Extracellular Route Research Articles

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Stimulated Raman scattering microscopy reveals a unique and steady nature of brain water dynamics

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

Biomimetic Synthesis of Nanoparticles: State-of-the-Art

A state-of-the-art review of biomimetic nanoparticle synthesis is presented. The technique’s origin has been traced to the studies, started over 150 years ago, on the hyperaccumulation of certain metals by different species of plants. How the initial intracellular method of nanoparticle synthesis evolved into the now widely used extracellular route has been described. The review then covers the gist of all the studies reported on the biomimetic synthesis of nanoparticles of different metals using extracts of different botanical species (plants). The synthesis mechanism is discussed, and the factors influencing the nanoparticles’ extent, shapes, and sizes are identified.

Chlamydia trachomatis Cell-to-Cell Spread through Tunneling Nanotubes.

Tunneling nanotubes (TNTs) are transient cellular connections that consist of dynamic membrane protrusions. They play an important role in cell-to-cell communication and mediate the intercellular exchanges of molecules and organelles. TNTs can form between different cell types and may contribute to the spread of pathogens by serving as cytoplasmic corridors. We demonstrate that Chlamydia (C.) trachomatis-infected human embryonic kidney (HEK) 293 cells and other cells form TNT-like structures through which reticulate bodies (RBs) pass into uninfected cells. Observed TNTs have a life span of 1 to 5 h and contain microtubules, which are essential for chlamydial transfer. They can bridge distances of up to 50 μm between connecting neighboring cells. Consistent with the biological role for TNTs, we show that C. trachomatis spread also occurs under conditions in which the extracellular route of chlamydial entry into host cells is blocked. Based on our findings, we propose that TNTs play a critical role in the direct, cell-to-cell transmission of chlamydia. IMPORTANCE Intracellular bacterial pathogens often undergo a life cycle in which they parasitize infected host cells in membranous vacuoles. Two pathways have been described by which chlamydia can exit infected host cells: lytic cell destruction or exit via extrusion formation. Whether direct, cell-to-cell contact may also play a role in the spread of infection is unknown. Tunneling nanotubes (TNTs) interconnect the cytoplasm of adjacent cells to mediate efficient communication and the exchange of material between them. We used Chlamydia trachomatis and immortalized cells to analyze whether TNTs mediate bacterial transmission from an infected donor to uninfected acceptor cells. We show that chlamydia-infected cells build TNTs through which the intracellular reticulate bodies (RBs) of the chlamydia can pass into uninfected neighboring cells. Our study contributes to the understanding of the function of TNTs in the cell-to-cell transmission of intracellular pathogens and provides new insights into the strategies by which chlamydia spreads among multicellular tissues.

For the road: calibrated maternal investment in light of extracellular symbiont transmission.

Faithful transmission of beneficial symbionts is critical for the persistence of mutualisms. Many insect groups rely on extracellular routes that require microbial symbionts to survive outside the host during transfer. However, given a prolonged aposymbiotic phase in offspring, how do mothers mitigate the risk of symbiont loss due to unsuccessful transmission? Here, we investigated symbiont regulation and reacquisition during extracellular transfer in the tortoise beetle, Chelymorpha alternans (Coleoptera: Cassidinae). Like many cassidines, C. alternans relies on egg caplets to vertically propagate its obligate symbiont Candidatus Stammera capleta. On average, each caplet is supplied with 12 symbiont-bearing spheres where Stammera is embedded. We observe limited deviation (±2.3) in the number of spheres allocated to each caplet, indicating strict maternal control over symbiont supply. Larvae acquire Stammera 1 day prior to eclosion but are unable to do so after hatching, suggesting that a specific developmental window governs symbiont uptake. Experimentally manipulating the number of spheres available to each egg revealed that a single sphere is sufficient to ensure successful colonization by Stammera relative to the 12 typically packaged within a caplet. Collectively, our findings shed light on a tightly regulated symbiont transmission cycle optimized to ensure extracellular transfer.

