Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics.

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

A Caenorhabditis elegans Male Pheromone Feminizes Germline Gene Expression in Hermaphrodites and Imposes Life-History Costs.

Sex pheromones not only improve the reproductive success of the recipients, but also impose costs, such as a reduced life span. The underlying mechanisms largely remain to be elucidated. Here, we show that even a brief exposure to physiological amounts of the dominant Caenorhabditis elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome is the upregulation of genes expressed during oogenesis and the downregulation of genes associated with male gametogenesis. This result reveals a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function with the presence of potential mating partners. We also found that exposure to ascr#10 increased the risk of persistent intestinal infections in hermaphrodites due to pathological pharyngeal hypertrophy. Thus, our study reveals ways in which the male pheromone can not only have beneficial effects on the recipients' reproduction, but also cause harmful consequences that reduce life span.

A C. elegans male pheromone feminizes germline gene expression in hermaphrodites and imposes life-history costs.

Sex pheromones improve reproductive success, but also impose costs. Here we show that even brief exposure to physiological amounts of the dominant C. elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome was the upregulation of genes expressed during oogenesis and downregulation of genes associated with male gametogenesis. Among the detrimental effects of ascr#10 on hermaphrodites is the increased risk of persistent infections caused by pathological pharyngeal hypertrophy. Our results reveal a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function to the presence of potential mating partners. They also show that the beneficial effects of the pheromone are accompanied by harmful consequences that reduce lifespan.

Genome-wide analysis of anterior-posterior mRNA localization in Stentor coeruleus reveals a role for the microtubule cytoskeleton.

Cells have complex and beautiful structures that are important for their function, but understanding the molecular mechanisms that produce these structures is a challenging problem due to the gap in size scales between molecular interactions and cellular structures. The giant ciliate Stentor coeruleus is a unicellular model organism whose large size, reproducible structure, and ability to heal wounds and regenerate has historically allowed the formation of structure in a single cell to be addressed using methods of experimental embryology. Such studies have shown that specific cellular structures, such as the oral apparatus, always form in specific regions of the cell, which raises the question: what is the source of positional information within this organism? By analogy with embryonic development, in which localized mRNA is often used to mark position, we asked whether position along the anterior-posterior axis of Stentor might be marked by specific regionalized mRNAs. By physically bisecting cells and conducting half-cell RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half. We repeated this analysis in cells in which a set of longitudinal microtubule bundles running down the whole length of the cell, known as KM-fibers, were disrupted by RNAi of b-tubulin. We found that many messages either lost their regionalized distribution or switched to an opposite distribution, such that anterior-enriched messages in control became posterior-enriched in the RNAi cells, or vice versa. This study indicates that mRNA can be regionalized within a single giant cell and that microtubules may play a role, possibly by serving as tracks for the movement of the messages.

A male pheromone that improves the quality of the oogenic germline.

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest.

When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.

Using Transcriptomes as Mutant Phenotypes Reveals Functional Regions of a Mediator Subunit in Caenorhabditis elegans.

Although transcriptomes have recently been used as phenotypes with which to perform epistasis analyses, they are not yet used to study intragenic function/structure relationships. We developed a theoretical framework to study allelic series using transcriptomic phenotypes. As a proof-of-concept, we apply our methods to an allelic series of dpy-22, a highly pleiotropic Caenorhabditis elegans gene orthologous to the human gene MED12, which encodes a subunit of the Mediator complex. Our methods identify functional units within dpy-22 that modulate Mediator activity upon various genetic programs, including the Wnt and Ras modules.

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements.

RNA-sequencing (RNA-seq) is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Also, quantifying and interpreting epistatic effects using expression profiles remains a challenge. We developed a single coefficient to quantify transcriptome-wide epistasis that reflects the underlying interactions and which can be interpreted intuitively. To demonstrate our approach, we sequenced four single and two double mutants of Caenorhabditis elegans From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes with HIF-1-dependent expression that have opposite changes in expression in mutants of two genes that cooperate to negatively regulate HIF-1 abundance; however, the double mutant of these genes exhibits suppression epistasis. This class violates the classical model of HIF-1 regulation but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.

The Caenorhabditis elegans Female-Like State: Decoupling the Transcriptomic Effects of Aging and Sperm Status.

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm.

Tissue enrichment analysis for C. elegans genomics.

