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Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shape, elongation, division, and sporulation (SEDS) proteins. Here, we have identified four phylogenetically distinct groups of bacteria that lack any identifiable aPBPs. We performed experiments on a panel of species within one of these groups, the Rickettsiales, and found that bacteria lacking aPBPs build a PG-like cell wall with minimal abundance and rigidity relative to cell walls of aPBP-containing bacteria. This reduced cell wall may have evolved to minimize the activation of host responses to pathogens and endosymbionts while retaining the minimal PG-biosynthesis machinery required for cell elongation and division. We term these “peptidoglycan-intermediate” bacteria, a cohort of host-associated species that includes some human pathogens. IMPORTANCE Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals. PG is normally polymerized by two groups of enzymes, aPBPs and bPBPs working together with shape, elongation, division, and sporulation (SEDS) proteins. We have identified a diverse set of host-associated bacteria that have selectively lost aPBP genes while retaining bPBP/SEDS and show that some of these build a minimal PG-like structure. It is expected that these minimal cell walls built in the absence of aPBPs improve the evolutionary fitness of host-associated bacteria, potentially through evasion of PG-recognition by the host immune system.

Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive., Species of Rickettsia (Alphaproteobacteria: Rickettsiales) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses. IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies.

The cat flea, Ctenocephalides fells, is widely recognized as a global veterinary pest and a vector of pathogenic bacteria. We recently reported on the C. felis nuclear genome, which is characterized by over 38% protein coding gene duplication, extensive tRNA gene family expansion, and remarkable gene copy number variation (CNV) between individual fleas. Herein, we describe the assembly of the C. felis mitochondrial genome, a novel resource for comparative genomics of fleas and other insects. The order and content of mitochondrial genes is highly consistent with four previously sequenced flea mitochondrial genomes, limiting CNV to siphonapteran nuclear genomes.

Rickettsia buchneri (formerly Rickettsia endosymbiont of Ixodes scapularis, or REIS) is an obligate intracellular endoparasite of the black-legged tick, the primary vector of Lyme disease in North America. It is noteworthy among the rickettsiae for its relatively large genome (1.8 Mb) and extraordinary proliferation of mobile genetic elements (MGEs), which comprise nearly 35% of its genome. Previous analysis of the R. buchneri genome identified several integrative conjugative elements named Rickettsiales amplified genomic elements (RAGEs); the composition of these RAGEs suggests that continued genomic invasions by MGEs facilitated the proliferation of rickettsial genes related to an intracellular lifestyle. In this study, we compare the genomic diversity at RAGE loci among sequenced rickettsiae that infect three related Ixodes spp., including two strains of R. buchneri and Rickettsia endosymbiont of Ixodes pacificus strain Humboldt, as well as a closely related species R. tamurae infecting Amblyomma testudinarium ticks. We further develop a novel multiplex droplet digital PCR assay and use it to quantify copy number ratios of chromosomal R. buchneri RAGE-A and RAGE-B to the single-copy gene gltA within natural populations of I. scapularis. Our results reveal substantial diversity among R. buchneri at these loci, both within individual ticks as well as in the I. scapularis population at large, demonstrating that genomic rearrangement of MGEs is an active process in these intracellular bacteria.

The soil microbiome plays an essential role in processing and storage of nitrogen (N) and carbon (C), and is influenced by vegetation above-ground through imparted differences in chemistry, structure, mass of plant litter, root physiology, and dominant mycorrhizal associations. We used shotgun metagenomic sequencing and bioinformatic analysis to quantify the abundance and distribution of gene families involved in soil microbial N and C cycling beneath three deciduous hardwood tree species: ectomycorrhizal (ECM)-associated Quercus rubra (red oak), ECM-associated Castanea dentata (American chestnut), and arbuscular mycorrhizal (AM)-associated Prunus serotina (black cherry). Chestnut exhibited the most distinct soil microbiome of the three species, both functionally and taxonomically, with a general suppression of functional genes in the nitrification, denitrification, and nitrate reduction pathways. These changes were related to low inorganic N availability in chestnut stands as soil was modified by poor, low-N litter quality relative to red oak and black cherry soils.IMPORTANCEPrevious studies have used field biogeochemical process rates, isotopic tracing, and targeted gene abundance measurements to study the influence of tree species on ecosystem N and C dynamics. However, these approaches do not enable a comprehensive systems-level understanding of the relationship between microbial diversity and metabolism of N and C below-ground. We analyzed microbial metagenomes from soils beneath red oak, American chestnut, and black cherry stands and showed that tree species can mediate the abundance of key microbial genes involved in N and (to a lesser extent) C metabolism pathways in soil. Our results highlight the genetic framework underlying tree species’ control over soil microbial communities, and below-ground C and N metabolism, and may enable land managers to select tree species to maximize C and N storage in soils.

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae,wCfeT andwCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA).wCfeT is basal to nearly all describedWolbachiasupergroups, whilewCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution ofWolbachiahost associations.wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across theWolbachiatree, indicating parallel evolution towards mutualism. Alternately,wCfeJ harbors a toxin-antidote operon analogous to thewPipcinABoperon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies.wCfeJcinBand immediate-5’ end genes are syntenic to large modular toxins encoded in CI-like operons of certainWolbachiastrains andRickettsiaspecies, signifying that CI toxins streamline by fission of larger toxins. Remarkably, theC.felisgenome itself contains two CI-like antidote genes, divergent from wCfeJcinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongstC.feliscolonies, though occasionallywCfeJ singly infects fleas in wild populations. Collectively, genomes ofwCfeT,wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.ImportanceMany arthropod and certain nematode species are infected with wolbachiae which are intracellular bacteria well known for reproductive parasitism (RP). Like other RP strategies,Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and frequency in host populations. Mutualism is another strategy employed by wolbachiae to maintain host infection, with some strains synthesizing and supplementing certain B vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novelWolbachiastrains that co-infect cat fleas (Ctenocephalides felis):wCfeT carries biotin synthesis genes, whilewCfeJ carries a CI-inducing toxin-antidote operon. Our analyses of these genes highlight their mobility across theWolbachiaphylogeny and source to other intracellular bacteria. Remarkably, theC.felisgenome also carries two CI-like antidote genes divergent from thewCfeJ antidote gene, indicating episodic RP in cat fleas. Collectively,wCfeT andwCfeJ inform on the rampant dissemination of diverse factors that mediateWolbachiastrategies for persisting in invertebrate host populations.

BackgroundFleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e. the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea,Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe.ResultsBy combining Illumina and PacBio sequencing with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly forC.felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ∼38% of protein-coding genes with two or more copies and over 4,000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) inC. felis. Similarly broad genome sizes were also calculated for individuals ofXenopsylla cheopis(Oriental rat flea), indicating that this remarkable “genome-in-flux” phenomenon could be a siphonapteran-wide trait. Finally, from theC. felissequence reads we also generated closed genomes for two novel strains ofWolbachia, one parasitic and one symbiotic, found to co-infect individual fleas.ConclusionRampant CNV inC.felishas dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novelWolbachiaendosymbionts – potential tools for blocking pathogen transmission – these oddities highlight a unique and underappreciated disease vector.

The soil microbiome plays an essential role in processing and storage of nitrogen (N) and carbon (C) and is influenced by vegetation above-ground through imparted differences in chemistry, structure, mass of plant litter, root physiology, and dominant mycorrhizal associations. We used shotgun metagenomic sequencing to quantify the abundance and distribution of gene families involved in soil microbial N and C cycling beneath three deciduous hardwood tree species: ectomycorrhizal (ECM)-associated Quercus rubra (red oak), ECM-associated Castanea dentata (American chestnut), and arbuscular mycorrhizal (AM)-associated Prunus serotina (black cherry). Chestnut exhibited the most distinct soil microbiome of the three species, both functionally and taxonomically, with a general suppression of functional genes in the nitrification, denitrification, and nitrate reduction pathways. These changes were related to low inorganic N availability in chestnut stands as soil was modified by poor, low-N litter quality relative to red oak and black cherry soils. Previous studies have used field biogeochemical process rates, isotopic tracing, and targeted gene abundance measurements to study the influence of tree species on ecosystem N and C dynamics. However, these approaches do not enable a comprehensive systems-level understanding of the relationship between microbial diversity and dynamics of N and C below-ground. We analyzed microbial metagenomes from soils beneath red oak, American chestnut, and black cherry stands and showed that tree species can mediate the abundance of key microbial genes involved in N and (to a lesser extent) C metabolism pathways in soil. We compare gene abundance values to measures of N and C flux from incubated soil. Our results highlight the genetic framework underlying tree species’ control over soil microbial communities, and below-ground C and N metabolism, and may enable land managers to select tree species to maximize C and N storage in soils.

Background Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. Results By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433–551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable “genome-in-flux” phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. Conclusion Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts—potential tools for blocking pathogen transmission—these oddities highlight a unique and underappreciated disease vector.

Extensive clonal T cell expansion is associated with response to immunotherapy., In both human and murine systems, we have developed an adoptive cellular therapy platform against medulloblastoma and glioblastoma that uses dendritic cells pulsed with a tumor RNA transcriptome to expand polyclonal tumor-reactive T cells against a plurality of antigens within heterogeneous brain tumors. We demonstrate that peripheral TCR Vβ repertoire analysis after adoptive cellular therapy reveals that effective response to adoptive cellular therapy is concordant with massive in vivo expansion and persistence of tumor-specific T cell clones within the peripheral blood. In preclinical models of medulloblastoma and glioblastoma, and in a patient with relapsed medulloblastoma receiving adoptive cellular therapy, an early and massive expansion of tumor-reactive lymphocytes, coupled with prolonged persistence in the peripheral blood, is observed during effective therapeutic response to immunotherapy treatment.

While typically a flea parasite and opportunistic human pathogen, the presence of Rickettsia felis (strain LSU-Lb) in the non-blood-feeding, parthenogenetically reproducing booklouse, Liposcelis bostrychophila, provides a system to ascertain factors governing not only host transitions but also obligate reproductive parasitism (RP). Analysis of plasmid pLbAR, unique to R. felis str. LSU-Lb, revealed a toxin–antitoxin module with similar features to prophage-encoded toxin–antitoxin modules utilized by parasitic Wolbachia strains to induce another form of RP, cytoplasmic incompatibility, in their arthropod hosts. Curiously, multiple deubiquitinase and nuclease domains of the large (3,841 aa) pLbAR toxin, as well the entire antitoxin, facilitated the detection of an assortment of related proteins from diverse intracellular bacteria, including other reproductive parasites. Our description of these remarkable components of the intracellular mobilome, including their presence in certain arthropod genomes, lends insight on the evolution of RP, while invigorating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens.

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component of Rickettsia biology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borne Rickettsia typhi , the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regarding R. typhi biology in flea vectors that, importantly, do not suffer lethality due to R. typhi infection. To address if fleas combat rickettsial infection, we characterized the cat flea ( Ctenocephalides felis ) innate immune response to R. typhi . Initially, we determined that R. typhi infects Drosophila cells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of the C. felis transcriptome identified homologs to all of the Drosophila immune deficiency (IMD) and Toll pathway components, an AMP gene expression profile in Drosophila cells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessed R. typhi -mediated flea IMD pathway activation in vivo using small interfering RNA (siRNA)-mediated knockdown. Knockdown of Relish and Imd increased R. typhi infection levels, implicating the IMD pathway as a critical regulator of R. typhi burden in C. felis . These data suggest that targeting the IMD pathway could minimize the spread of R. typhi , and potentially other human pathogens, vectored by fleas.

We present three cases of genetically confirmed Gorlin syndrome with desmoplastic medulloblastoma (DMB) in whom tumor recurred despite standard therapy. One patient was found to have a novel germline missense PTCH1 mutation. Molecular analysis of recurrent tumor using fluorescent in situ hybridization (FISH) revealed PTEN and/ or PTCH1 loss in 2 patients. Whole exome sequencing (WES) of tumor in one patient revealed loss of heterozygosity of PTCH1 and a mutation of GNAS gene in its non-coding 3' -untranslated region (UTR) with corresponding decreased protein expression. While one patient died despite high-dose chemotherapy (HDC) plus stem cell rescue (ASCR) and palliative radiotherapy, two patients are currently alive for 18+ and 120+ months respectively following retrieval therapy that did not include irradiation. Infants with DMB and GS should be treated aggressively with chemotherapy at diagnosis to prevent relapse but radiotherapy should be avoided. The use of molecular prognostic markers for DMB should be routinely used to identify the subset of tumors that might have an aggressive course.

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains (cf. R. felis str. URRWXCal2), which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice.

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia ( Alphaproteobacteria ; Rickettsiaceae ). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia , arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote-microbe interactions. Here, we used the genome of the primitive animal Trichoplax adhaerens (Metazoa: Placozoa), which is known to harbor an uncharacterized Gram-negative endosymbiont, to search for the presence of bacterial DNA sequences. Bioinformatic and phylogenomic analyses of extracted data from the genome assembly (181 bacterial coding sequences [CDS]) and trace read archive (16S rDNA) revealed a dominant proteobacterial profile strongly skewed to Rickettsiales (Alphaproteobacteria) genomes. By way of phylogenetic analysis of 16S rDNA and 113 proteins conserved across proteobacterial genomes, as well as identification of 27 rickettsial signature genes, we propose a Rickettsiales endosymbiont of T. adhaerens (RETA). The majority (93%) of the identified bacterial CDS belongs to small scaffolds containing prokaryotic-like genes; however, 12 CDS were identified on large scaffolds comprised of eukaryotic-like genes, suggesting that T. adhaerens might have recently acquired bacterial genes. These putative LGTs may coincide with the placozoan's aquatic niche and symbiosis with RETA. This work underscores the rich, and relatively untapped, resource of eukaryotic genome projects for harboring data pertinent to host-microbial interactions. The nature of unknown (or poorly characterized) bacterial species may only emerge via analysis of host genome sequencing projects, particularly if these species are resistant to cell culturing, as are many obligate intracellular microbes. Our work provides methodological insight for such an approach.

Many prokaryotes utilize type IV secretion systems (T4SSs) to translocate substrates (e.g. nucleoprotein, DNA, protein) across the cell envelope, and/or to elaborate surface structures (i.e. pili or adhesins). Among eight distinct T4SS classes, P-T4SSs are typified by the Agrobacterium tumefaciens vir T4SS, which is comprised of 12 scaffold components (VirB1-VirB11, VirD4). While most P-T4SSs include all 12 Vir proteins, some differ from the vir archetype by either containing additional scaffold components not analogous to Vir proteins or lacking one or more of the Vir proteins. In a special case, the Rickettsiales vir homolog (rvh) P-T4SS comprises unprecedented gene family expansion. rvh contains three families of gene duplications (rvhB9, rvhB8, rvhB4): RvhB9,8,4-I are conserved relative to equivalents in other P-T4SSs, while RvhB9,8,4-II have evolved atypical features that deviate substantially from other homologs. Furthermore, rvh contains five VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Our work herein focuses on the complexity underpinned by rvh gene family expansion. Furthermore, we describe an RvhB10 insertion, which occurs in a region that forms the T4SS pore. The significance of these curious properties to rvh structure and function is evaluated, shedding light on a highly complex T4SS.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent., This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (NIAID, NIH, DHHS) under contract numbers N01-AI30071, HHSN272200900007C, HHSN266200400001C and 5R01GM77117-5. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH. Additional grants and contracts supporting work described in this manuscript were from the NIH-NIAID (HHSN266200400039C and HHSN272200900039C) to F.H.C., and a subcontract under HHSN272200900039C to C.A.H. and J.M.M., the Australian Research Council Discovery Project (DP120100240) to S.C.B. and R.S., the Ministerio de Ciencia e Innovación of Spain (BFU2007–6292; BFU2010–15484) to J.R., BIO2009–07990 and BIO2012–37926 to J.V. NIH-1R01AI090062 to Y.P., L.S., and J.K., NIH 1R21AI096268 and NSF IOS-0949194 to R.M.R., the Xunta de Galicia of Spain (10PXIB918057PR) to J.M.C.T. and M.T., BFU2011–23896 and EU FP7 ANTIGONE (278976) to J.F., the USDA-NRI/CREES (2008-35302-18820) and Texas AgriLife Research Vector Biology grant to P.V.P. and European Research Council Starting Independent Researcher Grant (205202) to R.B., J.M.R was supported by the intramural program of the NIAID, R.M.W. by a Marie Curie International Outgoing Fellowship PIOF-GA-2011–303312, E.M.Z. by Swiss National Science Foundation awards 31003A-125350 and 31003A-143936, J.M.G. by an NIH-NCATS award TL1 TR000162 and NSF Graduate Research Fellowship (DGE 1333468), V.C. by a Boehringer Ingelheim Ph.D. Fellowship, F.G.V. by a Fundação para a Ciência e a Tecnologia, Portugal fellowship (SFRH/BD/22360/2005), C.J.P.G. and F.H. by The Lundbeck Foundation (Denmark), and J.J.G. by NIH awards HHSN272200900040C, R01AI017828 and R01AI043006. Support from the Broad Genomics Platform is gratefully acknowledged.

Cooper's Hawk (Accipiter cooperii) populations breeding in the northern portion of the species' range exhibit variation in morphological traits that conforms to predictions based on differences in prey size, tree stand density, and migratory behavior. We examined genetic structure and gene flow and compared divergence at morphological traits (PST) and genetic markers (FST) to elucidate mechanisms (selection or genetic drift) that promote morphological diversification among Cooper's Hawk populations. Cooper's Hawks appear to conform to the genetic pattern of an east-west divide. Populations in British Columbia are genetically differentiated from north-central populations (Wisconsin, Minnesota, and North Dakota; pairwise microsatellite FST= 0.031-0.050; mitochondrial DNA ΦST = 0.177-0.204), which suggests that Cooper's Hawks were restricted to at least two Pleistocene glacial refugia. The strength of the Rocky Mountains—Great Plains area as a barrier to dispersal is further supported by restricted...

Motivation: Infectious disease research is generating an increasing amount of disparate data on pathogenic systems. There is a growing need for resources that effectively integrate, analyze, deliver and visualize these data, both to improve our understanding of infectious diseases and to facilitate the development of strategies for disease control and prevention. Results: We have developed Disease View, an online host–pathogen resource that enables infectious disease-centric access, analysis and visualization of host–pathogen interactions. In this resource, we associate infectious diseases with corresponding pathogens, provide information on pathogens, pathogen virulence genes and the genetic and chemical evidences for the human genes that are associated with the diseases. We also deliver the relationships between pathogens, genes and diseases in an interactive graph and provide the geolocation reports of associated diseases around the globe in real time. Unlike many other resources, we have applied an iterative, user-centered design process to the entire resource development, including data acquisition, analysis and visualization. Availability and Implementation: Freely available at http://www.patricbrc.org; all major web browsers supported. Contact: cmao@vbi.vt.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Trichomoniasis is a digestive tract disease caused by ingestion of the protozoan Trichomonas gallinae. This disease can be a significant source of mortality. No deaths of nestlings could be attributed to trichomoniasis in Cooper's Hawks (Accipiter cooperii) breeding in urban and rural environs in Wisconsin, North Dakota, and British Columbia. We detected T. gallinae in four (5.2%) of 77 nestling Cooper's Hawks during 2006 and 2007 among 42 urban nests on new study areas in southeast Wisconsin and eastern North Dakota/western Minnesota. All four infected young fledged. We did not detect T. gallinae in 52 breeding adult Cooper's Hawks on two urban study sites, nor in 28 migrant hatching year (n = 24) and adult (n = 4) Cooper's Hawks at Hawk Ridge Nature Reserve, Duluth, Minnesota in 2006–2007. Overall, we detected T. gallinae in only 2.5% of 157 Cooper's Hawks in northcentral North America. These results suggest a low prevalence of T. gallinae in Cooper's Hawks in the northern part of this hawk...

Bacteria of the order Rickettsiales are Gram-negative, small, rod-shaped, and coccoid, with all described species existing as obligate intracellular parasites of a wide range of eukaryotic organisms. This chapter primarily focuses on the family Rickettsiaceae , which is currently comprised well-studied species in the genera Rickettsia and Orientia, as well as candidate species in several provisional genera that are poorly understood. The genus Rickettsiella , originally described as a member of Rickettsiales, comprises a group of intracellular pathogens of diverse arthropod species. Family Holosporaceae was first added to the Rickettsiales in 2001. The first sequenced genome of a rickettsial species, R. prowazekii , revealed numerous pseudogenes and a highly reduced genome relative to facultative intracellular and free-living bacterial species. Throughout modern history, the morbidity and mortality associated with rickettsial infections has been underestimated, primarily due to misdiagnosis. In the United States, a major concern is the changing ecology of endemic typhus, hereafter referred to as murine typhus.

Generation of patient-derived, autologous dendritic cells (DCs) is a critical component of cancer immunotherapy with ex vivo-generated, tumor antigen-loaded DCs. An important factor in the ability to generate DCs is the potential impact of prior therapies on DC phenotype and function. We investigated the ability to generate DCs using cells harvested from pediatric patients with medulloblastoma for potential evaluation of DC-RNA based vaccination approach in this patient population. Cells harvested from medulloblastoma patient leukapheresis following induction chemotherapy and granulocyte colony stimulating factor mobilization were cryopreserved prior to use in DC generation. DCs were generated from the adherent CD14+ monocytes using standard procedures and analyzed for cell recovery, phenotype and function. To summarize, 4 out of 5 patients (80 %) had sufficient monocyte recovery to permit DC generation, and we were able to generate DCs from 3 out of these 4 patient samples (75 %). Overall, we successfully generated DCs that met phenotypic requisites for DC-based cancer therapy from 3 out of 5 (60 %) patient samples and met both phenotypic and functional requisites from 2 out of 5 (40 %) patient samples. This study highlights the potential to generate functional DCs for further clinical treatments from refractory patients that have been heavily pretreated with myelosuppressive chemotherapy. Here we demonstrate the utility of evaluating the effect of the currently employed standard-of-care therapies on the ex vivo generation of DCs for DC-based clinical studies in cancer patients.

The Pathosystems Resource Integration Center (PATRIC) is the all-bacterial Bioinformatics Resource Center (BRC) (http://www.patricbrc.org). A joint effort by two of the original National Institute of Allergy and Infectious Diseases-funded BRCs, PATRIC provides researchers with an online resource that stores and integrates a variety of data types [e. g. genomics, transcriptomics, protein-protein interactions (PPIs), three-dimensional protein structures and sequence typing data] and associated metadata. Datatypes are summarized for individual genomes and across taxonomic levels. All genomes in PATRIC, currently more than 10 000, are consistently annotated using RAST, the Rapid Annotations using Subsystems Technology. Summaries of different data types are also provided for individual genes, where comparisons of different annotations are available, and also include available transcriptomic data. PATRIC provides a variety of ways for researchers to find data of interest and a private workspace where they can store both genomic and gene associations, and their own private data. Both private and public data can be analyzed together using a suite of tools to perform comparative genomic or transcriptomic analysis. PATRIC also includes integrated information related to disease and PPIs. All the data and integrated analysis and visualization tools are freely available. This manuscript describes updates to the PATRIC since its initial report in the 2007 NAR Database Issue. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Service [HHSN272200900040C] Funding for open access charge: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Service [Contract No. HHSN272200900040C].

This study was undertaken to determine how photosynthesis tolerates desiccation in an intertidal alga Fucus vesiculosus L. and a terrestrial sunflower Helianthus annuus L. Photosynthetic O2 evolution generally was inhibited at low water potentials (psiw) but more in sunflower leaves than in Fucus fronds at the same psiw. As psiw decreased, less carbon accumulated in an organic carbon store in Fucus. The inhibition of photosynthesis appeared to be mostly biochemical because it could not be prevented by supplying additional CO2 or by supplying CO2 from the internal organic carbon store. The inhibition of photosynthesis and carbon storage occurred after turgor disappeared and thus when solute concentrations were increasing in the cells. Solute concentrations were much higher in Fucus than in sunflower. After desiccation to the air-dry state (psiw below - 10 MPa), photosynthesis could not recover in sunflower but it recovered rapidly when Fucus was exposed to seawater. The lack of recovery in sunflower was associated with inability to recover turgor probably because of breaks in cell membranes. The ability to recover in Fucus was gradually lost during 1.5 d of desiccation at 45% relative humidity. At lower humidities, recovery was lost sooner as small amounts of water were removed. We conclude that photosynthesis tolerated desiccation more in Fucus than in sunflower because of differences in the molecular environment around the photosynthetic enzymes. Important aspects of this environment were features that prevented membrane breakage but promoted the retention of small amounts of water that were critical for viability.

Infections caused by oxacillin-resistant Staphylococcus aureus are a growing problem in the United States, accounting for considerable morbidity, mortality, and expense. The increasing incidence of these infections has led to frequent empiric use of broader spectrum antibiotics to treat these more resistant pathogens, contributing to further worsening of the resistance among gram-positive organisms. Three main mechanisms of oxacillin resistance in S aureus are described: (1) classic resistance, (2) modified S aureus phenotype, and (3) hyper-^-lactamase production. Correctly identifying the mechanism of resistance in S aureus isolates should lead to substantial changes in antibiotic use and in infection control protocols. These modifications in clinical practices could lead to cost savings, as well as a decreased potential for further antibiotic resistance.

We optimized a spectrophotometric method for measuring ribulose-l,5-bisphosphate carboxylase oxygenase (Rubisco) activity in crude extracts of the kelp Laminaria saccharina Lamour. Activity exhibited a decline during the assays, such that rates determined over a 10-min period were only 70% of activity during the first minute. Activity was significantly enhanced by addition of HCO3− to the extraction buffer and was stable for at least 4 h. Highest activities were determined using small extract loads (1–2.5 mg fresh weight·mL−1 of assay mixture); extract loads of 10 mg·mL−1caused a 25% decrease in activity. Rubisco activities determined for individual kelp plants using the spectrophotometric method were significantly correlated with total protein and gross photo synthetic capacity (Pmax) and were 1.5–2 times higher than Pmax. Low Rubisco activities relative to photosynthetic rates reported in many previous studies of macroalgae and microalgae may have resulted at least partly from underestimation of enzyme activity by radiometric assays using relatively large volumes of un-activated crude extract and assay periods longer than 1 min.

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species ( Alphaproteobacteria: Rickettsiales ), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae ( Orientia tsutsugamushi ) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi , REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ∼35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis , its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.

Funded by the National Institute of Allergy and Infectious Diseases, the Pat hosystems R esource I ntegration C enter (PATRIC) is a genomics-centric relational database and bioinformatics resource designed to assist scientists in infectious-disease research. Specifically, PATRIC provides scientists with (i) a comprehensive bacterial genomics database, (ii) a plethora of associated data relevant to genomic analysis, and (iii) an extensive suite of computational tools and platforms for bioinformatics analysis. While the primary aim of PATRIC is to advance the knowledge underlying the biology of human pathogens, all publicly available genome-scale data for bacteria are compiled and continually updated, thereby enabling comparative analyses to reveal the basis for differences between infectious free-living and commensal species. Herein we summarize the major features available at PATRIC, dividing the resources into two major categories: (i) organisms, genomes, and comparative genomics and (ii) recurrent integration of community-derived associated data. Additionally, we present two experimental designs typical of bacterial genomics research and report on the execution of both projects using only PATRIC data and tools. These applications encompass a broad range of the data and analysis tools available, illustrating practical uses of PATRIC for the biologist. Finally, a summary of PATRIC's outreach activities, collaborative endeavors, and future research directions is provided.

Hypoxia is a common feature of solid tumors. Up-regulation of hypoxia-inducing factor-1 (HIF-1) occurs in the majority of primary malignant tumors and in two-thirds of metastases, while most normal tissues are negative. HIF-1 induces the glycolytic phenotype, which creates an acidic extracellular microenvironment and associated pH gradient such that drugs that are weak acids are selectively taken up and retained in acidic tumors. 7-Butyl-10-amino-camptothecin (BACPT) is a prime example of an agent that can exploit the tumor pH gradient for enhanced selectivity. This study profiles the antitumor activity of BACPT in vitro and its water-soluble dipeptide ester, BACPTDP, in vivo. Antitumor activity was evaluated by proliferation assays in cancer cell lines and in murine xenograft models for human neuroblastoma (IMR-32), colon (HT29), ovarian (SK-OV-3), pancreatic (Panc-1), glioma (SF-295) and non-small-cell lung (NCI-H460) cancers. BACPT had superior antiproliferative activity compared to established drugs in monolayer cultures of human neuroblastoma and pancreatic tumor cell lines and in 3-dimensional histocultures of colon and primary ovarian cancer. Antitumor activity of BACPTDP was comparable to irinotecan in IMR-32, HT29, SF-295 and NCI-H460 xenografts, significantly greater in SK-OV-3 and in Panc-1 where complete regressions were observed. Combination of BACPT with gemcitabine produced additive to synergistic interactions in Panc-1 cells that were independent of drug ratio and optimal when gemcitabine was administered 24 h prior to BACPT. BACPTDP is a water-soluble camptothecin pro-drug that spontaneously generates the lipid-soluble active agent, BACPT. This topoisomerase inhibitor exploits solid tumor physiology for improved selectivity and activity against multiple tumor types with particular promise for use in treating pediatric neuroblastoma and pancreatic carcinoma.

tmRNA employs both tRNA-like and mRNA-like properties as it rescues stalled bacterial ribosomes, while targeting the defective mRNA and incomplete nascent protein for degradation. We describe variation of the tmRNA gene (ssrA) and how it informs tmRNA structure and function. Endosymbiont tmRNAs tend to lose secondary structure and length in the mRNA-like region as nucleotide composition drifts with that of the whole genome. A dramatic gene structure variation is circular permutation, which produces two-piece tmRNAs in three bacterial lineages; new sequences blur these lineages. We present evidence that Sinorhizobium two-piece tmRNA retains the 5'-triphosphate of transcriptional initiation and predict a new structure at the 5' end of cyanobacterial two-piece tmRNA precursor. ssrA is a target for some mobile DNAs and a passenger on others. It has been found interrupted (but not functionally disrupted) by mobile elements such as group I introns, genomic islands and palindromic elements. The alphaproteobacterial permuted genes are significantly less frequently interrupted by genomic islands than are their standard counterparts, yet are a hotspot for insertion or swapping of rickettsial palindromic elements, in contrast to other rickettsial loci that show steady decay of a single ancestral element. Bacteriophages, plasmids and genomic islands can carry tmRNA genes; we describe a native bacterial ssrA disrupted by insertion of a genomic island that carries its own ssrA, a genome encoding both one- and two-piece tmRNA, and a phage encoding a tmRNA variant lacking the mRNA-like function, which may counteract host tmRNA during infection.

Protein-protein interactions (PPIs) play a vital role in initiating infection in a number of pathogens. Identifying which interactions allow a pathogen to infect its host can help us to understand methods of pathogenesis and provide potential targets for therapeutics. Public resources for studying host pathogen systems, in particular PPIs, are scarce. To facilitate the study of host-pathogen PPIs, we have collected and integrated host-pathogen PPI (HP-PPI) data from a number of public resources to create the Pathogen Interaction Gateway (PIG). PIG provides a text based search and a BLAST interface for searching the HP-PPI data. Each entry in PIG includes information such as the functional annotations and the domains present in the interacting proteins. PIG provides links to external databases to allow for easy navigation among the various websites. Additionally, PIG includes a tool for visualizing a single HP-PPI network or two HP-PPI networks. PIG can be accessed at http://pig.vbi.vt.edu. National Institute of Allergy and Infectious Diseases [HHSN26620040035C]; NIH National Institute of Allergy and Infectious Diseases grant HHSN26620040035C. Funding for open access charge: NIH grant.

Most solid human tumors exist in an acidic microenvironment, due in part to inefficient vasculature and a higher intrinsic rate of glycolysis. This leads to a tumor-selective pH gradient, which can be exploited therapeutically with antitumor agents such as the camptothecins (CPTs). Previous work in this laboratory has shown that camptothecin activity is enhanced 40- to 60-fold in monolayer cell culture by reducing the extracellular pH to 6.8. Three-dimensional histoculture has been shown to be a technique that allows human tumor tissue to grow in an in vivo-like way with maintenance of tissue histology and function and drug sensitivity for long periods of time.In the current study, we utilized these features of histoculture to study new analogues of camptothecin that have superior pharmacological properties.We evaluated six CPT analogues in histocultures of human brain, neuroblastoma, breast, colon, and prostate tumors. Fragments were exposed to 10,11-methylenedioxy-CPT (MDC), 7-chloromethyl-MDC, SN-38, topotecan (TPT), 9-amino-CPT, 10-amino-CPT, paclitaxel, 5-fluorouracil, 4-hydroperoxycyclophosphamide and doxorubicin, and antitumor activity was assessed. For in vivo tumor outgrowth studies, fragments were treated in parallel, implanted into nude mice, and monitored for development of tumors. RESULTS. Against 15 of 16 tumor xenografts and all primary tumor samples tested, all compounds were cytotoxic at pH 7.4 (IC(50) range 13-921 microM). MDC, SN-38, TPT, and 9-amino-CPT achieved an average 5-fold increase in activity (range 3-14) at pH 6.8, while 7-chloromethyl-MDC was enhanced 8-fold (range 6-14). The most potentiated analogue was 10-amino-CPT at 27-fold (range 17-49). In contrast, the other agents were active against one or more tumor types but were not enhanced by acidic pH. Importantly, the toxicity of MDC in histoculture of D54 glioma xenografts strongly correlated with the outgrowth of treated fragments subsequently implanted in vivo.Evaluation of anticancer drug activity in native-state histoculture supports the concept that pH modulation may be an important approach to improve the selectivity and antitumor effectiveness of camptothecin-based chemotherapy.

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor α. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.

A retrospective review of methicillin-resistant Staphylococcus aureus isolates from the Naval Medical Center, San Diego, for the years 1994 through 1997, found that the annual number of community-acquired MRSA isolates increased during the period. These outpatient isolates were more likely than inpatient isolates to be sensitive to a greater number of antibiotics.

Apoptosis is a fundamental process of cell regulation whereby cells execute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to play roles in the regulation of apoptosis, including the activation of phospholipase A2 (PLA2), usually measured as release of 3H-labelled arachidonic acid (AA) from prelabelled cells. The current study was aimed at examining the role of PLA2 in regulating apoptosis in response to several inducers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [3H]fatty acids and the released radioactivity was characterized. These studies indicated that the AA release assay did not reflect release of non-esterified fatty acid via activation of the PLA2 pathway. Rather, studies using TLC and electron microscopy showed that AA release reflected a previously unsuspected shedding of a heterogeneous population of membrane vesicles and fragments, probably as components of apoptotic bodies. Further studies demonstrated that this process is an integral part of apoptosis. Overexpression of Bcl-2 or the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethane prevented the characteristic morphological changes of cell death, and completely inhibited the release of membrane vesicles and fragments. On the other hand, release of membrane vesicles and fragments was caused by various inducers of apoptosis, as measured by release of either 3H-labelled AA or palmitic acid. Thus the present study demonstrates that the release of membrane lipids during apoptosis defines a new assay for apoptosis and has allowed the investigation of the mechanisms regulating formation of apoptotic bodies.

Apoptosis Triggered by the Absence of an Integrin Signal: Role of the Sphingolipid Ceramide