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A prerequisite for creating animal models in which gene expression is spatially and temporally controlled is the development of promoters to target genetic switches to specific populations of cells. Here we used the dopaminergic biosynthetic enzyme, tyrosine hydroxylase (TH) to test various combinations of tetracycline (Tet) system elements to determine the optimal configuration for inducible, tissue-specific expression. The present study shows that the degree of expression and level of leakiness associated with the Tet transactivators rtTA, rtTA2S-M2, tTS/rtTA or tTS/rtTA2S-M2 was dependent upon both the promoter and cell type utilized. Specifically, CMV-driven tTS/rtTA2S-M2 exhibited the highest level of inducibility in HEK cells (∼1000-fold) versus the dopaminergic cell line, MN9D (∼70-fold). In contrast, TH-driven rtTA2S-M2 yielded the highest level of expression with the least background in dopaminergic cell types versus HEK cells. Moreover, the TH promoter could be combined with the bi-directional Tet response system, BiTetO, allowing for the co-expression and regulation of two genes in the same cell. To further test the feasibility of this system we replaced the reporter gene with human Bcl-2. Consistent with previous studies, induction of Bcl-2 expression in dopaminergic cell types attenuated cell death due to the neurotoxin, MPP+. Taken together, these data suggest that targeted, inducible gene expression can be achieved in dopaminergic cell types. [Copyright &y& Elsevier]

h.-s. park and j.j. kilbane II. 2004. To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the β-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. Thermus thermophilus PPKU was constructed, in which the β- gal gene was deleted from the chromosome. Two inducible promoters P dnaK (regulating the DnaK heat shock-inducible protein) and P arg (regulating expression of an arginine-inducible protein) and a carbon-regulated promoter, P scs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) were cloned upstream of the β- gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of β-galactosidase activity produced by the P dnaK promoter in pTEX7 was substantially above the background level of 0·3 U mg−1, and increased from 5·2 to 10·4 U mg−1 after heat-shock induction indicating that significant amounts of DnaK are produced even when T. thermophilus is grown at its optimum temperature. The P arg promoter was found to be maximally induced by 10–30 m m arginine, but was inhibited by higher concentrations. The P scs-mdh promoter was maximally active in the presence of malate while lower levels of activity were observed in the presence of succinate, pyruvate, glutamate, glucose and the presence of yeast extract or peptone. These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermus and that β-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus. The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus. The demonstration that β-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for β- gal-positive transformant colonies on agar plates will add to the convenience of performing genetic manipulations in T. thermophilus. Future studies of transcriptional regulation in Thermus will benefit from the β- gal host–vector system reported here. [ABSTRACT FROM AUTHOR]

Apoptosis (programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/- mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays' an important protective role against sepsis-induced pulmonary apoptosis. [ABSTRACT FROM AUTHOR]

Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal. [ABSTRACT FROM AUTHOR]

The impact of hypoxia in utero during delivery was correlated with the immunocytochemistry, expression and activity of the neuronal (nNOS) and inducible (iNOS) isoforms of the nitric oxide synthase enzyme as well as with the reactivity and expression of nitrotyrosine as a marker of protein nitration during early postnatal development of the cortex. The expression of nNOS in both normal and hypoxic animals increased during the first few postnatal days, reaching a peak at day P5, but a higher expression was consistently found in hypoxic brain. This expression decreased progressively from P7 to P20, but was more prominent in the hypoxic group. Immunoreactivity for iNOS was also higher in the cortex of the hypoxic rats and was more evident between days P0 and P5, decreasing dramatically between P10 and P20 in both groups of rats. Two nitrated proteins of 52 and 38 kDa, were also identified. Nitration of the 52-kDa protein was more intense in the hypoxic animals than in the controls, increasing from P0 to P7 and then decreasing progressively to P20. The 38-kDa nitrated protein was seen only from P10 to P20, and its expression was more intense in control than in the hypoxic group. These results suggest that the NO system may be involved in neuronal maturation and cortical plasticity over postnatal development. Overproduction of NO in the brain of hypoxic animals may constitute an effort to re-establish normal blood flow and may also trigger a cascade of free-radical reactions, leading to modifications in the cortical plasticity. [Copyright &y& Elsevier]