BACKGROUND: Over the last ten years, there has been explosive development in methods for measuring gene expression. These methods can identify thousands of genes altered between conditions, but understanding these datasets and forming hypotheses based on them remains challenging. One way to analyze these datasets is to associate ontologies (hierarchical, descriptive vocabularies with controlled relations between terms) with genes and to look for enrichment of specific terms. Although Gene Ontology (GO) is available for Caenorhabditis elegans, it does not include anatomical information. RESULTS: We have developed a tool for identifying enrichment of C. elegans tissues among gene sets and generated a website GUI where users can access this tool. Since a common drawback to ontology enrichment analyses is its verbosity, we developed a very simple filtering algorithm to reduce the ontology size by an order of magnitude. We adjusted these filters and validated our tool using a set of 30 gold standards from Expression Cluster data in WormBase. We show our tool can even discriminate between embryonic and larval tissues and can even identify tissues down to the single-cell level. We used our tool to identify multiple neuronal tissues that are down-regulated due to pathogen infection in C. elegans. CONCLUSIONS: Our Tissue Enrichment Analysis (TEA) can be found within WormBase, and can be downloaded using Python's standard pip installer. It tests a slimmed-down C. elegans tissue ontology for enrichment of specific terms and provides users with a text and graphic representation of the results.

Vocabulario expresivo en una muestra de niños preescolares con trastornos psiquiátricos y un grupo con desarrollo típico / Expressive vocabulary in a sample of preschoolers with psychiatric disorders and a group of children with typical development

Introduction Expressive language problems are common amongst preschoolers both in the general population (15-20%) and in clinical settings (50-75%); furthermore, these problems are often not detected. Language problems require attention since they are associated with severe developmental disorders such as autism (Au), Asperger's syndrome (AS), attention-deficit hyperactivity disorder (ADHD) and mental retardation. In theory, language development, specifically expressive vocabulary, associated to psychiatric disorders could be identified with a scale that measures expressive language. Objectives 1. To determine the frequency of language delay in a sample of Mexican children with typical development in the community. 2. To determine the vocabulary level for autism, Asperger's syndrome, ADHD and other psychiatric disorders through the use of the Language Development Survey (LDS). 3. To analyze if differences in vocabulary ratings among the clinical subgroups can be detected with this instrument. Materials and methods The sample consisted of: A community group with typical development (TDG) (n=302) and a clinical group (CG) (n=55); both groups had an age range of 2-5 years. The clinical group was subdivided into 4 clinical subgroups based on DSM-IV criteria for: autism, Asperger's syndrome, ADHD and other psychiatric disorders (OPD) (enuresis, encopresis, separation anxiety). Exclusion criteria were: deafness, hypoacusia and other sensorial disorders and mental retardation. A semi-structured interview based on DSM-IV criteria was designed ad hoc to diagnose: autism, Asperger's syndrome, ADHD (inattentive, combined or hyperactive impulsive varieties), specific phobia disorder, tics (transitory, chronic and Tourette's syndrome), dysthymic disorder, depression, enuresis, separation anxiety disorder based on parent information. The clinical evaluation included a semi-structured play session with age-appropriate didactic material. Discrepancies in diagnosis were resolved by consensus. All interviews were conducted by an experienced clinician. The number of bulbs in the household was used to measure socioeconomic status (SES). The LDS is a list of words that explores children's vocabulary based upon parental report. The original survey has a Cronbach's alpha coefficient of 0.99, test-retest coefficient of 0.97-0.99, and a sensitivity and specificity of 86-90%. Language delay (LD) was defined as ≤50 words, as recommended by several researchers. All parents signed an informed consent form and answered the LDS. Statistical analysis. Categorical data was analyzed using a χ2 analysis; continuous data such as age, socioeconomic status, and LDS score, were analyzed using t-tests. To analytically compare the LDS group medians, a Kruskal-Wallis test was used, since the variable distribution violated the normality distribution requirements for parametric tests. For the post hoc tests, a Tamhane analysis was used for groups of different sizes. Differences were considered statistically significant if they had a p<0.05. Results The groups were similar for variables such as child's age, parents' age and the LDS median between the normal development group and the clinical group t(355)=1.12, p=.26. The proportion of male children was greater in the clinical group (CG) than in the TDG, 76.4% vs. 53%, χ2(1,N=357)=10.63, p<.001. SES was higher for the TDG (M=7.2, SD=4.2) than for the CG (M=5.8, SD=3), p<.005. The father's age (r=.15, p<.009), the mother's age (r=.16, p<.003) and the SES (r=.13, p<.01) were correlated to the LDS score. Additionally, father's and mother's age were strongly correlated (r=.72, p<.0001) and the mother's age showed small correlations with the socioeconomic status (r=.15, p<.004). The mother's age was correlated with the child's vocabulary for both sexes (males: r=.16, p<.04, females: r=.16, p<.02), and vocabulary was significantly correlated with the SES, only for the males. Language delay frequency in the TDG was 21.2%, and 23.6% for the CG, χ2(1,N=352)=1.03, p<0.59. By sex, males in both groups exhibited a greater frequency of LD [TDG: 21.6% males vs. 20.7% females, χ2(1,N=302)=.154, p<0.926; CG: 26.2% males vs. 15.4% females, χ2(1,N=55)=.642, p<0.423]. The autism subgroup had the lowest vocabulary rating (M=85, SD=78.68), followed by the OPD subgroup (M=149, SD=121), whose rating was very similar to the typically development group (M=179, SD=105). The Asperger group (M=259, SD=27) had a similar score to the ADHD group (M=286, SD=100.2), which had the highest vocabulary score of all. The Kruskal-Wallis test for median differences was significant [H(4)=17.47, p<.002]. Multiple contrast comparisons and Tamhane's post hoc analysis showed that only the contrast between the autism and the ADHD subgroups (means: 85 vs. 286, respectively) was significant (ANOVA Tamhane post hoc, p<.01).