Staphylococcal Protein A with Engineered Cysteine: Comparison of Monomeric Content as a Critical Quality Attribute during Intracellular and Extracellular Expression

Background: The introduction of engineered cysteine in staphylococcal protein A (SPA-cys) for site-specific conjugation results in a substantial amount of dimerized SPA due to spontaneous oxidation during its production, leading to inaccessibility and thus rendering it unusable. Monomers are usually recovered from dimers by using reducing agents before conjugation in subsequent steps. However, this leads to low conjugation efficiency and increases overall cost and production time. This study aims to systematically compare and quantify the monomeric and dimeric content of SPA when produced through intracellular and extracellular routes in E. coli. Methods: Purified SPAs with and without cysteine from both intracellular and extracellular processes are compared for their monomeric content and efficiency to conjugate on solid support matrix with and without an additional pre-step of reduction. Results: The monomeric form of SPA-cys, which is a desired key quality attribute, is less than 50% when produced extracellularly. SPA-cys produced through the intracellular production process has high monomeric content (≥85%) and shows higher binding to solid support. Conclusion: The study demonstrates that the intracellular route for production of SPA-cys should be the preferred method, and the release assays for SPA-cys products should include the amount of monomeric content as one of the quality attributes. The abundance of monomeric content enhances the site-specific conjugation efficiency and density of SPA on the resin matrix.

Zinc Oxide Nanoparticles Bacterial Synthesis and Application

Attributed to their unique properties zinc oxide nanoparticles (ZnONPs) in recent years, have received considerable attention. Notably, research has demonstrated that zinc in living organisms is an essential nutrient. For example, both eukaryotes and prokaryotes including fungi, bacteria, and yeast by using protein, enzyme or microbial cells and other biomolecules compounds are exploited for the synthesis of ZnONPs in either an extracellular or intracellular route. However, the properties of nanoparticles (NPs) are depended upon their shape and size. ZnONPs demonstrated antimicrobial properties, making them unique for different applications. Therefore, in the current study Due to its extensive antimicrobial activity, eco-friendly and simplicity, ZnO Nanoparticles (ZnONPs) synthesis has gained much attention. So, this review was done to concentrate about the green synthesized of ZnONPs by bacteria and their application. Also, the study of their effect as biopesticides against the House Fly, Musca domestica L was mentioned.

Extracellular electron transfer routes in microbiologically influenced corrosion of X80 steel by Bacillus licheniformis

Bacillus licheniformis can take up electrons from X80 steel for nitrate reduction and accelerate corrosion. In this work, the routes B. licheniformis adopts for extracellular electron transfer (EET) were revealed using electrochemical tests, fluorescence staining, high performance liquid chromatography (HPLC) and weight loss tests, and their kinetics were also analyzed in detail. The results showed that B. licheniformis can take up electrons from steel surface via three routes: direct electron transfer by cytochromes, direct electron transfer by flavin-bound cytochromes and mediated electron transfer by diffusible flavin. B. licheniformis itself can only secrete traces of riboflavin which are mainly bound to the surface cytochromes assisting electron transfer. Adding flavins can increase the bound content, and with the binding sites of cytochrome becoming saturated the rest of flavins will mediate electron transfer through diffusion even more efficiently than the bound.

Linking GOLPH3 and Extracellular Vesicles Content-a Potential New Route in Cancer Physiopathology and a Promising Therapeutic Target is in Sight?

