Intracellular Granules Research Articles

The difference in the intracellular Arg/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2.

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme-inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators.

Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov., Two Acidophilic Bacteria Isolated from Decaying Wood, Hydrolyzing Cellulose and Producing Poly-3-hydroxybutyrate.

Two novel strains, HW T2.11T and HW T5.17T, were isolated from decaying wood (forest of Champenoux, France). Study of the 16S rRNA sequence similarity indicated that the novel strains belong to the genus Acidisoma. The sequence similarity of the 16S rRNA gene of HW T2.11T with the corresponding sequences of A. tundrae and A. sibiricum was 97.30% and 97.25%, while for HW T5.17T it was 96.85% and 97.14%, respectively. The DNA G+C contents of the strains were 62.32–62.50%. Cells were Gram-negative coccobacilli that had intracellular storage granules (poly-3-hydroxybutyrate (P3HB)) that confer resistance to environmental stress conditions. They were mesophilic and acidophilic organisms growing at 8–25 °C, at a pH of 2.0–6.5, and were capable of using a wide range of organic compounds and complex biopolymers such as starch, fucoidan, laminarin, pectin and cellulose, the latter two being involved in wood composition. The major cellular fatty acid was cyclo C19:0ω8c and the major quinone was Q-10. Overall, genome relatedness indices between genomes of strains HW T2.11T and HW T5.17T (Orthologous Average Nucleotide Identity (OrthoANI) value = 83.73% and digital DNA-DNA hybridization score = 27.5%) confirmed that they belonged to two different species. Genetic predictions indicate that the cyclopropane fatty acid (CFA) pathway is present, conferring acid-resistance properties to the cells. The two novel strains might possess a class IV polyhydroxyalcanoate (PHA) synthase operon involved in the P3HB production pathway. Overall, the polyphasic taxonomic analysis shows that these two novel strains are adapted to harsh environments such as decaying wood where the organic matter is difficult to access, and can contribute to the degradation of dead wood. These strains represent novel species of the genus Acidisoma, for which the names Acidisoma silvae sp. nov. and Acidisoma cellulosilytica sp. nov. are proposed. The type strains of Acidisoma silvae and Acidisoma cellulosilytica are, respectively, HW T2.11T (DSM 111006T; UBOCC-M-3364T) and HW T5.17T (DSM 111007T; UBOCC-M-3365T).

A Novel Magnetotactic Alphaproteobacterium Producing Intracellular Magnetite and Calcium-Bearing Minerals.

Magnetotactic bacteria (MTB) are prokaryotes that form intracellular magnetite (Fe3O4) or greigite (Fe3S4) nanocrystals with tailored sizes, often in chain configurations. Such magnetic particles are each surrounded by a lipid bilayer membrane, called a magnetosome, and provide a model system for studying the formation and function of specialized internal structures in prokaryotes. Using fluorescence-coupled scanning electron microscopy, we identified a novel magnetotactic spirillum, XQGS-1, from freshwater Xingqinggong Lake, Xi'an City, Shaanxi Province, China. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain XQGS-1 represents a novel genus of the Alphaproteobacteria class in the Proteobacteria phylum. Transmission electron microscopy analyses reveal that strain XQGS-1 forms on average 17 ± 3 magnetite magnetosome particles with an ideal truncated octahedral morphology, with an average length and width of 88.3 ± 11.7 nm and 83.3 ± 11.0 nm, respectively. They are tightly organized into a single chain along the cell long axis close to the concave side of the cell. Intrachain magnetic interactions likely result in these large equidimensional magnetite crystals behaving as magnetically stable single-domain particles that enable bacterial magnetotaxis. Combined structural and chemical analyses demonstrate that XQGS-1 cells also biomineralize intracellular amorphous calcium phosphate (2 to 3 granules per cell; 90.5- ± 19.3-nm average size) and weakly crystalline calcium carbonate (2 to 3 granules per cell; 100.4- ± 21.4-nm average size) in addition to magnetite. Our results expand the taxonomic diversity of MTB and provide evidence for intracellular calcium phosphate biomineralization in MTB. IMPORTANCE Biomineralization is a widespread process in eukaryotes that form shells, teeth, or bones. It also occurs commonly in prokaryotes, resulting in more than 60 known minerals formed by different bacteria under wide-ranging conditions. Among them, magnetotactic bacteria (MTB) are remarkable because they might represent the earliest organisms that biomineralize intracellular magnetic iron minerals (i.e., magnetite [Fe3O4] or greigite [Fe3S4]). Here, we report a novel magnetotactic spirillum (XQGS-1) that is phylogenetically affiliated with the Alphaproteobacteria class. In addition to magnetite crystals, XQGS-1 cells form intracellular submicrometer calcium carbonate and calcium phosphate granules. This finding supports the view that MTB are also an important microbial group for intracellular calcium carbonate and calcium phosphate biomineralization.