Introducción Aun cuando los problemas de lenguaje expresivo son muy comunes tanto en la población general (15-20%) como en la clínica (50-75%), su detección es insuficiente. Los problemas de lenguaje requieren atención debido a su comorbilidad con problemas graves del desarrollo como el autismo, el trastorno de Asperger, el trastorno por déficit de la atención e hiperactividad (TDAH) y el retraso mental. En teoría, el vocabulario asociado a estos trastornos psiquiátricos podría identificarse con un instrumento que midiera el vocabulario expresivo. Objetivos 1. Determinar la frecuencia de atraso del lenguaje (AL) (SDL ≤50 palabras) en un grupo con desarrollo típico de la comunidad. 2. Determinar el nivel de vocabulario para los subgrupos de: autismo, trastorno de Asperger (TA), TDAH y otros trastornos psiquiátricos (OTP) por medio del sondeo del desarrollo del lenguaje (SDL). 3. Analizar si el SDL puede discriminar entre los subgrupos clínicos. Sujetos y método La muestra estuvo compuesta por: un grupo de la comunidad con desarrollo típico (GDT) (n=302), y un grupo clínico (GC) (n=55), con un rango de edad de 2-5 años. Se formaron cuatro subgrupos clínicos: autismo, trastorno de Asperger, TDAH y un grupo de OTP (enuresis, encopresis, ansiedad de separación). El SDL es una lista de palabras que identifica el padre sobre el vocabulario de los niños que tiene un coeficiente de alpha de Cronbach de (.99), un test-retest de .97 a .99 y una sensibilidad y especificidad de 86-90%. Se utilizó la definición de atraso de lenguaje (AL) basada en un punto de corte de ≤50 palabras. Análisis estadístico. Los datos categóricos fueron analizados mediante la prueba de chi-cuadrada y para las medidas continuas como la edad, el MSE y el puntaje del SDL se usaron pruebas t de Student. Para el análisis del contraste de las medianas del SDL de los grupos se aplicó una prueba de Kruskal-Wallis. Resultados Los grupos fueron semejantes para las variables como edad del niño, edad de los padres y la media del SDL. La frecuencia de AL (≤50 palabras) fue de 21.2% para el GDT y de 23.6% para la población clínica. Por sexo, los varones presentaron mayor frecuencia de atraso de lenguaje (GDT): 21.6% masculino vs. 20.7% femenino (p<0.926), GC: 26.2% masculino vs. 15.4% femenino (p<0.423). El vocabulario del grupo de autismo fue el menor de todos (Mdn=85, DE=78.68) seguido del grupo de OTP (Mdn=149, DE=121.0) que presentó un desempeño muy semejante al grupo de la comunidad (GDT) (Mdn=179, DE=105.0). El grupo de Asperger (Mdn=259, DE=127) tuvo un puntaje cercano al grupo de TDAH (Mdn=286, DE=100.25). La prueba de Kruskal-Wallis para la diferencia en las medianas fue significativa (p<.002) pero sólo el contraste entre el grupo de autismo y de TDAH (Mdn=85 vs. Mdn=286, p<.01) fue significativo. Discusión La frecuencia de AL para el GDT fue de 21.6% y para el GC fue de 23.6%. El SDL fue sensible en la detección del nivel de vocabulario entre los grupos y los resultados fueron congruentes con el desempeño esperado con algunas excepciones. Los niños con TDAH expresaron un mayor número de palabras comparados con el GDT. El único contraste significativo fue la comparación entre el grupo de TDAH y el autismo. El vocabulario del grupo de Asperger fue mejor que el de autismo, pero esta diferencia no alcanzó significancia estadística. Conclusiones La versión mexicana del SDL es un instrumento de tamizaje útil para identificar el atraso del lenguaje en los niños preescolares. Este estudio muestra que el atraso de lenguaje en un niño preescolar con TDAH es una indicación para profundizar en el diagnóstico del autismo. Tampoco deben pasarse por alto otros trastornos que pueden acompañar o no el TDAH como los trastornos del lenguaje específicos (pronunciación, expresión, comprensión). El SDL mide el vocabulario y no identifica alteraciones del lenguaje cualitativas más complejas asociadas al trastorno de Asperger.

