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2021 ◽  
Author(s):  
◽  
Michelle Hedley Rich

<p><b>Bacterial nitroreductase enzymes, which exhibit the capacity to reduce a wide range of nitroaromatic drugs, antibiotics and environmental pollutants, have shown promise in the activation of prodrugs such as CB1954 and PR-104A. Use of these prodrugs in gene-directed enzyme prodrug therapy (GDEPT) cancer treatment would allow for targeted chemotherapy in tumour cells following specific delivery of nitroreductases to these cancerous tissues, using specialised bacterial or viral vectors. However, one key limitation in nitroreductase-based GDEPT is the current inability to rapidly and non-invasively determine vector localisation and gene delivery prior to systemic administration of prodrug. Dual-purpose nitroreductases that exhibit the ability to activate both GDEPT prodrugs and radioisotope-labelled PET imaging probes, in a manner that renders them temporarily cell-entrapped for detection using a PET scanner, would facilitate clinical development of this treatment.</b></p> <p>Previous attempts to repurpose hypoxia-activated 2-nitroimidazole PET imaging probes for nitroreductase detection have suffered from relatively high background activation under hypoxia alone. The design of nextgeneration 5-nitroimidazole PET imaging probes, by our collaborators at the Auckland Cancer Society Research Centre (ACSRC), has resulted in much lower levels of hypoxia activation in vivo.</p> <p>This thesis describes attempts to generate improved nitroreductases that can activate a bespoke 5-nitroimidazole PET-capable imaging probe, S33. A 58-membered library of nitroreductase candidates, including enzymes from many different bacterial species and oxidoreductase families, was heterologously over-expressed in E. coli screening strains. Microplate-based screening strategies were then used to identify enzymes that exhibited the most activity with S33, based on the ability of high levels of activated S33 to induce DNA damage and (at very high levels) E. coli cell death. Following this, site-targeted libraries of two different promising nitroreductase NfsA homologues were screened for S33 activity, with selected variants from eachlibrary showing improvement in S33 activation over the parent nitroreductase. In parallel I performed error-prone PCR mutagenesis of a top NfsA variant and top NfsB variant, subjecting each to two rounds of random mutagenesis, and selecting improved variants using a specialised E. coli screening strain and fluorescence-activated cell sorting (FACS). Selected variants from the NfsB (but not NfsA) nitroreductase candidate library showed substantially improved capacity to activate S33 over the parent enzyme.</p> <p>As an alternative means for developing improved nitroreductase variants, two different nitroaromatic ‘anti-prodrugs’, the anthelmintic niclosamide and the antibiotic chloramphenicol, whose cytotoxic effects on E. coli can be mitigated by the presence of an over-expressed active nitroreductase, were used to select for improved S33-activating enzymes from a site-targeted NfsA library. Variants were discovered that exhibited improved ability to active S33 as well as other nitroaromatic substrates of interest. Finally, attempts to discover novel nitroreductases from nature through the screening of cloned soil metagenomic fragments, were made utilising a novel cloning strategy to improve expression of the cloned gene fragments in E. coli. Screening and selection of nitroreductase gene ragments was conducted using niclosamide as well as nitroaromatic compounds that change colour upon activation.</p>


