significant effect on germination percentage. The study also provides valuable insights into the correlations between different germination indices. Additionally, as black cumin seeds age, their oleoresin and essential oil contents decrease across all three varieties. Proper seed storage practices are essential in maintaining the germination potential of black cumin seeds, particularly over ex-tended storage periods. Overall, this study provides insights into the impact of extended seed storage on black cumin seed quality and germination potential, highlighting the importance of proper seed management practices to support sustainable crop production.
ARTICLE | doi:10.20944/preprints202306.1887.v1
Subject: Biology And Life Sciences, Agricultural Science And Agronomy Keywords: Cactaceae; seed viability; seedling quality; salinity; PGRs; Hylocereus sp.
Online: 27 June 2023 (11:08:08 CEST)
Lack of water and salinity are common problems in many parts of the world. Therefore, some types of cacti can represent as promising crops. Therefore, the ability of cactus species to survive and adapt under natural stress conditions should be evaluated. In this study, it was aimed to evaluate the effect of salt stress on germination and emergence of pitaya seeds obtained from different fruit flesh colors, priming with plant growth regulators, namely salicylic acid (SA), oxalic acid (OA) and mepiquat chloride (MC). The experiment had a completely randomized design with a 2×4×3 factorial scheme corresponding to two pitaya cultivars (white and red fleshed), four NaCl concentrations (0, 2500, 5000 and 10000 ppm), and three PGRs (150 ppm/MC, SA, OC). The following variables were analyzed; germination percentage, mean germination time (MGT), germination stress tolerance ındex (GSTI), shoot length, seedling fresh weight and seedling dry weight ratio. According to the results, it was determined that the growing environment of the seed was important in salt stress resistance, while seeds matured in the environment with red fruit flesh were more tolerant to salt stress than white. Although Pitaya species are relatively salt tolerant, growth (about 30%) was significantly reduced above 2500 ppm and germination (about 45%) above 5000 ppm. MC was found promising as a priming agent in seeds obtained from fruits with white fruit flesh, and OA was found promising as a priming agent in seeds obtained from fruits with red fruit flesh in reducing NaCl-induced stress. Germination percentage stood out as the most important trait determining seed quality and was found to have positive effects on germination stress tolerance index (r: 0.63), seedling length (r: 0.74) and fresh seedling weight (r: 0.56). It is thought that the results obtained may contribute to pitaya cultivation and breeding.
ARTICLE | doi:10.20944/preprints202305.2097.v1
Subject: Engineering, Bioengineering Keywords: Cell viability; cytotoxicity; DLC coatings; genotoxicity; Tribocorrosion; Ti6Al4V; SS316L
Online: 30 May 2023 (09:29:55 CEST)
Stainless steel 316L (SS316L) and titanium-alloy Ti6AL4V exhibit properties for bio-medical applications; however their tribocorrosion associated with metallosis is still a major concern. Therefore smoothing DLC coatings aiming for the improvement of tri-bocorrosion and cell viability of stainless steel 316L (SS316L) and titanium-alloy Ti6AL4V with and without DLC coatings were compared. The motivation was due to many papers detailing metallosis caused by metal debris in the body's soft tissues. Nowadays, metallosis is responsible for almost of prosthesis replacement surgeries. DLC coatings were produced using the Plasma Enhanced Chemical Vapor Deposition (PECVD) technique; acetylene gas was used as a carbon precursor, and silane gas produced an amorphous silicon interface between the DLC coating and metal substrate. A combined setup of techniques thoroughly evaluated the coatings considering chemical composition, coatings adhesion at a metallic substrate, scratch resistance, and tribocorrosion susceptibility. The cytotoxicity and genotoxicity of metallic samples with and without DLC coatings were analyzed, considering cell viability and its correlation with coatings’ chemical composition
REVIEW | doi:10.20944/preprints202201.0012.v3
Subject: Environmental And Earth Sciences, Environmental Science Keywords: Nature-Positive; Quantified Benefit Assessment; Security; Wellness; Viability; Gain
Online: 21 April 2022 (17:37:36 CEST)
The needs for environmental reporting to include positive outcomes considering differences between creation of less harm, benefits and net benefits are explored. To become mainstream, nature-positive development needs positive messaging, measures and metrics to guide, plan and assess urban outcomes. With the accelerating climate crisis and negative messages getting the upper-hand, it’s important to avoid paralysis by bad news. Whilst striving for a nature-positive world, more effort should be on moving beyond zero to qualify and quantify benefits, gains, and regenerative outcomes instead of oscillating around damage and loss sticking points. Life Cycle Benefit Assessment (LCBA) is a method to measure gains in accelerating restoration and climate security. It enables a good news focus as its reach is to quantify and show positive gains beyond the negative and zero loss outcomes. The paper aims to clarify concepts, challenges and quantitative methods then review real-world third-party-certified case studies. Climate security, human wellness and resource viability gains inside safe operating space within planetary boundaries are quantified as positive benefits. Contrary to conventional Life Cycle Impact Assessment (LCIA) LCBA assigns damage and loss as negative debts and benefit as positive gains. It concludes that LCBA offers business and design a new environment assessment tool, with research needed on economic and other outcomes.