Golgi phosphoprotein 3 (GOLPH3), a highly conserved phosphatidylinositol 4-phosphate effector, is required for maintenance of Golgi architecture, vesicle trafficking, and Golgi glycosylation. GOLPH3 overexpression has been reported in several human solid cancers, including glioblastoma, breast cancer, colorectal cancer, nonsmall cell lung cancer, epithelial ovarian cancer, prostate cancer, gastric cancer, and hepatocellular carcinoma. Although the molecular mechanisms that link GOLPH3 to tumorigenesis require further investigation, it is likely that GOLPH3 may act by controlling the intracellular movement of key oncogenic molecules, between the Golgi compartments and/or between the Golgi and the endoplasmic reticulum. Indeed, numerous evidence indicates that deregulation of intracellular vesicle trafficking contributes to several aspects of cancer phenotypes. However, a direct and clear link between extracellular vesicle movements and GOLPH3 is still missing. In the past years several lines of evidence have implicated GOLPH3 in the regulation of extracellular vesicle content. Specifically, a new role for GOLPH3 has emerged in controlling the internalization of exosomes containing either oncogenic proteins or noncoding RNAs, especially micro-RNA. Although far from being elucidated, growing evidence indicates that GOLPH3 does not increase quantitatively the excretion of exosomes, but rather regulates the exosome content. In particular, recent data support a role for GOLPH3 for loading specific oncogenic molecules into the exosomes, driving both tumor malignancy and metastasis formation. Additionally, the older literature indirectly implicates GOLPH3 in cancerogenesis through its function in controlling hepatitis C virus secretion, which in turn is linked to hepatocellular carcinoma formation. Thus, GOLPH3 might promote tumorigenesis in unexpected ways, involving both direct and indirect routes. If these data are further confirmed, the spectrum of action of GOLPH3 in tumor formation will significantly expand, indicating this protein as a strong candidate for targeted cancer therapy.

Significant anisotropic neuronal refractory period plasticity

Refractory periods are an unavoidable feature of excitable elements, resulting in necessary time-lags for re-excitation. Herein, we measure neuronal absolute refractory periods (ARPs) in synaptic blocked neuronal cultures. In so doing, we show that their duration can be significantly extended by dozens of milliseconds using preceding evoked spikes generated by extracellular stimulations. The ARP increases with the frequency of preceding stimulations, and saturates at the intermittent phase of the neuronal response latency, where a short relative refractory period might appear. Nevertheless, preceding stimulations via a different extracellular route does not affect the ARP. It is also found to be independent of preceding intracellular stimulations. All these features strongly suggest that the anisotropic ARPs originate in neuronal dendrites. The results demonstrate the fast and significant plasticity of the neuronal ARP, depending on the firing activity of its connecting neurons, which is expected to affect network dynamics.

Mycoviruses in Biological Control: From Basic Research to Field Implementation.

Mycoviruses from plant pathogens can induce hypovirulence (reduced virulence) in their host fungi and have gained considerable attention as potential biocontrol tools. An increasing number of mycoviruses that induce fungal hypovirulence, from a wide variety of taxonomic groups, are currently being reported. Successful application of these viruses in disease management is greatly dependent on their ability to spread in the natural populations of the pathogen. Mycoviruses generally lack extracellular routes of transmission. Hyphal anastomosis is the main route of horizontal mycovirus transmission to other isolates, and conidia of vertical transmission to the progeny. Transmission efficiencies are influenced by both the fungal host and the infecting virus. Interestingly, artificial transfection methods have shown that potential biocontrol mycoviruses often have the ability to infect a variety of fungi. This expands their possible use to the control of pathogens others than those where they were identified. Mycovirus research is also focused on gaining insights into their complex molecular biology and the molecular bases of fungus-virus interactions. This knowledge could be exploited to manipulate the mycovirus and/or the host and generate combinations with enhanced properties in biological control. Finally, when exploring the use of mycoviruses in field conditions, the pathogen life style and the characteristics of the disease and crops affected will deeply impact the specific challenges to overcome, and the development of biocontrol formulations and delivery methods.

Drug Delivery with Extracellular Vesicles: From Imagination to Innovation

ConspectusExtracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications.As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and on subsequent vesicle activity.We try to meet this challenge by careful experimental design and development of tools that enable robust readouts. By engineering the surface and cargo of extracellular vesicles through chemical and biological techniques, favorable characteristics can be enforced while unfavorable qualities can be overruled or masked. This is coupled to the precise evaluation of the interaction of extracellular vesicles with cells to determine the extracellular vesicle uptake routes and intracellular routing. Sensitive reporter assays enable reproducible analysis of functional delivery.This systematic evaluation and optimization of extracellular vesicles improves our insight into the critical determinants of extracellular vesicle activity and should improve translation into clinical application of engineered extracellular vesicles as a new class of drug delivery systems.

Key aspects of PCSK9 inhibition beyond LDL lowering.

Our primary objective is to review the most recent findings on the biology of PCSK9 and on two key aspects of PCSK9 inhibition beyond LDL control of great clinical relevance: the regulation of lipoprotein (a) circulating levels by PCSK9 inhibitors and the putative diabetogenic effects of these novel therapies. The reality of two distinct extracellular and intracellular pathways by which PCSK9 decreases the abundance of the LDLR at the surface of many cell types, most importantly hepatocytes, has recently been established. In contrast, the exact mechanisms by which PCSK9 inhibitors lower the circulating levels of lipoprotein (a) remain a point of major dispute. Despite strong indications from genetic studies that PCSK9 inhibition should increase diabetes risk, no such effect has been observed in clinical trials, and in-vitro and in-vivo studies do not clarify this issue. The trafficking pathways by which PCSK9 enhance LDLR degradation via the endolysosomal extracellular route or via the Golgi-lysosomal intracellular route remain to be fully elucidated. The mechanisms by which PCSK9 inhibitors reduce lipoprotein (a) also merit additional research efforts. The role of PCSK9 on glucose metabolism should likewise be studied in depth.

Mechanisms of Pulmonary Escape and Dissemination by Cryptococcus neoformans

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

What Goes Around, Comes Around - HSV-1 Replication in Monocyte-Derived Dendritic Cells.

HSV-1 is a very successful human pathogen, known for its high sero-prevalence and the ability to infect a wide range of different cell types, including dendritic cells (DCs). As very potent antigen-presenting cells DCs play an important role in the induction of antiviral immune responses and therefore represent a strategic target for viral-mediated immune escape mechanisms. It is known that HSV-1 completes its gene expression profile in immature as well as in mature DCs, while lytic infection is only found in immature DCs (iDCs). Notably, HSV-1 infected mature DCs (mDCs) fail to release infectious progeny virions into the supernatant. Apart from HSV-1 dissemination via extracellular routes cell-to-cell spread counteracts a yet unknown mechanism by which the virus is trapped in mDCs and not released into the supernatant. The dissemination in a cell–cell contact-dependent manner enables HSV-1 to infect bystander cells without the exposure toward the extracellular environment. This supports the virus to successfully infect the host and establish latency. In this review the mechanism of HSV-1 replication in iDCs and mDCs and its immunological as well as virological implications, will be discussed.

Toward establishing minimum requirements for extracellular electron transfer in Geobacter sulfurreducens.

The highly redundant pathways for extracellular electron transfer in Geobacter sulfurreducens must be simplified for this microorganism to serve as an effective chassis for applications such as the development of sensors and biocomputing. Five homologs of the periplasmic c-type cytochromes, PpcA-E, offer the possibility of multiple routes of electron transfer across the periplasm. The presence of a large number of outer membrane c-type cytochromes allows G. sulfurreducens to adapt to disruption of an electron transfer pathway in the outer membrane. A strain in which genes for all five periplasmic cytochromes, PpcA-E, were deleted did not reduce Fe(III). Introducing ppcA under the control of an IPTG-inducible system in the quintuple deletion strain yielded a strain that reduced Fe(III) only in the presence of IPTG. A strain lacking known major outer membrane cytochromes, OmcB, OmcE, OmcS and OmcT, and putative functional homologs of OmcB, did not reduce Fe(III). Introduction of omcB in this septuple deletion strain restored the ability to reduce Fe(III). These results demonstrate that it is possible to trim redundancy from the extracellular electron transfer pathways in G. sulfurreducens in order to construct strains with defined extracellular electron transfer routes.

