Green Fluorescent Protein Reporter Research Articles

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering.

Nicotinate dehydrogenase (NDHase) is a membrane protein with three subunits (ndhS, ndhL, and ndhM), which is difficult to express in a functional form using common hosts such as Escherichia coli, Bacillus subtilis, or Pichia pastoris. Comamonas testosteroni is a suitable microbial chassis for expressing multi-subunit membrane proteins. However, the expression of NDHase in C. testosteroni is extremely low. We have developed a systematic approach to create an efficient protein expression system in C. testosteroni CNB-2 using multi-level N-terminal engineering. We selected a strong promoter for the Mmp1 system that enables control of transcriptional strength in unconventional bacteria. This enhanced the expression of a green fluorescent reporter protein threefold. Following modification of the N-terminal Shine-Dalgarno sequence and rearrangement of amino acid sequence in the starting area of the gene encoding NDHase, enzyme activity increased from 90.6 to 165 U/L. These optimized N-terminal Shine-Dalgarno and amino acid sequences were used to enhance the expression of ndhL subunit and improve the balance expression of three subunits of NDHase, resulting in enzyme activity of 192 U/L that far surpasses the previously reported level. These results highlight a promising strategy for the development of other heterologous expression systems for challenging proteins using unconventional bacteria.

Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry.

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

β3-Adrenergic receptor agonist treats rotator cuff fatty infiltration by activating beige fat in mice

Rotator cuff (RC) muscle atrophy and fatty infiltration (FI) are independent factors correlated with failure of attempted tendon repair in larger RC tears. However, there is no effective treatment for RC muscle atrophy and FI at this time. The recent discovery of beige adipose tissue (BAT) in adults shed light on a new avenue in treating obesity and excessive fat deposition by promoting BAT activity. The goal of this study was to define the role of intramuscular BAT in RC muscle FI and the effect of β3-adrenergic receptor agonists in treating RC muscle FI by promoting BAT activity. Three-month-old wild-type C57BL/6J, platelet derived growth factor receptor-alpha (PDGFRα) green fluorescent protein (GFP) reporter and uncoupling protein 1 (UCP-1) knockout mice underwent a unilateral RC injury procedure, which included supraspinatus (SS) and infraspinatus tendon resection and suprascapular nerve transection. To stimulate BAT activity, amibegron, a selective β3-adrenergic receptor agonist, was administered to C57BL/6J mice either on the same day as surgery or 6 weeks after surgery through daily intraperitoneal injections. Gait analysis was conducted to measure forelimb function at 6 weeks or 12 weeks (in groups receiving delayed amibegron treatment) after surgery. Animals were killed humanely at 6 weeks (or 12 weeks for delayed amibegron groups) after surgery. SS muscles were harvested and analyzed histologically and biochemically. Histologicanalysis of SS muscles from PDGFRα-GFP reporter mice showed that PDGFRα-positive fibroadipogenic progenitors in RC muscle expressed UCP-1, a hallmark of BAT during the development of FI after RC tears. Impairing BAT activity by knocking out UCP-1 resulted in more severe muscle atrophy and FI with inferior forelimb function in UCP-1 knockout mice compared with wild-type mice. Promoting BAT activity with amibegron significantly reduced muscle atrophy and FI after RC tears and improved forelimb function. Delayed treatment with amibegron reversed muscle atrophy and FI in muscle. Fat accumulated in muscle after RC tears possesses BAT characteristics. Impairing BAT activity results in worse RC muscle atrophy and FI. Amibegron reduces and reverses RC atrophy and FI by promoting BAT activity.

A Synthetic Reporter for Probing Mistranslation in Living Cells.

Aminoacyl-tRNA synthetases (AARSs) play key roles in maintaining high fidelity of protein synthesis. They charge cognate tRNAs with corresponding amino acids and hydrolyze mischarged tRNAs by editing mechanisms. Impairment of AARS editing activities can reduce the accuracy of tRNA aminoacylation to produce mischarged tRNAs, which cause mistranslation and cell damages. To evaluate the mistranslation rate of threonine codons in living cells, in this study, we designed a quantitative reporter derived from the green fluorescent protein (GFP). The original GFP has multiple threonine codons which could affect the accuracy of measurement, so we generated a GFP variant containing only one threonine residue to specifically quantify mistranslation at the threonine codon. To validate, we applied this single-threonine GFP reporter to evaluate mistranslation at the threonine codon with mutations or modifications of threonine-tRNA synthetase and compared it with other methods of mistranslation evaluation, which showed that this reporter is reliable and facile to use.

