Internal Budding Research Articles

Single cell expression and chromatin accessibility of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential ribosome regulon driver

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite’s unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

Co-dependent formation of the Toxoplasma gondii sub-pellicular microtubules and inner membrane skeleton.

One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In Toxoplasma gondii, this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, T. gondii tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite Sarcocystis neurona revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected T. gondii γ-tubulin and γ-tubulin complex protein 4 (GCP4). While γ-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the γ-tubulin complex in tubulin scaffold formation .

A systematic review of resprouting in woody plants and potential implications for the management of urban plantings.

Naturalistic plantings, such as meadow-style plantings, can improve the quality of urban green spaces through aesthetic, biodiversity and low maintenance features. Species selection for, and maintenance of naturalistic plantings are key to their success. While herbaceous and grassy meadows can be mowed, naturalistic plantings with woody plants require more intense maintenance to remove biomass and promote resprouting. We aim to understand woody plant responses to diverse disturbance regimes to potentially inform the selection and management of woody species in urban plantings. We conducted a quantitative systematic literature review of 72 papers and investigated what main external (climate, disturbance regime) and internal (buds, life stage, storage reserves) factors influence the resprouting response of woody plants. We found resprouting literature is geographically widespread for woody plants, but studies are skewed towards Temperate climates in USA and Australia, with a focus on high severity and high frequency fire disturbance. Resprouting response was mostly defined as a continuous response to disturbance dependent on disturbance regime, climate and plant traits. Maintenance and management of naturalistic woody plantings, through hard pruning techniques such as coppicing, may be informed by analogous high severity and high frequency disturbance studies. However, the literature on woody plant resprouting has several knowledge gaps for lower severity and lower frequency disturbance regimes and in more arid climates. Future research should evaluate the response of naturalistic woody plantings to disturbance in specific urban contexts.

The Chart Diagnostic System Improves the Diagnostic Accuracy of Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Purpose: To establish a diagnosis method based on imaging findings and histopathological factors associated with cervical lymph node metastasis. Methods: A total of 1587 cervical lymph nodes that were detected using imaging tools in 73 OSCC patients who underwent surgical treatment were enrolled to evaluate the association between imaging findings (long diameter, short diameter, long–short ratio, US findings (hilum and internal echo), contrast effect with enhanced CT, standardized uptake value (SUV) max and SUV average with 18F FDG-Positron Emission Tomography (PET)) and metastatic cervical lymph nodes. In 57 OSCC patients, biopsy specimens were evaluated for histopathologic factors (budding score, lymphatic invasion, vascular invasion, nerve invasion, and YK classification) and the presence of cervical lymph node metastases. Cervical lymph node metastasis was determined based on histopathological examination of the lymph nodes of patients with no metastasis observed 3 years after primary surgery. Results: In total, 22 of the 73 patients had cervical lymph node metastasis pathologically. In the comparison of the presence of metastatic lymph nodes, univariate analysis showed significant differences in cervical lymph node long and short diameter, long/short ratio, internal echo, rim enhancement, SUV max, SUV average, budding score, and vascular invasion. Multivariable analysis showed significant differences in internal echo, rim enhancement, SUV max, and budding score. Conclusions: We propose a chart diagnostic system that combines imaging and histopathological findings to improve the diagnosis of cervical lymph node metastasis.

Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses.

All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

The Modular Circuitry of Apicomplexan Cell Division Plasticity.

The close-knit group of apicomplexan parasites displays a wide variety of cell division modes, which differ between parasites as well as between different life stages within a single parasite species. The beginning and endpoint of the asexual replication cycles is a ‘zoite’ harboring the defining apical organelles required for host cell invasion. However, the number of zoites produced per division round varies dramatically and can unfold in several different ways. This plasticity of the cell division cycle originates from a combination of hard-wired developmental programs modulated by environmental triggers. Although the environmental triggers and sensors differ between species and developmental stages, widely conserved secondary messengers mediate the signal transduction pathways. These environmental and genetic input integrate in division-mode specific chromosome organization and chromatin modifications that set the stage for each division mode. Cell cycle progression is conveyed by a smorgasbord of positively and negatively acting transcription factors, often acting in concert with epigenetic reader complexes, that can vary dramatically between species as well as division modes. A unique set of cell cycle regulators with spatially distinct localization patterns insert discrete check points which permit individual control and can uncouple general cell cycle progression from nuclear amplification. Clusters of expressed genes are grouped into four functional modules seen in all division modes: 1. mother cytoskeleton disassembly; 2. DNA replication and segregation (D&S); 3. karyokinesis; 4. zoite assembly. A plug-and-play strategy results in the variety of extant division modes. The timing of mother cytoskeleton disassembly is hard-wired at the species level for asexual division modes: it is either the first step, or it is the last step. In the former scenario zoite assembly occurs at the plasma membrane (external budding), and in the latter scenario zoites are assembled in the cytoplasm (internal budding). The number of times each other module is repeated can vary regardless of this first decision, and defines the modes of cell division: schizogony, binary fission, endodyogeny, endopolygeny.