Early gallbladder carcinoma: a clinicopathologic study of 13 cases of intramucosal carcinoma.

We report the clinicopathologic features of 13 cases of intramucosal carcinoma (IMC) of the gallbladder. All IMCs were incidental findings in cholecystectomy specimens for cholelithiasis. However, one of the patients had a carcinoma of the pancreas, and the gallbladder incidentally removed during the Whipple procedure showed an IMC. Another patient had a small cell carcinoma of the gallbladder, and one of the sections showed an IMC. Of the IMCs, 10 were well-differentiated adenocarcinomas, 1 was a moderately differentiated adenocarcinoma, 1 was an undifferentiated carcinoma, and 1 was a squamous cell carcinoma. Of the patients, 8 were disease-free from 3 to 11 years, and 2 patients died, one as a result of the pancreatic ductal carcinoma and the other with disseminated metastases of the small cell carcinoma. The follow-up of another patient was too short to be significant. Two patients were lost to follow-up. Our findings suggest that a simple cholecystectomy is a curative procedure for IMCs of the gallbladder.

Cutaneous angiosarcoma. Analysis of 434 cases from the Surveillance, Epidemiology, and End Results Program, 1973-2007.

Cutaneous angiosarcoma is an aggressive malignant mesenchymal vasoformative neoplasm that accounts for 1% of all soft tissue sarcomas. Using data from the National Cancer Institute's Surveillance, Epidemiology, and End Results program, we analyzed the demographics and survival of cutaneous angiosarcoma. The Surveillance, Epidemiology, and End Results program recorded 434 cases of cutaneous angiosarcoma from 1973 to 2007. The incidence was nearly the same in men (222 cases) and women (212 cases). Most patients were white (88%) with a mean age of 73 years. African Americans made up only 4% of the cases. Two hundred seventy (62%) cases were tumors of the head and neck, whereas 106 (24%) cases arose in the skin of the trunk. Grade was recorded in 194 cases (45%): 28 were grade I, 44 were grade II, 60 were grade III, and 62 were grade IV. Survival rates of cutaneous angiosarcoma correlated with age, anatomical site, and stage of disease. Patients younger than 50 years had a 10-year relative survival rate of 71.7%, whereas patients 50 years and older had a 36.8% 10-year survival rate. Tumors of the scalp and neck resulted in a 13.8% 10-year relative survival rate, whereas tumors arising in the trunk resulted in a 75.3% 10-year survival rate. Tumors localized to the skin had better prognosis (53.6% 10-year relative survival rate) than those with regional or distant stage (19.0% and 6.2%). Twenty-six percent of patients with angiosarcoma had a prior primary. Cutaneous angiosarcomas arise predominantly in the head and neck of white individuals older than 60 years.