ABSTRACT Aims: To investigate the role of germination processes in the expression of the β-glucosidase enzyme in Bacillus subtilis ATCC 9372 spores. Methods and Results: Enzyme activity was monitored in germinating and non-germinating spores of B. subtilis ATCC 9372. The expression of β-glucosidase by spores of B. subtilis was further investigated in the presence of a germination inhibitor, d-alanine, and a transcription inhibitor, novobiocin. Detection of enzyme activity required the presence of the germinant, l-alanine, as well as the inducer, 4-methylumbelliferryl-β-d-glucoside. Furthermore, β-glucosidase synthesis was abolished in the presence of d-alanine or novobiocin. Conclusions: The data obtained in this study indicated that β-glucosidase was not pre-existing, or merely attached to the spore, but was synthesized de novo during spore germination. Significance and Impact of the Study: The requirement of functional germination processes for the detection of β-glucosidase activity makes this enzyme a good candidate for detection of spore viability. [ABSTRACT FROM AUTHOR]

Abstract: Commensurate with the increased frequency of cost-of-defense studies, the conventional wisdom that inducibility is a cost saving strategy is enjoying growing empirical support. However, there may be other advantages to inducibility that have yet to be confirmed. To identify them, I consider the disadvantages of high levels of constitutive defense in the context of herbivores differing in response to the defense and in the context of potential predators and parasitoids of herbivores. Among the potential benefits of inducibility are that it allows herbivore-specific targeting, reliable signaling to predators/parasitoids, avoidance of kairomonal use by specialist herbivores, and avoidance of adverse effects of direct defense on third trophic level organisms. Inducibility also enables local and systemic variation in defense responses. These factors may figure prominently in certain systems, but I predict that their quantification will prove to be a more daunting challenge than the quantifying of costs. Moreover, there may be constraints on the evolution of inducibility that limit the fine-tuning of responses necessary to realize many of these hypothetical benefits. Nevertheless, the growing body of detailed knowledge of the physiology of multiple induction pathways will provide opportunities for testing some of these hypotheses in different systems. Einhergehend mit der steigenden Zahl der Studien zu Kosten der pflanzlichen Abwehr bekommt die allgemeine Annahme, dass Induzierbarkeit von Abwehr eine kostensparende Strategie ist, immer mehr empirische Unterstützung. Aber vielleicht gibt es noch andere Vorteile der Induzierbarkeit als die, die bisher schon bestätigt werden konnten. Um solche, bisher noch nicht detektierten Vorteile herauszufinden, werde ich hier die Nachteile von starker konstitutiver Abwehr analysieren und dabei die unterschiedlichen Reaktionen von Herbivoren auf die pflanzliche Abwehr ebenso berücksichtigen wie potentielle Prädatoren und Parasitoide der Herbivoren. Die möglichen Vorteile von Induzierbarkeit sind u.a. folgende: gezielte, spezifische Abwehr gegen bestimmte Herbivoren, verlässliche Signalgebung an Prädatoren und Parasitoide, Vermeidung der Kairomonausnutzung durch spezialisierte Herbivore und Vermeidung von nachteiligen Effekten der direkten Abwehr auf die Organismen in der dritten trophischen Ebene. Induzierbarkeit ermöglicht auch lokale und systemische Variation bei Abwehrreaktionen. Diese Faktoren mögen in bestimmten Systemen eine herausragende Rolle spielen und dennoch prognostiziere ich, dass ihre Quantifizierung eine entmutigendere Aufgabe darstellen wird als die Quantifizierung der Kosten. Darüber hinaus könnten “constraints” bei der Evolution der Induzierbarkeit limitierend wirken auf die präzise, jeweils angriffsspezifische Feinabstimmung der Abwehrreaktionen, die aber notwendig ist, um die hypothetischen Vorteile zu realisieren. Dennoch können die wachsenden Kenntnisse zur Physiologie der multiplen Induktionswege uns Wege eröffnen, um einige der Hypothesen zu den Kosten der Induktion pflanzlicher Abwehr in verschiedenen Systemen zu testen. [Copyright &y& Elsevier]

