A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.RNA-interferenssin (RNAi) käyttö geenituotteiden toimintojen tutkimuksessa on vuosikymmenessä kehittynyt solubiologisen tutkimuksen merkittävimpien teknologioiden joukkoon. RNAi-menetelmät ovat mahdollistaneet myös eri solubiologisten signaalivälitysreittien kartoittamisen koko perimänlaajuisten geenitoimintojen suurtehoseulonnan avulla. Teknologian nopeasta kehityksestä ja laajamittaisesta hyödyntämisestä huolimatta, RNAi-menetelmien käyttö suurtehoseulontaan rajoittuu edelleen erikoislaboratorioihin huomattavien laite-, reagenssi- sekä tietotaitovaatimusten johdosta. Lisäksi yleisimmin käytetyt kuoppalevypohjaiset seulonta-menetelmät rajoittavat seulontatutkimusten laajuutta, eri analyysimenetelmien käyttöä sekä useiden mallisolulinjojen rinnakkaista seulontaa suuren reagenssikulutuksen takia. RNAi-suurtehoseulonnan kustannuksien ja laiteriippuvuuden alentamiseksi sekä monipuolisuuden lisäämiseksi tulevaisuudessa tarvitaan uusia vaihtoehtoisia seulontamenetelmiä. Tämän väitöskirjatyön lähtökohtana oli uuden kuoppalevyillä tehtäviä seulontamenetelmiä edullisemman ja monipuolisemman RNAi-suurtehoseulontamenetelmän kehittäminen. Tutkimuksessa kehitettiin solujen siRNA käänteistransfektioon perustuva solumikrosirumenetelmä, joka tarkasti rajattujen siRNA näytepisteiden avulla mahdollistaa jopa koko ihmisen perimänlaajuisten siRNA näytekirjastojen seulonnan yksittäisellä mittasirulla. Menetelmän suuri näytetiheys sekä sirujen pieni pinta-ala mahdollistavat myös erikoismittamenetelmien käytön suurtehoseulonnassa, jotka teknisistä tai kustannussyistä eivät sovellu käytettäväksi suurtehoseulontaan kuoppalevyillä. Väitöskirjan ensimmäinen osatyö kuvaa menetelmän kehittämistyön. Väitöskirjan toisessa, kolmannessa ja neljännessä osatyössä menetelmää on hyödynnetty syöpäbiologisissa seulontatutkimuksissa selvitettäessä geenejä jotka vaikuttavat eturauhassyöpäsolujen kasvuun, rintasyöpäsolujen solunjakautumiseen sekä integriini tarttumisreseptorien aktiivisuuden säätelyyn.Siirretty Doriast

The quantum phase slip phenomenon in superconducting nanowires with high-impedance environment

Quantum phase slip (QPS) is the particular manifestation of quantum fluctuations of the order parameter of a current-biased quasi-1D superconductor. The QPS event(s) can be considered a dynamic equivalent of tunneling through conventional Josephson junction containing static in space and time weak link(s). At low temperatures T<<Tc the QPS effect leads to finite resistivity of narrow superconducting channels and suppresses persistent currents in tiny nanorings. Here we demonstrate that the quantum tunneling of phase may result in Coulomb blockade: superconducting nanowire, imbedded in high-Ohmic environment, below a certain bias voltage behaves as an insulator.Comment: 3 pages, 3 figure

The gene expression landscape of breast cancer is shaped by tumor protein p53 status and epithelial-mesenchymal transition