2021 ◽  
Author(s):  
◽  
Michelle Hedley Rich

<p><b>Bacterial nitroreductase enzymes, which exhibit the capacity to reduce a wide range of nitroaromatic drugs, antibiotics and environmental pollutants, have shown promise in the activation of prodrugs such as CB1954 and PR-104A. Use of these prodrugs in gene-directed enzyme prodrug therapy (GDEPT) cancer treatment would allow for targeted chemotherapy in tumour cells following specific delivery of nitroreductases to these cancerous tissues, using specialised bacterial or viral vectors. However, one key limitation in nitroreductase-based GDEPT is the current inability to rapidly and non-invasively determine vector localisation and gene delivery prior to systemic administration of prodrug. Dual-purpose nitroreductases that exhibit the ability to activate both GDEPT prodrugs and radioisotope-labelled PET imaging probes, in a manner that renders them temporarily cell-entrapped for detection using a PET scanner, would facilitate clinical development of this treatment.</b></p> <p>Previous attempts to repurpose hypoxia-activated 2-nitroimidazole PET imaging probes for nitroreductase detection have suffered from relatively high background activation under hypoxia alone. The design of nextgeneration 5-nitroimidazole PET imaging probes, by our collaborators at the Auckland Cancer Society Research Centre (ACSRC), has resulted in much lower levels of hypoxia activation in vivo.</p> <p>This thesis describes attempts to generate improved nitroreductases that can activate a bespoke 5-nitroimidazole PET-capable imaging probe, S33. A 58-membered library of nitroreductase candidates, including enzymes from many different bacterial species and oxidoreductase families, was heterologously over-expressed in E. coli screening strains. Microplate-based screening strategies were then used to identify enzymes that exhibited the most activity with S33, based on the ability of high levels of activated S33 to induce DNA damage and (at very high levels) E. coli cell death. Following this, site-targeted libraries of two different promising nitroreductase NfsA homologues were screened for S33 activity, with selected variants from eachlibrary showing improvement in S33 activation over the parent nitroreductase. In parallel I performed error-prone PCR mutagenesis of a top NfsA variant and top NfsB variant, subjecting each to two rounds of random mutagenesis, and selecting improved variants using a specialised E. coli screening strain and fluorescence-activated cell sorting (FACS). Selected variants from the NfsB (but not NfsA) nitroreductase candidate library showed substantially improved capacity to activate S33 over the parent enzyme.</p> <p>As an alternative means for developing improved nitroreductase variants, two different nitroaromatic ‘anti-prodrugs’, the anthelmintic niclosamide and the antibiotic chloramphenicol, whose cytotoxic effects on E. coli can be mitigated by the presence of an over-expressed active nitroreductase, were used to select for improved S33-activating enzymes from a site-targeted NfsA library. Variants were discovered that exhibited improved ability to active S33 as well as other nitroaromatic substrates of interest. Finally, attempts to discover novel nitroreductases from nature through the screening of cloned soil metagenomic fragments, were made utilising a novel cloning strategy to improve expression of the cloned gene fragments in E. coli. Screening and selection of nitroreductase gene ragments was conducted using niclosamide as well as nitroaromatic compounds that change colour upon activation.</p>


Cell Research ◽  
2021 ◽  
Author(s):  
Minglei Zhi ◽  
Jinying Zhang ◽  
Qianzi Tang ◽  
Dawei Yu ◽  
Shuai Gao ◽  
...  

AbstractPig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1892
Author(s):  
Haojie Chen ◽  
Hao Ji ◽  
Xiangjun Kong ◽  
Pengyu Lei ◽  
Qinsi Yang ◽  
...  

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expressions of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. Globally, BGs have been used as vaccine delivery systems and vaccine adjuvants. There is an increasing interest in the development of novel delivery systems that are based on BGs for biomedical applications. Due to intact reservation of bacterial cell membranes, BGs have an inherent immunogenicity, which enables targeted drug delivery and controlled release. As carrier vehicles, BGs protect drugs from interference by external factors. In recent years, there has been an increasing interest in BG-based delivery systems against tumors, inflammation, and infection, among others. Herein, we reviewed the preparation methods for BGs, interactions between BGs and the host, and further highlighted research progress in BG development.


2021 ◽  
Author(s):  
Daria Krefft ◽  
Maciej Prusinowski ◽  
Paulina Maciszka ◽  
Aleksandra Skokowska ◽  
Joanna Zebrowska ◽  
...  

Abstract BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research and practical applications in biotechnology industry, including pharmaceutical drugs production. Various factors can highly affect successful clones construction and their stable maintenance as well as obtained biosynthesis levels. These include correct selection of recombinant hosts, expression systems, promoters regulation, repression level at uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics and chaperons presence/absence, among others. However, the optimization of the growth media compositions is often overlooked. We systematically evaluate this factor, which can have dramatic effect on biosynthesis of recombinant proteins, especially those, which are toxic to a recombinant host. Results Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed ORFs with wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28 – coding thermophilic endolysin (moderately toxic) and (iii) tthHB27IRM – coding for thermophilic restriction endonuclease-methyltransferase (REase-MTase) (very toxic). The use of plant-derived peptones (soy peptone and wheat extract) in culture media causes leakage of the T7-lac expression system. We show, that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of cultures, clones stability and biosynthesis levels.Conclusions The use of plant-derived peptones in culture media when using the T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins toxic to a recombinant host, this can result in the expression vector and/or cloned gene mutations, deletions, host’s death or highly decreased expression levels. This phenomenon is caused by certain saccharides content in plant peptones, some of which (galactosides) may act as T7-lac promoter inducers by theirinteraction with Lac repressors. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or use them with caution and to pilot-scale evaluate the derepression effect on the case-by-case basis.