Identification and Molecular Dissection of IMC32, a Conserved Toxoplasma Inner Membrane Complex Protein That Is Essential for Parasite Replication.

ABSTRACTThe inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites.

A Homolog of Structural Maintenance of Chromosome 1 Is a Persistent Centromeric Protein Which Associates With Nuclear Pore Components in Toxoplasma gondii.

Apicomplexa are obligate intracellular parasites which cause various animal and human diseases including malaria, toxoplasmosis, and cryptosporidiosis. They proliferate by a unique mechanism that combines physically separated semi-closed mitosis of the nucleus and assembly of daughter cells by internal budding. Mitosis occurs in the presence of a nuclear envelope and with little appreciable chromatin condensation. A long standing question in the field has been how parasites keep track of their uncondensed chromatin chromosomes throughout their development, and hence secure proper chromosome segregation during division. Past work demonstrated that the centromeres, the region of kinetochore assembly at chromosomes, of Toxoplasma gondii remain clustered at a defined region of the nuclear periphery proximal to the main microtubule organizing center of the cell, the centrosome. We have proposed that this mechanism is likely involved in the process. Here we set out to identify underlying molecular players involved in centromere clustering. Through pharmacological treatment and structural analysis we show that centromere clustering is not mediated by persistent microtubules of the mitotic spindle. We identify the chromatin binding factor a homolog of structural maintenance of chromosomes 1 (SMC1). Additionally, we show that both TgSMC1, and a centromeric histone, interact with TgExportin1, a predicted soluble component of the nuclear pore complex. Our results suggest that the nuclear envelope, and in particular the nuclear pore complex may play a role in positioning centromeres in T. gondii.

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes.

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a “binary fission” mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota.

The role of bud protection and bark density in frost resistance of savanna trees.

Frost events occur with a significant frequency in savannas of the Southern Hemisphere, especially in the Cerrados of Brazil. One of the main strategies to deal with such events is to invest in thick and dense bark, which can insulate internal branch tissues and protect buds, essential to ensure resprouting if frost damage causes plant canopy die-back. Such strategies may be fundamental to determine the persistence of savanna species in regions where low temperatures and frost events are recurrent. Here we describe bud protection and bark strategies of 53 woody species growing in typical savanna vegetation of central Brazil. In addition, we used an experimental approach exposing branches to 0°C to measure temperature variation in internal branch tissue and test its relationship to bud protection and bark properties. We found that the majority of species (69%) showed medium to high bud protection against extreme temperatures; however, the degree of bud protection was not clearly related to bark properties, such as bark thickness and density. Bark density is a fundamental trait in determining protection against low temperatures (0°C), since species with low bark density showed lower temperature variation in their internal branch tissues, independently of the bud protection degree. Bark properties and bud protection are two different (albeit related) strategies for the protection and persistence of savanna trees under extreme environmental temperatures and can explain ecological observations related to savanna tree responses after frost events.

A highly dynamic F-actin network regulates transport and recycling of micronemes in Toxoplasma gondii vacuoles

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.

The apical annuli of Toxoplasma gondii are composed of coiled-coil and signalling proteins embedded in the inner membrane complex sutures.

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signalling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

Effect of pruning strategy on 'Syrah' bud necrosis and fruitfulness in Brazilian subtropical Southeast

The change of wine grape harvest from wet season (summer) to dry season (winter) by changing the pruning management has improved quality of wines produced in the Brazilian Southeast. However, the vines need to be spur pruned twice a year, i.e. with a 1st pruning in August (winter pruning) for a vegetative cycle during the hot and wet summer, and a 2nd pruning in January (summer pruning) for a productive cycle during the cold and dry season. This double pruning strategy is made necessary by the fact that latent buds developed during the dry season cycle are not fruitful to support a productive cycle in the following year. This histological study, performed in the South of Minas Gerais State (Brazil), showed that annual single pruning done in the wet season (in January) displayed a high rate of necrosis on primary and secondary buds (bud necrosis – BN). In April, 99 days after summer pruning (DASP), the rates of BN were 40 % and 50 % at basal and apical node positions, respectively, reaching 80 % of BN in December (322 DASP). As a consequence of BN, bud potential fertility was drastically reduced from 0.5 inflorescence primordial (IP) per bud (in July) to 0.06 (in December) and bud burst in the next cycle from secondary and tertiary bud axes. Vines managed by double pruning system (submitted to summer and winter pruning) displayed a much higher fruitfulness potential, i.e. 1.46 IP per bud in December (112 days after winter pruning) and limited BN occurrence (20 %). On single pruned vines, we also observed a significant decrease of starch content in canes, trunks and roots. Internal bud anatomy showed that a random cell breakdown started 70 days DASP. At 211 DASP, all buds showed a large starch granule concentration, raphides and crystals of calcium oxalate inside idioblasts of leaf primordia and also in cortical parenchyma of the vegetative axis. The bud starch content was increased and a positive correlation between necrosis and starch accumulation was observed. The impact of carbohydrate availability on bud necrosis development was discussed. This study showed that the necrosis development towards secondary and tertiary axis of the dry season buds is the main reason of unfruitfulness in the vineyards managed by single pruning in the wet season, making the double pruning compulsory.

Reproduction of the anthozoan Anemonia sulcata (Pennant, 1777) in southern Spain: from asexual reproduction to putative maternal care

Anemonia sulcata (Pennant, 1777) is a common shallow water cnidarian from rocky platform and boulder beaches in southern Spain, where it is a popular seafood item with an increasing fishery. To aid in the management of a sustainable fishery, a study on the reproduction of A. sulcata in the littoral of Malaga (southern Spain) was performed from November 2014 to September 2015, using histological methods. A total of 123 specimens were examined, with a size range (as diameter of the pedal disc) from 1.1 to 48.2 mm. The sex ratio was significantly biased to females, with 1.7 females: 1 male (χ2 = 4.45, p < 0.01). The spermatozoids and oocytes arise from the endodermal cells. The mature oocytes receive nutritive filaments (trophonema) from the endoderm cells. There were zooxanthellae in the mesenteries, tentacles and also inside the oocytes. A gastrula was observed in one individual, as well as several planula larvae in different degree of development in others. Asexual reproduction by internal budding was observed in some individuals. The studied population showed an extended reproductive cycle with a peak of spawning in April. The size and weight of sexual maturity of the studied population were 21.5 mm and 16.5 g, respectively. A positive significant correlation was observed between size and weight of individuals. We suggest that the diameter of the pedal disc should be used as the legal parameter for the management of this fishery, as this measurement is easier to take by fishermen at sea than the weight, the current legal parameter.

Potential roles of smell and taste in the orientation behaviour of coral-reef fish larvae: insights from morphology.

An ontogenetic analysis of the olfactory organ and the number and distribution of internal taste buds was carried out in two neon gobies (Elacatinus lori and Elacatinus colini) with the goal of revealing morphological trends that might inform an understanding of the roles of olfaction and taste in larval orientation behaviour. The pattern of development of the olfactory organ is unremarkable and enclosure of the olfactory epithelium occurs concurrently with metamorphosis and settlement in both species. Like other gobies, juvenile and adult E. lori and E. colini lack complex olfactory lamellae, and lack the accessory nasal sacs present in some adult gobies that could facilitate active olfactory ventilation (i.e., sniffing). A small number of internal taste buds are present at hatch with most found in the caudal region of the buccal cavity (on gill arches, roof of buccal cavity). As taste bud number increases, they demonstrate an anterior spread to the lips, buccal valves and tongue (i.e., tissue covering the basihyal). In the absence of an active ventilatory mechanism for the olfactory organs, the water that moves through the buccal cavity with cyclic gill ventilation may provide chemical cues allowing the internal taste buds to play a role in chemical-mediated orientation and reef-seeking behavior in pelagic larval fishes.

A new microsporidium, Apotaspora heleios n. g., n. sp., from the Riverine grass shrimp Palaemonetes paludosus (Decapoda: Caridea: Palaemonidae).

We report a new microsporidium from a key species of the estuarine communities of the Gulf States, the Riverine grass shrimp, Palaemonetes paludosus. A milky-white shrimp was found in the Mobile Bay Delta, a large, oligohaline-freshwater wetland in Alabama, USA. Light microscopy of smears and thick sections of the abdominal tissues demonstrated infection with microsporidian spores enclosed in sporophorous vesicles (SVs) in sets of eight. Broadly oval spores measured 2.9 ± 0.06 × 1.7 ± 0.03 µm (2.5-3.3 × 1.6-1.9 µm, n = 11). SVs with a persistent membrane ranged from 4.4 to 5.6 µm in diameter. Subcuticular epithelium and underlying musculature were packed with sporonts, sporoblasts, and spores. Electron microscopy demonstrated diplokaryotic meronts that gave rise to sporont mother cells with a large single nucleus. The meront plasma membrane turned into a SV envelope, and the sporont wall segregated internally. The sporont nucleus underwent meiosis followed by two mitotic divisions accompanied by internal budding to produce four sporonts, each dividing in two uninucleate sporoblasts. Eight-spore SVs were filled with fibrillary-tubular secretions. Spores possessed 90-110-nm thick envelopes (exospore, 40-60 nm + endospore, 30-50 nm), a triangle-shaped nucleus, isofilar polar filament of 10-13 coils arranged in two-three rows, bipartite polaroplast, and a mushroom-shaped polar disk. The SSU rDNA sequence of the novel species was deposited in GenBank under Accession number MG 708238. SSU rDNA-based phylogenetic analysis indicated that the Riverine grass shrimp microsporidium was a new species and placed it in one branch with two species of Potaspora, xenoma-forming microsporidia from freshwater perciform fishes. Because morphological and developmental characters of the novel species did not fit the diagnosis of the genus Potaspora, and, based on SSU rDNA-inferred phylogenetic analyses, different host specificity, pathogenesis, and ecological considerations, we erect here the new genus Apotaspora for the Riverine grass shrimp microsporidium and name the new species Apotaspora heleios. Grouping together fish and crustacean parasites on SSU rDNA phylogenetic trees suggests that polyxenous life cycles might be a common feature of extinct and/or extant members of the studied lineage of the Microsporidia.

Labio-palatine Cleft, Morphological Substrate

Labio-palatin clefts are the most common type of craniofacial malformation encountered in practice and among the most common congenital abnormalities; it represent morphological and functional defects in newborns caused by genetically changes or environmental factors; those of the cervico-facial region have an increased incidence due to local embryological complexity and multifactorial etiology. The anomaly, with its variants, results from an inherent defect between the internal and external nasal buds, both from the frontal and upper maxillary bud. Labio-palatine clefts are characterized by a tissue deficiency as well as an abnormal disposition of the present tissues. It is believed that the labial clefts are consecutive to the lack of apoptosis of the ectoderm which envelops the internal maxillary and nasal buds, thus constituting a barrier to the fusion process of buds. Depending on the location and the extentension of the cleft, the labial cleft occurs, or the more frequent, the labial-alveolar cleft. The study group consisted of 22 patients with labio-palatine clefts, of which 16 boys (72.72%) and 6 girls (27.27%), who presented in the clinic for treatment. When the causative factor acts earlier, the clfet develops posteriorly, since mesodermization begins from the posterior to the anterior. The more disturbing factor acts, the greater the cleft. The increase in the incidence of labial-palatine cleft is believed to be mainly due to the increase in the number of women of older gestational age in women; women over 35 years of age have a double risk of having children with labio-palatine clefts, the risk being three times higher for mothers over the 39 years.

Differential carbohydrate gene expression during preplanting temperature treatments controls meristem termination and bulbing in garlic

Garlic is a sterile plant that reproduces vegetatively through cloves. We previously found that in ‘Shani’, a garlic cultivar adapted to Mediterranean climate, preplanting storage temperature alters plant development, architecture and yield during the growing period. To determine the possible mechanism underlying this phenomenon, the effects of two temperature treatments, 2 °C and 33 °C, were examined in the internal bud (IB) and storage leaf (SL) of garlic cloves using a transcriptomic approach. Higher representation of carbohydrate metabolism genes was found in cloves at 2 °C vs. 33 °C in both analyzed organs. Of the upregulated genes, 34% were common to IB and SL, whereas 15% and 51%, respectively, were organ-specific. Gene-ontology analysis confirmed that in both organs, carbohydrate metabolism-related genes are more highly expressed at 2 °C than at 33 °C. Chromatography analysis of fructan-degradation products sucrose and fructose in the IB and SL confirmed their higher availability at 2 °C vs. 33 °C. Expression analysis of garlic homologs of meristem-specific and flowering genes revealed differential expression patterns at 2 °C vs. 33 °C. RNA-Seq data were validated by real-time quantitative PCR of 10 candidate genes related to carbohydrate metabolism and flowering; results suggested that FLOWERING LOCUS T2 (FT2) is affected by carbohydrate metabolism. Overexpression of garlic FT2 in Arabidopsis resulted in an early-flowering phenotype and early termination of stem development. We suggest that preplanting environment temperature alters carbohydrate metabolism and consequently FT2 expression, affecting the timing of meristem termination and bulbing after replanting the garlic clove.

Abstract P6-05-02: Targeting vesicular trafficking machinery for breast cancer therapeutics

Abstract The Rab GTPase family is a key regulator of vesicular import, transport and export. Deregulation of vesicular trafficking promotes undesirable intercellular cross talk via exosomes that contribute to cancer pathophysiology. Preclinical and clinical studies underscore a role for exosomes and their cargo in breast cancer development, progression, metastasis, and resistance to therapy. While many studies have approached the role of exosomes in cancer pathophysiology, the 'rules' governing their production and cargo sorting represents a major unexplored gap in knowledge. Exosomes arise from internal budding of specific endosomal compartments. Rab11 GTPase family members comprised of Rab11a, Rab11b and Rab25 are critical regulators of endocytic cargo trafficking specifically of apical and recycling endosomes. Previously, our lab has shown that Rab25 is amplified and contributes to oncogenesis in both ovarian and Luminal B breast cancers. However, Rab25 is lost and when re-expressed ectopically, it inhibits invasion and migration in Claudin Low tumors suggesting a cell-lineage and context dependent function. Importantly, while in Luminal B cancer, Rab25 and its downstream effector RCP (Rab coupling protein), collaborate in driving oncogenesis, in Her2+ cancers, RCP expression is protective. Further our group recently uncovered the presence of a stroma enriched “reactive protein signature” in ER+/Her- group that is indicative of good outcome for patients. Indeed, many of the “reactive signature” proteins are reported in exosomal cargo by others. Taking together, our ongoing study utilizes a panel of breast cancer cell lines representing ER+ve/Her2+; ER+/Her-, ER-/Her2+, and ER-/Her- to evaluate the role of Rab11 family in modulating vesicular trafficking that alters the exosome biology and how that may contribute to tumor- stroma communications. Our preliminary results show that indeed over expression of Rab25 differentially alters size and number of extracellular vesicles in Luminal B cancers when compared to the Claudin Low subtype. Using a novel class of peptides that interrupt the cellular functions 25, we are investigating if Rab25 expression can be associated with an exosomal cargo profile in each subtype of breast cancer, which could then serve as a biomarker for tumor-stroma interaction. Citation Format: Mitra S, Vykoukal J, Jeong K, Mills G. Targeting vesicular trafficking machinery for breast cancer therapeutics [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-02.

Virus-Like Nanoparticle Vaccine Confers Protection against Toxoplasma gondii.

The inner membrane complex (IMC) of Toxoplasma gondii as a peripheral membrane system has unique and critical roles in parasite replication, motility and invasion. Disruption of IMC sub-compartment protein produces a severe defect in T. gondii endodyogeny, the form of internal cell budding. In this study, we generated T. gondii virus-like particle particles (VLPs) containing proteins derived from IMC, and investigated their efficacy as a vaccine in mice. VLP vaccination induced Toxoplasma gondii-specific total IgG, IgG1 and IgG2a antibody responses in the sera and IgA antibody responses in the feces. Upon challenge infection with a lethal dose of T. gondii (ME49), all vaccinated mice survived, whereas all naïve control mice died. Vaccinated mice showed significantly reduced cyst load and cyst size in the brain. VLP vaccination also induced IgA and IgG antibody responses in feces and intestines, and antibody-secreting plasma cells, mixed Th1/Th2 cytokines and CD4+/CD8+ T cells from spleen. Taken together, these results indicate that non-replicating VLPs containing inner membrane complex of T. gondii represent a promising strategy for the development of a safe and effective vaccine to control the spread of Toxoplasma gondii infection.