Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry

Objective

Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.

Methods

The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.

Results

Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABA_A) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

Conclusions

Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

     

MicroRNA profiles in Sorghum exposed to individual drought or heat or their combination

Sorghum is largely grown for food, fodder and for biofuel production in semi-arid regions where the drought or high temperature or their combination co-occur. Plant microRNAs (miRNAs) are integral to the gene regulatory networks that control almost all biological processes including adaptation to stress conditions. Thus far, plant miRNA profiles under separate drought or heat stresses have been reported but not under combined drought and heat. In this study, we report miRNA profiles in leaves of sorghum exposed to individual drought or heat or their combination. Approximately 29 conserved miRNA families represented by 80 individual miRNAs, 26 families represented by 47 members of less conserved or sorghum-specific miRNA families as well as 8 novel miRNA families have been identified. Of these, 25 miRNAs were found to be differentially regulated in response to stress treatments. The comparative profiling revealed that the miRNA regulation was stronger under heat or combination of heat and drought compared to the drought alone. Furthermore, using degradome sequencing, 48 genes were confirmed as targets for the miRNAs in sorghum. Overall, this study provides a framework for understanding of the miRNA-guided gene regulations under combined stresses.

     

Short and Straightforward Synthesis of Triphosphates of Artificial Nucleobase Pairs Displaying Unconventional Pairing Scheme

Concise, facile and efficient synthesis of 5′-O-triphosphates of 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one (dP) is reported using a one-pot synthetic procedure.     

Synthesis of Zidovudine Derivatives with Anti-HIV-1 and Antibacterial Activities

Twelve novel zidovudine derivatives were prepared by modifying 5 ′-hydroxyl group of sugar moiety (1–8) and 5-methyl group of thymidine nucleus (9–12) and characterized spectrally. The compounds were evaluated for anti-HIV-1, antitubercular and antibacterial activities. Compound (3-azido-tetrahydro-5- (3,4-dihydro-5-methyl-2,4-dioxopyrimidin- 1 (2H)-yl) furan-2-yl)methyl 7- (4- (2-phenylacetoyloxy) -3,5- dimethylpiperazin-1-yl) -5- (2-phenylacetoyloxyamino) -1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (5) was found to be the most potent anti-HIV-1 agent with EC₅₀ of 0.0012 μM against HIV-1_IIIB and CC₅₀ of 34.05 μM against MT-4 with selectivity index of 28,375. Compound 5 inhibited Mycobacterium tuberculosis with MIC of 1.72 μM and inhibited four pathogenic bacteria with MIC of less than 1 μM.     

Design,Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K_i and IC₅₀ values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC₅₀ values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).     

Behavioural Lateralization in Budgerigars Varies with the Task and the Individual

Handedness/footedness and side biases are a well-known phenomenon in many animals, including humans. However, these so-called biases have mostly been studied at the population level - individual biases have received less attention, especially with regard to consistency over different tasks. Here we investigate behavioral lateralization in 12 male Budgerigars, Melopsittacus undulatus, a social parrot inhabiting the Australian bushlands. We performed 5 types of experiments to investigate lateralization, in tasks that involved climbing onto a perch, or landing on perches arranged in various configurations. The birds displayed highly significant, individually varying biases. The bias displayed by any particular individual varied with the task, in strength as well as polarity. Analysis of the data revealed that the preferred foot used for climbing did not coincide with the foot that was used while landing. Thus, landing choices are probably not determined by foot bias. Furthermore, these individual preferences were overridden completely when a bird had to perform a task simultaneously with another bird.     

The Histone Variant His2Av is Required for Adult Stem Cell Maintenance in the Drosophila Testis

Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. Here, we show that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function was dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.