Insulin-like growth factor-I (IGF-I) has important growth-promoting and metabolic effects and is expressed in virtually every tissue of the body. The highest expression is found in the liver, but the physiological role of liver-derived IGF-I is unknown. It has been difficult to separate the endocrine effects of liver-derived IGF-I from the autocrine/paracrine effects of locally produced IGF-I in peripheral tissues. Therefore, we have developed a mouse model with a liver-specific inducible deletion of the IGF-I gene (LI-IGF-I-/- mouse). The LI-IGF-I-/- mouse has dramatically reduced (>80%) serum IGF-I levels, demonstrating that the major part of serum IGF-I is liver-derived. Surprisingly, LI-IGF-I-/-mice demonstrate a normal appendicular skeletal growth up to at least 12 mo of age despite the dramatic decrease in circulating IGF-I levels, indicating that liver-derived IGF-I is not required for appendicular skeletal growth. However, the adult axial skeletal growth is reduced in the LI-IGF-I-/- mice. Furthermore, the amount of cortical bone is reduced due to decreased radial growth of the cortical bone, while the trabecular bone mineral density is unchanged in the LI-IGF-I-/-mice. The decreased levels of circulating IGF-I are associated with increased serum levels of growth hormone (GH), indicating a role for liver-derived IGF-I in the negative-feedback regulation of GH secretion. Measurements of factors regulating GH secretion in the pituitary and in the hypothalamus revealed an increased expression of GH-releasing-hormone (GHRH) and GH-secretagogue (GHS) receptors in the pituitary of LI-IGF-I-/-mice. This in turn results in an increased sensitivity to systemically administered GHRH and GHS, demonstrating that the regulatory action of liver-derived IGF-I on GH secretion is at the pituitary rather than at the hypothalamic level. The liver is an important metabolic organ and LI-IGF-I-/- mice are markedly hyperinsulinemic and yet normoglycemic, consistent with an adequately compensated insulin resistance. Interestingly, LI-IGF-I-/- mice display a reduced age-dependent fat mass accumulation compared with control mice. Furthermore, LI-IGF-I-/- mice have increased blood pressure attributable to increased peripheral resistance indicating a role for liver-derived IGF-I in the regulation of blood pressure. In conclusion, liver-derived IGF-I is important for carbohydrate and lipid metabolism and for the regulation of GH secretion at the pituitary level. Furthermore, it regulates adult axial skeletal growth and cortical radial growth while it is not required for appendicular skeletal growth. [ABSTRACT FROM AUTHOR]

Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application. [Copyright &y& Elsevier]

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ERTM) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ERTM is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration. [Copyright &y& Elsevier]

In order to accurately analyze gene function in transgenic mice, as well as to generate credible murine models of human diseases, the ability to regulate temporal- and spatial-specific expression of target genes is absolutely critical. Pioneering work in inducible transgenics, begun in the 1980s and continuing to the present, has led to the development of a variety of different inducible systems dedicated to this goal, the shared basis of which is the accurate conditional expression of a given transgene. Recent advances in inducible transgene expression in mice are discussed. [Copyright &y& Elsevier]

Nitric oxide (NO) plays a role in the secondary damage after spinal cord (SC) injury. NO is produced by the activity of two classes of enzymes: calcium-dependent constitutive nitric oxide synthase (NOS) and calcium-independent inducible NOS. To determine the time course of both NOS activities after SC injury, 50 Wistar rats were submitted to severe SC contusion. NOS activities were assayed at the site of SC injury at several times after lesion. Results showed a significant increase of 138 and 96% in the constitutive NOS activity at 4 and 8 h after the lesion, respectively, as compared to sham-operated rats. iNOS activity was increased 72 h after lesion by 103% (P<0.05). In conclusion, both isoforms of NOS increase their activity at different time periods after SC injury. [Copyright &y& Elsevier]

