Transcriptome changes and cAMP oscillations in an archaeal cell cycle

Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 uM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusions The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6% - 28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression

Simultaneous evaluation of multiple microarray surface chemistries through real-time interferometric imaging.

Surface chemistry is a crucial aspect for microarray modality biosensor development. The immobilization capability of the functionalized surface is indeed a limiting factor for the final yield of the binding reaction. In this work, we were able to simultaneously compare the functionality of protein ligands that were locally immobilized on different polymers, while on the same solid support, therefore demonstrating a new way of multiplexing. Our goal was to investigate, in a single experiment, both the immobilization efficiency of a group of reactive polymers and the resulting affinity of the tethered molecules. This idea was demonstrated by spotting many reactive polymers on a Si/SiO2 chip and depositing the molecular probes on the spots immediately after. As a proof of concept, we focused on which polymers would better immobilize a model protein (α-Lactalbumin) and a peptide (LAC-1). We successfully showed that this protocol is applicable to proteins and peptides with a good efficiency. By means of real-time binding measurements performed with the interferometric reflectance imaging sensor (IRIS), local functionalization proved to be comparable to the classical flat coating solution. The final outcome highlights the multiplexing power of this method: first, it allows to characterize dozens of polymers at once. Secondly, it removes the limitation, related to coated surfaces, that only molecules with the same functional groups can be tethered to the same solid support. By applying this protocol, many types of molecules can be studied simultaneously and immobilization for each probe can be individually optimized.766466 (INDEX) - Horizon 2020 Framework Programmehttps://s3-eu-west-1.amazonaws.com/itempdf74155353254prod/8976347/Simultaneous_Evaluation_of_Multiple_Microarray_Surface_Chemistries_Through_Real-Time_Interferometric_Imaging_v1.pdfFirst author draf

Multiplex STR amplification sensitivity in a silicon microchip

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (mu PCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 mu l each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the mu PCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both mu PCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Microchips and their significance in isolation of circulating tumor cells and monitoring of cancers

In micro-fluid systems, fluids are injected into extremely narrow polymer channels in small amounts such as micro-, nano-, or pico-liter scales. These channels themselves are embedded on tiny chips. Various specialized structures in the chips including pumps, valves, and channels allow the chips to accept different types of fluids to be entered the channel and along with flowing through the channels, exert their effects in the framework of different reactions. The chips are generally crystal, silicon, or elastomer in texture. These highly organized structures are equipped with discharging channels through which products as well as wastes of the reactions are secreted out. A particular advantage regarding the use of fluids in micro-scales over macro-scales lies in the fact that these fluids are much better processed in the chips when they applied as micro-scales. When the laboratory is miniaturized as a microchip and solutions are injected on a micro-scale, this combination makes a specialized construction referred to as "lab-on-chip". Taken together, micro-fluids are among the novel technologies which further than declining the costs; enhancing the test repeatability, sensitivity, accuracy, and speed; are emerged as widespread technology in laboratory diagnosis. They can be utilized for monitoring a wide spectrum of biological disorders including different types of cancers. When these microchips are used for cancer monitoring, circulatory tumor cells play a fundamental role

Separation of On-Column Labeled Model Proteins with Packed Capillary Electrophoresis

Protein drugs are increasingly developed in the pharmaceutical company. Under the regulation of FDA, high purity of therapeutic proteins needs to be maintained. Before putting those drugs in the market, fast and efficient method is in need to achieve homogeneity. Traditionally, polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), and size-exclusion chromatography (SEC) are used for the purification process. These methods have the disadvantages of low time and cost efficiency, and this quality assurance process has become the bottleneck of production. In our group, sub-micron silica colloidal particles with polyacrylamide layer on the surface are packed inside capillaries to increase the separation efficiency. In this particular project, NanoOrange dye is non-covalently associated with the protein sample to best reserve their native conformation. Proteins were separated at a distance as short as 8.2 mm in 85 seconds with extremely low plate height. The efficiency can be improved by decrease the silica particle size. This high throughput separation has a potential to be adopted in industries. Part II of this thesis describes the cost-effective DNA microarray project. Microarray is a technology evolved from southern blotting, and it is a common tool to measure the expression levels of the DNA samples. To quantify the DNA samples, fluorescently labeled target strands hybridize with the DNA probe which binds to a solid surface. DNA microarray is frequently utilized in clinical studies, and the efficiency is highly desired to be improved. When attach the probes on a smooth surface, the amount of DNA captured will be limited. In this research, the microarray sensitivity is increased by layering silica colloidal particles on the solid surface. Silica particles obtain face-centered cubic packing which increases the surface area to bind to the DNA probe, thus improve the microarray sensitivity. To reduce the cost of the microarray and to make it more point-of-care feasible, transparent plastic sheets is going to be researched to replace quartz silica plates. This new sensitive and cost effective microarray has a great possibility to be developed into point-of-care which detects a variety of diseases

Microreactor Array Device

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.The final version of this article, as published in SCIENTIFIC REPORTS, can be viewed online at: http://dx.doi.org/10.1038/srep0873

An optimized microarray platform for assaying genomic variation in Plasmodium falciparum field populations

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination

Effects of negative energy balance on liver gene and protein expression during the early postpartum period and its impacts on dairy cow fertility

End of project reportNegative energy balance (NEB) is a severe metabolic affecting high yielding dairy cows early post partum with both concurrent and latent negative effects on cow fertility as well as on milk production and cow health. The seasonal nature of Irish dairy production necessitates high cow fertility and a compact spring calving pattern in order to maximise grass utilisation. Poor dairy cow reproductive performance currently costs the Irish cattle industry in excess of €400 million annually. High milk yields have been associated with lower reproductive efficiency, and it has been suggested that this effect is probably mediated through its effects on the energy balance of the cow during lactation. The modern high genetic merit dairy cow prioritises nutrient supply towards milk production in early lactation and this demand takes precedence over the provision of optimal conditions for reproduction. In this study we used the bovine Affymetrix 23,000 gene microarray, which contains the most comprehensive set of bovine genes to be assembled and provides a means of investigating the modifying influences of energy balance on liver gene expression. Cows in severe negative energy balance (SNEB) in early lactation showed altered hepatic gene expression in metabolic processes as well as a down regulation of the insulin-like growth factor (IGF) system, where insulin like growth factor-1 (IGF-1), growth hormone receptor variant 1A (GHR1A) and insulin-like growth factor binding protein-acid labile subunit (IGFBP-ALS) were down regulated compared to the cows in the moderate negative energy balance MNEB group, consistent with a five-fold reduction in systemic concentrations of IGF1 in the SNEB group.Cows in SNEB showed elevated expression of key genes involved in the inflammatory response such as interleukin-8 (IL-8). There was a down regulation of genes involved in cellular growth in SNEB cows and moreover a negative regulator of cellular proliferation (HGFIN) was up regulated in SNEB cows, which is likely to compromise adaptation and recovery from NEB. The puma method of analysis revealed that 417 genes were differentially regulated by EB (P<0.05), of these genes 190 were up-regulated while 227 were down-regulated, with 405 genes having known biological functions. From Ingenuity Pathway Analysis (IPA), lipid catabolism was found to be the process most affected by differences in EB status