Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

BACKGROUND: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. OBJECTIVE: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of Immobilized Metal Ion Affinity Chromatography (IMAC) for producing 3Cpro. METHODS: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. RESULTS: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. CONCLUSION: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.

Print-and-Grow within a Novel Support Material for 3D Bioprinting and Post-Printing Tissue Growth.

3D bioprinting holds great promise for tissue engineering, with extrusion bioprinting in suspended hydrogels becoming the leading bioprinting technique in recent years. In this method, living cells are incorporated within bioinks, extruded layer by layer into a granular support material followed by gelation of the bioink through diverse cross-linking mechanisms. This approach offers high fidelity and precise fabrication of complex structures mimicking living tissue properties. However, the transition of cell mass mixed with the bioink into functional native-like tissue requires post-printing cultivation in vitro. An often-overlooked drawback of 3D bioprinting is the nonuniform shrinkage and deformation of printed constructs during the post-printing tissue maturation period, leading to highly variable engineered constructs with unpredictable size and shape. This limitation poses a challenge for the technology to meet applicative requirements. A novel technology of "print-and-grow," involving 3D bioprinting and subsequent cultivation in κ-Carrageenan-based microgels (CarGrow) for days is presented. CarGrow enhances the long-term structural stability of the printed objects by providing mechanical support. Moreover, this technology provides a possibility for live imaging to monitor tissue maturation. The "print-and-grow" method demonstrates accurate bioprinting with high tissue viability and functionality while preserving the construct's shape and size.

SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner.

SOX9 is upregulated in the majority of pancreatic ductal adenocarcinoma cases. It is hypothesized that the increased expression of SOX9 is necessary for the formation and maintenance of tumor phenotypes in pancreatic cancer cells. In our research, we studied six pancreatic cancer cell lines, which displayed varying levels of differentiation and a range of oncogenic mutations. We chose the method of downregulation of SOX9 expression via siRNA transfection as the main method for investigating the functional role of the SOX9 factor in pancreatic cancer cells. We discovered that the downregulation of SOX9 expression in the cell lines leads to cell-line-specific changes in the expression levels of epithelial and mesenchymal protein markers. Additionally, the downregulation of SOX9 expression had a specific effect on the expression of pancreatic developmental master genes. SOX9 downregulation had the greatest effect on the expression levels of the protein regulators of cell proliferation. In three of the four cell lines studied, the transfection of siSOX9 led to a significant decrease in proliferative activity and to the activation of proapoptotic caspases in transfected cells. The acquired results demonstrate that the SOX9 protein exerts its multiple functions as a pleiotropic regulator of differentiation and a potential promoter of tumor growth in a cell-specific manner in pancreatic cancer cells.

Boosting extracellular vesicle secretion.

In recent years, extracellular vesicles (EVs), specifically exosomes, have emerged as a promising strategy for treating a wide spectrum of pathologies, such as cancer and COVID-19, as well as promoting tissue regeneration in various conditions, including cardiomyopathies and spinal cord injuries. Despite the great potential of EV-based therapies, poor yield and unscalable production of EVs remain big challenges to overcome to translate these types of treatment to clinical practices. Here, we review different strategies for enhancing EV yield by physical, biological or chemical means. Some of these novel approaches can lead to about 100-fold increase in EV production yield, thus bringing closer the clinical translation with regard to scalability and efficiency.

Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos.

The 3C protease is a key factor in picornavirus-induced pathologies with a comprehensive action on cell targets. However, the effects induced by the enzyme have not been described at the organismic level. Here, the model of developing Danio rerio embryos was used to analyze possible toxic effects of the 3C protease of human hepatitis A virus (3Cpro) at the whole-body level. The transient 3Cpro expression had a notable lethal effect and induced a number of specific abnormalities in Danio rerio embryos within 24 h. These effects are due to the proteolytic activity of the enzyme. At the same time, the 3Cpro variant with reduced catalytic activity (3Cmut) increased the incidence of embryonic abnormalities; however, this effect was smaller compared to the native enzyme form. While the expression of 3Cmut increased the overall rate of abnormalities, no predominance of specific ones was observed. The data obtained point to a presence significant impact of picornavirus 3Cprotease at the whole-organism level and make contribution to the study of the infectious process caused by human hepatitis A virus.

The protealysin operon encodes emfourin, a prototype of a novel family of protein metalloprotease inhibitors.

Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (Ki = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases.

Alemtuzumab mediates the CD39+ T-regulatory cell response via CD23+ macrophages.