Monitoring pilot-scale polyhydroxyalkanoate production from fruit pulp waste using near-infrared spectroscopy

Given the current plastic disposal global crisis, biodegradable polymers may have an important role in leading the way towards a more sustainable plastic industry. Polyhydroxyalkanoates (PHA) are biobased and biodegradable aliphatic polyesters synthesized by bacteria and stored as intracellular granules. In this study, PHA was produced at pilot scale by a mixed microbial culture (MMC) fed with fermented fruit pulp waste. The PHA accumulation reactor was monitored at line by near-infrared (NIR) spectroscopy to develop and optimize partial least squares (PLS) calibration models for predicting bulk PHA concentration and intracellular PHA content. The PLS models were subjected to internal cross-validation (62 spectra) and the ones with best performance were validated with an external test set (12 spectra). For bulk PHA concentration, a root mean squared error of prediction (RMSEP) of 0.69 g/L and a coefficient of determination (R2) of 0.89 were attained, while intracellular PHA content was predicted with a RMSEP of 14.6% and a R2 of 0.86. These NIR-based calibration models demonstrated a great potential for the real-time monitoring of the MMC pilot-scale PHA production from a complex fruit pulp waste substrate. This approach can be used for in situ control of the pulse-wise feeding strategy of the PHA accumulation stage, minimizing PHA consumption triggered by carbon source depletion and contributing for an improved global process efficiency and productivity.

A review on the potential of polyhydroxyalkanoates production from oil-based substrates

Polyhydroxyalkanoate (PHA) is a type of polyesters produced in the form of accumulated intracellular granules by many microorganisms. It is viewed as an environmentally friendly bioproduct due to its biodegradability and biocompatibility. The production of the PHA using oil substrates such as waste oil and plant oil, has gained considerable attention due to the high product yield and lower substrate cost. Nevertheless, the PHA fermentation using oil substrate is complicated due to the heterogenous fatty acid composition, varied bio-accessibility and possible inhibitory effect on the bacterial culture. This review presents the current state-of-the-art of PHA production from oil-based substrates. This paper firstly discusses the technical details, such as the choice of bacteria strain and fermentation conditions, characteristic of the oil substrate as well as the PHA composition and application. Finally, the paper discusses the challenges and prospects for up-scaling towards a cleaner and effective bioprocess. From the literature review, depending on the cell culture and the type of PHA produced, the oil platform can have a PHA yield of 0.2–0.8 g PHA/g oil substrate, with PHA content mostly from 40 to 90% of the cell dry weight. There is an on-going search for more effective oil-utilising PHA producers and lower cost substrate for effective PHA production. The final application of the PHA polymer influences the treatment needed during downstream processing and its economic performance. PHA with different compositions exhibits varied decomposition behaviour under different conditions, requiring further insight towards its management towards a sustainable circular economy.

Electrospinning nanofibers of microbial polyhydroxyalkanoates for applications in medical tissue engineering

AbstractDifferently to most chemically synthesized medical materials, polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage granules, which is a family of natural bio‐polymers synthesized by microorganism's materials. Due to excellent biocompatibility reasonable biodegradability and versatile material difference, PHAs are well medical biomaterials candidates for applications in tissue engineering and drug delivery, including commercial PHB, PHBV, PHBHHx, PHBVHHx, P34HB and few uncommercial PHAs. Electrospinning nanofibers with the size of 10–10,000 nm can improve the mechanical properties and decrease the crystallinity of PHA, meanwhile simulate the structure and function of native extracellular matrix of cells. Hence, PHAs electrospinning nanofibers as engineered scaffolds have been widely used for tissue engineering scaffolds in cardiovascular, vascular, nerve, bone, cartilage and skin; also, as carriers for application in drug delivery system. In this review, we highlight the extraction and properties of medical PHAs from natural or engineered microorganism, and microstructure, current manufacturing techniques and medical applications of electrospinning nanofibers of PHAs. Moreover, the current challenges and prospects of PHAs electrospinning nanofibers are discussed rationally, providing an insight into developing vibrant fields of PHAs electrospinning nanofibers‐based biomedicine.