Downregulating the P2X3 receptor in the carotid body to reduce blood pressure via acoustic gene delivery in canines

The purinergic P2X3 receptor in the carotid body (CB) is considered a new target for treating hypertension, although approaches for targeted regulating P2X3 receptor expression are lacking. Here, we explored the feasibility of targeted P2X3 receptor down-regulation in CBs by localized low-intensity focused ultrasound (LIFU)-mediated gene delivery to reduce the blood pressure. Thirty-two Kunming canines were randomly assigned to the treatment group (n = 14), negative control group (n = 10), LIFU + cationic microbubbles group (n = 4), and LIFU-only group (n = 4). Plasmid-loaded cationic microbubbles were injected and bilateral CBs were irradiated with a LIFU-based transducer. Flow cytometry showed that 33.15% of transfected cells expressed the green fluorescent protein reporter gene. T7 endonuclease I assays showed an insertion-deletion rate of 8.30%. The P2X3 receptor mRNA- and protein-expression levels in CBs decreased by 56.31% and 45.10%, respectively, in the treatment group. Mean systolic (152.5 ± 3.0 vs 138.0 ± 2.9 mm Hg, P = 0.003) and diastolic (97.8 ± 1.5 vs 87.2 ± 2.3 mm Hg, P= 0.002) blood pressures reduced on day 14 in the treatment group, compared with the baseline values, whereas no effects were observed with LIFU treatment or cationic microbubbles injection alone. Canines treated with this strategy exhibited no local or systemic adverse events. Thus, LIFU-mediated gene delivery to CBs successfully modulated CB function and reduced blood pressure in a canine model, suggesting a new possibility for treating hypertension and further clinical translation.

Development and optimization of N-acetylneuraminic acid biosensors in Bacillus subtilis.

Transcriptional factor (TF)-based metabolite-responsive biosensors are important tools for screening engineered enzymes with desired properties and for the dynamic regulation of biosynthetic pathways. However, TF-based biosensor construction is often constrained by undesired effects of TF-binding site sequence insertion on gene expression and unpredictable optimal TF expression levels. In the present study, a stepwise TF-based biosensor construction approach was developed using an N-acetylneuraminic acid (NeuAc) biosensor for Bacillus subtilis, as a case study. Specifically, 12 promoters with various strengths were selected as the first promoter library. Next, binding site sequences for the NanR were inserted into various positions of the selected promoter sequences to develop the second promoter library, resulting in 6 engineered promoters containing TF-binding site sequences (NanO), without major effects on promoter strength. NanR expression cassettes with different expression levels were further integrated to construct the biosensor library, yielding 9 NeuAc biosensors with efficient repression in the absence of NeuAc. Finally, biosensor activation was characterized by testing fold changes in expression levels of the green fluorescent protein reporter in the presence of NeuAc in vivo, which revealed 61-fold activation when NeuAc was present. The NeuAc biosensor developed in this study can be used for screening engineered enzymes for enhanced NeuAc biosynthesis in B. subtilis.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

The Efficiency of Gene Electrotransfer in Breast-Cancer Cell Lines Cultured on a Novel Collagen-Free 3D Scaffold.

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p < 0.01) and 24% versus 17% (p < 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p < 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation

Мета. Дослідити експресію генів gus та gfp в калюсних експлантах амфідиплоїдної пшениці спельти ( Triticum spelta L.) після Agrobacterium -опосередкованої генетичної трансформації. Методи. Для трансформації було обрано сорт пшениці спельти озимої ‘Європа’. Як експланти використовували калюси, отримані зі зрілих зародків. Прекультивацію калюсів здійснювали на живильному середовищі МС (Мурасіге–Скуга), доповненому 2 мг/л 2,4-Д (2,4-дихлорфеноксиоцтова кислота) та 10 мг/л нітратом срібла. Для генетичної трансформації використовували Agrobacterium tumefaciens Conn., штам GV3101, та генетичну конструкцію з репортерними генами gus (ген бета-глюкуронідази (-glucuronidase)) та gfp (ген зеленого флюоресцентного білка (Green Fluorescent Protein, GFP)). Калюси трансформували шляхом інокуляції з агробактеріями та вакуумною інфільтрацією. Далі їх ко-культивували на середовищі МС із 2 мг/л 2,4-Д та 10 мг/л AgNO 3 , але без антибіотиків. Експресію гена gus перевіряли за допомогою гістохімічного, а гена gfp – візуального аналізу (флуоресценція білка GFP в УФ світлі). Рівні експресії генів gfp та gus оцінювали за допомогою програмного забезпечення ImajeJ. Інтеграцію генів gfp та gus в геном спельти перевіряли методом ПЛР . Результати. Генетична трансформація калюсних експлан­тів спельти шляхом їхньої інокуляції в живильному середовищі з агробактеріями та вакуумною інфільтрацією відбувалась з різною частотою. Рівень експресії гена gus за вакуумної інфільтрації становив 4,66 ± 0,74%, за інокуляції – 4,00 ± 0,91%; а гена gfp за вакуумної інфільтрації – 3,66 ± 0,74%, за інокуляції – 4,66 ± 1,39%. Рівень експресії гена gfp був вищим у разі використання інокуляції з агробактеріями, а гена gus – при ва­ку­умній інфільтрації. За допомогою ПЛР-аналізу було під­тверджено інтеграцію генів gfp та gus в геном калюсів спельти. Довжина ПЛР продукту із праймерами до гена gus становила 240 п. н., а до гена gfp – 717 п. н. Висновки. Використання методів вакуумної інфільтрації та інокуляції для генетичної трансформації спельти дали різні результати. Частота генетичної трансформації коливалась від 3,66 до 4,66%. Agrobacterium -опосередкована генетична трансформація амфідиплоїдної пшениці спельти дозволяє дослідити експресію репортерних генів gus та gfp за використання калюсних експлантів, отриманих із зрілих зародків.

