ARTICLE | doi:10.20944/preprints202208.0059.v1
Subject: Biology, Other Keywords: Staphylococcus epidermidis; metabolic network validation; minimal cut sets; knock-outs; systems biology
Online: 2 August 2022 (09:33:09 CEST)
Increasingly, systems biology is gaining relevance in basic and applied research. The combination of computational biology with wet lab produces a synergy that results in an exponential increase in knowledge of biological systems. The study of microorganisms such as Staphylococcus epidermidis RP62A enables the researcher to understand better its metabolic network, which allows the design of effective strategies to treat infections caused by this species or others. S. epidermidis is the second cause of infection in patients with joint implants, so treating its proliferation seems vital for public health. There are different approaches to the analysis of metabolic networks. Flux Balance Analysis (FBA) is one of the most widespread streams. It allows the study of large metabolic networks, their structural properties, the optimization of metabolic flux, and the search for intervention strategies to modify the state of the metabolic network. This work presents the validation of the Staphylococcus epidermidis RP62A metabolic network model elaborated by Díaz-Calvo et al.. Then, we elaborate further on the network analysis’s essential reactions, classifying them. Finally, we introduce some proposals to intervene in the network and design knock-outs.
ARTICLE | doi:10.20944/preprints202205.0377.v1
Subject: Engineering, Energy & Fuel Technology Keywords: detonation; super-knock; CSP; explosive dynamics
Online: 27 May 2022 (09:48:29 CEST)
The occurrence and intensity of the detonation phenomenon at spark-ignition (SI) engine conditions is investigated, with the objective to successfully predict super-knock and to elucidate the effect of kinetics and transport at the ignition front. The computational singular perturbation (CSP) framework is employed in order to investigate the chemical and transport mechanisms of deflagration and detonation cases in the context of 2D high-fidelity numerical simulations. The analysis revealed that the detonation development is characterized by: (i) stronger explosive dynamics and (ii) enhanced role of convection. The role of chemistry was also found to be pivotal to the detonation development which explained the stronger explosive character of the system, the latter being an indication of the system's reactivity. The role of convection was found to be enhanced at the edge of the detonating front, thereby suggesting that it is the result and not the cause of the detonation onset. Moreover, the increased contribution of convection was found to be related mainly to heat convection. Remarkably, the detonation front was mainly characterized by dissipative and not explosive dynamics. Finally, diffusion was found to have negligible role to both examined cases.
CONCEPT PAPER | doi:10.20944/preprints202003.0321.v1
Subject: Life Sciences, Biotechnology Keywords: bookmark; CRISPR/Cas9; complementation; clostridium; knock-out
Online: 23 March 2020 (00:50:23 CET)
Phenotypic complementation of gene knock-outs is an essential step in establishing function. Here we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9, but could be designed for any CRISPR/Cas system. For proof of concept, 9 bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by incorporation of ‘watermark’ sequences into the complementing genes.
ARTICLE | doi:10.20944/preprints202005.0028.v1
Online: 3 May 2020 (07:27:40 CEST)
This paper aims to determine the insecticidal activity of Panyawan (Tinospora rumphii) leaf extract against American cockroach (Periplaneta americana). A quantitative experimental research design was employed in the study. Cockroach strains were collected through trapping and hand catch methods. Panyawan leaf was collected and extracted to achieve seven different concentrations. The concentrations and controls were tested against the strains through the standard contact glass jar bioassay. Results revealed that the shortest knock-down time was achieved by the positive control at 1.08 minutes while the Extracts of Panyawan leaf reported a 1.42 to 20.25 minutes knock-down time. Probit Regression reported that the lethal concentration 50 of Panyawan leaf extract was 15.836%. One-Way ANOVA revealed that there is a significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls (p<0.01). Homogeneous subsets derived from Post Hoc Test using Tukey’s Honest Significant Difference reported that there is no significant difference between positive control and Extract 100% (p>0.01).