Polyhydroxyalkanoate (PHA) Production in Pseudomonas sp. phDV1 Strain Grown on Phenol as Carbon Sources.

Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

Reticulated platelets – a new risk factor of atherothrombosis?

In this review we described the properties of reticulated platelets (RP) and showed how variations of their content might influence platelet activity, efficacy of antiplatelet drugs and the rate of thrombotic events in patients with cardiovascular diseases. RP represent a minor platelet fraction containing residual RNA from megakaryocytes. Platelets have no nucleus and do not synthesize RNA de novo, and RNA of megakaryocytic origin is destroyed during their circulation. That is why only recently produced “young” platelets contain RNA. In healthy donors RP are identified by staining with the RNA specific fluorescent dyes by flow cytofluorimetry or using standard protocols in modern flow haematological analyzers. RP content in blood reflects the level of thrombocytopoesis in the bone marrow. RP on average amounted from 3 to 10% of all platelets in the circulation depending on the method applied for their determination. RP absolute amount and/or their percentage is changed in haematological diseases associated with the alterations of megakaryocyte productive properties. RT measurements in patients with cardiovascular diseases have shown that their content is increased in acute coronary syndrome patients. RP are larger and functionally more active in comparison with not reticulated forms. They more frequently incorporate into the platelet aggregates and contain more intracellular granules. Increase of RT content in the circulation correlates with the increase of the average size and functional activity in the whole platelet population. High RP content in patients with cardiovascular diseases reduces antiaggregative effects of aspirin and P2Y12 APD receptor antagonists and increases the risk of atherothrombotic events.

Isolation and Characterization of Polyhydroxyalkanoates (PHAs) Producers from Kg Batu Melintang hotspring

The increasing awareness on the negative environmental impact of petroleum-based plastics has driven industries to explore more efficient biodegradable polymers for production of bioplastic. Polyhydroxyalkanoates (PHAs) is one of the potential biodegradable polymers to replace petroleum-based plastic. It is synthesized and accumulated as intracellular granules in microorganism. In this study, polyhydroxyalkanoates (PHAs) producing bacteria were successfully isolated from sediment collected from Kg. Batu Melintang hotspring. Isolation process was carried on Minimal Salt Medium (MSM) agar supplemented with excess glucose as a carbon source. Potential PHA producers were screened by using Nile Blue staining plate assay. Out of 144 bacterial isolates, 12 bacterial isolates which showed strong orange fluorescence under ultraviolet (UV) light (365nm) were selected for further identification by morphological characterization and biochemical analysis. Based on the result obtained, possible species for Gram positive rod shape bacteria B75 and B87 is Corynebacterium kutsceri meanwhile Gram negative rod shape bacteria A4, A12, A50, A68, B2, B13, B22, B31, B73 and C3 showed affiliation to Citrobacter sp., Enterobacter sp., Erwinia sp., Klebsiella sp., Proteus sp., Salmonella sp., Serratia sp., Shigella sp., and Yersinia sp.

Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by microorganisms as intracellular energy reservoirs under stressful environmental conditions. PHA synthase (PhaC) is the key enzyme responsible for PHA biosynthesis, but the importance of its N- and C-terminal ends still remains elusive. Six plasmid constructs expressing truncation variants of Aquitalea sp. USM4 PhaC (PhaC1As) were generated and heterologously expressed in Cupriavidus necator PHB−4. Removal of the first six residues at the N-terminus enabled the modulation of PHA composition without altering the PHA content in cells. Meanwhile, deletion of 13 amino acids from the C-terminus greatly affected the catalytic activity of PhaC1As, retaining only 1.1–7.4% of the total activity. Truncation(s) at the N- and/or C-terminus of PhaC1As gradually diminished the incorporation of comonomer units, and revealed that the N-terminal region is essential for PhaC1As dimerization whereas the C-terminal region is required for stabilization. Notably, transmission electron microscopy analysis showed that PhaC modification affected the morphology of intracellular PHA granules, which until now is only known to be regulated by phasins. This study provided substantial evidence and highlighted the significance of both the N- and C-termini of PhaC1As in regulating intracellular granule morphology, activity, substrate specificity, dimerization and stability of the synthase.