Alemtuzumab (ALM) effectively prevents relapses of multiple sclerosis (MS). It causes lymphocyte depletion with subsequent enhancement of the T-regulatory cell population. Direct administration of ALM to T cells causes cytolysis. However, the T cells may be indirectly affected by monocyte-derived cells, which are resistant to ALM cytotoxicity. We aimed to examine whether ALM modulates monocytes and whether the crosstalk between monocytes and lymphocytes previously exposed to ALM would result in anti-inflammatory effects. The CD14+ monocytes of 10 healthy controls and 10 MS (treatment naive) patients were isolated from peripheral blood mononuclear cells (PBMCs), exposed to ALM and reintroduced to PBMCs depleted of CD14+ cells. The macrophage profile was assessed and T-cell markers were measured. ALM promoted M2 anti-inflammatory phenotype as noted by an increased percentage in the populations of CD23+ , CD83+ and CD163+ cells. The CD23+ cells were the most upregulated (7-fold, P = 0.0002), and the observed effect was higher in patients with MS than in healthy subjects. ALM-exposed macrophages increased the proportion of T-regulatory cells, without affecting the proportion of T-effector cells. Neutralizing the CD23+ monocytes with antibodies reversed the effect specifically on the CD4+ CD39+ T-regulatory cell subpopulation but not on the CD4+ CD25hi CD127lo FOXP3+ subpopulation. ALM induces the conversion of monocytes into anti-inflammatory macrophages, which in turn promotes T-regulatory cell enhancement, in a CD23-dependent manner. These findings suggest that the mechanism of action of ALM is relevant to aspects of MS pathogenesis.

"Flora of Russia" on iNaturalist: a dataset.

BACKGROUND: The "Flora of Russia" project on iNaturalist brought together professional scientists and amateur naturalists from all over the country. Over 10,000 people were involved in the data collection. NEW INFORMATION: Within 20 months, the participants accumulated 750,143 photo observations of 6,857 species of the Russian flora. This constitutes the largest dataset of open spatial data on the country's biodiversity and a leading source of data on the current state of the national flora. About 87% of all project data, i.e. 652,285 observations, are available under free licences (CC0, CC-BY, CC-BY-NC) and can be freely used in scientific, educational and environmental activities.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Lipoprotein receptor loss in forebrain radial glia results in neurological deficits and severe seizures.

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.

Functional efficiency of PCR vectors in vitro and at the organism level.

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.

PDX1, a key factor in pancreatic embryogenesis, can exhibit antimetastatic activity in pancreatic ductal adenocarcinoma.

Background: In cancer biology, metastasizing is one of the most poorly studied processes. Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastasis, which is the leading cause of death. The PDX1 protein is crucial for the development of cancer, and its low levels are characteristic of the most aggressive PDAC tumors. The PDX1 is a mediator of initiation and progression of PDAC. However, further studies are needed to elucidate the role of PDX1 in the cancer metastasis. Purpose: To confirm the hypothesis that PDX1 in PDAC plays suppressor role of epithelial-mesenchymal transition (EMT), and to study its possible ability to inhibit metastasis. Methods: A PDX1-overexpressing PDAC cell line was obtained by lentiviral transduction of PANC-1 cells. PDX1 overexpression was confirmed by RT-PCR and Western blotting. Effects of PDX1 ectopic expression on cell proliferation and motility were determined in PANC-1 cells using MTS, cell cycle analysis, transwell and wound-healing assay. EMT genes expression was analyzed in PDX1-overexpressing and Control PANC-1. Finally, the migration potential of pancreatic cancer cells expressing PDX1 was evaluated using a zebrafish embryo model. Results: The motility of human PDAC cells PANC-1 considerably decreased at ectopic expression of PDX1. The decreased expression of ZEB1, the key factor of EMT, and almost unchanged expression of the genes that characterize the epithelial state suggest a decrease in the EMT ability. Suppression of PDX1 expression by siRNA knockdown restored the PANC1 motility. Conclusion: The results obtained suggest a possible therapeutic use of PDX1 delivery into PDAC patients with a reduced or absent expression of PDX1 in the most aggressive tumors.

Differential expression patterns of sodium potassium ATPase alpha and beta subunit isoforms in mouse brain during postnatal development.

