Plug and play: Is “directed endosymbiosis” of chloroplasts possible?

The origin of mammalian mitochondria and plant chloroplasts is thought to be endosymbiosis. Millennia ago, a bacterium related to typhus-causing bacteria may have been consumed by a proto-eukaryote and over time evolved into an organelle inside eukaryotic cells, known as a mitochondrion. The plant chloroplast is believed to have evolved in a similar fashion from cyanobacteria. This project attempted to use “directed endosymbiosis” (my term) to investigate if chloroplasts can be taken up by a land animal and continue to function. It has been shown previously that mouse fibroblasts could incorporate isolated chloroplasts when co-cultured. Photosynthetic bacteria containing chloroplasts have been successfully injected into zebrafish embryos, mammalian cells, and ischemic rodent hearts. The photosynthetic alga Chlamydomonas reinhardtii (C. reinhardtii) has also been injected into zebrafish embryos. However, to the best of my knowledge, injection of isolated chloroplasts into a land animal embryo has not been attempted before.In four pilot experiments, solutions of chloroplasts in PBS were microinjected into Drosophila melanogaster (D. melanogaster) embryos to determine if the embryos would tolerate the foreign protein. Interestingly, results indicated that a portion of the D. melanogaster embryos appeared to tolerate the injections and survive to adulthood. To determine if chloroplasts had indeed been transferred, larvae were placed under fluorescent microscopy. Chlorophyll (serving as the reporter) was found to be present in several larvae and to decline in amount over time. To investigate if the chloroplasts still functioned, a radiotracer food intake assay was performed. It was hypothesized that if the chloroplasts were generating ATP (and possibly glucose), the larvae might need less food. Results indicated a decrease in intake, however this might have occurred for other reasons.

Production leptospirosis vaccine with included strain of Leptospira interrogans of serogroup Canicola

The leptospirosis vaccine is the main method of preventing the occurrence and spread of leptospirosis. Compliance with the standards of manufacturing, labeling, and storage is mandatory for immunological preparations. All stages of vaccine production must comply with the rules established by the Ministry of Industry and Trade and ensure its safety for humans. The article presents epidemiological data on leptospirosis in the Russian Federation in the period from 2013 to 2018. A method for producing a vaccine against human leptospirosis is described. The leptospirosis vaccine is polyvalent using membrane technologies and semi-synthetic culture media. It eliminates the use of foreign protein and does not require cleaning. The vaccine is an opalescent liquid with sediment and a pH of 7.2-7.6 and it is not allowed to contain live leptospira. Four strains are used and a new strain has been developed and implemented. Vaccination is carried out according to epidemiological indicators. Leptospirosis suspension forms specific immunity for 1 year. During the production of the updated vaccine, it was necessary to study the virulent properties of the strains. Moreover, analyze the formation of specific antibodies to leptospira in the new vaccine and in the vaccine currently used. From 2018 to 2020, 5 series of experimental vaccines in the form of a 0.5 ml suspension were produced.

Engineering of Cytolethal Distending Toxin B by Its Reducing Immunogenicity and Maintaining Stability as a New Drug Candidate for Tumor Therapy; an In Silico Study

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.

Review of testing for foreign horse and pig DNA in meats in Croatia

Several years after the food industry scandal when horsemeat was found in products sold in Europe as beef products in 2013, Croatia began testing food for the presence of foreign protein. For the time being, these tests are not part of routine monitoring, but the result of examining the situation on the market in the city of Zagreb. Namely, in recent years, central Croatia has been trying to establish itself as a tourist destination, and Zagreb hosted hundreds of thousands of tourists from all over the world before the COVID-19 pandemic. The eating habits of the various groups that came to Zagreb were different, and the larger hotel chains recognized the seriousness of the services and sought help to ensure that the food offered was consistent with their declarations and would not conflict with religious requirements. One of these requirements was the testing for foreign proteins such as horse and pork in foods where they were not declared. Although horse and pork are safe for human consumption, they are not part of the eating habits in all countries. The Dr. Andrija Štampar Teaching Institute for Public Health introduced methods for detection of horse and pig DNA in food samples.

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PLacUV5), which produces more T7 RNAP than wild-type lac promoter (PLacWT) to promote the production of recombinant proteins. However, there is a resource allocated limitation between cell growth and protein production when producing autolytic proteins or membrane proteins. T7 RNAP is the key factor to solve this problem. Hence, we replaced respectively PLacUV5 with other inducible promoters: arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet) to optimize the production of recombinant protein by regulating the transcription level of T7 RNAP. Compared with BL21 (DE3), the constructed engineering strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the glucose dehydrogenase (GDH) production, the engineered strains BL21 (DE3::tet) exhibited great biomass, cell survival rate and foreign protein expression level. In addition, these engineered strains had been successfully applied to the production of other membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and E. coli F-ATPase subunit b (Ecb). The engineering strains constructed in this paper provided more host choices for the production of recombinant proteins.