Single‐shot quantitative phase imaging as an extension of differential interference contrast microscopy

Various studies have been conducted to obtain quantitative phase information based on differential interference contrast (DIC) microscopy. As one such attempt, we propose in this study a single-shot quantitative phase imaging (QPI) method by combining two developments. First, an add-on optical system to a commercialized DIC microscope was developed to perform quantitative phase gradient imaging (QPGI) with single image acquisition using a polarization camera. Second, an algorithm was formulated to reconstitute QPI from the obtained QPGI by reducing linear artifacts which arise in simply integrated QPGI images. To demonstrate the applicability of the developed system in cell biology, the system was used to measure various cell lines and compared with fluorescence microscopy images of the same field of view. Consistent with previous studies, nucleoli and lipid droplets can be imaged by the system with greater optical path lengths (OPL). The results also implied that combining fluorescence microscopy and the developed system might be more informative for cell biology research than using these methods individually. Exploiting the single-shot performance of the developed system, time-lapse imaging was also conducted to visualize the dynamics of intracellular granules in monocyte-/macrophage-like cells. Our proposed approach may accelerate the implementation of QPI in standard biomedical laboratories.

Autophagy of mucin granules contributes to resolution of airway mucous metaplasia

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

Isolation of Two Bacterial Species from Argan Soil in Morocco Associated with Polyhydroxybutyrate (PHB) Accumulation: Current Potential and Future Prospects for the Bio-Based Polymer Production.

The environmental issues caused by the impacts of synthetic plastics use and derived wastes are arising the attention to bio-based plastics, natural polymers produced from renewable resources, including agricultural, industrial, and domestic wastes. Bio-based plastics represent a potential alternative to petroleum-based materials, due to the insufficient availability of fossil resources in the future and the affordable low cost of renewable ones that might be consumed for the biopolymer synthesis. Among the polyhydroxyalkanoates (PHA), the polyhydroxybutyrate (PHB) biopolymer has been synthesized and characterized with great interest due to its wide range of industrial applications. Currently, a wide number of bacterial species from soil, activated sludge, wastewater, industrial wastes, and compost have been identified as PHB producers. This work has the purpose of isolating and characterizing PHB-producing bacteria from the agricultural soil samples of Argania spinosa in the south region of Morocco where the plant species is endemic and preserved. During this research, four heat-resistant bacterial strains have been isolated. Among them, two species have been identified as endospore forming bacteria following the Schaffer-Fulton staining method with Malachite green and the Methylene blue method. Black intracellular granules have been appreciated in microscopy at 100× for both strains after staining with Sudan black B. The morphological and biochemical analyses of the isolates, including sugar fermentation and antibiotic susceptibility tests, preliminarily identified the strains 1B and 2D1 belonging to the genus Serratia and Proteus, respectively.

Innate immune cells in the tumor microenvironment.

The tumor immune microenvironment (TIME) is a complex ecosystem that contains adaptive and innate immune cells that have tumor-promoting and anti-tumor effects. There is still much to learn about the diversity, plasticity, and functions of innate immune cells in the TIME and their roles in determining the response to immunotherapies. Experts discuss recent advances in our understanding of their biology in cancer as well as outstanding questions and potential therapeutic avenues.

BIOSYNTHESIS OF POLY (HYDROXYALKANOATES)

Bioplastics are biodegradable polymers that are made by bacterial cells, whose molecules have biochemical properties similar to petrochemical polymers as they are concentrated inside intracellular granules as a carbon and energy sources, and which then degrade in the environment as they get exposed to light. Bioplastics have desirable biodegradable and renewable qualities, sustainable qualities as well as alternatives to petroleum-based plastics, and is used due to much of its biodegradability and durability. Due to their vast usefulness, Poly Hydroxy Alkanoate (PHAs) and their derivatives are used in many different sectors, PHAs are the most biodegradable bioplastics found throughout the world. These polymers are generated by microorganisms through metabolic pathways that start with hydroxy-acyl-A and end with different types of acyl-A. Bioplastic polymers have chemical characteristics that differ in structural and physical properties which also differ due to the source of their polymerization microorganisms.

Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy.

Haloferax mediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

Eosinophil Response Against Classical and Emerging Respiratory Viruses: COVID-19.

Eosinophils were discovered more than 140 years ago. These polymorphonuclear leukocytes have a very active metabolism and contain numerous intracellular secretory granules that enable multiple effects on both health and disease status. Classically, eosinophils have been considered important immune cells in the pathogenesis of inflammatory processes (eg, parasitic helminth infections) and allergic or pulmonary diseases (eg, asthma) and are always associated with a type 2 immune response. Furthermore, in recent years, eosinophils have been linked to the immune response by conferring host protection against fungi, bacteria, and viruses, which they recognize through several molecules, such as toll-like receptors and the retinoic acid-inducible gene 1-like receptor. The immune protection provided by eosinophils is exerted through multiple mechanisms and properties. Eosinophils contain numerous cytoplasmatic granules that release cationic proteins, cytokines, chemokines, and other molecules, all of which contribute to their functioning. In addition to the competence of eosinophils as effector cells, their capabilities as antigen-presenting cells enable them to act in multiple situations, thus promoting diverse aspects of the immune response. This review summarizes various aspects of eosinophil biology, with emphasis on the mechanisms used and roles played by eosinophils in host defence against viral infections and response to vaccines. The review focuses on respiratory viruses, such as the new coronavirus, SARS-CoV-2.