2021 ◽  
Vol 24 (5) ◽  
pp. 161-169
Author(s):  
Enny Ratnaningsih ◽  
Lousiana Dwinta Utami ◽  
Nurlaida Nurlaida ◽  
Rindia Maharani Putri

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Irene Kim ◽  
Hyomin Park ◽  
Injoo Hwang ◽  
Dodam Moon ◽  
Hyunji Yun ◽  
...  

Abstract Background The homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still don’t know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver. Methods and results When we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector. Conclusions Chemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.


2021 ◽  
Vol 20 (2) ◽  
pp. 102-111
Author(s):  
Kumarss Amini ◽  
◽  
Maryam Akbari ◽  

Background and Objectives: Serratia is a gram-negative bacterium. The pigmentation property of Serratia Marcescens is used as a marker of dust particles in the environment and in the hospital. Today biopigments are also widely used in the manufacture and production of pharmaceutical products. Prodigiosin is a promising drug due to its reported properties of antifungal immunosuppressive and anti-proliferative activities. In the present study, cloning of pig gene- isolated from Serratia Marcescens in Ecoli XL1blue was performed. Subjects and Methods 60 Samples were taken from clinical sources of patients hospitalized with urinary tract infections in Saveh Hospitals. Serratia Marcescens were identified and isolated by different tests. The pig gene was cloned by T-A cloning using PTG-19 vector into the Escherichia coli XL1blue as host. Expression of cloned gene in recombinant colonies was evaluated by Real time PCR. The phylogenetic tree was plotted using clustalX and Mega5 software Results Screening of samples identified 12 isolates of Serratia Marcescens from then 4 isolates had pig gene. Expression of Pig gene in Escherichia coli XL1blue was confirmed by Real-Tima PCR. As a result of phylogenetic studies, some close relatives of serratia have been identified as candidates for further studies Conclusion Serratia Marcescens can be considered as a rich source of pigments with many applications and can be used as indigenous strains to produce Prodigiosin.


Author(s):  
Jakeer Husen A Olekar ◽  
Ashish Kumar ◽  
Ambika Sharma ◽  
Kranti Dev

Background: Luteinizing hormone (LH) plays a critical role in ovulation and maintenance of pregnancy in female and gamete production in male during fertile phase of life. Physiological disturbance of this hormone leads to conditions like delayed ovulation, anovulation, cystic ovarian disease and lack of sexual desire in male. Since, there had been no report of molecular characterization of β-subunit of luteinizing hormone (LH) of Indian goat, the present study aimed to clone and characterize genomic DNA encoding LHβ subunit.Methods: Genomic DNA was extracted from goat blood and amplified using specific LHβ gene primers. After cloning and transformation, plasmids were isolated from randomly selected white colonies. Presence of insert was confirmed by restriction enzyme digestion of plasmids. After confirmation by PCR, plasmids were sent for DNA sequencing. Result: Analysis of sequence revealed an insert of 1006 bp size as expected. Comparison of nucleotide sequence revealed the cloned gene to be is LHβ encoding 141 amino acids. It showed 97.3 and 91.7% similarity with sheep and cattle respectively. Inferred amino acid sequence showed absolute similarity (100%) with sheep and buffalo. The common and essential features such as twelve cysteine molecules, a single potential N-glycosylation site, the CAGY region and another tetrapeptide CGPC are all found in the goat sequence too.


2021 ◽  
Vol 12 ◽  
Author(s):  
Mariko Okamoto ◽  
Masahiko Kumagai ◽  
Hiroyuki Kanamori ◽  
Daisuke Takamatsu

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.


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