ARTICLE | doi:10.20944/preprints202107.0113.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Probiotics; food matrices; food stress; cell viability; model digestion
Online: 5 July 2021 (15:59:51 CEST)
The aim was to evaluate the influence of model (alcohol, sugar, salt, protein and acid) and real foods and beverages on the viability of probiotics during incubation and artificial digestion. Viability of monocultures Lactobacillus acidophilus CCM4833 and Bifidobacterium breve CCM7825T and commercial mixture of 9 probiotic bacterial strains were tested by cultivation assay and flow cytometry. In model foods, the best viability was determined in the presence of 0.2 g/L glucose, 10% albumin and 10% ethanol. As the most suitable real food for probiotic survival complex protein and carbohydrate substrates were found, such as beef broth, potato salad with pork, chicken with rice, chocolate spread, porridge and yoghurt. The best liquid was milk and meat broth, followed by coca-cola, beer and coffee. Viability of probiotics was higher when consumed with meals than with beverages only. Addition of prebiotics increased the viability of probiotics especially in presence of instant and fast foods. Generally, the highest viability of probiotics during artificial digestion was observed in mixed culture in presence of protein, sugar and fat or their combination. The increase of cell viability observed in such foods during model digestion may further contribute to the positive effect of probiotics on human health.
ARTICLE | doi:10.20944/preprints202008.0441.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: Furin; ppFurin; Breast cancer; Calcium; SOCE; TRPC6; Viability; Migration.
Online: 20 August 2020 (07:41:14 CEST)
The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we show that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced SOCE through TRPC6 activation that associated reduced cells malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments, revealed that the ppFurin-expressing cells are unable to process adequately the PC substrates proVEGF-C and proIGF-1R. Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 significantly induces Ca2+ entry which is impaired by silencing of TRPC6 expression. Analysis of TRPC6 activation revealed its up-regulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. The expression of ppFurin in MDA-MB-231 cells reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization leading to breast cancer cells malignant phenotype repression and reduction of their resistance to treatments.
ARTICLE | doi:10.20944/preprints202304.0758.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: marigold; callus; cell suspension; growth parameters; cell viability; stem cell
Online: 23 April 2023 (04:05:25 CEST)
Plant stem cell cultures have so far been established in only a few plant species using cambial meristematic cells. The presence of stem cells or stem cell-like cells in other organs and tissues of the plant body, as well as the possibility of de novo generation of meristematic cells from differentiated cells, allows to consider the establishment of stem cell cultures in a broader range of species. Therefore, this study aimed to establish a stem cell culture of the medicinal plant Calendula officinalis L. Callus tissues were induced from explants taken from leaves and roots. Appropriate combinations of plant growth regulators and light parameters for in vitro cultivation were selected. Already at this stage, stem and dedifferentiated cells could be identified, and cell suspension cultures were established from specific parts of the formed callus. Cell suspension cultures with a high proportion of stem cells originating from roots (92–93%) or leaves (72–73%) were developed. The amount of stem cells derived from the roots in the cell suspension reached a plateau at a level of 5.60–5.72105.
ARTICLE | doi:10.20944/preprints201608.0052.v1
Subject: Medicine And Pharmacology, Medicine And Pharmacology Keywords: acinar cells; PLP; amylase; insulin secretion; FACS; viability; PDX-1
Online: 5 August 2016 (08:49:33 CEST)
Aims: To study the effects of pyridoxal phosphate (PLP) on oxidative stress in isolated pancreatic acinar cells. We have previously shown that PLP has cytoprotective and insulinotropic effects on mice islet cells in vivo and in vitro studies. Main methods: Acinar cells were isolated from three months old WNIN male rats and were cultured in vitro for a period of 24 h in CO2 incubator. Later the cells were divided into four groups as untreated (group 1), H2O2 treatment (group 2), PLP treatment (group 3) and PLP followed by H2O2 treatment (group 4). Cell viability was confirmed using MTT assays, oxidative stress levels were measured with ROS assay, change in different protein levels were recorded by flow cytometry. The acinar cells insulin secretion assay was performed with ELISA. The amylase protein expression was assessed using confocal microscopy. Key findings: The cell viability of acinar cell in group 1 was considered as 100%, while in group 2 it was reduced to 82% due to H2O2 effect, and in group 3 (99.8%) and group 4 (99.5%) were near to group 1 due to the cytoprotective effect of PLP. The ROS levels were increased by 1.47 folds in group 2, while PLP decreased to 1.02 fold in group 4, which was comparable with the changes in group 1. Beneficial effects of PLP were also observed from the increased expression levels of acinar cells are amylase -2.01, neurogenin-3-9.51, PDX-1- 23.6 and insulin-13.5 in group 3 compared to group 1. The specificity of PLP’s response was confirmed by amino oxy acetic acid (AOAA), a specific PLP inhibitor. The increased amylase protein localization with PLP was confirmed by confocal microscopy. Insulin secretion efficiency of acinar cells was observed to be 6.13 folds higher at basal and 24.63 fold higher at stimulated levels in group3 compared to group1. Significance: Our results advocate the antioxidant and cytoprotective effects of PLP on the pancreatic acinar cell along with increased pancreatic marker expressions of amylase,PDX-1, neurogenin-3 and insulin proteins.
ARTICLE | doi:10.20944/preprints202304.1197.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: B-cells, hypoxia, miRNA, CRISPR/Cas9, leukemia, gene expression, cell viability
Online: 29 April 2023 (03:34:08 CEST)
Hypoxia represents one of the key factors that stimulate the growth of leukemic cells in their niche. Leukemic cells in hypoxia are forced to reprogram their original transcriptome, miRNome, and metabolome. How the coupling of miRNAs/mRNAs helps to maintain or progress the leukemic status is still not fully described. MicroRNAs (miRNAs) regulate practically all biological processes within cells and play a crucial role in leukemia development/progression. Here we worked with the human cell line MEC-1 (human chronic lymphocytic leukemia (CLL)) where we deleted mature miR-155 by CRISPR/Cas9. MEC-1 cells in hypoxia showed a higher proliferation rate with a relatively low level of apoptosis. Interestingly, miR-155 deficiency decreased the proliferation rate in both, normoxia and hypoxia. Besides common hypoxia-related genes (HIF1α, EGLN1, VHL, HK1, and HK2), we also measured, genes for glucose transporters SLC2A1 (GLUT1) and SLC2A3 (GLUT3). Surprisingly, only SLC2A3 (GLUT3) showed significant overexpression in hypoxia conditions in miR-155 deficient MEC-1cells which points to the possible novel target of miR-155 in CLL. Increased glucose uptake and decreased lactate support our hypothesis that miR-155 deficiency and hypoxia transform cell metabolism. To conclude, miR-155 deficiency and hypoxia-impaired glucose metabolism stimulate GLUT3 in CLL cells.
ARTICLE | doi:10.20944/preprints202208.0364.v1
Subject: Biology And Life Sciences, Cell And Developmental Biology Keywords: TMPyP4 1; Thymoquinone 2; cell viability 3; cell adhesion 4; telomerase
Online: 19 August 2022 (10:46:07 CEST)
G‐quadruplexes (G4) are structures formed at the ends of the telomere, these are rich in guanines and were stabilized by molecules that bind to specific sites. TMPyP4 and Thymoquinone (TQ) are small molecules that bind to the G4, they have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines LC‐HK2 (NSCLC) and RPE‐1 were treated with TMPyP4 (5μM) and TQ (10μM). Both inhibitors were effective in decreasing telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S‐phase. TMPyP4 changed the cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a dose‐dependent manner, increasing the frequency of senescent cells, and inducing cell cycle arrest in the G1. In conclusion, the effects of both drugs on LC-HK2 and RPE-1 cell lines were different although both are telomerase inhibitors, because TMPyP4 decreased proteins of cell adhesion and TQ induces a decrease in cell viability.
ARTICLE | doi:10.20944/preprints202105.0671.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Folate-targeted nanoparticles; BSA/Alginate nanocarriers; paclitaxel; cellular uptake; cell viability.
Online: 27 May 2021 (13:55:13 CEST)
Among the different ways to reduce the secondary effects of antineoplastic drugs in cancer treatment, the use of nanoparticles has demonstrated good results due to the protection of the drug and the possibility of releasing compounds to a specific therapeutic target. The α-isoform of folate receptor (FR) is overexpressed on a significant number of human cancers; therefore, folate-targeted crosslinked nanoparticles based on BSA and alginate mixtures and loaded with paclitaxel (PTX) have been prepared to maximizing the proven antineoplastic activity of the drug against solid tumors. Nanometric-range sized particles (169 ± 28nm - 296 ± 57nm), with negative Z-potential values (between -0.12 ± 0.04 and -94.1± 0.4), were synthesized. The loaded PTX (2.63±0.19 - 3.56 ±0.13 µg PTX/mg Np) was sustainably released along 23 and 27h. Three cell lines (MCF-7, MDA-MB-231 and HeLa) were selected to test the efficacy of the folate-targeted PTX-loaded BSA/ALG nanocarriers. The presence of FR on cell membrane led to a significant larger uptake of BSA/ALG-Fol nanoparticles regarding to the equivalent nanoparticles without folic acid on its surface. The cell viability results demonstrated a cytocompatibility of unloaded nanoparticle-Fol and a gradual decrease in cell viability after treatment with PTX-loaded nanoparticles-Fol due to the sustainable PTX release.
ARTICLE | doi:10.20944/preprints202012.0571.v1
Subject: Environmental And Earth Sciences, Atmospheric Science And Meteorology Keywords: Fragaria X ananassa; planting spacing; photosynthetic efficiency; productive viability; fruit quality
Online: 23 December 2020 (08:59:51 CET)
In the search for more efficient production systems, many changes have occurred in the strawberry production sector. Planting density is one of the management techniques that most interferes with the quality of fruits and production aspects. This study aimed to evaluate the effect of different planting densities on the photosynthetic characteristics, fruit quality, and production of the strawberry cultivar Pircinque. The study was conducted in the 2018/2019 and 2019/2020 harvests in Lages, Santa Catarina, Brazil. The treatments consisted of plant spacing of 5, 10, 15, 20, 25, and 30 cm. The experimental design was in randomized blocks, with four blocks and plots of 20 plants. Plant spacing interfered with fruit quality, photosynthetic efficiency, production, productivity, and economic return. Due to the fruits of 'Pircinque' with higher quality than other cultivars, the planting spacing between 5 and 15 cm allows meeting the fruit's main production and quality requirements. However, it is up to the producer to adapt the management if opting for higher planting densities, which allow greater economic viability of the business.
HYPOTHESIS | doi:10.20944/preprints201910.0248.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Vibrio cholerae; cholera toxin gene; low ionic strength aquatic solution; viability
Online: 22 October 2019 (04:00:54 CEST)
It has been regarded that Vibrio cholerae O1 inhabit in environmental water. As many cholera patients emerge in Kolkata, it has been thought that V. cholerae O1 is easily detected in environmental water in Kolkata. However, the detection of V. cholerae O1 is rare, though other V. cholerae (NAG Vibrio) is constantly detected. To clear the reason for the difference of the detection rate of two Vibrios, we examined the viability of V. cholera O1 and NAG Vibrios in low ionic strength aquatic medium. We observed greater declining viability of V. cholerae O1 possessing cholera toxin gene (ctx) in low ionic strength solution, but the decline of NAG Vibrios non-possessing ctx is small. To evaluate the concerning of ctx in the viability, we examined the viabilities of V. cholerae O1which do not possess ctx and NAG Vibrios possessing ctx under the same condition. The result indicated that the existence of the ctx induces the decrease the viability of the host in low ionic strength solution. The decrease observed in this experiment might relate with the low detection of V. cholerae O1 possessing ctx in environmental water, though NAG Vibrio is constantly detected.
ARTICLE | doi:10.20944/preprints202301.0150.v2
Subject: Biology And Life Sciences, Cell And Developmental Biology Keywords: oral; mucosa; mucins; tight junctions; wound-healing; E-liquids; cytotoxicity; viability; confluency.
Online: 3 April 2023 (03:33:37 CEST)
Background: Expansion of OKF6/TERT-2 oral epithelial cells in vitro is important for studying the molecular biology of disease and pathology affecting the oral cavity. Keratinocyte Serum-Free Medium (KSFM) is the medium of choice for this cell line. This study compares three media for OKF6/TERT-2 cultures: KSFM, Dulbecco’s Modified Eagle Medium/Nutrient Mixture of Hams F-12 (DMEM/F12) and a composite medium comprised of DMEM/F-12 and KSFM (1:1 v/v), referred as DFK. The toxicological effects of electronic cigarette liquids (E-liquids) on OKF6/TERT-2 cells cultured in these media were also compared. Methods: Cells were cultured in KSFM, DMEM/F12 or DFK and cellular morphology, growth, wound healing and gene expression of mucins and tight junctions were evaluated. Additionally, cytotoxicity was determined after E-liquid exposures. Results: Switching from KSFM to DMEM/F12 or DFK 24-hours post-seeding leads to typical cellular morphologies, and these cultures reach confluency faster than those in KSFM. Wound-healing recovery occurred fastest in DFK. Except for claudin-1, there is no difference in expression of the other genes tested. Additionally, E-liquid cytotoxicity appears to be amplified in DFK cultures. Conclusions: DMEM/F12 and DFK are alternative media for OKF6/TERT-2 cell culture to study molecular biology of disease and pathology, provided cells are initially seeded in KSFM.
ARTICLE | doi:10.20944/preprints202211.0114.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: UVC; shoe sole decontaminator; ESKAPE-E pathogens; multidrug resistance; disinfection; cell viability; SEM.
Online: 7 November 2022 (09:48:19 CET)
Healthcare-associated infections (HAI) worldwide includes infections by ESKAPE-E pathogens. Environmental surfaces and fomites are important components in HAI transmission dynamics, and shoe soles are vectors of HAI. Ultraviolet (UV) disinfection is an effective method to inactivate pathogenic microorganisms. In this study, we investigated whether the SANITECH UVC shoe sole decontaminator equipment that provides germicidal UVC radiation could effectively reduce this risk of different pathogens. Six standard strains and four clinical MDR strains in liquid and solid medium were exposed to a UVC System at specific concentrations at other times. Bacterial inac-tivation (growth and cultivability) was investigated using colony counts and resazurin as meta-bolic indicators. SEM was performed to assess the membrane damage. Statistically significant reduction in cell viability for all ATCCs strains occurred after 10 sec of exposure to the UVC sys-tem, except for S enterica, which only occurred at 20 sec. The cell viability of P. aeruginosa (90.9%), E. faecalis and A. baumannii (85.3%), S. enterica (82.9%), E. coli (79.2%) and S. aureus (71.9%) was re-duced considerably at 20 sec. In colony count, after 12 sec of UVC exposure, all ATCC strains showed a 100% reduction in CFU counts, except for A. baumannii, which reduced 97.7%. A sub-stantial reduction of colonies above 3 log10 was observed at 12 and 20 sec in all bacterial strains tested, except for A. baumannii ATCC 19606 (12 sec). The exposure of ATCCs bacterial strains to the UVC system for only 2 sec was able to reduce 100% in the CFU count in all ATCCs strains, S. au-reus, P. aeruginosa, E. coli, A. baumannii, E. faecalis, except the S. enterica strain which had a statis-tically significant reduction of 99.7%. In ATCC strains, there was a substantial decrease in colonies after 4 sec (sec) of exposure to the UVC system, with a reduction ranging from 3.78-4.15 log10 CFU/mL. This reduction was observed in MDR/ESKAPE-E strains within 10 sec, showing that UVC could eliminate above 3.84 log10 CFU/mL. SEM showed a reduction of pili-like appendages after UVC treatment in all strains except for E. coli (ATCC 25922). The Sanitech UVC shoe sole decontaminator equipment from Astech Co. effectively killed in vitro a series of ATCCs and MDR/ESKAPE-E bacteria of sanitary interest, commonly found in the hospital environment.
ARTICLE | doi:10.20944/preprints202012.0213.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: bael; buffalo milk; Lactobacillus rhamnosus GG; non-cow milk; probiotic yoghurt; probiotic viability
Online: 9 December 2020 (09:52:03 CET)
Recently, there is a trend among food manufacturers in producing functional foods containing fruit and milk. On the other hand, there is an increasing demand for buffalo milk as an alternative to cow milk. Therefore, the aim of this study was to investigate the impact of adding bael fruit extract on microbial, physiochemical and sensory characteristics of buffalo yoghurt containing the EPS-producing probiotic Lactobacillus rhamnosus GG (LGG) during 21 days of refrigerated storage. Four formulations of buffalo yoghurt: a control yoghurt manufactured with conventional yoghurt culture (CON); a probiotic yoghurt containing Lb. rhamnosus GG plus yoghurt culture (PY); a probiotic yoghurt containing 5% (w/v) bael (PY5); and 10% (w/v) bael (PY10) were evaluated for changes in pH, syneresis, hardness, probiotic viability and sensory attributes during the storage. Control yoghurt experienced significant post acidification and a higher rate of syneresis. Addition of probiotics had a positive effect on post acidification and syneresis rate. Addition of bael did not affect post-acidification, but significantly decreased the level of syneresis. All probiotic formulations maintained LGG counts of >107 cfu/mL and the highest counts were observed in 5% (w/v) bael incorporated yoghurt. Results showed that buffalo yoghurt is an ideal matrix to deliver LGG and 5% bael incorporation would be ideal or symbiotic product development.
ARTICLE | doi:10.20944/preprints202306.2091.v1
Subject: Social Sciences, Geography, Planning And Development Keywords: High-efficient irrigation-system; hydraulic ram pump; water use efficiency; economic viability; solar pump
Online: 29 June 2023 (12:44:28 CEST)
Glacial meltwater has been lifeline of agriculture in Gilgit-Baltistan for centuries which is being effected by the climatic changes and associated challenges. The government line institutions and development agencies have been investing to sustain and further extend the cultivated areas by leveraging the available glacial meltwater source. However, approximately 13% arable land, laying mainly along river banks, has limited access to glacial meltwater. Moreover, already cultivated areas also need alternate and sustainable irrigation water supplies to insurefood security. Hence, there is need to explore more potential options to address these challenges and bringing arable land under irrigation for regional prosperity and environmental gains. The International Centre for Integrated Mountain Development (ICIMOD) and its partners piloted solutions for aforementioned issues in Gojal, District Hunza. These solutions included river water lifting through solar and Hydraulic Ram Pumps, uphill water storages and application through drip irrigation systems. In order to assess the technical and economical viabilities of these piloted systems, a semi-structured questionnaire survey, field observation visits to collect data and economic assessment studies were carried out in Morkhun and Khyber. Both piloted systems have been proven as climate resilient, adaptable, environment friendly and economically viable. The cost to benefit ratio for solar powered drip irrigation and Hydraulic Ram Pump drip irrigation systems are 2.96 and 4.21 respectively, with payback period of 9.21 and 8.25 years respectively. The drip irrigation systems at Khyber and Morkhun sites have exhibited excellent hydraulic performance with Coefficient of Uniformity 92.61 and 93 % respectively. However, the field application efficiency of drip irrigation systems estimated for Khyber and Morkhun sites are 85.24 and 88.88% which are lower than the optimal value mainly due to lesser water holding capacity. The Hydraulic Ram Pump can lift four times more water to greater height, while comparing to solar pump of approximately similar capacity and higher capital costs. Both water lifting systems have some limitations and requires local customization for sustainable use.
ARTICLE | doi:10.20944/preprints201704.0070.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: genistein; soy isoflavone extracts; anti-menopausal dietary supplements; bacterial growth; viability of human cells
Online: 13 April 2017 (05:33:06 CEST)
Flavonoids, compounds present in many dietary supplements, affect growth of different bacterial species when tested as purified or synthetic substances. Here, we asked if soy isoflavone extracts, commonly used in many products sold as anti-menopausal dietary supplements, influence bacterial growth similarly to synthetic isoflavone, genistein. Four commercially available products were tested in amounts corresponding to genistein concentrations causing inhibition of growth of Vibrio harveyi (a model bacterium sensitive to this isoflavone) and Escherichia coli (a model bacterium resistant to genistein). Differential effects of various extracts on V. harveyi and E. coli growth, from stimulation, through no changes, to inhibition, were observed. Moreover, contrary to genistein, tested extracts caused a decrease (to different extends) in viability of human dermal fibroblasts. These results indicate that effects of various soy isoflavone extracts on bacterial growth and viability of human cells are different, despite similar declared composition of the commercially available products.
ARTICLE | doi:10.20944/preprints202210.0223.v1
Subject: Biology And Life Sciences, Virology Keywords: Drug discovery; Systems biology; SARS-CoV-2; Dengue; Influenza A; RSV; cell viability analysis; antivirals
Online: 17 October 2022 (03:42:37 CEST)
The SARS-CoV-2 pandemic has reemphasized the urgent need to develop broad-spectrum antiviral therapies. Wedeveloped a computational pipeline that uses scRNA-Seq data to reconstruct the metabolic state of cells and tissuesduring viral infection. Using this pipeline, we investigated the cellular capacity to produce SARS-CoV-2 virions invarious tissues and disease conditions. Subsequently, we expanded our analysis to influenza A and dengue virus andidentified several metabolic targets and their inhibitors for broad-spectrum antiviral treatment. Phenformin, an inhibitorof NADH:ubiquinone oxidoreductase, suppressed SARS-CoV-2 and dengue virus replication. Using Atpenin A5 toblock the succinate dehydrogenase inhibited SARS-CoV-2, dengue virus, influenza A virus and respiratory syncytialvirus with superior therapeutic indices. Thus, our work establishes host metabolism as druggable for broad antiviraltherapy. Moreover, our pipeline, the identified targets, and inhibitors are invaluable tools for pandemic preparedness.
ARTICLE | doi:10.20944/preprints202112.0228.v1
Subject: Biology And Life Sciences, Biophysics Keywords: lipofuscin; retina; retinal pigment epithelium; docosahexaenoate; docosahexaenoic acid; fluorescence; photodegradation; photobleaching; cell viability; endocytic activity
Online: 14 December 2021 (11:41:14 CET)
Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE) where its fluorescence properties are used to assess the retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE, and lipofuscin-laden-cells to visible light, and determine whether an abundant component of lipofuscin, docosahexaenoate (DHA) can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible leads to a decrease of its long-wavelength fluorescence at about 610 nm with concomitant growth of the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure to light of lipofuscin-laden cells leads to loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes of fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra together with quantitation of intensity of long-wavelength fluorescence can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, thereby useful for screening the retina for increased oxidative damage and early AMD-related changes.
ARTICLE | doi:10.20944/preprints202108.0177.v1
Subject: Medicine And Pharmacology, Dentistry And Oral Surgery Keywords: Antibacterial composites; Antiadhesive composites; Poly-Pore; Split-mouth; Clinical trial; Live/dead staining; Bacterial viability
Online: 9 August 2021 (07:53:57 CEST)
The purpose of the study was to investigate the bacterial viability of the initial biofilm on the surface of experimental modified dental resin composites. Twenty-five healthy individuals with good oral hygiene were included in this study. In a split-mouth design, they received acrylic splints with five experimental composite resin specimens. Four of them were modified with either a novel polymeric hollow beads delivery system or methacrylated polymerizable Irgasan (Antibacterial B), while one specimen served as unmodified control (ST). The delivery system based on Poly-Pore® was loaded with one of the active agents Tego® Protect 5000 (Antiadhesive A), Dimethicone (Antiadhesive B) or Irgasan (Antibacterial A). All study subjects refrained from toothbrushing during the study period. Specimens were detached from the splints after 8h and given a live/dead staining before fluorescence microscopy. Friedman test and post-hoc Nemenyi test were applied with significance level at p < 0.05. In summary all materials but Antibacterial B showed a significant antibacterial effect compared to ST. In conclusion dental resin composites with Poly-Pore loaded active agents show antibacterial effectiveness in situ.
ARTICLE | doi:10.20944/preprints202203.0060.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: carnosine; glioblastoma; fibroblasts; imidazole-containing compounds; cell viability; high per-formance liquid chromatography coupled mass spectrometry
Online: 3 March 2022 (10:31:32 CET)
The naturally occurring dipeptide carnosine (β alanyl L histidine) specifically attenuates tumor growth. Here, we asked whether other small imidazole containing compounds also affect viability of tumor cells without affecting non-malignant cells, and whether formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L alanyl L histidine (LA-LH), ß alanyl L alanine, L histidine, histamine, imidazole, β alanine and L alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L histidine and formation of histamine was investigated by High Performance Liquid Chromatography coupled Mass Spectrometry. Whereas carnosine and LA LH inhibited tumor cell growth with minor effects on fibroblasts, L-histidine, histamine and imidazole affected viability in both cell types. Compounds without imidazole moiety did not diminish viability. In the presence of LA LH but not in the presence of carnosine a significant rise of intracellular amounts of histidine was detected in all cells. Formation of histamine was not detectable in the presence of carnosine, LA LH or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA LH. Despite histamine had a strong effect on cell viability, formation of histamine is not responsible for the effects on cell viability of carnosine, LA LH and histidine.