Characteristics of normal human retinal pigment epithelium cells with extremes of autofluorescence or intracellular granule count.

Background:Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation.Results:Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell.Conclusions:In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscińs role in aging and AMD further into perspective.

Mitochondria Are Potential Targets for the Development of New Drugs Against Neutrophilic Inflammation in Severe Pneumonia Including COVID-19.

A high neutrophil-to-lymphocyte ratio is associated with disease severity and poor prognosis in pneumonia progressing to acute respiratory distress syndrome (ARDS) (Steinberg et al., 1994) including COVID-19 (Coronavirus Disease-19) caused by the novel SARS-CoV-2 coronavirus (Wang et al., 2020a; Mehta et al., 2020). Extensive infiltration of neutrophils into the pulmonary capillaries as well as their extravasation into the alveolar space have been described in bronchoalveolar lavage (Steinberg et al., 1994) and in autopsy specimens (Barnes et al., 2020; Wang et al., 2020b). Neutrophils are the first line of defense against invading pathogens in the foci of inflammation, where they use effector functions such as phagocytosis, degranulation, and the formation of reactive oxygen species (ROS). Excessive activation of neutrophils results in the release of neutrophil extracellular traps (NETs) consisting of decondensed chromatin, «decorated» with myeloperoxidase (MPO), neutrophil elastase (NE), and other bactericidal proteins derived from intracellular granules (Brinkmann et al., 2004). The formation of NETs is usually accompanied by cell death, therefore, this process is called NETosis. Examination of patients with severe pneumonia (Twaddell et al., 2019) as well as patients infected with SARS-CoV-2 (Zuo et al., 2020) revealed an increased level of NETosis markers, such as cell-free DNA, MPO-DNA-complexes, citrullinated histone H3, and a marker of cell death lactate dehydrogenase. In the serum from patients with COVID-19 the concentration of cell-free DNA correlated with the content of neutrophils, the marker of the acute phase of inflammation C-reactive protein, and the marker of thrombosis D-dimer (Zuo et al., 2020). This serum induced NETosis in healthy donor blood in an in vitro system (Barnes et al., 2020; Zuo et al., 2020). One of the manifestations of COVID-19 is Kawasaki syndrome, a vasculitis that occurs in children and is accompanied by excessive NETosis (Yoshida et al., 2020). NETosis in COVID-19 can be caused by epithelial and endothelial cells affected with the virus, by activated platelets, and by inflammatory cytokines. At the same time, excessive NETosis is involved in the development of the «cytokine storm» and immunothrombosis, which are the main cause of severe complications associated with COVID-19 (Wang et al., 2020a; Barnes et al., 2020), as well as with H1N1 influenza and some other viral infections (Cantan et al., 2019).

Single- or double-membrane-bound vesicles and P, Ca, and Fe-containing granules in Xanthomonas citri cultured on a solid medium

The organelle-like structures of Xanthomonas citri, a bacterial pathogen that causes citrus canker, were investigated using an analytical transmission electron microscope. After high-pressure freezing, the bacteria were then freeze-substituted for imaging and element analysis. Miniscule electron-dense structures of varying shapes without a membrane enclosure were frequently observed near the cell poles in a 3-day culture. The bacteria formed cytoplasmic electron-dense spherical structures measuring approximately 50 nm in diameter. Furthermore, X. citri produced electron-dense or translucent ellipsoidal intracellular or extracellular granules. Single- or double-membrane-bound vesicles, including outer-inner membrane vesicles, were observed both inside and outside the cells. Most cells had been lysed in the 3-week X. citri culture, but they harbored one or two electron-dense spherical structures. Contrast-inverted scanning transmission electron microscopy images revealed distinct white spherical structures within the cytoplasm of X. citri. Likewise, energy-dispersive X-ray spectrometry showed the spatial heterogeneity and co-localization of phosphorus, oxygen, calcium, and iron only in the cytoplasmic electron-dense spherical structures, thus corroborating the nature of polyphosphate granules.