Simulation of COVID-19 Symptoms in a Genetically Engineered Mouse Model

The ongoing infectious viral disease pandemic (also known as the coronavirus disease-19; COVID-19) by a constantly emerging viral agent commonly referred as the severe acute respiratory syndrome corona virus 2 or SARS-CoV-2 has revealed unique pathological findings from infected human beings, and the postmortem observations. The list of disease symptoms, and post-mortem observations is too long to mention; however, a few notable ones are worth mentioning to put into a perspective in understanding the malignity of this pandemic starting with respiratory distress or dyspnea, chest congestion, muscle or body aches, malaise, fever, chills, etc. We opine that further improvement for delivering highly effective treatment, and preventive strategies would be benefited from validated animal disease models. In this context, we designed a study and show that a genetically engineered mouse expressing the human angiotensin converting enzyme 2; hACE2 (the receptor used by SARS-CoV-2 agent to enter host cells) represents an excellent investigative resource in simulating important clinical features of the COVID-19 infection. The hACE2 mouse model (which is susceptible to SARS-CoV-2) when administered with a recombinant SARS-CoV-2 spike (S) protein intranasally exhibited a profound cytokine storm capable of altering the physiological parameters including significant changes in in vivo cardiac function along with multi-organ damage that was further confirmed via histological findings. More importantly, visceral organs from SARS-CoV-2 spike (S) treated mice revealed thrombotic blood clots as seen during postmortem examination of the mice. Thus, the hACE2 engineered mouse appears to be a suitable model for studying intimate viral pathogenesis paving the way for further identification, and characterization of appropriate prophylactics as well as therapeutics for COVID-19 management.

Expression of phenylalanine ammonia lyases in Synechocystis sp. PCC 6803 and subsequent improvements of sustainable production of phenylpropanoids

Background Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Results A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L−1, respectively, after 4 days. Conclusions Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.

Second messenger c-di-GMP modulates exopolysaccharide Pea-dependent phenotypes via regulating eppA expression in Pseudomonas putida

Exopolysaccharides (EPSs) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida . The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through unknown mechanism(s). Herein, we found that c-di-GMP modulated wrinkly colony morphology via regulating expression of eppA ( PP_5586 ), a small individually transcribed gene with 177 base pairs, and this gene was adjacent to the upstream of pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with the loss of the entire pea operon. EppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blot revealed that EppA was located on cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on transcriptomic profile of P. putida . Bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes, and that c-di-GMP modulates Pea-dependent phenotypes via regulating eppA expression in P. putida . IMPORTANCE Microbe-secreted EPSs are high molecular weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes, and provide possible pathway to construct genetically engineered strain for high Pea production.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity. Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes. We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically cold. Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

The development in cryobiology in animal breeding had revolutionized the field of reproductive medicine. The main objective to preserve animal germplasm stems from variety of reasons such as conservation of endangered animal species, animal diversity, and an increased demand of animal models and/or genetically modified animals for research involving animal and human diseases. Cryopreservation has emerged as promising technique for fertility preservation and assisted reproduction techniques (ART) for production of animal breeds and genetically engineered animal species for research. Slow rate freezing and rapid freezing/vitrification are the two main methods of cryopreservation. Slow freezing is characterized by the phase transition (liquid turning into solid) when reducing the temperature below freezing point. Vitrification, on the other hand, is a phenomenon in which liquid solidifies without the formation of ice crystals, thus the process is referred to as a glass transition or ice-free cryopreservation. The vitrification protocol applies high concentrations of cryoprotective agents (CPA) used to avoid cryoinjury. This chapter provides a brief overview of fundamentals of cryopreservation and established methods adopted in cryopreservation. Strategies involved in cryopreserving germ cells (sperm and egg freezing) are included in this chapter. Last section describes the frontiers and advancement of cryopreservation in some of the important animal models like rodents (mouse and rats) and in few large animals (sheep, cow etc).

Crown disease with extrainterinal manifestations in the form of granulematous alveolitis

Objective: To present a clinical description of the observation of a patient with Crohn’s disease (CD) with extraintestinal manifestations in the form of granulomatous alveolitis.Materials and Methods: A brief review of the literature on the current understanding of the prevalence and clinical manifestations of lung lesions in inflammatory bowel diseases (IBD) and CD, as well as a description of a patient with this pathology with the results of autopsy is presented.Results: The rare presence of granulomatous lung lesions in a patient with CD was proved.Conclusion: Difficulties in the differential diagnosis and treatment of CD lung lesions are shown. In particular, suspicion of tuberculous lesion, lung abscess creates potential risks of generalization of the process when using such therapeutic effects as glucocorticosteroids, cytostatics, immunosuppressants, biological genetically engineered drugs and active surgical intervention. The authors hope that the described observation will alert doctors in terms of possible systemic pulmonary lesions in CD.

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

Prospective Application of Aspergillus Species: Focus on Enzyme Production Strategies, Advances and Challenges

Fungal enzymes that catalyze different types of biochemical reactions play a significant role in modern industry by improving existing processes. Also, the use of enzymes to replace some traditional toxic chemical or mechanical approaches helps decrease energy demand and environmental pollution. However, enzymes must be able to compete commercially with relatively low-priced traditional approaches. Meeting economical and commercial feasibility criteria depends on a number of enzymatic properties including the specificity to the substrate, stability in industrial enzymatic reaction conditions and catalytic efficiency. Fungi used as an enzyme manufacture host should be appropriate for industrial scale fermentation. Aspergillus species are being developed as one of the best enzyme manufacture factories due to their capability to secrete high quantities of enzymes suitable for industrial applications. The industrial importance of Aspergillus species also includes the progress and commercialization of new products derived from genetically engineered modified strains. Hence, the main aim of this chapter investigation is to analyze the secreted and cellular proteins from Aspergillus species and their application in industries.