The sodium potassium ATPase (Na+/K+ ATPase) is essential for the maintenance of a low intracellular Na+ and a high intracellular K+ concentration. Loss of function of the Na+/K+ ATPase due to mutations in Na+/K+ ATPase genes, anoxic conditions, depletion of ATP or inhibition of the Na+/K+ ATPase function using cardiac glycosides such as digitalis, causes a depolarization of the resting membrane potential. While in non-excitable cells, the uptake of glucose and amino acids is decreased if the function of the Na+/K+ ATPase is compromised, in excitable cells the symptoms range from local hyper-excitability to inactivating depolarization. Although several studies have demonstrated the differential expression of the various Na+/K+ ATPase alpha and beta isoforms in the brain tissue of rodents, their expression profile during development has yet to be thoroughly investigated. An immunohistochemical analysis of postnatal day 19 mouse brain showed ubiquitous expression of Na+/K+ ATPase isoforms α1, ß1 and ß2 in both neurons and glial cells, whereas α2 was expressed mostly in glial cells and the α3 and ß3 isoforms were expressed in neurons. Furthermore, we examined potential changes in the relative expression of the different Na+/K+ ATPase isoforms in different brain areas of postnatal day 6 and in adult 9 months old animals using immunoblot analysis. Our results show a significant up-regulation of the α1 isoform in cortex, hippocampus and cerebellum, whereas, the α2 isoform was significantly up-regulated in midbrain. The ß3 isoform showed a significant up-regulation in all brain areas investigated. The up-regulation of the α3 isoform matched that of the ß2 isoform which were both significantly up-regulated in cortex, hippocampus and midbrain, suggesting that the increased maturation of the neuronal network is accompanied by an increase in expression of α3/ß2 complexes in these brain structures.

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector.

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²âº homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²âº concentration ([Ca²âº]i ) and Fura-2 fluorescence quenching by Mn²âº within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²âº entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²âº]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²âº homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²âº removal from the cytosol during the DCD latency period.

A LewisX glycoprotein screen identifies the low density lipoprotein receptor-related protein 1 (LRP1) as a modulator of oligodendrogenesis in mice.

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

Differential expression of micro-heterogeneous LewisX-type glycans in the stem cell compartment of the developing mouse spinal cord.

Complex glycan structures and their respective carrier molecules are often expressed in a cell type specific manner. Thus, glycans can be used for the enrichment of specific cell types such as neural precursor cells (NPCs). We have recently shown that the monoclonal antibodies 487(LeX) and 5750(LeX) differentially detect the LewisX (LeX) glycan on NPCs in the developing mouse forebrain. Here, we analysed the staining pattern of both antibodies during late embryonic mouse spinal cord development. At E13.5 both antibodies strongly label the central canal region. Along these lines they detect the LeX glycan primarily on Nestin-positive NPCs at that age. Moreover, we show that spinal cord NPCs cultured as free floating neurospheres display a high immunoreactivity to both antibodies. In that context, we also demonstrate that the 487(LeX) antibody can be used to deplete a subpopulation of neurosphere forming NPCs from a mixed E13.5 spinal cord cell suspension. Towards the end of embryogenesis the overall immunoreactivity to both antibodies increases and the staining appears very diffuse. However, the 5750(LeX) antibody still labels the central canal region. The increase in immunoreactivity correlates with an expression increase of the extracellular matrix molecules Tenascin C and Receptor Protein Tyrosine Phosphatase ß/ζ, two potential LeX carrier proteins. In line with this, immunoprecipitation analyses confirmed Tenascin C as a LeX carrier protein in the embryonic mouse spinal cord. However, the immunoreactivity to both antibodies appears only to be marginally affected in the absence of Tenascin C, arguing against Tenascin C being the major LeX carrier. In conclusion our study gives some novel insights into the complex expression of LeX glycans and potential carrier proteins during the development of the mouse spinal cord.

Derivation, characterization, and stable transfection of induced pluripotent stem cells from Fischer344 rats.

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.

Involvement of propeptides in formation of catalytically active metalloproteinase from Thermoactinomyces sp.

The metalloproteinase from Thermoactinomyces sp. 27a (Mpr) represents secretory thermolysin-like metalloproteinases of the M4 family. The Thermoactinomyces enzyme is synthesized as a precursor consisting of a signal peptide, N-terminal propeptide, mature region, and C-terminal propeptide. The functional molecule lacks the signal peptide, N-terminal propeptide, and C-terminal propeptide, which indicates the processing of these regions. Until now, it remained unclear if the N-terminal propeptide is involved in the formation and functioning of Mpr, and the role of the C-terminal propeptide was also unclear. In this work, a Bacillus subtilis AJ73 strain expressing Mpr without the C-terminal propeptide- encoding region being involved has been obtained. The absence of the Mpr C-terminal propeptide had no significant effect on the formation of the functional molecule and did not interfere with the protease secretion in B. subtilis AJ73 cells. Strains producing the N-terminal propeptide, mature region, and mature region covalently bound to the N-terminal propeptide were generated from Escherichia coli BL-21(DE3) cells. Functionally active Mpr forms could be produced only in the presence of the N-terminal propeptide, either covalently bound to the mature region (in cis) or as a separate molecule (in trans). Thus, the Mpr three-dimensional structure is formed according to the propeptide-assisted mechanism with no requirement of a covalent bond between the N-terminal propeptide and mature region. Moreover, Mpr variants generated in cis and in trans differed in their specificity for certain synthetic substrates.