Introduction: Gene expression data derived from clinical cancer specimens provide an opportunity to characterize cancer-specific transcriptional programs. Here, we present an analysis delineating a correlation-based gene expression landscape of breast cancer that identifies modules with strong associations to breast cancer-specific and general tumor biology. Methods: Modules of highly connected genes were extracted from a gene co-expression network that was constructed based on Pearson correlation, and module activities were then calculated using a pathway activity score. Functional annotations of modules were experimentally validated with an siRNA cell spot microarray system using the KPL-4 breast cancer cell line, and by using gene expression data from functional studies. Modules were derived using gene expression data representing 1,608 breast cancer samples and validated in data sets representing 971 independent breast cancer samples as well as 1,231 samples from other cancer forms. Results: The initial co-expression network analysis resulted in the characterization of eight tightly regulated gene modules. Cell cycle genes were divided into two transcriptional programs, and experimental validation using an siRNA screen showed different functional roles for these programs during proliferation. The division of the two programs was found to act as a marker for tumor protein p53 (TP53) gene status in luminal breast cancer, with the two programs being separated only in luminal tumors with functional p53 (encoded by TP53). Moreover, a module containing fibroblast and stroma-related genes was highly expressed in fibroblasts, but was also up-regulated by overexpression of epithelial-mesenchymal transition factors such as transforming growth factor beta 1 (TGF-beta1) and Snail in immortalized human mammary epithelial cells. Strikingly, the stroma transcriptional program related to less malignant tumors for luminal disease and aggressive lymph node positive disease among basal-like tumors. Conclusions: We have derived a robust gene expression landscape of breast cancer that reflects known subtypes as well as heterogeneity within these subtypes. By applying the modules to TP53-mutated samples we shed light on the biological consequences of non-functional p53 in otherwise low-proliferating luminal breast cancer. Furthermore, as in the case of the stroma module, we show that the biological and clinical interpretation of a set of co-regulated genes is subtype-dependent

Turbulence Properties of Interplanetary Coronal Mass Ejections in the Inner Heliosphere: Dependence on Proton Beta and Flux Rope Structure

Interplanetary coronal mass ejections (ICMEs) have low proton beta across a broad range of heliocentric distances and a magnetic flux rope structure at large scales, making them a unique environment for studying solar wind fluctuations. Power spectra of magnetic field fluctuations in 28 ICMEs observed between 0.25 and 0.95 au by Solar Orbiter and Parker Solar Probe have been examined. At large scales, the spectra were dominated by power contained in the flux ropes. Subtraction of the background flux rope fields reduced the mean spectral index from $-5/3$ to $-3/2$ at $kd_i \leq 10^{-3}$. Rope subtraction also revealed shorter correlation lengths in the magnetic field. The spectral index was typically near $-5/3$ in the inertial range at all radial distances regardless of rope subtraction, and steepened to values consistently below $-3$ with transition to kinetic scales. The high-frequency break point terminating the inertial range evolved approximately linearly with radial distance and was closer in scale to the proton inertial length than the proton gyroscale, as expected for plasma at low proton beta. Magnetic compressibility at inertial scales did not show any significant correlation with radial distance, in contrast to the solar wind generally. In ICMEs, the distinctive spectral properties at injection scales appear mostly determined by the global flux rope structure while transition-kinetic properties are more influenced by the low proton beta; the intervening inertial range appears independent of both ICME features, indicative of a system-independent scaling of the turbulence.Comment: 12 pages, 5 figures; accepted for publication in the Astrophysical Journal Letters 2023 September 2

Biogeochemical cycling and ecological thresholds in a High Arctic lake (Svalbard)

Lakes are a dominant feature of the Arctic landscape and a focal point of regional and global biogeochemical cycling. We collected a sediment core from a High Arctic Lake in southwestern Svalbard for multiproxy paleolimnological analysis. The aim was to find linkages between the terrestrial and aquatic environments in the context of climate change to understand centennial-long Arctic biogeochemical cycling and environmental dynamics. Two significant thresholds in elemental cycling were found based on sediment physical and biogeochemical proxies that were associated with the end of the cold Little Ice Age and the recent warming. We found major shifts in diatom, chironomid and cladoceran communities and their functionality that coincided with increased summer temperatures since the 1950s. We also discovered paleoecological evidence that point toward expanded bird (Little Auk) colonies in the catchment alongside climate warming. Apparently, climate-driven increase in glacier melt water delivery as well as a prolonged snow- and ice-free period have increased the transport of mineral matter from the catchment, causing significant water turbidity and disappearance of several planktonic diatoms and clear-water chironomids. We also found sedimentary accumulation of microplastic particles following the increase in Little Auk populations suggesting that seabirds potentially act as biovectors for plastic contamination. Our study demonstrates the diverse nature of climate-driven changes in the Arctic lacustrine environment with increased inorganic input from the more exposed catchment, larger nutrient delivery from the increased bird colonies at the surrounding mountain summits and subsequent alterations in aquatic communities.Peer reviewe