Integrated Strategies for Enhancing the Expression of Chitosanase AqCoA in the Pichia Pastoris by A Novel High-Copy Plasmid PMC-GAP

In our previous study, the chitosanase AqCoA and its products chitooligosaccharides exhibited significant application in fungal disease protection. In this study, to enhance the expression of AqCoA, we obtained various strains with multi-copy by a novel plasmid pMC-GAP with stable transformation ability in Pichia pastoris and built an integrated model combining the gene copy number, the chaperones and protein production of AqCoA. In terms of gene dosage, the highest enzyme activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that in strain with only one copy (0.18 U/ml). In addition, we found the chaperone like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased by 61%, 31% and 42% in expression levels of the strain GAP-1AqCoA. Finally, we investigated the effect co-expression of gene copy and chaperones on protein expression. The maximum activity reached 2.319 U/ml by the strain with six chaperones intergrant plus sixteen copies, which was 13-fold higher than that by the control strain with only one copy (GAP-1AqCoA). Co-expression of gene dosage and chaperones significantly enhanced expression levels of AqCoA, which presented a powerful tool to improve foreign protein expression.

How Does a COVID mRNA vaccine really work?

The most potent weapon against COVID-19 is a vaccine based on messenger RNA (mRNA). The first of these vaccines authorized for use was developed by the German company BioNTech in cooperation with Pfizer, closely followed by the (U.S.-produced) Moderna vaccine. These vaccines send a piece of mRNA into cells of a host. The mRNA instructs the cells to produce masses of the same spike protein that also occurs on the shell of the real coronavirus. The immune system responds by learning to destroy anything showing that protein: if the real virus arrives, the immune system will attack it immediately. This much has been reported widely by the media. But important questions remain. How is mRNA actually synthesized as a transcription of the spike-producing segment of the virus' RNA? How is the selection and replication done? How does mRNA enter a host cell, and how long will it stay there? Will it produce the spike protein forever? Is it perhaps dangerous? And the biggest question of all: How does the immune system record the structure of the foreign protein, how does it recognize the invader, and how is the immune response cranked up? To answer these questions, we bring you a conversation between Ubiquity editor Walter Tichy and his daughter Dr. Evelyn Tichy, an infectious disease expert.

An inactivated recombinant rabies virus displaying the Zika virus prM-E induces protective immunity against both pathogens

The global spread of Zika virus (ZIKV), which caused a pandemic associated with Congenital Zika Syndrome and neuropathology in newborns and adults, prompted the pursuit of a safe and effective vaccine. Here, three kinds of recombinant rabies virus (RABV) encoding the prM-E protein of ZIKV were constructed: ZI-D (prM-E), ZI-E (transmembrane domain (TM) of prM-E replaced with RABV G) and ZI-F (signal peptide and TM domain of prM-E replaced with the region of RABV G). When the TM of prM-E was replaced with the region of RABV G (termed ZI-E), it promoted ZIKV E protein localization on the cell membrane and assembly on recombinant viruses. In addition, the change in the signal peptide with RABV G (termed ZI-F) was not conducive to foreign protein expression. The immunogenicity of recombinant viruses mixed with a complex adjuvant of ISA 201 VG and poly(I:C) was tested in BALB/c mice. After immunization with ZI-E, the anti-ZIKV IgG antibody lasted for at least 10 weeks. The titers of neutralizing antibodies (NAbs) against ZIKV and RABV at week 6 were all greater than the protective titers. Moreover, ZI-E stimulated the proliferation of splenic lymphocytes and promoted the secretion of cytokines. It also promoted the production of central memory T cells (TCMs) among CD4+/CD8+ T cells and stimulated B cell activation and maturation. These results indicate that ZI-E could induce ZIKV-specific humoral and cellular immune responses, which have the potential to be developed into a promising vaccine for protection against both ZIKV and RABV infections.

Hamster Polyomavirus Research: Past, Present, and Future

Hamster polyomavirus (Mesocricetus auratus polyomavirus 1, HaPyV) was discovered as one of the first rodent polyomaviruses at the end of the 1960s in a colony of Syrian hamsters (Mesocricetus auratus) affected by skin tumors. Natural HaPyV infections have been recorded in Syrian hamster colonies due to the occurrence of skin tumors and lymphomas. HaPyV infections of Syrian hamsters represent an important and pioneering tumor model. Experimental infections of Syrian hamsters of different colonies are still serving as model systems (e.g., mesothelioma). The observed phylogenetic relationship of HaPyV to murine polyomaviruses within the genus Alphapolyomavirus, and the exclusive detection of other cricetid polyomaviruses, i.e., common vole (Microtus arvalis polyomavirus 1) and bank vole (Myodes glareolus polyomavirus 1) polyomaviruses, in the genus Betapolyomavirus, must be considered with caution, as knowledge of rodent-associated polyomaviruses is still limited. The genome of HaPyV shows the typical organization of polyomaviruses with an early and a late transcriptional region. The early region encodes three tumor (T) antigens including a middle T antigen; the late region encodes three capsid proteins. The major capsid protein VP1 of HaPyV was established as a carrier for the generation of autologous, chimeric, and mosaic virus-like particles (VLPs) with a broad range of applications, e.g., for the production of epitope-specific antibodies. Autologous VLPs have been applied for entry and maturation studies of dendritic cells. The generation of chimeric and mosaic VLPs indicated the high flexibility of the VP1 carrier protein for the insertion of foreign sequences. The generation of pseudotype VLPs of original VP1 and VP2–foreign protein fusion can further enhance the applicability of this system. Future investigations should evaluate the evolutionary origin of HaPyV, monitor its occurrence in wildlife and Syrian hamster breeding, and prove its value for the generation of potential vaccine candidates and as a gene therapy vehicle.