Srigokul Upadhyayula

Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications.

This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.

Seipin is required for converting nascent to mature lipid droplets

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.001

Coatings of Polyethylene Glycol for Suppressing Adhesion between Solid Microspheres and Flat Surfaces

This article describes the development and the examination of surface coatings that suppress the adhesion between glass surfaces and polymer microspheres. Superparamagnetic doping allowed for exerting magnetic forces on the microbeads. The carboxyl functionalization of the polymer provided the means for coating the beads with polyethylene glycol (PEG) with different molecular weight. Under gravitational force, the microbeads settled on glass surfaces with similar polymer coatings. We examined the efficacy of removing the beads from the glass surfaces by applying a pulling force of ~1.2 pN. The percent beads remaining on the surface after applying the pulling force for approximately 5 s served as an indication of the adhesion propensity. Coating of PEG with molecular weight ranging between 3 and 10 kDa was essential for suppressing the adhesion. For the particular substrates, surface chemistry and aqueous media we used, coatings of 5 kDa manifested optimal suppression of adhesion: that is, only 3% of the microbeads remained on the surface after applying the pulling magnetic force. When either the glass or the beads were not PEGylated, the adhesion between them was substantial. Addition of a noncharged surfactant, TWEEN, above its critical micelle concentrations (CMCs) suppressed the adhesion between noncoated substrates. The extent of this surfactant-induced improvement of the adhesion suppression, however, did not exceed the quality of preventing the adhesion that we attained by PEGylating both substrates. In addition, the use of surfactants did not significantly improve the suppression of bead-surface adhesion when both substrates were PEGylated. These findings suggest that such surfactant additives tend to be redundant and that covalently grafted coatings of PEGs with selected chain lengths provide sufficient suppression of nonspecific interfacial interactions.

Electrochemical Reduction of Quinones: Interfacing Experiment and Theory for Defining Effective Radii of Redox Moieties

Using cyclic voltammetry, we examined the dependence of the reduction potentials of six quinones on the concentration of the supporting electrolyte. An increase in the electrolyte concentration, resulting in an increase in the solution polarity, caused positive shifts of the reduction potentials. We ascribed the observed changes in the potentials to the dependence of the solvation energy of the quinones and their anions on the media polarity. Analysis of the reduction potentials, using the Born solvation energy equation, yielded unfeasibly small values for the effective radii of the quinone species, that is, the experimentally obtained effective radii were up to 4-fold smaller than the radii of the solvation cavities that we calculated for the quinones. The nonspherical shapes of the quinones, along with the uneven charge density distribution in their anions, encompassed the underlying reasons for the discrepancies between the obtained experimental and theoretical values for the radii of these redox species. The generalized Born approach, which does not treat the solvated species as single spheres, provided means for addressing this discrepancy and yielded effective radii that were relatively close to the measured values.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

Lattice light-sheet microscopy is used to examine two problems in membrane dynamics—molecular events in clathrin-coated pit formation and changes in cell shape during cell division. This methodology sets a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

Anthranilamides as Bioinspired Molecular Electrets: Experimental Evidence for a Permanent Ground-State Electric Dipole Moment

As electrostatic equivalents of magnets, organic electrets offer unparalleled properties for impacting energy conversion and electronic applications. While biological systems have evolved to efficiently utilize protein α-helices as molecular electrets, the synthetic counterparts of these conjugates still remain largely unexplored. This paper describes a study of the electronic properties of anthranilamide oligomers, which proved to be electrets based on their intrinsic dipole moments as evident from their spectral and dielectric properties. NMR studies provided the means for estimating the direction of the intrinsic electric dipoles of these conjugates. This study sets the foundation for the development of a class of organic materials that are de novo designed from biomolecular motifs and possess unexplored electronic properties.

Effect of polyethylene glycol coatings on uptake of indocyanine green loaded nanocapsules by human spleen macrophages in vitro

Near-infrared (NIR) optically active nanoparticles are promising exogenous chromophores for applications in medical imaging and phototherapy. Since nanoparticles can be rapidly eliminated from the body by cells of the reticuloendothelial system, a thriving strategy to increase their blood circulation time is through surface modification with polyethylene glycol (PEG). We constructed polymeric nanocapsules loaded with indocyanine green (ICG), an FDA-approved NIR dye, and coated with aldehyde-terminated PEG. Using optical absorbance spectroscopy and flow cytometry, we investigated the effect of PEG coating and molecular weight (MW) of PEG [5000 and 30,000 Daltons (Da)] on the phagocytic content of human spleen macrophages incubated with ICG-containing nanocapsules (ICG-NCs) between 15 to 360 min. Our results indicate that surface coating with PEG is an effective method to reduce the phagocytic content of ICG-NCs within macrophages for at least up to 360 min of incubation time. Coating the surface of ICG-NCs with the low MW PEG results in lower phagocytic content of ICG-NCs within macrophages for at least up to 60 min of incubation time as compared to ICG-NCs coated with the high MW PEG. Surface coating of ICG-NCs with PEG is a promising approach to prolong vasculature circulation time of ICG for NIR imaging and phototherapeutic applications.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms

Continuing the resolution revolution The living cell contains dynamic, spatially complex subassemblies that are sensitive to external perturbations. To minimize such perturbations, cells should be imaged in their native multicellular environments, under as gentle illumination as possible. However, achieving the spatiotemporal resolution needed to follow three-dimensional subcellular processes in detail under these conditions is challenging: Sample-induced aberrations degrade resolution and sensitivity, and high resolution usually requires intense excitation. Liu et al. combined noninvasive lattice light-sheet microscopy with aberration-correcting adaptive optics to study a variety of delicate subcellular events in vivo, including organelle remodeling during mitosis and growth cone dynamics during spinal cord development. Science, this issue p. eaaq1392 Adaptive optical lattice light-sheet microscopy permits delicate 3D subcellular processes to be viewed natively in vivo. INTRODUCTION Organisms live by means of the complex, dynamic, three-dimensional (3D) interplay between millions of components, from the molecular to the multicellular. Visualizing this complexity in its native form requires imaging at high resolution in space and time anywhere within the organism itself, because only there are all the environmental factors that regulate its physiology present. However, the optical heterogeneity of multicellular systems leads to aberrations that quickly compromise resolution, signal, and contrast with increasing imaging depth. Furthermore, even in the absence of aberrations, high resolution and fast imaging are usually accompanied by intense illumination, which can perturb delicate subcellular processes or even introduce permanent phototoxic effects. RATIONALE We combined two imaging technologies to address these problems. The first, lattice light-sheet microscopy (LLSM), rapidly and repeatedly sweeps an ultrathin sheet of light through a volume of interest while acquiring a series of images, building a high-resolution 3D movie of the dynamics within. The confinement of the illumination to a thin plane insures that regions outside the volume remain unexposed, while the parallel collection of fluorescence from across the plane permits low, less perturbative intensities to be used. The second technology, adaptive optics (AO), measures sample-induced distortions to the image of a fluorescent “guide star” created within the volume—distortions that also affect the acquired light-sheet images—and compensates for these by changing the shape of a mirror to create an equal but opposite distortion. RESULTS We applied AO-LLSM to study a variety of 3D subcellular processes in vivo over a broad range of length scales, from the nanoscale diffusion of clathrin-coated pits (CCPs) to axon-guided motility across 200 μm of the developing zebrafish spinal cord. Clear delineation of cell membranes allowed us to computationally isolate and individually study any desired cell within the crowded multicellular environment of the intact organism. By doing so, we could compare specific processes across different cell types, such as rates of CCP internalization in muscle fibers and brain cells, organelle remodeling during cell division in the developing brain and eye, and motility mechanisms used by immune cells and metastatic breast cancer cells. Although most examples were taken from zebrafish embryos, we also demonstrated AO-LLSM in a human stem cell–derived organoid, a Caenorhabditis elegans nematode, and Arabidopsis thaliana leaves. CONCLUSION AO-LLSM takes high-resolution live-cell imaging of subcellular processes from the confines of the coverslip to the more physiologically relevant 3D environment within whole transparent organisms. This creates new opportunities to study the phenotypic diversity of intracellular dynamics, extracellular communication, and collective cell behavior across different cell types, organisms, and developmental stages. High-resolution in vivo cell biology. AO-LLSM permits the study of 3D subcellular processes in their native multicellular environments at high spatiotemporal resolution, including (clockwise from upper left) growth of spinal cord axons; cancer cell metastasis; collective cellular motion; endocytosis; microtubule displacements; immune cell migration; and (center) organelle dynamics. True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration

The ratio of ATP:ADP is highest at perinuclear sites, where mitochondria are dense, and dissipates toward the periphery. Miro1 positions mitochondria toward the cortical cytoskeleton. Deletion of Miro1 results in perinuclear clustering of mitochondria, altering intracellular ATP:ADP gradients, and impairs energy-expensive cell migratory processes.