We constructed and tested a Cre- loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) in the gut has attracted increasing interest as a regulatory factor for a wide variety of intestinal functions. This study was performed to evaluate some methodological aspects and jejunal sources for NO synthesis. Bench side evaluations and an animal model using chloralose-anaesthetized pigs were used. Immunohistochemistry was performed on samples from pig intestine and direct measurements of intestinal NO formation were performed using intraluminal tonometry. Tonometric measurements were quantitatively accurate and with high reproducibility. A substantial NO formation was assessed which was markedly inhibited by luminal administration of the non-selective NOS inhibitor L-NAME. Intravenous administration of L-NAME also reduced jejunal NO formation but to a lesser extent. Immunohistochemistry revealed staining for the inducible type of NOS in the mucosal surface epithelium whereas endothelial and neuronal subtypes were located in deeper layers of the jejunal wall. The study argues for that the source of jejunal NO production, as measured by intraluminal tonometry, is located in close proximity with the intestinal mucosa. The NOS in this compartment is predominantly of the inducible type. [ABSTRACT FROM AUTHOR]

A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising l-phosphonoalanine as carbon, nitrogen and phosphorus source. The activity was induced only in the presence of phosphonoalanine, did not require phosphate starvation for induction and was uniquely specific for phosphonopyruvate, producing equimolar quantities of pyruvate and inorganic phosphate. The native enzyme had a molecular mass of some 232 kDa and showed activation by metal ions in the order Co2+ > Ni2+ > Mg2+ > Zn2+ > Fe2+ > Cu2+. Temperature and pH optima in crude cell extracts were ¶50 °C and 7.5, respectively, and activity was inhibited by EDTA, phosphite, sulfite, mercaptoethanol and sodium azide. Phosphonopyruvate hydrolase is the third bacterial C-P bond cleavage enzyme reported to date that proceeds via a hydrolytic mechanism. [ABSTRACT FROM AUTHOR]

Background: Although chronic urticaria (CU) is a common, cause of medical consulting both in general practitioners and allergist specialists worldwide, there is little information about its behavior and management in Latin America. Currently, national and international guidelines recommend using Omalizumab for cases refractory to management with antihistamines. Despite advances in the knowledge of Omalizumab for the management of CU, although there are few studies in underdeveloped countries, there are many studies evaluating the impact of Omalizumab treatment. There is not clinical information related with CSU-Omalizumab in patient settled in the Caribbean area. This research aims to evaluate the management of CU with Omalizumab in a real-life scenario in Colombia., Methodology: We conducted an observational, descriptive, and retrospective study with patient recruitment between 2014 and 2017 of individuals diagnosed with Chronic Urticaria (CU) treating allergology specialists in five Colombian cities. We included patients with CU who failed to achieve disease control after treatment for 4 weeks with fourfold doses of second-generation H1-antihistamines, as recommended by the EAACI/GA 2 LEN/EDF/WAO guidelines and who received treatment with Omalizumab., Results: We included 123 patients, 73.1% ( n = 90) were women. The mean age was 47.1 years (Standard Deviation, SD: 16.2). The median of the total months of disease evolution was 30 (IQR = 13-58). 81.3 % ( n = 100) of patients were diagnosed with chronic spontaneous urticarial (CSU). 4.8% ( n = 6) had inducible CU (CIndU), and 13.8% ( n = 17) reported mixed urticaria (spontaneous CU with at least one inducible component). Regarding emotional factors, 34.9% ( n = 43) of subjects indicated anxiety symptoms, 34.1% ( n = 42) had exacerbations associated with stress, and 14.6% ( n = 18) manifested episodes of sadness. The percentage of patients with CSU controlled according to medical criteria at 3 months with Omalizumab were 80% ( n = 80/100) and at 6 months 87% ( n = 87/100). The frequency of adverse events was 29.2% ( n = 36), with headache being the most frequent adverse event., Conclusions: This real-life study with Omalizumab at CU describes percentages of effectiveness and safety similar to those observed in pivotal and real-life studies conducted in other regions around the world., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 García-Gómez, Chapman, García-Paba, Ocampo-Gómez, Egea-Bermejo, Garavito-De Egea, Fang, Sarrazola, Sánchez-Caraballo, Serrano-Reyes, Silva-Espinosa, Rojas-Mejía and Moreno.)

The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila , spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila ., Competing Interests: CM, AH, GA, KB, AE, AD, CY, AB, TS No competing interests declared, (© 2022, McClure et al.)

There is evidence that high penetrance hereditary genes cause a number of relatively uncommon tumors in the familial setting, whereas common cancers are influenced by multiple loci that alter susceptibility to cancer and other conditions. The latter category of genes are involved in the metabolism of carcinogens (activation, detoxification) as well as those that interact with dietary exposure. This paper will consider some of the basic principles in studying susceptibility genes and provide a few examples in which they interact with dietary components. [ABSTRACT FROM AUTHOR]

Intestinal toxicity exerted by indomethacin was compared to that induced by copper-indomethacinate, free or associated to zwitterionic phospholipids. A single high dose of indomethacin (15 or 20 mg/kg), copper-indomethacinate (15 or 20 mg/kg), or copper-indomethacinate liposomes or nanocapsules (15 mg/kg) was orally administered. Then 24 hr later jejunoileal tissue was taken for macroscopic observation, ex vivo nitrite production, and determination of myeloperoxydase and iNOS activities. Antiinflammatory activity of the drugs was investigated using the carrageenan-induced paw edema model. Indomethacin induced penetrating ulcerations of the intestine that were maximal at hour 24. Copper-indomethacinate induced significantly less ulceration than indomethacin with no significant difference in MPO and iNOS activities. The injurious action of indomethacin on the small intestine was further reduced when copper-indomethacinate was administered as the phospholipid-associated state while similar anti-inflammatory action was observed on rat paw edema. The antiulcerogen effect of copper-indomethacinate seems to be linked to its free radical scavenging effect without any modification of nitric oxide release. [ABSTRACT FROM AUTHOR]

Abstract Background The use of the Cre/loxP system for gene targeting has been proven to be a powerful tool for understanding gene function. The purpose of this study was to create and characterize an inducible, skeletal muscle-specific Cre transgenic mouse strain. Methods To achieve skeletal muscle-specific expression, the human α-skeletal actin promoter was used to drive expression of a chimeric Cre recombinase containing two mutated estrogen receptor ligand-binding domains. Results Western blot analysis, PCR and β-galactosidase staining confirmed that Cre-mediated recombination was restricted to limb and craniofacial skeletal muscles only after tamoxifen administration. Conclusions A transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult skeletal muscle of different developmental origins. This new mouse will be of great utility to the skeletal muscle community.

Rats treated with daily injection of DL-isoproterenol for 10 consecutive days (25 mg kg1 body weight) showed marked induction of a proline-rich glycoprotein (GPRP) of 220 kDa. Proteinase K digestion of GPRP produced a homogeneous glycopeptide with an average chemical composition as follows (residues per mol): Pro4, Glx3, Asx2, Gly1, His1, Thr1, Arg1, GlcNAc5, GalNac1, Man3, Gal2–3, and Fuc1. The structural analysis of the asparagine-linked carbohydrate unit was performed by methylation, periodate oxidation and enzymatic degradation. Methylation studies indicated that the three mannosyl residues were substituted at 1,2-, 1,2,4-, and 1,3,6-positions. Fucose, N-acetylgalactosamine, 1.5 residues of galactose and 0.35 residues of N-acetylglucosamine were terminally located and one galactose residue was 1,4-substituted. Approximately four of the 5 N-acetylglucosamine residues were substituted at 1,4-position and approximately 1 residue of N-acetylglucosamine was substituted at 1,4,6-positions. Periodate oxidation studies and exoglycosidase results were consistent with the methylation data. Based on the results of Smith degradation, methylation and sequential exoglycosidase digestions a triantennary oligosaccharide structure having terminal N-acetylgalactosamine in one of the branches is proposed for the major Asn-linked carbohydrate moiety of GPRP. [ABSTRACT FROM AUTHOR]

The microalga Chlamydomonas reinhardtii offers a rapid, scalable and low-cost platform for recombinant protein production. Its chloroplast provides a particularly robust expression system for high yield production of complex proteins requiring challenging post-transcriptional modifications. Controlled transgene expression provides an important advantage supporting many applications. Currently, however, only three inducible systems exist for algal chloroplast expression regulation. Here, we present the nuclear encoded translation enhancer TDA1 that regulates transgene expression in the chloroplast, as a component of an inducible system that is designed for high-yield production. Specifically, the C-terminus of TDA1 (cTDA1) promotes translation of the atpA transcript by interacting with its 5′-UTR, which has been shown to support high levels of recombinant protein expression. cTDA1 under the control of the inducible promoter HSP70A-RBCS2 was introduced into a chloroplast mutant expressing GFP under the control of the atpA 5′-UTR. These cTDA1/GFP mutants were grown under optimised illumination regimes and subjected to specific heat-shock treatments, after which cTDA1 and GFP expression levels were measured. A ~1.9-fold increase was detected in induced samples grown under optimised production conditions (4 days cultivation period under 6 h·d−1 illumination at 200 μE·m−2·s−1 followed by 2 h light exposure (200 μE·m−2·s−1) after the end of the 4 day period; 30 min heat-shock at 40 °C with subsequent 5 h incubation at RT (200 μE·m−2·s−1 illumination)). Western Blot analysis confirmed that after induction, the cTDA1 and GFP accumulation correlated. This demonstrates proof-of-concept that expression of the nuclear cTDA1 regulates atpA 5′-UTR mediated chloroplast transgene expression and thus provides the basis for a next generation nuclear inducible chloroplast regulation systems. • Presentation of cTDA1 as regulator for inducible chloroplasts transgene expression. • Successful over-expression of cTDA1 through the HSP70A-RBCS2 inducible promoter. • cTDA1 upregulation increased atpA 5′-UTR mediated plastid transgene expression. • Induced mutants yielded ~1.9-fold increase in GFP levels compared to control. • Proof-of-concept for an inducible system geared for high-yield expression. [ABSTRACT FROM AUTHOR]

The hexanucleotide G 4 C 2 repeat expansion in the first intron of the C9ORF72 gene explains the majority of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) cases. Numerous studies have indicated the toxicity of dipeptide repeats (DPRs) which are produced via repeat-associated non-AUG (RAN) translation from the repeat expansion and accumulate in the brain of C9FTD/ALS patients. Mouse models expressing the human C9ORF72 repeat and/or DPRs show variable pathological, functional, and behavioral characteristics of FTD and ALS. Here, we report a new Tet-on inducible mouse model that expresses 36x pure G 4 C 2 repeats with 100bp upstream and downstream human flanking regions. Brain specific expression causes the formation of sporadic sense DPRs aggregates upon 6 months dox induction but no apparent neurodegeneration. Expression in the rest of the body evokes abundant sense DPRs in multiple organs, leading to weight loss, neuromuscular junction disruption, myopathy, and a locomotor phenotype within the time frame of four weeks. We did not observe any RNA foci or pTDP-43 pathology. Accumulation of DPRs and the myopathy phenotype could be prevented when 36x G 4 C 2 repeat expression was stopped after 1 week. After 2 weeks of expression, the phenotype could not be reversed, even though DPR levels were reduced. In conclusion, expression of 36x pure G 4 C 2 repeats including 100bp human flanking regions is sufficient for RAN translation of sense DPRs and evokes a functional locomotor phenotype. Our inducible mouse model suggests early diagnosis and treatment are important for C9FTD/ALS patients., (© 2021. Published by The Company of Biologists Ltd.)

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way to condition the activation of most CRISPR-TRs on the presence of a user defined inducer. In this chapter, I cover the know-how relating to the design and synthesis of iSBH-sgRNAs, as well as the implementation in mammalian cells of inducible CRISPR-TR strategies based on this technology.

Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a "hot" expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus., Results: A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600 bp DNA region containing a putative silica-inducible promoter. β-galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to 73 kDa of β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Truncation of the putative silica-inducible promoter region in Thermus expression vector improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100 bp DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment., Conclusion: We successfully expressed the thermostable β-galactosidase and PisGDH in T. thermophilus as active and soluble forms and achieved with our system the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.

Iron-catalyzed, free radical-mediated lipid peroxidation may play a major role in tumor cell killing by photodynamic therapy (PDT), particularly when membrane-localizing photosensitizers are employed. Many cancer cells exploit endogenous iNOS-generated NO for pro-survival/expansion purposes and for hyper-resistance to therapeutic modalities, including PDT. In addition to inhibiting the pro-oxidant activity of Fe(II) via nitrosylation, NO may intercept downstream lipid oxyl and peroxyl radicals, thereby acting as a chain-breaking antioxidant. We investigated this for the first time in the context of PDT by using POPC/Ch/PpIX (100:80:0.2 by mol) liposomes (LUVs) as a model system. Cholesterol (Ch or [ 14 C]Ch) served as an in-situ peroxidation probe and protoporphyrin IX (PpIX) as photosensitizer. PpIX-sensitized lipid peroxidation was monitored by two analytical methods that we developed: HPLC-EC(Hg) and HPTLC-PI. 5α-hydroperoxy-Ch (5α-OOH) accumulated rapidly and linearly with irradiation time, indicating singlet oxygen ( 1 O 2 ) intermediacy. When ascorbate (AH - ) and trace lipophilic iron [Fe(HQ) 3 ] were included, 7α/7β-hydroperoxy-Ch (7-OOH) accumulated exponentially, indicating progressively greater membrane-damaging chain lipid peroxidation. With AH - /Fe(HQ) 3 present, the NO donor SPNO had no effect on 5α-OOH formation, but dose-dependently inhibited 7-OOH formation due to NO interception of chain-carrying oxyl and peroxyl radicals. Similar results were obtained when cancer cells were PpIX/light-treated, using SPNO or activated macrophages as the NO source. These findings implicate chain lipid peroxidation in PDT-induced cytotoxicity and NO as a potent antagonist thereof by acting as a chain-breaking antioxidant. Thus, unless NO formation in aggressive tumors is suppressed, it can clearly compromise PDT efficacy.

Lactobacillus species are attractive hosts for the expression of heterologous proteins, antigens, vaccines, and drugs due to their GRAS (generally recognized as safe) status. The bioengineering techniques open new possibilities of improving Lactobacillus strains. In this regard, the control of the gene expression in Lactobacillus strains through the adequate native or engineered promoters acquires a key role in the development of biotechnological applications and for their function as probiotic bacteria. Depending on the objective sought, the protein produced and the strain used, inducible or constitutive promoters can be chosen. Whereas, when a fine-tuning of gene expression is required, the development of synthetic promoter libraries could be the best approach. In this work, we revise the main constitutive and inducible natural promoters from Lactobacillus strains or from other genus that have been applied in Lactobacillus, as well as the few engineered promoters developed for these bacteria.

Neurological diseases comprise more than a thousand ailments that adversely affect the brain and nervous system. When grouped together, these neurological conditions impact an estimated 100 million individuals in the United States and up to a billion people worldwide, making drug discovery efforts imperative. However, recent research and development efforts for these neurological diseases, including Alzheimer's disease and amyotrophic lateral sclerosis, have been exceedingly disappointing and typify the challenges associated with translating in vitro and cell-based discoveries to successful preclinical models and subsequent human clinical trials. Our viewpoint is that neuronal progenitor cells and neurons derived from inducible pluripotent stem cells afford an innovative translational bridge, with higher pathological relevancy than previous cellular models. We outline some of the opportunities and challenges associated with their evolving usage in drug discovery and development.

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)-inducible Krt19 CreERT allele is available. The current study describes the characterization of Krt19 CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle-aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19 CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam-treated Krt19 CreERT/+ ; R26 tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19 CreERT allele for genetic characterization of NP cells at different stages of the mouse life., (© 2019 Wiley Periodicals, Inc.)