Attosecond pulse shaping using a seeded free-electron laser.

Attosecond pulses are central to the investigation of valence- and core-electron dynamics on their natural timescales1-3. The reproducible generation and characterization of attosecond waveforms has been demonstrated so far only through the process of high-order harmonic generation4-7. Several methods for shaping attosecond waveforms have been proposed, including the use of metallic filters8,9, multilayer mirrors10 and manipulation of the driving field11. However, none of these approaches allows the flexible manipulation of the temporal characteristics of the attosecond waveforms, and they suffer from the low conversion efficiency of the high-order harmonic generation process. Free-electron lasers, by contrast, deliver femtosecond, extreme-ultraviolet and X-ray pulses with energies ranging from tens of microjoules to a few millijoules12,13. Recent experiments have shown that they can generate subfemtosecond spikes, but with temporal characteristics that change shot-to-shot14-16. Here we report reproducible generation of high-energy (microjoule level) attosecond waveforms using a seeded free-electron laser17. We demonstrate amplitude and phase manipulation of the harmonic components of an attosecond pulse train in combination with an approach for its temporal reconstruction. The results presented here open the way to performing attosecond time-resolved experiments with free-electron lasers.

The role of crinophagy in quality control of the regulated secretory pathway.

In specialized secretory cells that produce and release biologically active substances in a regulated fashion, tight control of both the quantity and quality of secretory material is of paramount importance. During crinophagy, abnormal, excess or obsolete secretory granules directly fuse with lysosomes to yield crinosomes, in which the delivered secretory material is degraded. Crinophagy maintains the proper intracellular pool of secretory granules, and it is enhanced when secretory material accumulates because of compromised secretion. Recent studies highlight that it can even degrade newly formed, nascent secretory granules that shed from the trans-Golgi network. This implies that crinophagy provides a quality control checkpoint acting at the formation of secretory vesicles, and this degradation mechanism might survey secretory granules throughout their maturation. Of note, a plethora of human disorders is associated with defective lysosomal clearance of secretory material via crinophagy or similar pathways, including macro- or micro-autophagic degradation of secretory granules (referred to here as macro- and micro-secretophagy, respectively). In our Review, we summarize key recent advances in this field and discuss potential links with disease.

Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila.

Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 µm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.

Wave packet dynamics and control in excited states of molecular nitrogen.

Wave packet interferometry with vacuum ultraviolet light has been used to probe a complex region of the electronic spectrum of molecular nitrogen, N2. Wave packets of Rydberg and valence states were excited by using double pulses of vacuum ultraviolet (VUV), free-electron-laser (FEL) light. These wave packets were composed of contributions from multiple electronic states with a moderate principal quantum number (n ∼ 4-9) and a range of vibrational and rotational quantum numbers. The phase relationship of the two FEL pulses varied in time, but as demonstrated previously, a shot-by-shot analysis allows the spectra to be sorted according to the phase between the two pulses. The wave packets were probed by angle-resolved photoionization using an infrared pulse with a variable delay after the pair of excitation pulses. The photoelectron branching fractions and angular distributions display oscillations that depend on both the time delays and the relative phases of the VUV pulses. The combination of frequency, time delay, and phase selection provides significant control over the ionization process and ultimately improves the ability to analyze and assign complex molecular spectra.

Rab26 controls secretory granule maturation and breakdown in Drosophila.

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.

Different Metabolism and Toxicity of TRANS Fatty Acids, Elaidate and Vaccenate Compared to Cis-Oleate in HepG2 Cells.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.

Time-resolved photoelectron imaging of complex resonances in molecular nitrogen.

We have used the FERMI free-electron laser to perform time-resolved photoelectron imaging experiments on a complex group of resonances near 15.38 eV in the absorption spectrum of molecular nitrogen, N2, under jet-cooled conditions. The new data complement and extend the earlier work of Fushitani et al. [Opt. Express 27, 19702-19711 (2019)], who recorded time-resolved photoelectron spectra for this same group of resonances. Time-dependent oscillations are observed in both the photoelectron yields and the photoelectron angular distributions, providing insight into the interactions among the resonant intermediate states. In addition, for most states, we observe an exponential decay of the photoelectron yield that depends on the ionic final state. This observation can be rationalized by the different lifetimes for the intermediate states contributing to a particular ionization channel. Although there are nine resonances within the group, we show that by detecting individual photoelectron final states and their angular dependence, we can identify and differentiate quantum pathways within this complex system.

The Role of Deubiquitinating Enzymes in the Various Forms of Autophagy.

Deubiquitinating enzymes (DUBs) have an essential role in several cell biological processes via removing the various ubiquitin patterns as posttranslational modification forms from the target proteins. These enzymes also contribute to the normal cytoplasmic ubiquitin pool during the recycling of this molecule. Autophagy, a summary name of the lysosome dependent self-degradative processes, is necessary for maintaining normal cellular homeostatic equilibrium. Numerous forms of autophagy are known depending on how the cellular self-material is delivered into the lysosomal lumen. In this review we focus on the colorful role of DUBs in autophagic processes and discuss the mechanistic contribution of these molecules to normal cellular homeostasis via the possible regulation forms of autophagic mechanisms.

Complete Characterization of Phase and Amplitude of Bichromatic Extreme Ultraviolet Light.

Intense, mutually coherent beams of multiharmonic extreme ultraviolet light can now be created using seeded free-electron lasers, and the phase difference between harmonics can be tuned with attosecond accuracy. However, the absolute value of the phase is generally not determined. We present a method for determining precisely the absolute phase relationship of a fundamental wavelength and its second harmonic, as well as the amplitude ratio. Only a few easily calculated theoretical parameters are required in addition to the experimental data.

Reflectance variation in the blue tit crown in relation to feather structure.

Structural plumage colour is one of the most enigmatic sexually selected traits. The information content of structural colour variation is debated, and the heterogeneity of the findings is hard to explain because the proximate background of within-species colour differences is very scarcely studied. We combined measurements of feather macrostructure and nanostructure to explain within-population variability in blue tit crown reflectance. We found that sexual dichromatism in aspects of crown reflectance was explained only by feather macrostructure, whereas nanostructural predictors accounted for some of the age-related differences in reflectance. Moreover, we found that both mean reflectance and spectral shape traits reflected a combination of quantity and regularity aspects in macrostructure and nanostructure. This rich proximate background provides ample scope for reflectance to convey various types of information on individual quality.

The role of K63-linked polyubiquitin in several types of autophagy.

Lysosomal-dependent self-degradative (autophagic) mechanisms are essential for the maintenance of normal homeostasis in all eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified, based on how the cellular self material can incorporate into the lysosomal lumen. Ubiquitination, a well-known and frequently occurred posttranslational modification has essential role in all cell biological processes, thus in autophagy too. The second most common type of polyubiquitin chain is the K63-linked polyubiquitin, which strongly connects to some self-degradative mechanisms in the cells. In this review, we discuss the role of this type of polyubiquitin pattern in numerous autophagic processes.

Liquid-cooled modular gas cell system for high-order harmonic generation using high average power laser systems.

We present the design and implementation of a new, modular gas target suitable for high-order harmonic generation using high average power lasers. To ensure thermal stability in this high heat load environment, we implement an appropriate liquid cooling system. The system can be used in multiple-cell configurations, allowing us to control the cell length and aperture size. The cell design was optimized with heat and flow simulations for thermal characteristics, vacuum compatibility, and generation medium properties. Finally, the cell system was experimentally validated by conducting high-order harmonic generation measurements using the 100 kHz high average power HR-1 laser system at the Extreme Light Infrastructure Attosecond Light Pulse Source (ELI ALPS) facility. Such a robust, versatile, and stackable gas cell arrangement can easily be adapted to different experimental geometries in both table-top laboratory systems and user-oriented facilities, such as ELI ALPS.

Ecdysone receptor isoform specific regulation of secretory granule acidification in the larval Drosophila salivary gland.

Bulk production and release of glue containing secretory granules takes place in the larval salivary gland during Drosophila development in order to attach the metamorphosing animal to a dry surface. These granules undergo a maturation process to prepare glue for exocytosis, which includes homotypic fusions to increase the size of granules, vesicle acidification and ion uptake. The steroid hormone 20-hydroxyecdysone is known to be required for the first and last steps of this process: glue synthesis and secretion, respectively. Here we show that the B1 isoform of Ecdysone receptor (EcR), together with its binding partner Ultraspiracle, are also necessary for the maturation of glue granules by promoting their acidification via regulation of Vha55 expression, which encodes an essential subunit of the V-ATPase proton pump. This is antagonized by the EcR-A isoform, overexpression of which decreases EcR-B1 and Vha55 expression and glue granule acidification. Our data shed light on a previously unknown, ecdysone receptor isoform-specific regulation of glue granule maturation.

Facile isolation and analysis of sporopollenin exine from bee pollen.

We present facile methods to obtain purified sporopollenin exine capsules, and provide mass balances for classical and novel purification procedures. An ionic liquid, tetrabutyl phosphonium hydroxide turned out to be the most effective in removing the intine wall. The sporopollenin capsules were investigated by fluorescent microscopy, AFM, solid-state NMR and infrared Raman spectroscopy. The latter two methods showed that sunflower and rape exines have different proportions of O-aliphatic and aromatic constituents. Purified exine capsules were coated with functionalized fluorophores. The procedures presented in this paper could contribute to further spread of the applications of this hollow, and chemically highly resistant material.

Crinophagy mechanisms and its potential role in human health and disease.

Autophagic-lysosomal degradation is essential for the maintenance of normal homeostasis in eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified. From these, probably the least known autophagic process is crinophagy, during which unnecessary or obsolete secretory granules directly fuse with late endosomes/lysosomes as a means of rapid elimination of unused secretory material from the cytoplasm. This process was identified in 1966, but we are only beginning to understand the molecular mechanisms and regulation of crinophagy. In this review, we summarize the current examination methods and possible model systems, discuss the recently identified factors that are required for crinophagy, and give an overview of the potential medical relevance of this process.

Biomanufacturing of Tomato-Derived Nanovesicles.

Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato (Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 1015 particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

On the Fly: Recent Progress on Autophagy and Aging in Drosophila.

Autophagy ensures the lysosome-mediated breakdown and recycling of self-material, as it not only degrades obsolete or damaged intracellular constituents but also provides building blocks for biosynthetic and energy producing reactions. Studies in animal models including Drosophila revealed that autophagy defects lead to the rapid decline of neuromuscular function, neurodegeneration, sensitivity to stress (such as starvation or oxidative damage), and stem cell loss. Of note, recently identified human Atg gene mutations cause similar symptoms including ataxia and mental retardation. Physiologically, autophagic degradation (flux) is known to decrease during aging, and this defect likely contributes to the development of such age-associated diseases. Many manipulations that extend lifespan (including dietary restriction, reduced TOR kinase signaling, exercise or treatment with various anti-aging substances) require autophagy for their beneficial effect on longevity, pointing to the key role of this housekeeping process. Importantly, genetic (e.g., Atg8a overexpression in either neurons or muscle) or pharmacological (e.g., feeding rapamycin or spermidine to animals) promotion of autophagy has been successfully used to extend lifespan in Drosophila, suggesting that this intracellular degradation pathway can rejuvenate cells and organisms. In this review, we highlight key discoveries and recent progress in understanding the relationship of autophagy and aging in Drosophila.

Understanding the importance of autophagy in human diseases using Drosophila.

Autophagy is a lysosome-dependent intracellular degradation pathway that has been implicated in the pathogenesis of various human diseases, either positively or negatively impacting disease outcomes depending on the specific context. The majority of medical conditions including cancer, neurodegenerative diseases, infections and immune system disorders and inflammatory bowel disease could probably benefit from therapeutic modulation of the autophagy machinery. Drosophila represents an excellent model animal to study disease mechanisms thanks to its sophisticated genetic toolkit, and the conservation of human disease genes and autophagic processes. Here, we provide an overview of the various autophagy pathways observed both in flies and human cells (macroautophagy, microautophagy and chaperone-mediated autophagy), and discuss Drosophila models of the above-mentioned diseases where fly research has already helped to understand how defects in autophagy genes and pathways contribute to the relevant pathomechanisms.

Sec20 is Required for Autophagic and Endocytic Degradation Independent of Golgi-ER Retrograde Transport.

Endocytosis and autophagy are evolutionarily conserved degradative processes in all eukaryotes. Both pathways converge to the lysosome where cargo is degraded. Improper lysosomal degradation is observed in many human pathologies, so its regulatory mechanisms are important to understand. Sec20/BNIP1 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) is a BH3 (Bcl-2 homology 3) domain-containing SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) protein that has been suggested to promote Golgi-ER retrograde transport, mitochondrial fission, apoptosis and mitophagy in yeast and vertebrates. Here, we show that loss of Sec20 in Drosophila fat cells causes the accumulation of autophagic vesicles and prevents proper lysosomal acidification and degradation during bulk, starvation-induced autophagy. Furthermore, Sec20 knockdown leads to the enlargement of late endosomes and accumulation of defective endolysosomes in larval Drosophila nephrocytes. Importantly, the loss of Syx18 (Syntaxin 18), one of the known partners of Sec20, led to similar changes in nephrocytes and fat cells. Interestingly. Sec20 appears to function independent of its role in Golgi-ER retrograde transport in regulating lysosomal degradation, as the loss of its other partner SNAREs Use1 (Unconventional SNARE In The ER 1) and Sec22 or tethering factor Zw10 (Zeste white 10), which function together in the Golgi-ER pathway, does not cause defects in autophagy or endocytosis. Thus, our data identify a potential new transport route specific to lysosome biogenesis and function.

Drosophila Arl8 is a general positive regulator of lysosomal fusion events.

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.