Continuous flux measurements of VOCs using PTR-MS — reliability and feasibility of disjunct-eddy-covariance, surface-layer-gradient, and surface-layer-profile methods

Peer reviewe

Transgenerational Effects of Heavy Metal Pollution on Immune Defense of the Blow Fly Protophormia terraenovae

Recently environmental conditions during early parental development have been found to have transgenerational effects on immunity and other condition-dependent traits. However, potential transgenerational effects of heavy metal pollution have not previously been studied. Here we show that direct exposure to heavy metal (copper) upregulates the immune system of the blow fly, Protophormia terraenovae, reared in copper contaminated food. In the second experiment, to test transgenerational effects of heavy metal, the parental generation of the P. terraenovae was reared in food supplemented with copper, and the immunocompetence of their offspring, reared on uncontaminated food, was measured. Copper concentration used in this study was, in the preliminary test, found to have no effect on mortality of the flies. Immunity was tested on the imago stage by measuring encapsulation response against an artificial antigen, nylon monofilament. We found that exposure to copper during the parental development stages through the larval diet resulted in immune responses that were still apparent in the next generation that was not exposed to the heavy metal. We found that individuals reared on copper-contaminated food developed more slowly compared with those reared on uncontaminated food. The treatment groups did not differ in their dry body mass. However, parental exposure to copper did not have an effect on the development time or body mass of their offspring. Our study suggests that heavy metal pollution has positive feedback effect on encapsulation response through generations which multiplies the harmful effects of heavy metal pollution in following generations

Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs

The aim of this study was to evaluate antimicrobial resistance in canine staphylococci, Escherichia coli and enterococci, which were isolated from 22 dogs with pyoderma and a history of previous antibiotic treatment, compared to bacterial isolates from 56 non-treated control dogs. Two isolates of each bacterial species per dog were investigated, if detected. Staphylococcal isolates from dogs with pyoderma (35 isolates) were more resistant to sulphatrimethoprim than the isolates from controls (56 isolates) (57% vs. 25%, p < 0.004). Multiresistance in staphylococci was also more common in dogs with pyoderma (29% vs. 9%, p = 0.02). A similar trend among isolates of E. coli was detected (24 and 74 isolates from treated and control dogs, respectively), but the differences were not significant. Resistance for macrolide-lincosamides was approximately 20% among staphylococci in both groups. Resistance to ampicillin among enterococci was 4%–7%. The age of the dogs might have an impact on resistance: multiresistance among staphylococcal isolates from younger dogs (≤5 years) was more common than in older dogs (≥6 years) (24%, vs. 0%, 63 and 27 isolates, respectively, p = 0.02). Staphylococci in younger dogs were more resistant to tetracycline (48% vs. 11%, p < 0.001) and sulphatrimethoprim (48% vs. 15%, p < 0.01) than those in older dogs. In contrast, the isolates of E. coli from older dogs tended to be more resistant, although a significant difference was detected only in resistance to tetracycline (13% vs. 2% of 40 and 50 isolates respecthely, p = 0.04)). The results of this small study indicate that resistance in canine staphylococci in the capital area of Finland is comparable with many other countries in Europe. Resistance in indicator bacteria, E. coli and enterococci, was low

Personalized drug sensitivity screening for bladder cancer using conditionally reprogrammed patient-derived cells

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals’ drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies