Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration. Germ plasm is a maternally supplied substance comprised of RNAs, proteins, and organelles that are amassed in oocytes and later is sequestered during the first embryonic cleavages within a few cells that will become the primordial germ cells (PGCs). In the next century, the identification of conserved germline-specific markers and the germ cell accumulation of alkaline phosphatase made it possible to trace the origins of the germline from its earliest emergence through PGC migration to the presumptive gonad where they differentiate as male or female gametes. Such lineage tracing revealed that indeed some animals establish their germline by inheritance of maternal factors and post-transcriptional regulation in the context of a silenced genome but that others do not. In the latter case, these animals lack detectable maternal germ plasm and induce their germ cells by zygotic transcription factors. Genetic and overexpression screens to identify germ cell inducers have uncovered only a few factors with the capacity to generate ectopic PGCs.Oskar3 andbucky ball4 are “drivers” of germline fate among animals that use a maternal inheritance mode of PGC specification, but these genes are specific to different subsets of species7, and it remains to be determined whether their mechanisms of action and specific activities are conserved 8, but in mouse no germ cell inducer has been identified. This suggests that only a few genes possess germ cell-inducing activity or that the coordinated action of multiple genes is required to establish the germline or both. Consistent with this notion, many factors involved in germ cell development are involved in RNA regulation, including Vasa, a universal marker of and regulator of germ cell development26. Recently, investigators proposed a “last cell standing model”, whereby early PGC determination, as occurs in maternal germ plasm inducers, is not an innovation to protect germline traits. Instead, they proposed that germ plasm provides a means to specify the germline lineage earlier, before gastrulation, and thereby liberate the somatic cells of the embryo to more rapidly evolve27. According to this model, innovation in animals that do not specify the germline early—prior to or in early gastrulation either by maternal germ plasm or inductive modes—but instead specify PGCs late in gastrulation by zygotic inductive modes are constrained by signaling and morphogenesis requirements associated with germ layer specification and gastrulation27. Significantly, many of the same or related genes regulate PGC development independently of specification mode. This review focuses on the earliest events of PGC specification in the zygote, the mechanisms that induce early PGC-like cells in culture.In 1892, August Weismann challenged the notion that the germline (reproductive cells) was derived from the soma (cells of the body). Instead, Weismann proposed that the germ cells possessed a special immortal substance called “ancestral germ plasm” that was inherited from germ cells of one generation to the next in his “theory of the continuity of the germ plasm”nserved . In humaWeismann recognized the germ plasm as a peculiar and complicated structure, but at that time the molecular components were not known. Since then, germ cell-specific accumulations have been detected in germ cells at all stages of the germline cycle. Further adding to the complexity, germ cell substances that have been identified throughout the germline cycle have been referred to by a variety of terms—nuage, germ plasm, and germ granules, or P-granules—at different developmental stages and in different animals. In recent years, molecular identification of the components of these germ cell substances has revealed that the molecular components overlap somewhat and thus nuage, germ plasm, and germ granules have at times been treated as largely equivalent. This assumption has made it challenging to decipher the functions of these germ cell manifestations, particularly when attempting to compare studies of these germ cell substances conducted at different developmental stages and in different organisms. To facilitate comparisons of functional studies between species, definitions of nuage, germ plasm, and germ granules in the context of this review are provided below.28. Although nuage occupies a distinct subcellular space, it is generally not asymmetric in its distribution. In some animals, a subset of nuage components become asymetrically localized to a specific subcellular location in oocytes where the germ plasm that is transmitted from oocytes to the embryo forms . piRNAs are components of a gonad-specific RNA silencing pathway that is thought to protect genome integrity by counteracting transposable or selfish genetic elements that promote their own transmission at the expense of other elements  of germ plasm that can impart germ cell identity, the germ plasm nucleators or assemblers, have included isolation and transplantation of cytoplasm from oocytes to embryos to identify the substance with PGC-inducing activity in model systems such asDrosophila65, zebrafish66, andXenopus67. To date, only a limited number of factors that can induce germ cells have been identified, and how the germ plasm assembles remains a key question in the field.In primary oocytes of some animals, maternal germ plasm first assembles within an ancient perinuclear oocyte structure known as the Balbiani bodyates59 . Expressates59 ,59,60Drosophila because of the powerful genetic screens which led to the identification of key factors of germline assembly and germ cell development70, including the germ plasm assembler,oskar71. Alternative translation generates two forms of Oskar with distinct activities72. The short form of Oskar is required for germ plasm assembly and function, whereas long Oskar lacks the assembly activity and instead is required to anchor germ plasm74. Until recently, Oskar (Osk) was viewed as a scaffolding protein that gathered germ plasm via interactions with a myriad of partners94. The regions of Oskar mediating its association with partners such as Vasa defined Oskar functional domains73. The recently elucidated Oskar crystal structure revealed that both Oskar dimerization and interaction with Vasa are mediated via the Osk N-terminal LOTUS domain, named after Limkain, Oskar and Tudor domain-containing proteins 5 and 783. LOTUS is a globular domain present in several germ plasm/granule components95, including the conserved Tudor family, first discovered for Tudor’s function in germ plasm assembly inDrosophila68. This finding is in contrast to previous work that mapped the Vasa interaction to Oskar’s C-terminus73. In the new study, Vasa-Oskar interactions were tested without RNAs to exclude RNA-mediated association and this may explain the different binding sites. These findings further support interaction between Vasa and Oskar and raise new questions and models to explain how these different Oskar complexes promote germ plasm formation and activity.The molecular constituents of the germ plasm are best understood in83. SGNH hydrolases are a large enzyme family with thousands of members that are found in all life forms. An interesting property among some bacterial SGNH domain-containing proteins that may be of relevance to germ plasm assembly is their propensity to oligomerize to form amorphous aggregates or amyloid-like fibrils97. In addition, proteins encoded by some LINE (long interspersed nuclear elements) mobile genetic elements contain SGNH-like domains and mediate RNP assembly99. For example, the zebrafish LINE protein ZfL2-1 ORF1p forms multimers, binds nucleotides, and like Oskar possesses an SGNH-like domain that lacks overt RNA-binding domain structure98. In addition, ORF1ps, including ZfL2-1, have been shown to function as chaperones. In this context, ORF1ps interact with the LINE RNA and are postulated to mediate rearrangement of the RNA into a stable conformation that protects the RNA from degradation, but later can be reversed to facilitate reverse transcription98. Based onin vitro structure function studies, ZfL2-1 is postulated to mediate RNP assembly via interactions with positively charged peptides that bind RNA structural elements99. It remains to be determined whether Oskar has similar chaperone functions, but it is easy to imagine how such an activity could apply to germ plasm RNAs, which must be translationally silent and protected from degradation during transport but later are translated in a specific96 subcellular location. The unique functional domains and distinct interaction properties of Oskar isoforms discussed above provide new models to test the mechanism by which Oskar could promote RNP and germ plasm assembly either directly via its RNA-binding domain or directly or indirectly via its disordered LOTUS and interaction with Vasa.Another exciting aspect of the recent study is the evidence that short, but not long, Oskar associates directly with RNA. Surprisingly, this binding interaction is not via a canonical RNA-binding motif, but instead through a domain that resembles an enzymatically inactive SGNH hydrolase domainoskar and instead have a vertebrate-specific gene calledbucky ball (buc) orvegetally localized 1 (velo1)100. Buc shares features with Oskar in that both are localized in oocytes as RNAs and proteins103, both are required for and can organize germ plasm4, both have been viewed as unstructured proteins, both display complex post-transcriptional regulation at the level of splicing104 and RNA localization105, and both have properties of self-assembling aggregates and interact with RNA-binding proteins and other factors at the RNA and protein levels94. Whether or not Buc protein, like Osk, can directly bind RNAs is unknown. Based on homology searches, Buc lacks identifiable functional domains and thus the mechanisms by which it promotes Balbiani body formation, germ plasm assembly, and oocyte axis specification are not clear and have relied on identifying Buc interaction partners and mapping their interaction sites on Buc106. However, Bucky ball and Oskar germ plasm factors also have some differences -dependent mechanism106. Moreover, the ability of Buc to induce ectopic PGCs requires Mkif5Ba and Buc protein localization to the cleavage furrows106. Similarly, formation of germ plasm aggregates inXenopus requires the Kinesin-like protein Xklp2108, indicating a conserved role for microtubule motor-dependent assembly of germ plasm in these vertebrates. Because germ plasm is present in zebrafish Mkif5Ba mutant embryos but germ cells are absent, this mutant provides evidence that inheritance of maternal germ plasm from oocytes alone is not sufficient to specify PGCs. Moreover, germline establishment requires proper spatiotemporal localization of PGC determinants.Much less is understood about the molecular regulators of maternal germ plasm specification in vertebrates; however, recently, endogenous Buc protein was shown to localize to the cleavage furrows of early embryosDrosophila65, zebrafish66, andXenopus67, in which transplantation of the germ plasm can induce PGCs. In contrast, when germ granules are not properly segregated inCaenorhabditis elegans mutants, excess PGCs do not form nor do all cells adopt germline fates; instead, the cells take somatic fates109. This example suggests that germ granules are not sufficient for PGC fate. These differences in germ cell induction capacity between granules and plasm raise the question of whether germ plasm and granules are based on their shared enrichment of RNA-binding proteins (RNAbps), RNAs, and other overlapping component110 manifestations of the same mechanism/structure operating at different stages or instead are distinct entities with unique properties. Alternatively, not all granules are equivalent. The maternal germ plasm associated with the maternal inheritance specification mode is continuous from oocyte to embryo, whereas perinuclear germ granules assemble only in PGCs, even in zygotically induced/discontinuous modes of germline specification. Thus, maybe these germline entities are not functionally equivalent. If this is the case, germ plasm from oocytes should contain germ cell-inducing factors, which germ granules of PGCs lack. Consistent with this notion, the perinuclear nuage inDrosophila oocytes is distinct from the polar granules nucleated by Oskar111 during specification of pole cells/PGCs65. Moreover, recent studies inDrosophila,C. elegans, and zebrafish provide evidence that germ granules are heterogeneous. InDrosophila, single RNA fluorescentin situ hybridization (FISH) analyses of granules revealed their organized architecture of “core” germ plasm proteins  distributed throughout granules and specific RNAs spatially arranged within granules112. Significantly, although Oskar protein is present in granules,oskar RNA is not113 (osk with ananos 3’ untranslated region (UTR) is directed to granules, germ cell numbers and Vasa expression are reduced113, indicating thatosk segregation from granules is needed for normal PGC development. Taken together, these findings are consistent with plasm and granules being distinct entities inDrosophila. Similarly, endogenous Buc protein localizes to germ plasm of oocytes and early embryos but is not detected in germ granules of PGCs106. Moreover,buc RNA is a component of germ plasm of oocytes but not embryos, and even when overexpressed, Buc protein, but notbuc RNA, localizes to germ granules of zebrafish PGCs106 , or both, are essential for PGC identity. Accordingly, splitting determinants among sister cells, as occurs inC. elegans maternal-effect sterile (mes-1) mutants123, would produce two cells lacking sufficient factors for germline fate. Notably, granules are also heterogeneous and dynamic structures inC. elegans125. Finally, other evidence fromC. elegans shows that specific germ granule factors, including PGL-1 and PGL-3, induce aggregate/granule formation in non-germline cells without converting those cells to PGCs, further indicating that granules alone are not sufficient for germ cell identity/specification127.In addition to their varied composition discussed above, other evidence indicates that germ plasm and germ granules may not be functionally equivalent. For example, the germ plasm and germ granules occupy distinct subcellar locations. In zebrafish andnanos andpgc, which promote patterning and pole cell development in flies130, and Buc must be localized to induce PGCs in zebrafish106. Consistent with an essential role for granules in germ cells, depletion of one or more germ granule components inC. elegans causes sterility133. Recently, maternaldazap2 was shown to maintain germ granules of zebrafish PGCs by acting epistatic to Tudor-7 and antagonistic to Dynein activity78. Because PGCs are specified, and granules form but later are not maintained in PGCs lacking maternal Dazap2, Mdazap2 mutant germ cells provide an opportunity to explore potential roles of granules in maintenance of vertebrate PGCs. The historical view of nuage, germ plasm, and germ granules was that each of these entities would promote the germ cell fate beginning with their specification to maintenance of germline identity. Efforts to gain a deeper understanding of the components of germ granules and functional assessment of these conserved elements of PGCs have provided strong evidence for mechanisms to preserve or protect germline identity; therefore, it is worth considering the possibility that the germ granules of specified embryonic PGCs contribute to a mechanism that preserves germline identity rather than specification of PGC fate. Understanding the contribution of granules to germ cell development and fertility remains an active area of investigation.Germ granules are enriched for RNA-binding proteins and proteins with other roles in post-transcriptional regulation; therefore, if germ granules do impart germline identity, it is possible that post-transcriptional regulation of other factors that may or may not be granule components is involved. If so, granules could impart germline fate only to cells that already express that factor; for example, Osk recruits components that regulate localization and translation of two germ granule components137 and developmental pluripotency-associated 3 (DPP3/Stella), are required to maintain rather than specify the PGCs138. Strikingly, nearly all cells of the mouse pregastrula epiblast can express Prdm1 or Prdm14 when induced by ubiquitous expression of the bone morphogenetic protein ligands Bmp4139 and Bmp8b140. Importantly, induced cells in culture can reconstitute functional sperm to germ cell-depleted neonates141. However,in vivo, only a few cells within the posterior epiblast that are positioned proximal to the extra-embryonic ectoderm become PGCs142. A Wnt3 signal from the epiblast primes these cells to respond to BMP produced by the extra-embryonic ectoderm141. The mechanisms that limit germ cell induction in anterior regions are not fully understood. However, PGC formation involves anterior endoderm factors and additional BMP family members that repress Blimp1 and thus promote acquisition of pluripotency and PGC development specifically in the posterior epiblast141. Other factors, such as microRNAs and their antagonists, contribute to PGC development 143. However, the functions of these molecules are not confined to PGCs and so they do not represent germline determinants. Significant unresolved questions are how and what factors limit selection of just a few cells within this region to become PGCs. Moreover, the identity of the factor or factors that specify them remains unknown.In mammals, the germ cells are not specified by inheritance of maternal cytoplasm (germ plasm), but instead are specified later by inductive signals. In the mouse embryo just before gastrulation, signals from extra-embryonic ectoderm and visceral endoderm are necessary to specify germ cells within the posterior epiblast that is adjacent to the forming primitive streak . Unlike 144. Functional studies of the earliest stages of PGC specification in humans would require manipulation and analysis of embryos within the first month following fertilization and this is not feasible. As an alternative toin vivo mammalian contexts, more tractablein vitro systems to study specification of mouse and human PGCs have been sought. In mice, key advances in PGC reprogramming paradigms have been facilitated by the development of germline reporters to identify and select for the lineage, including a mouse Vasa homolog (MVH) transgenic embryonic stem (ES) cell line, wherein the MVH promoter drives GFP reporter expression145 and a germ cell-specific gcOct4-GFP146. These tools enabled identification of germ cell-like cells in culture within just a few days. With markers in hand and using the germline-promoting factors defined from developmental studies, it was not long before mouse sperm147 and egg146 cells were derived from long-term ES cells. As germline stem cells (GSCs), PGCs are expected to generate cells that ultimately develop as functional gametes, sperm, or eggs that can produce a normal healthy fertile animal. The first animals generated from PGC-like cells (PGCLCs) were abnormal148, a limitation to using such cells to study normal germline development or infertility. However, in 2011, healthy offspring were produced from male-derived PGCLCs building on the observation that nearly all of the pregastrula cells in the mouse could express key PGC factors Blimp1 (Prdm1) and Prdm14 in response to BMP4149. In that study, the authors recapitulated gametogenesisin vitro by first reprogramming cells to a pregastrula epiblast state before exposure to germ cell-differentiation cues (in vitro fertilization150, indicating that thein vitro produced cells could develop as functional female gametes in the correct environment.PGCs in humans, as in mice, are specified in extragonadal regions around the time of gastrulation onset. However, there are important differences in the timing and development of extra-embryonic tissues and likely PGC specification between human and mouse. During the second week of development, the human embryo is composed of epiblast and primitive endoderm, which gives rise to the yolk sac . Owing ton cues . Later, in vitro models of mouse PGC generation, several groups pursuedin vitro models of human PGC development. With the mouse methods based on developmental paradigms in hand, the opportunity to discover the molecular programs responsible for producing the human germline seemed imminent. However, initial attempts to derive human PGCs quickly revealed that the mouse programming strategies were not effective, suggesting that despite conserved germ cell factors there must be differences in their mechanisms to generate germline cells (151 (122, and the second8 imparted pluripotency by using a combination of four inhibitors and selected for germline cells by using a reporter for the conserved PGC-specific protein Nanos154. Interestingly, these studies indicate that PGC specification is somewhat more direct in human cells compared with mouse cells. In human cells, Sox17 induces and is necessary to specify hPGCLCs, with Blimp1 acting downstream to promote PGC gene expression and repress expression of mesendodermal, neuronal, and epigenetic reprogramming genes as the cells differentiate into hPGCLCs122. In mice, ES cells first transition to an epiblast state, and Blimp1, but not Sox17156, represses somatic programs in nascent mPGCLCs149 (122, indicating distinct PGC specification programs between mouse and human despite their reliance on shared signaling molecules. Notably, the hPGCLCs generated so far resemble early hPGCs and thus provide an unprecedented opportunity to study the earliest events in hPGC specification. However, late-stage hPGCLCs have not been obtained on the basis of the absence of markers that are normally expressed after PGCs migrate to the gonad, indicating that further differentiation or refinement of the protocols is required to obtain and study development of these later stages. Recapitulating development from stem cell to mature germ cell remains a challenge and an important step to realize the full potential of hPGCLCs to provide insight into human diseases, including infertility with a genetic basis or environmental/toxicological basis, as well as developmental disorders.Encouraged by the success in establishinge cells . Thus, ss (151 . SimilarLCs149 . In bothC. elegans, must travel from their extragonadal site of specification to the presumptive gonad where the PGCs will differentiate into sperm in males or oocytes in females (reviewed inDrosophila159,C. elegans160, and zebrafish161, andnanos orthologs have a conserved role from invertebrates to humans in GSC maintenance166. Despite conserved requirements fornanos orthologs, the relevant RNA targets ofnanos genes and downstream mechanisms promoting germ cell survival are not fully understood. In non-mammalian animals, GSCs persist into adulthood and continue to produce new gametes throughout the reproductive life of the animal. In mammals, GSCs maintain spermatogenesis throughout the lifetime of adult males, but whether or not female mammals have the capacity to generate new oocytes after birth has been highly controversial and an area of active study (reviewed in2. Clearly, the existence of GSCs, either naturally occurring in adult mammalian females orin vitro produced, has important therapeutic implications for the field of reproductive medicine; however, further development of tools and studies of the mechanisms to specify and maintain stem cell niches and ovarian reserve in diverse systems are essential for an improved understanding of germline development and reproductive health.Once specified, PGCs in flies, fish, and mammals, but not


Human coronaviruses (HCoVs) are large RNA viruses that infect the human respiratory tract. The emergence of both Severe Acute Respiratory Syndrome and Middle East Respiratory syndrome CoVs as well as the yearly circulation of four common CoVs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. Various animal models have been established to investigate HCoV infection, including mice and non-human primates. To establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. Currently, different cell culture systems are available to recapitulate the human airways, including the Air-Liquid Interface (ALI) human airway epithelium (HAE) model. Tracheobronchial HAE cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. These organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. Additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. No commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. These morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition. Respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. Currently, they are studied in various model systems, ranging from cell lines to animal models. Originally, the importance of HCoVs in the burden of human disease was underestimated and as a result, no general therapy exists to treat coronavirus induced disease in humans. Furthermore, no commercial vaccine is available leaving the human population vulnerable to emerging coronavirus infections. Both the Severe Acute Respiratory Syndrome and Middle East Respiratory Syndrome coronaviruses have recently crossed the species barrier and entered the human population to cause severe disease. In this review, we summarize the current knowledge on human coronavirus infection emphasizing the usefulness of organotypic human airway cultures as a model system.Coronaviridae family, are positive strand RNA viruses with the largest genome of all known RNA viruses (≥27 Kb). The genomic RNA is capped, polyadenylated and associated with nucleocapsid proteins within an enveloped virion. The envelope is covered by the characteristic surface glycoprotein that gives the virus particles their characteristic crown-like (latin: corona) appearance , 7675, 7Alveolar epithelial ALI cultures Fig.  can alsoWhen compared to traditional two dimensional cell cultures, the HAE cultures are more cumbersome and their preparation is time consuming but they do have an advantage over traditional monolayer cell cultures when it comes to virus-host interaction studies. Different types of ALI cultures used for virus research are summarized in Table Within the respiratory epithelium the innate immune system has a major protective role as the first line of defense against respiratory pathogens. In particular, the interferon (IFN) system orchestrates hundreds of different cellular effector proteins that (i) protect the epithelial barrier by altering the physiological and cellular environment, (ii) impair virus propagation, spread and transmission, and (iii) shape the host’s adaptive immune response. Recent publications have demonstrated that the innate immune system is functional in the HAE cell culture system and that most pathogen recognition receptors are expressed and up-regulated upon treatment with exogenous stimuli , 81.In general, HCoVs do not elicit a strong innate immune response in primary target cells of the human airway early during infection. Despite the presence of all major pathogen recognition receptors, no elevated expression of IFN beta, pro-inflammatory cytokines or interferon stimulated genes can be observed up to 12 h post-infection in HAEs infected with HCoV-229E, MERS- or SARS-CoVs . This isIn addition to the non-structural proteins, various CoV accessory proteins have been discovered to inhibit interferon signaling at different stages of the host innate immune response. For example, MERS-CoV accessory protein 4a inhibits innate antiviral signaling by suppressing the activation of MDA5 and RIGI , 87 wherDespite that respiratory infections with HCoVs can result in severe respiratory disease there are currently no effective prophylactic or therapeutic treatment options available. However, the emergence of novel coronaviruses has emphasized the need to develop effective treatment options. For example, vaccines using the spike proteins of both SARS- and MERS-CoVs have proven protective in animal models , 92 suggAdditionally, various drugs that inhibit HCoV infection at different stages of the replication cycle have been reported and some could potentially serve as treatment options for HCoV associated severe respiratory disease. For example, patients presenting with severe respiratory disease, caused by SARS- or MERS-CoVs, are generally treated with steroids and interferon, sometimes in combination with the antiviral drug Ribavirin –96. ThisHCoV infection can also be inhibited by pre-treating HAE cultures with either recombinant IFN alpha or lambda . SimilarScreenings of compound libraries have also resulted in the identification of some HCoV specific antivirals. For example, a novel small compound inhibitor (K22) has been identified, and showed to be effective against a broad spectrum of CoVs and could inhibit both HCoV-229E and MERS-CoV in HAE cultures . AdditioTo date, most treatment and inhibitor studies have been conducted in HCoV susceptible cell lines. However, the HAE cultures represent an ideal system to test the application and efficacy of those already identified, and new, antiviral compounds against HCoVs in cells that represent the primary site of replication. Furthermore, the HAE cultures are heterogenous, containing many different cellular sub-populations, and would allow for the evaluation of compound toxicity and effect in a differentiated layer similar to human airway epithelium. Compounds already shown to inhibit HCoVs in cell lines should be applied to HAE cultures as well before any animal or human trials.HCoV induced respiratory diseases are of both medical and socio-economic importance. The emergence of SARS- and MERS-CoV and the yearly circulation of the four common HCoVs highlight the importance of elucidating the different mechanisms employed by HCoVs to evade the host immune system as well as identifying antiviral compounds and human vaccine candidates. The HAE culture system is based on primary human cells offering a unique platform to study respiratory viruses in cells representing the primary entry point of these viruses, bronchial epithelial cells, or investigate the interaction of HCoVs and the distal airways, in type I and II alveolar cells. Additionally, the inclusion of airway epithelial cultures for other species enables the study of zoonosis and animal-to-human transmission. Currently, many aspects of HCoV infection and pathogenesis remain to be determined. The HAE culture system, both tracheobronchial and alveolar, represents a unique platform to study virus-host interaction in natural target cells at the molecular level. These cultures are becoming more common and more relevant to HCoV research. Especially, for those viruses for which there is no animal model, as they provide an organotypic substitute for virus – host interaction studies.


Anaplasmataceae family. Survival requires that the parasite fundamentally alter the native functions of the cell to allow its entry, intracellular replication, and transmission to a hematophagous arthropod. The small genomic repertoires encode several eukaryotic-like proteins, including ankyrin A (AnkA) of Anaplasma phagocytophilum and Ank200 and tandem-repeat containing proteins of Ehrlichia chaffeensis that localize to the host cell nucleus and directly bind DNA. As a model, A. phagocytophilum AnkA appears to directly alter host cell gene expression by recruiting chromatin modifying enzymes such as histone deacetylases and methyltransferases or by acting directly on transcription in cis. While cis binding could feasibly alter limited ranges of genes and cellular functions, the complex and dramatic alterations in transcription observed with infection are difficult to explain on the basis of individually targeted genes. We hypothesize that nucleomodulins can act broadly, even genome-wide, to affect entire chromosomal neighborhoods and topologically associating chromatin domains by recruiting chromatin remodeling complexes or by altering the folding patterns of chromatin that bring distant regulatory regions together to coordinate control of transcriptional reprogramming. This review focuses on the A. phagocytophilum nucleomodulin AnkA, how it impacts host cell transcriptional responses, and current investigations that seek to determine how these multifunctional eukaryotic-like proteins facilitate epigenetic alterations and cellular reprogramming at the chromosomal level.Obligate intracellular pathogenic bacteria evolved to manipulate their host cells with a limited range of proteins constrained by their compact genomes. The harsh environment of a phagocytic defense cell is one that challenges the majority of commensal and pathogenic bacteria; yet, these are the obligatory vertebrate homes for important pathogenic species in the  In order to infect mammalian hosts, bacterial pathogens evolved an array of mechanisms that serve to create an environment conducive for survival, replication, and spread. While many bacterial species survive in an extracellular environment, intracellular pathogens must be capable of both entering their host cells undetected and altering the cellular milieu in order to replicate. Traditionally, the ability of bacterial-derived proteins to induce disruptions of cell signaling or major cellular processes such as NF-κB, MAPK, and JAK/STAT pathways, are the predominant focus of host–pathogen interaction studies . RecentlEukaryotic DNA is highly organized and gene expression tightly regulated by an orchestrated network of proteins, RNAs, and other modulators. Histone octamers, or nucleosomes, organize DNA by acting as a bobbin on which the DNA winds. The charge of the histone proteins can be covalently modified in order to more tightly or loosely associate with DNA. Histone acetylation, which imparts a negative charge, is predominantly associated with an open configuration where promoters are easily accessed by RNA polymerases. Histone methylation or phosphorylation which impart a positive charge, cause DNA to more tightly associate with histone proteins, reducing promoter accessibility to transcription activating machinery. The process of modulating open and closed chromatin is further complicated by (i) the residue(s) of which histone protein(s) is/are modified, (ii) methylation of cytosine residues in DNA, and (iii) non-coding RNAs, and by the manner in which these mechanisms are intertwined. It therefore is no surprise that bacterial-derived proteins have evolved to interfere with host gene expression that improves bacterial fitness.Listeria monocytogenes, Chlamydia trachomatis, Shigella flexneri, and others, were found to target the host cell nucleus  that de-phosphorylates MAPKs in the nucleus resulting in a lack of histone H3 phosphorylation at serine 10 at a number of NF-κB-dependent genes , blocks binding of heterochromatin inducing protein BAHD1 at interferon-stimulated genes resulting in upregulated expression  protein effectors that when expressed, bind plant DNA and alter transcription and disease susceptibility (Anaplasmataceae family. Ankyrin A (AnkA) of Anaplasma phagocytophilum, as well as Ank200 and several tandem-repeat containing proteins (TRPs) from Ehrlichia chaffeensis, have been shown to enter the nucleus and bind DNA, and interact with host epigenetic machinery or alter nearby histone octamers  in 1990  that allows protein effectors to translocate into infected host cells where they likely act by mechanisms similar to those of other bacterial secreted effectors that target cytosolic pathways such as signal transduction, cytoskeletal rearrangements, intracellular trafficking, etc.  and AnkA, the latter of which was found by CYBB, encoding the gp91phox component of phagocyte oxidase  and special-AT rich binding protein-1  to investigate histone marks at defense gene promoters, acetylation of histone H3 was dramatically reduced, a finding often associated with silenced gene transcription .Of considerable interest is the interplay between chromatin structure and DNA methylation. Histone modifications, DNA methylation at CpG islands, and binding of methyl CpG DNA-binding proteins (MeCPs) via their methyl-DNA binding domains (MBDs) occur in concert during biological responses including neoplasia and cellular differentiation . Cytosin stimuli . MBDs ca stimuli . General stimuli . This coCYBB promoter during myeloid differentiation where it represses the transcription of CYBB, is involved in maintaining expression of BCL2 . In keeping with the original observations of Borjesson et al., most clusters were upregulated; however, the analysis also demonstrated a similar pattern of downregulated genes over long linear regions. Of interest was chromosome 17 with a 9.4 Mb cluster containing myeloid peroxidase (MPO), eosinophil peroxidase (EPX), and lactoperoxidase (LPO) which showed marked downregulation, and a 3.2 Mb cluster containing multiple upregulated chemokine genes . In addition to reduced transcript levels, MPO and EPX promoters were previously shown to be deacetylated upon A. phagocytophilum infection  was upregulated . The chromosomal landscape of the MHC locus is well documented with respect to chromatin looping via MARS and histone marks  complexes] or additional bacterial-derived nucleomodulins that further contribute to the dramatic changes observed. To examine this, we designed a genome-level bioinformatics approach to predict nucleomodulins based on the combination of secretory and NLSs and studied the genomes of 12 phylogenetically diverse intracellular prokaryotic pathogens, including for each . Among tfunction . ElucidaA. phagocytophilum and OspF of S. flexneri to alter inflammatory responses could provide opportunities for engineering of therapeutic agents which interfere with cellular responses in inflammatory diseases or other conditions where epigenetic factors control or contribute to disease.Understanding the role of prokaryotic control over complex eukaryotic transcription machinery in lieu of the bottleneck that would occur with single effector targets at signaling pathways will allow for better understanding of the essential components of whole cell transcriptional re-programming. However, this has implications not only for host–pathogen or host–symbiont interactions, but across biology. The ability of proteins like AnkA of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Focused ultrasound (FUS)-mediated opening of the blood-brain barrier (BBB) has been used to promote drug delivery to the brain. However, in a mouse model of Alzheimer’s disease, FUS has proven to be effective for reducing pathology and improving cognition, even in the absence of exogenous drug application. We have explored single and repeated FUS treatments in mice at both early and late stages of plaque pathology using a transgenic (Tg) mouse model of Alzheimer’s disease.Mice received single or weekly FUS treatments using a 1.68 MHz transducer, . Definity microbubble contrast agent was delivered at the onset of sonication. Effective BBB opening was confirmed using contrast enhanced MR images. Following FUS treatments, mice were evaluated using cognitive tests and histology.In the Y maze with novel arm, untreated Tg mice were found to spend significantly less time in the novel arm compared to their non-Tg littermates.Following FUS treatment, Tg mice performed as well as their non-Tg counterparts. These results indicate that repeated FUS treatments are safe and may improve cognitive behavior, even in a model of late-stage AD. To support these findings, mice treated with FUS also had reduced plaque burden in the brain and increased plasticity in the hippocampus, both of which have been correlated to improved learning and memory. Together this data demonstrates that FUS-mediated BBB opening can improve cognition related even in the absence of exogenous drug delivery and suggests that FUS should be considered part of the overall therapeutic strategy for treatment of Alzheimer’s disease.


Children with severe neuro-disability are at increased risk of feeding problems resulting in approximately half of such children being undernourished with growth failure. While gastrostomy tube feeding in such patients has been shown to improve weight gain, there is uncertainty to its impact on survival, respiratory complications, parental and child quality of life, cost, and consequently leads to potentially avoidable variability in practice. The issue of lack of standardized outcomes for this population could be addressed through the development of a standardized core outcome set (COS). We aim to develop an evidenced based COS for children 0-18 years with severe neuro-disability and dependent gastrostomy.A systematic review was undertaken to identify all outcome measures used in studies on children with severe neuro-disability dependent on gastrostomy tube feeding. PRISMA guidelines were followed. MEDLINE, EMBASE and Cochrane Register databases were searched from their inception until March 2015. Studies included systematic reviews with/without meta-analyses, randomised controlled trials, and observational studies. After initial screening of titles and abstracts, disagreements on the eligibility of studies were resolved through discussion. Data were extracted on study characteristics, outcomes measured, designated primary and secondary outcome(s), method of measurement, and time points at which they were measured. A thematic content analysis was undertaken to map the outcomes against the OMERACT framework that included four pre-specified outcome “domains”: mortality, pathophysiological manifestations, patient reported outcomes and healthcare utilization. To date, 8725 titles and abstracts are screened. A total of 1460 studies were found eligible. Final results will be presented at the meeting.Outcomes identified through this systematic review will be used to help stakeholders reach consensus on a COS for research in children with severe neuro-disability and feeding tube dependency which could be used to enhance future research, knowledge synthesis and inform clinical practice.


Prostate cancer (PCa) is the most commonly diagnosed cancer and kills about 28,000 American men annually. Although progress has been made in understanding the molecular features of different forms of the disease, PCa is considered incurable when it becomes resistant to standard therapies. Prostate specific antigen (PSA) test has been a gold standard of diagnosis for PCa, however, it can result in lead to the unnecessary biopsies and treatment of indolent cancers due to the low specificity. Thus, the limitations of PSA screening for PCa have prompted much focus on strategies how to enhance the accuracy of PSA for distinction between aggressive and indolent cancers.Studies of miRNAs in PCa patients have suggested differentially expressed miRNAs between healthy controls and those with PCa, providing potential biomarker candidates using body fluids including urine and blood. Virus infection has been considered to associate with PCa incidence. Virus infected PCa cells may shed extracellular vesicles and communicate with neighboring cells, which were not infected yet, however, no mechanistic approaches were performed to understand the biology. The miRNAs composition in the shedding extracellular vesicles, and its role in PCa are completely undefined. In the near future, new insights to connect between the viral derived miRNAs and PCa progression might provide an opportunity to diagnose, risk prediction and therapeutic strategies.The goal of this debate article is to provide a short review on miRNAs, virus infection and viral encoded miRNAs in PCa, with a primary focus on circulating miRNAs as potential non-invasive biomarkers for PCa patients. Prostate cancer (PCa) remains one of the most commonly diagnosed malignant tumor in men and the second leading cause of death from cancer –3. SeverThis brief debate article is to discuss ad seek to characterize the circulating microRNAs (miRNAs) in urine with regard to PCa. We aim to provide our audience the current knowledge, in particular, focused on clinical implication as a liquid biopsy with a clinically satisfactory degree of sensitivity and specificity.Small non-coding microRNA (miRNA) molecules are known as post-transcriptional regulators involved in the regulation of gene and protein expression by interfering in the post-transcriptional level, resulting in the degradation and translation inhibition of mRNAs. Approximately more than 2,000 mature human miRNAs have been reported so far. These miRNAs are short (19–24 nt) and low molecular weight RNAs. miRNAs are derived from hairpin-like precursor transcripts (pre-miRNAs) and taken out of nucleus by a mediator protein, exportin 5. Pre-miRNA is then cleaved by Dicer (a ribonuclease III enzyme) to excise the mature miRNAs in the form of siRNA-like duplexes and asymmetrical assembling of the mature miRNA strands. Since miRNAs interact with multiple messenger RNAs by binding the pairing of bases of the mRNAs and repress target gene expression, and regulate mRNA cleavage and mRNA decay initiated by the miRNA-guided rapid deadenylation, miRNAs have a wide variety of functions contributing to various pathological conditions including prostate cancer .Many of miRNAs play roles in cell proliferation and apoptosis processes, thus miRNA expression profiles can be considered as useful biomarkers monitoring many types of cancer progression and treatment responses . PreviouIn the accumulated literature the roles of miRNAs in the pathobiology of PCa were found in cell cycle, apoptosis, epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) states, invasion and metastasis, PCa stem cell, and androgen receptor (AR) pathway. PTEN-PI3 Kinase-AKT, EGFR and AR pathways are considered as the most important signaling pathways in PCa. Upregulation of miR-221/-222 in PCa suggest a mechanistic link to PCa since miR-221/-222 plays a role in AR pathway regulation, c-kit, PTEN, and TIMP3 et al. miR-124 was characterized to have a direct targeting of AR thereby inducing down-regulation of miR-125b and up-regulation of p53. When miR-125b is suppressed miR-125b effectors  were downregulated. The regulation of miR-125b on apoptotic proteins  and NCOR2 (a co-repressor of AR) was well known. Another PCa-associated miRNA, miR-let-7c regulates AR pathway and involves in the conversion of hormone dependent PCa to CRPC.Several cancer-associated miRNAs were found in circulating body fluids such as urine, which make it possible to develop non-invasive biomarkers mainly due to their ease of access and stability. Only few studies for PCa-associated miRNA in urine were reported. Five of the miRNAs that were differentially quantified in PCa patients compared to controls  were successfully quantified in urine of men with cancer, which were much higher in PCa patients than that of healthy volunteers. Such reports provide evidence that circulating miRNAs might be a next-generation biomarker and contribute to cancer screening in non-invasive liquid biopsy. Two additional promising miRNAs, miR-141 and miR-375 were found in the patient blood. In particular, data from this study showed that metastatic PCa patients have approximately 50 fold higher miR-141 levels, compared to the healthy individuals.Among the various viruses, herpes virus is one of the viruses most commonly related to carcinogenesis. Several epidemiological studies evaluated the association between herpes virus infection and prostate cancer risk, although results were inconsistent. Herpes virus plays an important role in the pathogenesis of cancer via the inhibition of cell apoptosis and stimulation of DNA synthesis, which may ultimately lead to PC. Previous meta-analysis indicated that infection by herpes simplex virus type 2 (HSV-2) or human herpesvirus 8 (HHV-8) may be associated with a higher prostate cancer risk. However, there has not been a study to elucidate the potential mechanism of HSV-2 infection underlying viral PC carcinogenesis. A potential link between HPV infection and PCa risk was attempted to study the causal role of HPV-16 in prostate carcinogens. A significant increase of PCa risk related with HPV-16 infection was observed. However, this is still contradictory since other epidemiological studies could not find same association –25. TherOur recent findings demonstrated the overexpression of herpes virus-encoded miRNAs in urine samples from prostate cancer patients, compared to those of control subjects . InteresViruses also encode their own sets of miRNAs, which they use to control the expression of either the host's genes and/or their own. In the past few years’ evidence of the presence of cellular miRNAs in extracellular human body fluids such as serum, plasma, saliva, and urine has been accumulated , 27. FurThe first viral-encoded miRNAs was found in cells infected with EBV , 28. TheCells can release different types of vesicles, which have been difficult to categorize in a definitive manner. The transmission of vesicles from cancer cells to other cell types has been the subject of intensive studies in recent years. It is a process that can make it possible a sophisticated form of cellular communication through the delivery of highly complex and dynamic cargo. As a delivery vehicle between cells, extracellular vesicles (EV) have been considered as a molecular cargo mediating a communication between cells in the microenvironment. Although EV were originally considered to be a means for exclusion of garbage molecules from cells, it is now clear that EV alter signaling pathways and have a biological influence in neighboring cells . Also, sThere are multiple types of EV, the small sealed membrane vesicles that are produced from cells differing in size, biology, contents, and secretory mechanisms. Exosomes and microparticles (MPs) are two distinct groups of EV. Exosomes are originated from the inward budding of the limiting membrane of multivesicular bodies (MVBs), while MPs are in bigger size vesicles formed by the outward membrane budding. Vesicles that are produced from cells during cell death are apoptotic bodies.It is known that PCa patients had higher levels of urinary exosomes than the healthy donors. The membranes of exosomes are resistant to the osmeolytic and proteolytic activity of urine, indicating that exosomes are quite stable in urine. PC cell-derived EV can deliver various genetic factors such as nucleic acids, including DNA, mRNA, miRNA, and small non-coding RNAs, as well as oncoproteins and metabolites, leading to the horizontal transfer of oncogenic information to neighboring immune cells, vessel cells, or cancer cells , 34. EV-3H-thymidine) incorporation assay revealed that EV contain miRNAs , which are transferred to tumor microenvironment leading to proliferative inhibition of immune cells. Other interesting findings suggest immune response may be mediated by miRNAs regulating Toll-like receptors (TLRs), which play an important role in immune response and inflammation including tissue repair and tissue injury-induced inflammation. In PCa cell lines, several miRNAs  were upregulated by TLR3 activation, leading to antitumoral effects on PCa. EV derived from dendritic cells contain miR-155 (a promoter of inflammatory responses) and miR-146a (a mediator of immune suppression), which are known as important players to regulate inflammation, alter the gene expression of inflammation related targets and reprogramm the response to endotoxin [Role of immune response caused/mediated by miRNA was suggested by a series of studies showing that EV-derived miRNAs participate in the regulation of inflammatory responses. A recent report from our group demonstrated that EV derived from amoeboid phenotype of prostate cell may influence the immune response of the tumor microenvironment. A tritiated thymidine , utilize the cellular vesiculation pathway for budding and assembly, immune evasion, and intercellular communication. It is also well known that many virally infected cells secrete vesicles  containing various viral proteins and genetic materials such as RNAs. Studies on herpes simplex virus-infected cells demonstrated that virus communicate via vesicles. In the case of Epstein-Barr virus, recent reports suggested that herpes virus utilizes exosomes, one kind of extracellular vesicles, as a mechanism of cell-to-cell communication and transfers signaling competent proteins and functional miRNAs to uninfected neighbor cells. For example, exosomes released from infected cells have been shown to contain co-receptors for HIV, which can enhance virus entry into cells. Proteins in the secreted exosomes from virus-infected cells can induce apoptosis in CD4 T cells, and contribute innate immune response.EV-mediated communication would allow the virus to respond to the cellular microenvironment. Virus-infected cells continuously shed and transfer EV to uninfected neighboring cells. Throughout EV shedding and secretion to extracellular space, the virus-encoded miRNAs were delivered to other cells, leading to alteration of miRNA-mediated gene repression and intercellular communication. Thus, the presence of viral-encoded miRNAs in EV suggests that virus-infected cells perturb gene expression in the surrounding tissue, resulting in destruction of the immune system. Exosomes secreted from HIV-infected alveolar macrophages have been identified to carry viral miRNA (vir-miRNA) such as vmiR88, vmiR99, and vmiR-TAR. It has not been clearly identified how vir-miRNA composition is decided and recruited to EV. Vir-miRNAs might have a role in cancer initiation by blocking of major tumor suppressors  or acceleration of cancer development by evading cellular immune response .These findings still raise a number of exciting questions. Does virus-infected prostate cells secrete miRNAs using EV as a cargo? What are the target genes of the abundantly secreted viral miRNAs? Are extracellular vesicles shed only in the absence of viral replication? Is there any evidence for functional miRNA delivery in vivo? If so, what’s the biological mechanism? In addition, the detail mechanism is currently lacking whether virus infection promotes miRNAs sorting into EV, or whether this secretion is a selective and specific process.In PCa biomarker development, the greatest unmet need remains: a biomarker that stratifies men at risk of aggressive PCa or a biomarker that identify the early stage of patients who need active surveillance, eventually leading to a reduction of unnecessary interventions. Although miRNAs might have many useful clinical applications for patients with PCa, many additional studies are warranted to clarify their function and regulation during tumorigenesis and tumor progression. The studies to determine the role of circulating miRNAs during PCa progression would have the potential that PCa patients can be molecularly stratified based on their miRNAs profile in urine samples. In addition, the studies could uncover important clues about underlying disease mechanisms.Urine analyses have great potential to be adapted in clinical practice, based on its non-invasiveness. Identification of non-invasive clinical indicators of PCa would be one of the most important advances achievable in this field. Our current mechanistic understanding of shedding vesicle biology and function, especially in the context of virus infection and communication with host, is not mature enough. Identification of potential candidates of the circulating miRNAs signature in patient urine samples would have significant implications for an alternative and/or supportive diagnostic tool for PCa.Unnecessary diagnostic procedures would be minimized for patients with early stage of PCa, contributing to the easier diagnostic assessment and to the reducing associated public health and economic burden to patients. In addition, the studies to understand role and biology of the circulating miRNAs have significant clinical relevance to public health since it will improve accuracy of predicting patient survival, identifying responsiveness candidates of PCa patients. Furthermore, we envision that this study will also provide the mechanistic data to address the long-term goal of the PCa field identifying new treatments tailoring more specific and effective therapies for PCa, thus it holds great promise for the treatment of high risk PCa patients.


A class of small non-coding RNAs, the microRNAs (miRNAs), have recently attracted great attention in cancer research since they play a central role in regulation of gene-expression and miRNA aberrant expression is found in almost all types of human cancer. The discovery of circulating miRNAs in body fluids and the finding that they are often tumor specific and can be detected early in tumorigenesis has soon led to the evaluation of their possible use as cancer biomarkers and treatment-response predictors. The evidence that tumor cells communicate via the secretion and delivery of miRNAs packed into tumor-released microvesicles has prompted to investigate miRNA contribution as signaling molecules to the establishment and maintenance of the tumor microenvironment and the metastatic niche in cancer. In this review we highlight the recent advances on the role of exosomal miRNAs as mediators of cancer cell-to-cell communication. Thousands of human protein-coding genes are regulated by miRNAs, indicating that miRNAs are master regulators of diverse biological systems, facilitating a rapid response of body physiology. miRNAs have been shown to regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation and apoptosis. In addition, the biogenesis and function of miRNAs are related to the molecular mechanisms of various diseases including cancer. The intensive research of the past years have demonstrated that miRNAs are not only contained into cells, but are also detectable outside them, secreted in various body fluids  routinely examined in patients ,2. It isThanks to their unique properties and functions, miRNAs play a crucial role in human cancer development and tumorigenesis as their expression is frequently deregulated in many types of tumors, functioning either as tumor suppressors or as oncogenes (oncomiRs). Furthermore, miRNAs were shown to be responsible for cancer-related inflammation, cancer drug resistance, and regulation of cancer stem cells. Therefore, miRNAs have generated great interest as a novel strategy in cancer diagnosis and therapy .The first observations that mature miRNAs are present in cell-free blood plasma and serum was made in 2008 by several independent research groups -8. LaterExosomes are membrane vesicles of an average 30–100 nm diameter, are formed within the multivesicular bodies (MVBs), also known as late endosomes, and released upon fusion of MVBs with the plasma membrane  from manhttp://www.exocarta.org/). A recent study on exosomes released from human colon carcinoma cells and purified by immunocapture with different antibodies revealed two different exosome populations distinct for both protein and miRNA composition [The profile of miRNAs in exosomes is specific, since particular repertoires of miRNAs are selectively sorted, while others are usually excluded. Moreover, profiling studies have revealed that exosomes of different cellular origin contain a unique expression profile of mRNAs and miRNAs, which may also differ from the signatures of their parent cells ,34,35. Hposition . Interesposition .The exosome composition is crucial in determining the outcome of the communication between donor and recipient cells. The mechanism of exosome-mediated cell-to-cell communication is particularly important in cancer, since tumor cells constitutively secrete exosome, which can target locally adjacent cells of the same type (autocrine effect), neighboring cells of different types (paracrine effect), or reach cells located at distant organs after entering the blood stream (endocrine effect). These exosomes play a key role in the modulation of the immune response against the tumor -41, the Exosome biogenesis ,46, mateIt has been suggested that sorting of pre-miRNAs into exosomes could also be occurring in a sequence-dependent manner and, indeed, pre-miRNA as well as mature miRNAs are found in exosomes , depending on target genes and cancer types. Furthermore, a particular miRNA can exploit both tumor-suppressive and oncogenic functions depending on the cellular context of its target genes in different cancers .Table Exosomes have been found to act as vehicles for suppressive signals and have suppressive effects on antitumor immune responses ,69. For In some cases, exosomal pathways might discard tumor-suppressor miRNAs that restrict metastatic progression. For example, the let-7 miRNA family was shown to be expressed at higher levels in exosomes derived from metastatic gastric cancer cells, compared to non-metastatic parental cells, suggesting that the exosomal mechanism may be used by metastatic cells to eliminate miRNAs having tumor suppressive functions, thus strengthening their aggressive behavior . Anotherin vitro and tumor growth in vivo in a murine xenograft model, accompanied by enhanced apoptosis [However, most literature reports a tumor promoting role of exosomal miRNAs, likely because tumor suppressor miRNAs are downregulated in many tumors, as well as in cancer stem cells. Many oncomiRs have been described in the past decade. For example, IL-4 activated macrophages have been shown to regulate invasiveness of breast cancer cells through exosome-mediated delivery of the miR-223 highlighting a novel communication mechanism between tumor-associated macrophages and cancer cells . miR-21 poptosis . Interespoptosis .It is becoming clear that tumor released exosomes contribute to both progression of primary tumors and metastases. The central role of exosomes in tumor promotion has been recently highlighted by the discovery that breast cancer exosomes can perform cell-independent miRNA biogenesis and stimulate non-tumorigenic epithelial cells to form tumors, by altering their transcriptome in a Dicer-dependent manner . The metIt was recently reported that melanoma exosomes can modify distant lymph nodes to facilitate melanoma growth and metastasis even in the local absence of tumor cells . ExosomaExosomal miRNAs can also participate in cancer metastasis by adapting the tumor niche cells. miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in non-metastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects . It has The contribution of exosome in induction of angiogenesis to promote cancer metastasis is also described. For instance, it was shown that miRNA-enriched exosomes released by CD105 cancer stem cells from renal carcinomas may modify the tumor microenvironment by triggering angiogenesis and may promote formation of a pre-metastatic niche . SpecifiThe exosomal miRNA profiling of sera from cancer patients versus healthy individuals has often revealed important differences in relation to tumor progression, highlighting a possible use of these miRNAs as disease prognostic biomarkers ,102. In in vitro models [Intensive research effort is spent to find out new cancer diagnostic and prognostic tools. Representing a non-invasive approach, measurement of extracellular miRNAs in biological fluids might prove a valuable strategy. In addition, since dysregulation of miRNAs is fundamental to the pathogenesis of many cancers, they are attractive candidates for clinical application as therapeutic targets in both solid and hematological malignancies . Down-reo models ,114,115.


The most common cause of acute kidney injury (AKI) in hospitalized patients is sepsis. However, the molecular pathways and mechanisms that mediate septic AKI are not well defined. Experiments performed over the past 20 years suggest that there are profound differences in the pathogenesis between septic and ischemic AKI. Septic AKI often occurs independently of hypoperfusion, and is mediated by a concomitant pro- and anti-inflammatory state that is activated in response to various pathogen-associated molecular patterns, such as endotoxin, as well as damage-associated molecular patterns. These molecular patterns are recognized by Toll-like receptors (TLRs) found in the kidney, and effectuate downstream inflammatory pathways. Additionally, apoptosis has been proposed to play a role in the pathogenesis of septic AKI. However, targeted therapies designed to mitigate the above aspects of the inflammatory state, TLR-related pathways, and apoptosis have failed to show significant clinical benefit. This failure is likely due to the protean nature of septic AKI, whereby different patients present at different points along the immunologic spectrum. While one patient may benefit from targeted therapy at one end of the spectrum, another patient at the other end may be harmed by the same therapy. We propose that a next important step in septic AKI research will be to identify where patients lie on the immunologic spectrum in order to appropriately target therapies at the inflammatory cascade, TLRs, and possibly apoptosis. Acute kidney injury (AKI) is a very common and especially formidable clinical problem in the ICU, where mortality rates approach 25% and soar to 50 to 60% when severe enough to require renal replacement therapy . These sIn this review, we will first summarize the findings of published human studies that support the concept that the pathogenesis of septic AKI in humans should not be exclusively viewed in the context of distributive shock-associated ischemia, but rather also within the context of a dysregulated and ill-defined inflammatory response to septic stimuli. We then discuss why previous attempts at modulating the inflammatory response in septic AKI may have failed, and highlight more recent trials and therapies aimed at targeted treatment based on where patients lie along the immunologic spectrum. We also discuss the role Toll-like receptors (TLRs) have been shown to play in endotoxemic models of AKI, and again focus on why agents used to block TLR-related pathways may have failed to show significant clinical benefit. Finally, we review the data supporting and refuting a central role for apoptosis in septic AKI, and argue that much of the clinical manifestations in septic AKI occur prior to significant cell death secondary to apoptosis or necrosis.The morphologic changes that are seen in classic ischemic and toxic acute tubular necrosis are often lacking ,6 or arePerhaps an even greater distinction between non-septic AKI and septic AKI might be made when examining the role of overall vascular hemodynamics. Traditionally, septic AKI has been thought to be secondary to a demand and perfusion mismatch, whereby renal blood flow was diminished in the midst of increased metabolic demand ,10. FurtNevertheless, the data directly measuring overall kidney blood flow during sepsis suggest that flow is increased in the septic physiologic state. Despite a plethora of conflicting animal studies - some of which show overall increased kidney blood flow while others show overall decreased kidney blood flow ,13 - LanEscherichia coli infusion as a source of infection, regional redistribution of blood flow from the medulla to the cortex was absent during sepsis. Local inhibition of endothelial nitric oxide synthase by liposaccharide (LPS) may play a role in the decreased GFR observed in endotoxemic models of AKI, at least in rats [Despite the above human studies that suggest an increase in overall renal blood flow in septic AKI, the complex interplay between regional microcirculation and hemodynamics may play a much more important role in overall kidney function during sepsis. As Langenberg and colleagues  and Moli in rats . In a hy in rats  likewise in rats  observed in rats . These s in rats .An in-depth discussion about renal microcirculation in septic AKI is beyond the scope of this review. As mentioned above, there is a dearth of human data on this important topic and most of the animal data are conflicting and difficult to interpret, given the different models used and measurement modalities employed. While it is clear from above that ischemic and microvascular dysfunction may contribute to septic AKI, septic AKI can occur independently of overall renal hypoperfusion or ischemia . Indeed,For many years, organ damage and other deleterious sequelae from sepsis were thought to result from a hyper-inflammatory ‘cytokine storm,’ triggered in response to either an infectious pathogen or major physiologic insult . Thus, aAs a consequence of these failed trails, new ideas regarding the pathogenesis and pathophysiology of sepsis emerged . UtiliziIndeed, the early trials focusing on inhibiting TNF-α or IL-1β did not risk-stratify their patients in terms of their individual inflammatory status or genetic predilections towards certain types of phenotypic responses ,29. It sin vitro and rat in vivo studies have demonstrated that the broad reduction of inflammatory cytokines by hemoadsorption can protect against renal and liver injury [As an alternative to targeted treatment, some researchers have proposed implementing treatment strategies that are able to broadly regulate the overall inflammatory response in sepsis by using extra-corporeal blood purification. Multiple r injury , improver injury  and meanr injury , decreasr injury , and impr injury ,37. Hower injury . Renal Sin vivo animal studies cited above have shown that broad regulation of cytokine levels is possible with the use of cytokine-reducing resins, modulation of cytokine levels by continuous hemodialysis in humans has not been clearly demonstrated. Despite the very short half-life of many inflammatory cytokines, small initial studies suggested that high-volume hemofiltration might be able to modulate overall cytokine profiles in humans [Although the n humans ,41. Howen humans , nor hasn humans -44. Whiln humans . This reFinally, the use of agents to augment immune function in patients suffering from sepsis-associated immunosuppression has shown clinical promise. As mentioned above, sepsis represents a complex inflammatory state with both pro- and anti-inflammatory characteristics. Thus, identifying and treating septic patients who are on the more immunosuppressed spectrum of sepsis with immune augmentation may improve outcomes. Meisel and associates  administInitial molecular research on sepsis suggested that the generalized inflammatory state described above is initiated when the innate immune system recognizes highly conserved pathogen-associated molecular patterns (PAMPs), such as LPS . Howeverin vivo experiments [TLRs have provided a link between host and pathogen ,51, as weriments , in renaeriments ,60, and eriments . TLR-4 eeriments ,60, but eriments ,63. The eriments .Figure 1NF-κB is a highly expressed transcription factor that is often activated in response to cellular stresses and induces the synthesis and release of TNF-α, IL-1, IL-6, and IL-8 . NF-κB-mHowever, a recent randomized controlled trial examining the effect of an MD2-TLR-4 antagonist failed to show any mortality benefit in human patients suffering from severe sepsis . In thisIndeed, multiple studies have shown that in polymicrobial and cecal ligation and puncture models the mechanism of damage induced by sepsis may be very different than in LPS-induced models , might bThe traditional model of apoptosis in kidney and other cells is similar . Processin vitro and in vivo studies examining the presence or role of apoptosis in septic AKI, there were major methodological shortcomings. LPS was used as a model for sepsis in most of the studies cited above [However, in most of the ed above -81, as wed above  and moused above . As mented above -8. Moreoed above ,8 has aled above , electroed above . In the ed above , they faed above , its rolPerhaps one of the reasons for the discrepancy between histologic data and functional performance in septic AKI is that most of the insults occur prior to significant cell death from apoptosis and necrosis. Indeed, apoptosis and necrosis are end-stage manifestations of cellular insults, and significant impairment of cellular function can occur before the histologic and genomic changes that are measured in most apoptosis and necrosis assays. Despite a measured decrease in kidney perfusion in their septic mouse model, Tran and associates found no elevation in hypoxia-inducible factor-1α, a transcription factor that is significantly upregulated  in hypoxWhile the relative contributions that ischemia, TLR-4-mediated pathways, apoptosis, and other subcellular processes have towards overall organ dysfunction are currently unclear, the abundance of research in the area has shed a significant amount of light on the basic mechanisms that drive septic AKI. Perhaps the most important finding is that therapeutic approaches targeted at a single mediator for all patients may not succeed given the protean nature of the disease. Indeed, targeted inhibition of cytokines such as TNF-α and IL-1 as well as TLR antagonists have failed to show consistent and significant clinical benefit, most likely because only certain subsets of septic patients fit an immunologic profile that would benefit from those therapies. An important next step in sepsis research should be to identify immunologic markers in patients that can help tailor specific therapies for patients based on where they lie along the immunologic spectrum.


Anticancer treatments aiming at killing malignant cells have been applied for decades but have been unsuccessful at curing the disease. The modern concept of tumor microenvironment, especially angiogenesis, suggests that the tumor is not only composed of malignant cells, but also consists of other groups of cells that work together. Recently, genetic message transfer has been revealed between tumor cells and their microenvironment. The latest cell-derived vector, extracellular membrane microvesicles (EMVs), has been found to provide membrane protection and allowed to deliver genetic information beyond the cells. Additionally, EMV-associated microRNAs are involved in a variety of cellular pathways for tumor initiation and progression. Previous published reviews have focused on miRNA that included EMVs as a sensitive marker for tumor monitoring in clinical applications that are based on the alteration of their expression levels in conjunction with disease occurrence and progression. From the aspect of cellular crosstalk, this article will review the role of EMV-mediated microRNA transfer in tumor pathogenesis, including tumor treatment obstacles, history and features, and current research in inflammatory/immune pathologies, as well as in solid tumors and hematological malignancies. This nascent crosstalk model will provide a novel insight into complementing the classic mechanisms of intercellular communication and contribute to the potential therapeutic strategy via small RNA molecule-carrying EMVs for multimodality treatment of cancer. Cancer is a leading cause of death in developed nations and a growing worldwide epidemic. Previous understanding of tumor pathogenesis mainly focused on the tumor itself, including the identification of oncogenes and suppressor genes. Accordingly, current anticancer therapies are mainly by means of surgery, radiation, and chemotherapy, which directly target the malignant compartment. However, cancer, the majority of which is still incurable, has evolved to be one of the most remarkable global problems. There are unexpected obstacles which prevent conventional treatments by using tumor-targeted toxins and resection to be curative. Thereby, much effort has been made in other aspects on exploring tumor pathogenesis in depth. The latest perspectives in oncology have begun to change from autonomous mechanisms of malignant cells to crosstalks with other nonmalignant adjacent components. A new concept, tumor microenvironment, is being placed forward to explain the reciprocal causation between tumors and their surrounding components, and this may potentially overcome the existing therapeutic limitations.National Cancer Institute (NCI) Dictionary of Cancer Terms, a tumor microenvironment represents a well-orchestrated integration where the tumor exists and is constituted by surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and the extracellular matrix. This concept was based on the ‘seed and soil’ hypothesis to explain the non-random pattern of metastasis by the English surgeon Stephen Paget in 1889. Then, it was presented to describe tumor cell-host cell interactions for the first time by the American scholar Lord EM, et al. in the 1970s [Tumor microenvironment was firstly described by Judah Folkman in 1971 [he 1970s . These ehe 1970s  and paveThe role of angiogenesis in the tumor microenvironment has always retained an essential focus in preclinical medical research on tumor progression. It was presented by Willis in 1948 and originated from the hypothesis of angiogenesis-dependent tumor, as tumor cells were observed to acquire a new phenotype to participate in the formation of blood channels . AlthougThe discovery of the tumor microenvironment, angiogenesis, raises an important question on the interplay between cancer cells and their neighboring components; how do cancer cells deliver messages to the remaining normal endothelial capillaries? In addition to that, the neovasculogenesis, the tumor microenvironment, mainly harbors genetic arrangement abnormalities. Significantly, the consensus tumor-specific genetic aberrations were found in both tumor cells and the corresponding microvascular endothelial cells -9. Two cExtracellular membrane microvesicles (EMVs), new performers participating in microenvironment conformation, are circular fragments of membrane released from the endosomal compartments as exosomes with diameters of 30 to 100 nm or shedding from the surface membranes of most cell types as microvesicles with diameters of 50 to 2,000 nm -13. The As a novel class of regulators, miRNAs are the core elements in EMVs. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which miRNAs were the most abundant, making up over 76% of all mappable reads . miRNAs The characteristics of EMV-mediated miRNA transfer will be summarized in this section. 1) EMVs function as the genetic messengers in intercellular communication but differ from conventional cell-to-cell communication. The circulating miRNAs have been found to be relatively stable in the extracellular milieu and are resistant to plasma ribonucleases (RNase) with a long half-life, even in the unfavorable physiological conditions such as freeze-thawing and extreme pH and room temperature -34. Thes EMVs funThe contribution of EMV-mediated miRNA transfer on various pathological conditions has been addressed, namely, trauma, inflammation, infection, and systematic diseases. The transfer of genetic information from injured cells, confirmed by the altered circulating levels of vascular and inflammation-associated miRNAs in inflammatory patients , may expEMV-miRNA transfer plays a crucial role in tumor pathogenesis and may be useful in clinical practice. These aspects can be summarized as four key points. 1) Tumor-derived EMVs can promote malignant transformation via horizontal propagation of selective oncogenes. miRNAs contribute to carcinogenesis not only as oncogenes, (miR-9 detected in EMVs) ,55, but  Tumor-deEMVs are secreted by many cell types but cancer cells have a higher production of them. Prostate cancer (PC) is the first model which has been profoundly studied in this area ,63. LehmSince the initial studies, serum EMVs were revealed and may be potential biomarkers in other tumors Table  includinEtiologically, Yamada et al. confirmed that the bovine leukemia virus proteins were released with milk EMVs and may be transferred into recipient cells of calves via milk EMVs as an alternative route without requiring a virus infection . In chroCancer cells can utilize EMV-mediated miRNA transduction to constitute their microenvironments in stimulating angiogenesis. Tumor EMVs, prostate, breast, and ovarian cancers and some hematological neoplasms, may convey phenotypic transforming signals to non-malignant cells and may acquire tumor-supporting characteristics, namely, vasculogenic and epithelial markers. Zhang et al. showed that human leukemia cell line THP-1 cells selectively packaged miR-150 into multivesicular bodies and actively secreted them into the extracellular environment to enhance target endothelial cell migration . KeiichiEMVs are novel and unique effectors carrying out many biological messages. Aside from their conventional basal intracellular communications, EMVs can also actively participate in triggering signal pathways and exclusively transfer nucleic acids as miRNA clusters. Due to its characteristics, microvesicle-mediated miRNA transduction is one of the first autogenous mediators to exchange specific and endogenous tumor-related genetic signals among multiple types of cells, which has attracted more attention on malignant cell-endothelial cell interaction in the tumor microenvironment. Therapy by microvesicle-mediated miRNA transduction may aid in drug resistance and inhibition of tumor invasion. Collectively, these studies suggest that this novel intracellular model complements the classic cell-to-cell communication and provides new therapeutic strategies for both solid cancers and hematological disorders.


Whereas in young mice satellite cells could be activated in response to muscle degeneration, their number and self-renewal ability rapidly decreased with age of the mice. The age-dependent decline in satellite cell number and activity was linked with defects in Notch signalling, an evolutionarily conserved pathway involved in muscle stem cell function. Interestingly, artificial activation of Notch signalling resulted in successful restoration of the self-renewal ability of mdx satellite cells. This study implicates Notch signalling as a key pathway for preserving stem cell function in dystrophic muscles, and suggests that this pathway might be modulated to improve long-term satellite regenerative function in DMD. 997Page Duchenne muscular dystrophy (DMD) is an inherited degenerative muscle disease for which there is currently no cure. It is caused by mutations in the gene encoding dystrophin, a cytoskeletal protein involved in maintaining the integrity of sarcolemma (muscle membrane) during muscle contraction. Defects in dystrophin lead to fragility of sarcolemma, which results in muscle dysfunction and degeneration. In normal conditions, satellite cells – a muscle-specific stem cell population – proliferate in response to damage and drive muscle regeneration; however, these cells are progressively depleted in DMD, hampering muscle repair capability. In this study, Shihuan Kuang and colleagues used


Various arthropod-borne and hemorrhagic fever viruses should be considered when Lassa fever is suspected.  Sierra Leone in West Africa is in a Lassa fever–hyperendemic region that also includes Guinea and Liberia. Each year, suspected Lassa fever cases result in submission of ≈500–700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory in eastern Sierra Leone. Generally only 30%–40% of samples tested are positive for Lassa virus (LASV) antigen and/or LASV-specific IgM; thus, 60%–70% of these patients have acute diseases of unknown origin. To investigate what other arthropod-borne and hemorrhagic fever viral diseases might cause serious illness in this region and mimic Lassa fever, we tested patient serum samples that were negative for malaria parasites and LASV. Using IgM-capture ELISAs, we evaluated samples for antibodies to arthropod-borne and other hemorrhagic fever viruses. Approximately 25% of LASV-negative patients had IgM to dengue, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola, and Marburg viruses but not to Crimean-Congo hemorrhagic fever virus. Arenaviridae family. Lassa fever is a severe, often fatal, hemorrhagic illness; the virus causes 100,000–300,000 infections and 5,000 deaths each year in the region , a member of the In 2002, Sierra Leone emerged from a brutal 11-year civil war that left the country with little infrastructure and much of its formal economy destroyed. Today Sierra Leone is undergoing substantial economic growth; however, poverty, unemployment, and inadequate health care remain major challenges. Through the Mano River Union–Lassa Fever Network, a variety of organizations are building diagnostic capacity for Lassa fever that will lead to better understanding of the disease and its treatment . The LasEach year, suspected Lassa fever infections result in submission of ≈500–700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory . Samples come from throughout the Lassa fever–hyperendemic region and initially are screened for malaria by thick blood smear and, if negative, are tested for LASV. LASV infection is determined by the presence of virus detected by an antigen-detection ELISA and by the presence of IgM determined by using an IgM-capture ELISA. Generally only 30%–40% of samples tested are positive for LASV antigen and/or LASV-specific IgM; therefore, 60%–70% of patients have acute diseases of unknown origin. We investigated what other arthropod-borne and hemorrhagic fever viral diseases might be causing serious illness in the region and confounding the diagnosis of Lassa fever. We tested samples from these patients using IgM-capture ELISAs to virus pathogens that could occur in the region and mimic Lassa fever. We tested for IgM to dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Rift Valley fever virus (RVFV), chikungunya virus (CHIKV), Ebola virus (EBOV), Marburg virus (MBGV), and Crimean-Congo hemorrhagic fever virus (CCHFV). Follow up analyses included IgG ELISAs and/or confirmatory plaque-reduction neutralization tests (PRNTs). This study provides a better understanding of the differential diagnoses for Lassa fever in the region, which can lead to improved diagnostic capability and disease treatment.Blood samples collected during October 2006–October 2008 from patients with suspected Lassa fever were submitted to the Lassa Diagnostic Laboratory. Most samples were from acutely ill patients from eastern Sierra Leone; some were submitted from Liberia and a few from Guinea. Samples were from patients whose illness met a surveillance case definition for Lassa fever  and werewww.hhs.gov/ohrp/humansubjects/guidance/belmont.html). All data were gathered and human subjects research was conducted under an institutional review board–approved protocol (no. HP-09-32).Research on human subjects was conducted in compliance with US Department of Defense, federal, and state statutes and regulations relating to the protection of human subjects, and adhered to principles identified in the Belmont Report (1979) (6 rad) and safety tested to ensure inactivation. Optimal dilutions of antigens were determined by checkerboard titrations against virus-specific antibodies. Negative or mock antigens, both supernatant and cell lysate, used as negative controls, were prepared from uninfected cell monolayers as described above.Viruses used in production of ELISA antigenic materials were the 4 DENVs   were IgM positive only . Of the Malaria parasites and LASV are known to occur in the region, and we excluded samples with evidence of either because of our interest in undiagnosed acute febrile illnesses. However, the sample group was tested for LASV IgG to better understand the prevalence of Lassa fever. Of 237 patients, 25.5% were positive for LASV-specific IgG (data not shown). Because of the high number of IgG-positive patients, we retested the samples for LASV IgM and demonstrated that 7 (3.0%) of the patients tested had LASV-specific IgM.Antibodies detected by ELISA cross-react, especially within a genus and particularly for antibodies elicited by alphaviruses and flaviviruses. Immunodiagnosis conventionally is confirmed by virus isolation or a rise in PRNT titer . Four ofComparative PRNTs for the ebolaviruses used EBOV, SUDV, and TAFV, all of which are known to have circulated in Africa. Eighteen samples were IgM positive only by ELISA. Of these, 14 had sufficient volume for PRNT against all 3 ebolaviruses . Eight oIn West Africa, as in many regions of Africa, infectious disease is part of everyday life. The cause of disease is often unknown or incompletely understood because of nonspecific clinical features, lack of diagnostic laboratory support, or little or no knowledge about disease prevalence in a region ; the bunyavirus RVFV; the alphavirus CHIKV; and the filoviruses EBOV and MBGV . We testPRNT is the laboratory standard for immunologic assays. It measures in vitro virus neutralization and is the most virus-specific serologic test to confirm immunologic test results. Testing CHIKV-positive serum demonstrated that the patients were more likely to have been infected by ONNV, a related but separate virus species. The viruses can be distinguished genetically by sequence analysis but with greater difficulty by serologic testing . Most paAedes species mosquitoes (Several arthropod-borne viruses are known to circulate in West Africa (Phlebovirus genus, is endemic to East and South Africa, but may not be established in West Africa (Nairovirus genus, circulates in West Africa, but we found no evidence of CCHFV infections in any patient samples tested (RVFV, a bunyavirus in the Our presumptive results provide some insight into the other viruses causing acute disease in the patients whose samples were submitted to the Lassa Diagnostic Laboratory. Although our results are not definitive, they demonstrate arthropod-borne and hemorrhagic fever viruses that should be considered when Lassa fever is suspected. These continued studies will add to the body of knowledge for Lassa fever and other arthropod-borne diseases and hemorrhagic fevers that occur naturally within Sierra Leone and West Africa.


Penetrating brain trauma commonly results in occult neurovascular injury. Detailed cerebrovascular imaging can evaluate the relationship of intracranial foreign bodies to major vascular structures, assess for traumatic pseudoaneurysms, and ensure hemostasis during surgical removal. We report a case of a self-inflicted intracranial nail gun injury causing a communicating ventricular tract hemorrhage upon removal, as well as a delayed pseudoaneurysm. Pre- and post-operative vascular imaging, as well as intra-operative endovascular assistance, was critical to successful foreign body removal in this patient. This report demonstrates the utility of endovascular techniques for the assessment and treatment of occult cerebrovascular injuries from intracranial foreign bodies. Penetrating brain trauma is less common than closed head injury, with outcomes dependent on the penetrating object , 2. GunsWe report here clinical and neuroimaging findings from a patient with an intracranial nail gun injury, a communicating ventricular tract hemorrhage upon nail removal, and a delayed pseudoaneurysm, whose treatment was augmented with neuroendovascular techniques.A 62-year-old male with a history of depression was found several hours after a self-inflicted nail gun injury to the left temple. He was brought to an area hospital awake but exhibiting severe expressive aphasia. He was sedated, intubated, and transferred to our facility for further management.Broad-spectrum antibiotics and anti-seizure medications were started. Head CT demonstrated a large nail horizontally traversing the left cerebral hemisphere, with its tip terminating near the body of the right lateral ventricle. Subarachnoid hemorrhage was seen in the bilateral frontoparietal sulci, sylvian fissures, and the basilar and prepontine cisterns, with a small amount of intraventricular hemorrhage in the occipital horns of the lateral ventricles. Vascular imaging demonstrated a nail trajectory immediately subjacent to a left M3 branch, with no aneurysms, pseudoaneurysms, vessel occlusions/transections, or active extravasation Figure .Nail removal was planned in the angiography suite with endovascular assistance for rapid hemostasis if needed. Following baseline angiograms of the left anterior circulation, a microcatheter and guidewire were positioned in the petrous segment of the left internal carotid artery for rapid selective embolization.The nail head was then identified following a small skin incision and superficial dissection and removed from the cranium using a double-action Leksell rongeur. An angiogram was subsequently obtained. Immediate injection of the left internal carotid revealed active contrast extravasation from a small M3 branch arising from the inferior trunk in the area of the anterior parietal artery and extending down the puncture tract into the left lateral ventricle Figures A–D. Surgvia branch sacrifice with n-butyl cyanoacrylate (n-BCA) liquid embolic glue  can be safely extracted with careful surgical planning and post-operative follow-up to assess for and treat cerebrovascular injury.The decision to remove intracranial foreign bodies is multifactorial. Conservative management is typically pursued with objects that are deeply situated or in eloquent tissue, are irregular and would cause significant injury with extraction, or are high velocity  and thereby cause more extensive injuries and theoretic  bactericidal heating upon entry , 9. ConvStaphylococcus aureus, Gram-negative facultatively aerobic bacteria, or mixed. A course of prophylactic broad-spectrum antibiotics is thus recommended following penetrating intracranial trauma (Infections following intracranial penetrating trauma of all types are nonetheless on the order of 2–5% for bullet or shrapnel wounds, and slightly higher for traumas from other causes . Causatil trauma  and was Cerebrovascular injuries at the time of injury and during foreign body removal can be life-threatening. In this case, the patient had no obvious pre-operative vascular insults. The nail was nonetheless removed on a biplane fluoroscopic table with a microcatheter positioned for rapid embolization in the event of hemorrhage from an occult vascular injury. The hemorrhage that occurred would not have been otherwise visible, or self-tamponaded given the communication of the nail tract with the lateral ventricle. While the injection of Surgiflo down the nail tract led to hemostasis before embolization was required, the utility of rapid endovascular embolization in this setting is clear. Surgical treatment, while previously described for removal of intracranial nails impacting the cerebrovasculature , would hImmediate or delayed pseudoaneurysm development is also described following traumatic or iatrogenic cerebral artery injury and can be successfully managed with endovascular techniques , 14–16. In conclusion, this case highlights the use of endovascular approaches to help identify and treat occult vascular injuries associated with intracranial foreign bodies.Written consent was obtained for all clinical procedures as per University of California protocol.RR, JAS, JS, JP, and AK contributed to the clinical care of this patient, as well as the drafting and editing of the manuscript.The authors have no disclosures concerning the materials or methods used in this study or the findings specified in this paper. The reviewer NM and handling Editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


The mammalian neocortex generally has a repetitious, laminar structure and performs functions integral to higher cognitive processes, including sensory perception, memory, and coordinated motor output. What computations does this circuitry subserve, that might connect these unique structural elements with their biological function? Potjans and Diesmann  parameteThis population statistic approach lends itself to quickly analyzing the response properties of population-scale dynamics of neural tissue. We use this strategy to examine the input-output relationship of the Potjans and Diesmann column model , in an