Localization microscopy is an imaging technique in which the positions of individual point emitters (e.g. fluorescent molecules) are precisely determined from their images. This is a key ingredient in single/multiple-particle-tracking and super-resolution microscopy. Localization in three-dimensions (3D) can be performed by modifying the image that a point-source creates on the camera, namely, the point-spread function (PSF). The PSF is engineered to vary distinctively with emitter depth, using additional optical elements. However, localizing multiple adjacent emitters in 3D poses a significant algorithmic challenge, due to the lateral overlap of their PSFs. Here, we train a neural network to localize multiple emitters with densely overlapping PSFs over a large axial range. Furthermore, we then use the network to design the optimal PSF for the multi-emitter case. We demonstrate our approach experimentally with super-resolution reconstructions of mitochondria and volumetric imaging of fluorescently labeled telomeres in cells.

Background: Acetylxylan esterases are enzymes that remove acetyl groups from the hemicellulolytic polymer xylan. Results: The axe2 gene product in Geobacillus stearothermophilus removes acetyl groups from acetylated xylan and xylosaccharides. Conclusion: Axe2 represents a new serine carbohydrate esterase family. Significance: The findings may provide new routes for the efficient utilization of biomass as a renewable energy source.

Phosphonates (Pn) are diverse organic phosphorus (P) compounds containing C-P bonds and comprise up to 25% of the high-molecular weight dissolved organic P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary production in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a number of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn-as well as inorganic phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the number of Pn transporters with different specificities towards phosphite and different Pns.

Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples , viral photosystem I (vPSI) genes have so far only been detected in environmental samples . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

Geobacillus stearothermophilus T6 is a thermophilic, Gram-positive soil bacterium that possesses an extensive and highly regulated hemicellulolytic system, allowing the bacterium to efficiently degrade high-molecular-weight polysaccharides such as xylan, arabinan and galactan. As part of the xylan-degradation system, the bacterium uses a number of side-chain-cleaving enzymes, one of which is Axe2, a 219-amino-acid intracellular serine acetylxylan esterase that removes acetyl side groups from xylooligosaccharides. Bioinformatic analyses suggest that Axe2 belongs to the lipase GDSL family and represents a new family of carbohydrate esterases. In the current study, the detailed three-dimensional structure of Axe2 is reported, as determined by X-ray crystallography. The structure of the selenomethionine derivative Axe2-Se was initially determined by single-wavelength anomalous diffraction techniques at 1.70 Å resolution and was used for the structure determination of wild-type Axe2 (Axe2-WT) and the catalytic mutant Axe2-S15A at 1.85 and 1.90 Å resolution, respectively. These structures demonstrate that the three-dimensional structure of the Axe2 monomer generally corresponds to the SGNH hydrolase fold, consisting of five central parallel β-sheets flanked by two layers of helices (eight α-helices and five 310-helices). The catalytic triad residues, Ser15, His194 and Asp191, are lined up along a substrate channel situated on the concave surface of the monomer. Interestingly, the Axe2 monomers are assembled as a `doughnut-shaped' homo-octamer, presenting a unique quaternary structure built of two staggered tetrameric rings. The eight active sites are organized in four closely situated pairs, which face the relatively wide internal cavity. The biological relevance of this octameric structure is supported by independent results obtained from gel-filtration, TEM and SAXS experiments. These data and their comparison to the structural data of related hydrolases are used for a more general discussion focusing on the structure-function relationships of enzymes of this category.

Edited by Miguel De la RosaKeywords: b-L-Arabinopyranosidase Glycoside hydrolase family 27 Arabinopyranose Sugar beet arabinan Larch arabinogalactan 13 C NMR a b s t r a c tIn this study we demonstrate that the abp gene in Geobacillus stearothermophilus T-6 encodes a family 27 glycoside hydrolase b-L-arabinopyranosidase. The catalytic constants towards the chromogenic substrate pNP-b-L-arabinopyranoside were 0.8 ± 0.1 mM, 6.6 ± 0.3 s À1 , and 8.2 ± 0.3 s À1 mM À1for K m , k cat and k cat /K m , respectively. 13C NMR spectroscopy unequivocally showed that Abp is capable of removing b-L-arabinopyranose residues from the natural arabino-polysaccharide, larch arabinogalactan. Most family 27 enzymes are active on galactose and contain a conserved Asp residue, whereas in Abp this residue is Ile67, which shifts the specificity of the enzyme towards arabinopyranoside.

Refractometry, namely, the measurement of refractive index (RI), provides information about various sample properties such as concentrations and molecular structure. One physical phenomenon which enables precise determination of a sample’s RI in a microscope is the supercritical-angle fluorescence. This effect is observed when the fluorescence from an emitter near a glass-medium interface is captured by an objective lens with a high numerical aperture. The materials’ index mismatch creates a distinguishable transition in the intensity pattern at the back focal plane of the objective that changes proportionally to the RI of the media. Here, we present a refractometry approach in which the fluorophores are preattached to the bottom surface of a microfluidic channel, enabling highly sensitive determination of the RI using tiny amounts of liquid (picoliters). With this method, we attained a standard deviation of 3.1 × 10–5 and a repeatability of 2.7 × 10–5 RI units. We first determine the capabilities of the device for glycerol-water solutions and then demonstrate the relevance of our system for monitoring changes in biological systems. As a model system, we show that we can detect single bacteria (Escherichia coli) and measure population growth.

ChIP-seq Flow cytometry MRI-based neuroimaging Eukaryotic cell lines Policy information about cell lines Cell line source(s) Jurkat cells for nanoparticle uptake measurement cells gift from D Yablonski. More information in Methods section. AuthenticationThe Jurkat cells used were authenticated to be those from which the Jurkat Clone E6.1 was originated (Cellosaurus no. CVCL_0367). Authentication was performed using the Promega GenePrint 24 System in order to determine short tandem repeat (STR) profile of 23 loci plus Amelogenin for gender determination (X or XY) and analyzed using the 3500xl Genetic Analyzer (Life Technologies) and GeneMapper IDX software. Mycoplasma contaminationCell lines tested were confirmed not to contain mycoplasma contamination. Commonly misidentified lines (See ICLAC register)No commonly misidentified cell lines were used.