Regulatory circuits controlling Spx levels in Streptococcus mutans.

Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2tail fusion protein. Addition of acidic amino acids to the GFP::SpxA2tail chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1tail . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.

The Tapetal Major Facilitator NPF2.8 Is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic β-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.

Exploring resveratrol dimers as virulence blocking agents \u2013 Attenuation of type III secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa

Bacterial infections continue to threaten humankind and the rapid spread of antibiotic resistant bacteria is alarming. Current antibiotics target essential bacterial processes and thereby apply a strong selective pressure on pathogenic and non-pathogenic bacteria alike. One alternative strategy is to block bacterial virulence systems that are essential for the ability to cause disease but not for general bacterial viability. We have previously show that the plant natural product (-)-hopeaphenol blocks the type III secretion system (T3SS) in the Gram-negative pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa. (-)-Hopeaphenol is a resveratrol tetramer and in the present study we explore various resveratrol dimers, including partial structures of (-)-hopeaphenol, as T3SS inhibitors. To allow rapid and efficient assessment of T3SS inhibition in P. aeruginosa, we developed a new screening method by using a green fluorescent protein reporter under the control of the ExoS promoter. Using a panel of assays we showed that compounds with a benzofuran core structure i.e. viniferifuran, dehydroampelopsin B, anigopreissin A, dehydro-δ-viniferin and resveratrol-piceatannol hybrid displayed significant to moderate activities towards the T3SS in Y. pseudotuberculosis and P. aeruginosa.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Is the erythropoietin receptor the key to the identification of the central macrophage in erythroblastic islands?

Is the erythropoietin receptor the key to the identification of the central macrophage in erythroblastic islands?

Establishment and characterization of a new cell line derived from half-smooth tongue sole Cynoglossus semilaevis kidney

In this study, we established a new cell line (Cynoglossus semilaevis kidney cells, CSK) from kidney of the half-smooth tongue sole (Cynoglossus semilaevis). The cells were subcultured over 1 000 days and passaged for more than 100 times. Additionally, CSK cells were optimally maintained in Dulbecco’s modified Eagle medium nutrient mixture F-12 supplemented with HEPES, antibiotics, fetal bovine serum, 2-mercaptoethanol, and basic fibroblast growth factor. The optimum growth temperature for CSK cells was 25°C, and the cells showed a fibroblast-like phenotype. Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2n=42. CSK cells were susceptible to Grouper nervous necrosis virus (NNV), and cytopathic effects were observed at 3–5 days postinfection. The NNV sensitivity of CSK cells was related to the high abundance of virions in the cytoplasm, as observed by electron microscopy. Additionally, CSK cells could be successfully transfected with a green fluorescent protein reporter plasmid, and fluorescent signals were easily observed. Finally, immunocytochemistry analysis showed that CSK cells were supporting cells. Overall, we established this new cell line, which may have potential applications in the identification of viral pathogens affecting the half-smooth tongue sole.

Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans.

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult "hidden" in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

Cholesterol grafted cationic lipopolymers: Potential siRNA carriers for selective chronic myeloid leukemia therapy.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

Exploring Target Genes Involved in the Effect of Quercetin on the Response to Oxidative Stress in Caenorhabditis elegans.

Quercetin is one the most abundant flavonoids in the human diet. Although it is well known that quercetin exhibits a range of biological activities, the mechanisms behind these activities remain unresolved. The aim of this work is to progress in the knowledge of the molecular mechanisms involved in the biological effects of quercetin using Caenorhabditis elegans as a model organism. With this aim, the nematode has been used to explore the ability of this flavonoid to modulate the insulin/insulin-like growth factor 1(IGF-1) signaling pathway (IIS) and the expression of some genes related to stress response. Different methodological approaches have been used, i.e., assays in knockout mutant worms, gene expression assessment by RT-qPCR, and C. elegans transgenic strains expressing green fluorescent protein (GFP) reporters. The results showed that the improvement of the oxidative stress resistance of C. elegans induced by quercetin could be explained, at least in part, by the modulation of the insulin signaling pathway, involving genes age-1, akt-1, akt-2, daf-18, sgk-1, daf-2, and skn-1. However, this effect could be independent of the transcription factors DAF-16 and HSF-1 that regulate this pathway. Moreover, quercetin was also able to increase expression of hsp-16.2 in aged worms. This observation could be of particular interest to explain the effects of enhanced lifespan and greater resistance to stress induced by quercetin in C. elegans, since the expression of many heat shock proteins diminishes in aging worms.