The photorespiratory intermediate glycerate is known to be shuttled between the peroxisome and chloroplast. Here, localization of NPF8.4 in the tonoplast, together with the reduced vacuolar glycerate content displayed by an npf8.4 mutant and the glycerate efflux activity detected in an oocyte expression system, identifies NPF8.4 as a tonoplast glycerate influx transporter. Our study shows that expression of NPF8.4 and most photorespiration-associated genes, as well as the photorespiration rate, is upregulated in response to short-term nitrogen (N) depletion. We report growth retardation and early senescence phenotypes for npf8.4 mutants specifically upon N depletion, suggesting that the NPF8.4-mediated regulatory pathway for sequestering the photorespiratory carbon intermediate glycerate in vacuoles is important to alleviate the impact of an increased C/N ratio under N deficiency. Thus, our study of NPF8.4 reveals a novel role for photorespiration in N flux to cope with short-term N depletion.

Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades. However, limited information is currently available on the formation of autolysosomes, which may partially be attributed to fewer methods to study the formation of autolysosomes than that of autophagosomes. Abemaciclib (Abe) and vacuolin-1 (Vac) are drugs that suppress the progression of breast cancer and induce characteristic vacuole formation in cells. Since Abe-induced vacuoles have the appearance of autolysosomes, they may be used to examine the formation of autolysosomes. However, it remains unknown whether Abe-/Vac-induced vacuoles are regulated by autophagosome-lysosome fusion. Markers for endosomes, lysosomes, and autophagosomes (Rab7, LAMP1, and mRFP-GFP-LC3, respectively) indicated that Abe-/Vac-induced vacuoles were autolysosomes. Abe and Vac failed to induce vacuolation in ATG16L1-deficient autophagy-null cells. Furthermore, Abe-/Vac-induced vacuolation was suppressed by bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, whereas it was facilitated by rapamycin and the overexpression of Beclin-1, inducers of autophagosome-lysosome fusion. Moreover, vacuole formation was inhibited by the knockdown of progranulin (PGRN), a regulator of autophagosome-lysosome fusion, and promoted by its overexpression. The present results suggest the potential of Abe-/Vac-induced vacuole-like autolysosomes as a tool for evaluating autophagosome-lysosome fusion and examining the effects of PGRN in autophagy.

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA\'s complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, β-Amyrin and 11-oxo-β-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters\' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = -10.9, -10.6, -10.9 kcal/mol for GA, β-amyrin and 11-oxo-β-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae.

We hypothesize that intracellular trafficking pathways are altered in chlamydial infected cells to maximize the ability of Chlamydia to scavenge nutrients while not overtly stressing the host cell. Previous data demonstrated the importance of two eukaryotic SNARE proteins, VAMP4 and syntaxin 10 (Stx10), in chlamydial growth and development. Although, the mechanism for these effects is still unknown. To interrogate whether chlamydial infection altered these proteins\' networks, we created BirA*-VAMP4 and BirA*-Stx10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between Stx10 and VAPB, we also identified caveats in using the BioID system to study the impact of infection by an obligate intracellular pathogen on SNARE protein networks. The addition of the BirA* altered the localization of VAMP4 and Stx10 during infection with Chlamydia trachomatis serovars L2 and D and Coxiella burnetii Nine Mile Phase II. We also discovered that BirA* traffics to and biotinylates Coxiella-containing vacuoles and, in general, has a propensity for labeling membrane or membrane-associated proteins. While the BioID system identified a novel association for Stx10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.

Human prion and non-prion neurodegenerative diseases share pathogenic mechanisms and neuropathological features. The lesion profile of a particular entity results from specific involvement of vulnerable neuron populations and connectivity circuits by a pathogenic protein isoform with strain-like properties. The lesion profile of the medial temporal lobe (MTL) was studied in postmortem tissue of 143 patients with human prion disease (HPD) including sporadic, genetic, and acquired forms. Most cases (90%) were classified according to PrPres type and/or PRNP codon 129 status, in addition to a full neuropathological profile. Mixed histotypes represented 29.4% of total sporadic Creutzfeldt-Jakob disease (sCJD) cases. An intensity score of involvement including spongiosis and astrogliosis was determined for the amygdala, presubiculum, subiculum, entorhinal cortex, CA1 to CA4 sectors of the hippocampal cortex, and dentate gyrus. Connectivity hubs within the MTL presented the highest scores. Diverse lesion profiles were obtained for different types and subtypes of HPD. Impact of mixed PrPres types on the MTL lesion profile was higher for sCJDMV2K cases than in other histotypes. Differences between MTL profiles was globally consistent with current evidence on specific strains in HPD. These results may be relevant for the analysis of possible strain effects in focal non-prion neurodegenerative conditions limited to the MTL.

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm\'s canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2-6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV\'s maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.

Chlamydia trachomatis is the leading cause of infectious blindness and a sexually transmitted infection. All chlamydiae are obligate intracellular bacteria that replicate within a membrane-bound vacuole termed the inclusion. From the confines of the inclusion, the bacteria must interact with many host organelles to acquire key nutrients necessary for replication, all while promoting host cell viability and subverting host defense mechanisms. To achieve these feats, C. trachomatis delivers an arsenal of virulence factors into the eukaryotic cell via a type 3 secretion system (T3SS) that facilitates invasion, manipulation of host vesicular trafficking, subversion of host defense mechanisms and promotes bacteria egress at the conclusion of the developmental cycle. A subset of these proteins intercalate into the inclusion and are thus referred to as inclusion membrane proteins. Whereas others, referred to as conventional T3SS effectors, are released into the host cell where they localize to various eukaryotic organelles or remain in the cytosol. Here, we discuss the functions of T3SS effector proteins with a focus on how advances in chlamydial genetics have facilitated the identification and molecular characterization of these important factors.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the \'high\' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

In this experimental study we used for the first time Tiprotec® as a solution for corneal preservation and cold storage. We compared the resultant endothelial cell morphology and viability with this obtained after preservation of the ex-vivo corneas with both usual standard techniques: conventional cold storage (using Eusol-C) and organ culture. This prospective, in vitro, 3-armed parallel study was performed with the use of 90 porcine corneas (examined for their endothelial quality and transparency) randomly selected for preservation in three storage methods (each 30 corneas): organ culture, standard cold storage (Eusol-C) and experimental cold storage (Tiprotec®) Endothelium cell quantity and quality as well as corneal opacification were assessed. The degree of endothelial transparency was significantly reduced over time with all preservation media, without any significant difference among the three groups at any point of time. A reduction in endothelial cell density was also observed with all three preservation media after 30 days of storage without statistically significant differences between groups. The number of hexagonal and pentagonal endothelium cells was significantly reduced overtime in all media with significantly more hexagonal and pentagonal in the organ culture group compared to the cold storage groups. We could show that the cryopreservation medium Tiprotec®, used until now for the preservation of vascular grafts, was of similar quality compared to the medium Eusol-C for the hypothermic storage of corneal tissue for an extended period of time up to 30 days. In comparison to organic culture with culture medium KII, both Tiprotec® and Eusol-C were found less effective in preserving endothelial cell quality, as assessed by the morphometric analysis, and viability, as assessed by the degree of vacuolization at least up to the 30th day of storage. However, both, Tiprotec®- and Eusol-C-preserved corneas demonstrated a certain capacity to recover after their submission in organ culture.

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome-like compartments called Coxiella-containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once-neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.