CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Background

Super resolution (SR) microscopy enabled cell biologists to visualize subcellular details up to 20 nm in resolution. This breakthrough in spatial resolution made image analysis a challenging procedure. Direct and automated segmentation of SR images remains largely unsolved, especially when it comes to providing meaningful biological interpretations.

Results

Here, we introduce a novel automated imaging analysis routine, based on Gaussian, followed by a segmentation procedure using CellProfiler software (www.cellprofiler.org). We tested this method and succeeded to segment individual nuclear pore complexes stained with gp210 and pan-FG proteins and captured by two-color STED microscopy. Test results confirmed accuracy and robustness of the method even in noisy STED images of gp210.

Conclusions

Our pipeline and novel segmentation procedure may benefit end-users of SR microscopy to analyze their images and extract biologically significant quantitative data about them in user-friendly and fully-automated settings.

     

Lessons Learned from Crowdsourcing Complex Engineering Tasks

Crowdsourcing

Crowdsourcing is the practice of obtaining needed ideas, services, or content by requesting contributions from a large group of people. Amazon Mechanical Turk is a web marketplace for crowdsourcing microtasks, such as answering surveys and image tagging. We explored the limits of crowdsourcing by using Mechanical Turk for a more complicated task: analysis and creation of wind simulations.

Harnessing Crowdworkers for Engineering

Our investigation examined the feasibility of using crowdsourcing for complex, highly technical tasks. This was done to determine if the benefits of crowdsourcing could be harnessed to accurately and effectively contribute to solving complex real world engineering problems. Of course, untrained crowds cannot be used as a mere substitute for trained expertise. Rather, we sought to understand how crowd workers can be used as a large pool of labor for a preliminary analysis of complex data.

Virtual Wind Tunnel

We compared the skill of the anonymous crowd workers from Amazon Mechanical Turk with that of civil engineering graduate students, making a first pass at analyzing wind simulation data. For the first phase, we posted analysis questions to Amazon crowd workers and to two groups of civil engineering graduate students. A second phase of our experiment instructed crowd workers and students to create simulations on our Virtual Wind Tunnel website to solve a more complex task.

Conclusions

With a sufficiently comprehensive tutorial and compensation similar to typical crowd-sourcing wages, we were able to enlist crowd workers to effectively complete longer, more complex tasks with competence comparable to that of graduate students with more comprehensive, expert-level knowledge. Furthermore, more complex tasks require increased communication with the workers. As tasks become more complex, the employment relationship begins to become more akin to outsourcing than crowdsourcing. Through this investigation, we were able to stretch and explore the limits of crowdsourcing as a tool for solving complex problems.     

Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.     

Biosynthesis,Characterization, and Evaluation of the Cytotoxic Effects of Biologically Synthesized Silver Nanoparticles from Cyperus conglomeratus Root Extracts on Breast Cancer Cell Line MCF-7

Biological Trace Element Research - Over recent years, metal nanoparticles have largely been investigated due to their potential activities. This study focused on synthesizing silver nanoparticles...     

Assessment of In Ovo Administration of Bifidobacterium bifidum and Bifidobacterium longum on Performance,Ileal Histomorphometry,Blood Hematological,and Biochemical Parameters of Broilers

Probiotics and Antimicrobial Proteins - Bifidobacterium is one of the most promising probiotics which was recently used as an alternative growth promoter in poultry. This trial was considered to...     

Isolation,identification, and biochemical characterization of a brown rot fungus capable of textile dye decolorization

Twenty-two local brown rot fungal isolates were obtained from 5 different environmental sources. Fourteen isolates were presumptively identified as Aspergillus sp. and eight as Penicillium sp. using mycelium and spore morphology. All the fungal isolates were screened for their ability to decolorize Isolan Red and colored waste water and to produce oxidase activity. Aspergillus isolate 2 was chosen for further study because it could decolorize both dyes and produce oxidase. A 400 bp fragment of the 18S rRNA gene from isolate 2 was analyzed by nucleotide sequence analysis. Blastn analysis of sequence data demonstrated 100% identity to Aspergillus sp. and isolate 2 was assigned the strain designation Aspergillus sp. EL-2 (Accession number: HM140797). EL-2 could remove up to 80% of Disperse Blue in waste water effluent within 48 h in submerged shake culture. The decolorization process was energy dependent, growth related, and required viable biomass. EL-2 was able to grow and decolorize waste water over a broad pH range. Addition of inducers and inhibitors of specific enzymes or families of enzymes demonstrated the involvement of phenol oxidase (laccase), cytochrome p-450 oxygenase and hydroxyl radicals in the decolorization process. The data also suggest that it may be practical to enhance decolorizing activity of Aspergillus sp. EL-2 through the metabolic control of fungal degradative pathways by altering media composition.     

The HSV-1 Exonuclease, UL12, Stimulates Recombination by a Single Strand Annealing Mechanism

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.