ARTICLE | doi:10.20944/preprints202306.0315.v1
Subject: Computer Science And Mathematics, Other Keywords: Financial Viability; Life Cycle Cost Analysis; LPWAN; Pragmatic Computational Tools; Design Science Research, Data-driven decision making
Online: 5 June 2023 (12:00:41 CEST)
This paper focuses on quantifying the economic and financial viability of NB-IoT and LoRaWAN technologies, two low-power wide-area network (LPWAN) technologies with unique characteristics that make them suitable for IoT applications. The purpose of the study is to propose an artefact for performing life cycle cost analysis and demonstrate its application to these technologies. The methodology uses pragmatic computational tools to facilitate the analysis and considers all relevant economic and financial factors, such as operating costs, equipment costs, and revenue potential. The main finding of the study is that NB-IoT and LoRaWAN technologies have different cost structures and revenue potentials, which may affect their economic and financial viability for different IoT applications. Ultimately, the study concludes that a comprehensive life cycle cost analysis is critical to making informed decisions about technology adoption, and that the proposed methodology can be applied to other IoT technologies to gain insight into their economic and financial viability.
ARTICLE | doi:10.20944/preprints202309.1412.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Zymomonas mobilis; respiratory chain; Type II NADH dehydrogenase; oxidative phosphorylation; starvation; viability; make-accumulate-consume strategy; ethanol stress
Online: 21 September 2023 (07:18:07 CEST)
Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, including also the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with a low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing culture on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. In the present work we show that in respiring, non-growing Z. mobilis cells, receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. Accordingly, no improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, unable to oxidize ethanol. The ethanol effect is hampered also by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v) ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters in aerobically growing culture under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease of the respiratory chain activity mitigates the production of reactive oxygen species.
HYPOTHESIS | doi:10.20944/preprints202010.0199.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: CRISPR interference; error-prone polymerase chain reaction; ribosomal proteins; ribosome; viability assay; Bacillus subtilis; Escherichia coli; sequence space; conformational space; mutational map
Online: 9 October 2020 (12:24:14 CEST)
Ribosomes are macromolecular complexes important to protein translation, and thus, essential to life. Comprising an ensemble of ribosomal proteins and RNA molecules, ribosomes are conserved in structure and function across all domains of life, but recent structural studies have revealed differentiated structures of ribosomes from bacterial, archaea and eukaryotes. Additionally, unique ribosomal protein mass fingerprints have been found for individual species; thereby, indicating that ribosomes are differentiated in structure amongst different species. Given that structure defines function, differentiated function likely exists amongst ribosomes of different species, which could manifest as differences in translation efficiency that could impact on cell growth rate. But ribosomal proteins also hold phylogenetic significance in informing the evolutionary trajectory of each species. Such ribosomal proteins are thus not highly conserved and offers sufficient sequence space for the evolution of differentiated structure and function in different species. Using ribosomal proteins that hold phylogenetic significance as templates, this study sought to understand the mutational and conformational limits that define functional ribosomes. Specifically, ribosomal proteins in Bacillus subtilis that hold phylogenetic cues would be mutated through error-prone polymerase chain reaction to generate variants that are subsequently transformed into Escherichia coli. To help assess the functional properties of the heterologous ribosomal proteins, endogenous ribosomal protein genes would be inactivated by multiplex CRISPR interference. Since variants in ribosomal proteins would likely impact on ribosome function and translation efficiency, live/dead screening on LB agar would be effective as a preliminary screen for functional mutants. These mutants would subsequently be inoculated into liquid LB medium in 96 well plates to quantify relative growth rates between different strains harbouring different heterologous variants of ribosomal proteins. Plasmids containing different ribosomal protein mutants would be extracted from each functional strain and subjected to Sanger sequencing for determining the specific mutations involved. Collection of such mutations would provide a comprehensive mutational map that define the limits of ribosomal protein sequence space important to ribosome function. Furthermore, biochemical isolation of ribosomal proteins and their structural characterization by X-ray crystallography or cryo-electron microscopy would further illuminate the structural significance of each mutation on ribosome structure and function; thereby, elucidating the structural tolerance space for functional ribosomes. Overall, generating a diverse pool of mutant ribosomal proteins in viability assays followed by sequencing and structural characterization would help define the mutational and conformational limits of a functional and efficient ribosome.