ARTICLE | doi:10.20944/preprints202204.0150.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: Ttc21a; Ttc21b; mouse knock-out; jaw; facial bone; viscerocranium; Hh signaling
Online: 16 April 2022 (03:36:54 CEST)
Ciliopathies are genetic syndromes that link skeletal dysplasias to dysfunction of primary cilia. Primary cilia are sensory organelles synthesized by intraflagellar transport (IFT) - A and B complexes, which traffic protein cargo along a microtubular core. We have reported that deletion of IFT-A gene, Thm2, together with a null allele of its paralog, Thm1, causes a small skeleton with small mandible or micrognathia in juvenile mice. Using micro-computed tomography, here we quantify the craniofacial defects of Thm2-/-;Thm1aln/- triple allele mutant mice. At postnatal day 14, triple allele mutant mice exhibit micrognathia, maxillary hypoplasia, and a decreased facial angle due to shortened maxilla, premaxilla, and nasal bones, reflecting altered development of facial anterior-posterior elements. In contrast, other ciliopathy-related craniofacial defects, such as cleft lip and/or palate, hypo-/hypertelorism, broad nasal bridge, craniosynostosis, and facial asymmetry, were not observed, suggesting development of the facial transverse dimension is intact. Calvarial-derived osteoblasts of triple allele mutant mice showed reduced bone formation in vitro that was ameliorated by Hedgehog agonist, SAG. Together, these data indicate that Thm2 and Thm1 genetically interact to regulate bone formation and sculpting of the postnatal face. The triple allele mutant mice present as a novel model to study craniofacial bone development.
ARTICLE | doi:10.20944/preprints201711.0110.v1
Subject: Life Sciences, Biotechnology Keywords: Fluorescent reporter; E2-Crimson; mouse embryonic stem cells; knock-in; in vivo imaging
Online: 16 November 2017 (17:46:53 CET)
Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over 9 days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus.
ARTICLE | doi:10.20944/preprints202212.0442.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: P21; CDKN1A; glioblastoma; senescence; cancer senescence; gene overexpression; gene knock-in; CRISPR/Cas9; dCas; dCas-VPR
Online: 23 December 2022 (04:10:22 CET)
High-grade gliomas are the most common and aggressive adult primary brain tumors with a median survival of only 12-15 months. Current standard therapy consists of maximal safe surgical resection followed by DNA-damaging agents, such as irradiation and chemotherapy that can delay but not prevent inevitable recurrence. Some have interpreted glioma recurrence as evidence of glioma stem cells which persist in a relatively quiescent state after irradiation and chemotherapy, before the ultimate cell cycle re-entry and glioma recurrence. Conversely, latent cancer cells with a therapy-induced senescent phenotype have been shown to escape senescence, giving rise to more aggressive stem-like tumor cells than those present in the original tumor. Therefore, approaches are needed to either eliminate or keep these glioma-initiating cells in a senescent state for a longer time to prolong survival. In our current study, we demonstrate that the radiation-induced cell cycle inhibitor P21 can provide a powerful route to induce cell death in short-term explants of PDXs derived from three molecularly diverse human gliomas. Additionally, cells not killed by P21 overexpression were maintained in a stable senescent state for longer than control cells. Collectively, these data suggest that P21 activation may provide an attractive therapeutic target to improve therapeutic outcomes.
ARTICLE | doi:10.20944/preprints202104.0788.v1
Subject: Life Sciences, Biochemistry Keywords: SARS-CoV-2; COVID-19; Flux Balance Analysis (FBA); Genome-Scale Metabolic Models; Target Identification; Reaction Knock-Out; Structural Proteins; Purine Metabolism; Pyrimidine Metabolism; B.1.1.7; B.1.351
Online: 30 April 2021 (15:14:06 CEST)
The current SARS-CoV-2 pandemic is still threatening humankind. Despite first successes in vaccine development and approval, no antiviral treatment is available for COVID-19 patients. The success is further tarnished by the emergence and spreading of mutation variants of SARS-CoV-2, for which some vaccines are not effective anymore. This highlights the urgent need for antiviral therapies even more. This article describes how the Genome-Scale Metabolic Model (GEM) of the host-virus interaction of human alveolar macrophages and SARS-CoV-2 was refined by incorporating the latest information about the virus’s structural proteins and the mutant variants B.1.1.7 and B.1.351. We confirmed the initially identified guanylate kinase as a potential antiviral target with this refined model and identified further potential targets from the purine and pyrimidine metabolism. The model was further extended by incorporating the virus’ lipid requirements. This opened new perspectives for potential antiviral targets in the altered lipid metabolism. Especially the phosphatidylcholine biosynthesis seems to play a pivotal role in viral replication. The guanylate kinase is even a robust target in all investigated mutation variants currently spreading worldwide. These new insights can guide laboratory experiments for the validation of identified potential antiviral targets. Only the combination of vaccines and antiviral therapies will effectively defeat this ongoing pandemic.