Orchestrating rapid long‐distance signaling in plants with Ca2+, ROS and electrical signals

Plants show a rapid systemic response to a wide range of environmental stresses, where the signals from the site of stimulus perception are transmitted to distal organs to elicit plant-wide responses. A wide range of signaling molecules are trafficked through the plant, but a trio of potentially interacting messengers, reactive oxygen species (ROS), Ca2+ and electrical signaling ('trio signaling') appear to form a network supporting rapid signal transmission. The molecular components underlying this rapid communication are beginning to be identified, such as the ROS producing NAPDH oxidase RBOHD, the ion channel two pore channel 1 (TPC1), and glutamate receptor-like channels GLR3.3 and GLR3.6. The plant cell wall presents a plant-specific route for possible propagation of signals from cell to cell. However, the degree to which the cell wall limits information exchange between cells via transfer of small molecules through an extracellular route, or whether it provides an environment to facilitate transmission of regulators such as ROS or H+ remains to be determined. Similarly, the role of plasmodesmata as both conduits and gatekeepers for the propagation of rapid cell-to-cell signaling remains a key open question. Regardless of how signals move from cell to cell, they help prepare distant parts of the plant for impending challenges from specific biotic or abiotic stresses.

Physiological roles of claudins in kidney tubule paracellular transport.

The paracellular pathways in renal tubular epithelia such as the proximal tubules, which reabsorb the largest fraction of filtered solutes and water and are leaky epithelia, are important routes for transepithelial transport of solutes and water. Movement occurs passively via an extracellular route through the tight junction between cells. The characteristics of paracellular transport vary among different nephron segments with leaky or tighter epithelia. Claudins expressed at tight junctions form pores and barriers for paracellular transport. Claudins are from a multigene family, comprising at least 27 members in mammals. Multiple claudins are expressed at tight junctions of individual nephron segments in a nephron segment-specific manner. Over the last decade, there have been advances in our understanding of the structure and functions of claudins. This paper is a review of our current knowledge of claudins, with special emphasis on their physiological roles in proximal tubule paracellular solute and water transport.

Lefty Glycoproteins in Human Embryonic Stem Cells: Extracellular Delivery Route and Posttranslational Modification in Differentiation.

Lefty is a member of transforming growth factor-beta (TGF-β) superfamily and a potent antagonist of the TGF-β/Nodal/Activin signaling pathway. Lefty is critical in sustaining self-renewal/pluripotency status, and implicated in the differentiation of embryonic stem cells (ESCs). However, emerging studies depict Lefty as a multifaceted protein involved in myriad cellular events. Lefty proteins (human Lefty A and B) are secreted glycoproteins, but their mode of secretion and the significance of their "glycan" moiety remain mostly unexplored. By employing an in vitro system of human ESCs (hESCs), we observed that Lefty protein(s) are encased in exosomes for extracellular release. The exosomal- and cell-associated Lefty diverge in their proteolytic processing, and possess N-glycan structures of high mannose and complex nature. Differentiation of hESCs to mesenchymal cells (MSCs) or neuronal progenitor cells (NPCs) entails distinct changes in the Lefty A/Lefty B gene(s), and protein expression. Specifically, the proteolytic cleavage and N-glycan composition of the cell-associated and exosomal Lefty differ in the differentiated progenies. These modifications affected Lefty's inhibitory effect on Nodal signaling in aggressive melanoma cells. The microheterogeneity in the processing and glycosylation of Lefty protein(s) between hESCs, MSCs, and NPCs could present efficient means of diversifying the endogenous functions of Lefty. Whether Lefty's diverse functions in embryonic patterning, as well as its diffusion range in the extracellular environment, are similarly affected remains to be determined. Our studies underscore the potential relevance of Lefty-packaged exosomes for combating debilitating diseases such as cancer.