To determine if melatonin, via its MT1 G protein-coupled receptor (GPCR), impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT1-Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud (TEB) formation. Elevated MT1 receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT1 expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha (ERα), progesterone receptors (PR) A and B, and milk proteins β-casein and whey acidic protein (WAP). Estrogen and progesterone stimulated mammary gland development was repressed by elevated MT1 receptor expression and exogenous melatonin administration. These studies demonstrate that the MT1 melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.
Melatonin; MT1 Receptor; AKT; Stat5; Mammary Gland Development
The authors investigated perceptions of parents with children in the Head Start program about the processes of detection and intervention for developmental concerns.
Descriptive, qualitative study.
A large, urban Head Start agency, operating 14 centers and annually serving more than 1200 predominantly Latino children. During 2008–2009, a collaborative partnership with academicians from UCLA was created to evaluate their model of developmental screening and referrals.
Participants and Procedures
We conducted 5 focus groups with a total of 30 parents of Head Start children with developmental concerns. Parents were asked about where they go for information when they have concerns, how they perceived the developmental screening process and services, and how children and families have changed after being in the Head Start program. Focus groups were recorded, transcribed and translated into English, then coded in ATLAS. ti using the domains above and sorted into themes for analysis.
Parents perceived the screening process as both diagnostically and therapeutically important, with multiple benefits ranging from closer parent-teacher relationships to improved parenting and understanding of developmental interventions. Families focused their discussion on the importance of social-emotional and behavioral development, with school readiness and improved expressive language as important but secondary outcomes.
For families of children with developmental and behavioral risks or concerns, a structured developmental screening process in a preschool setting, such as that provided by Head Start, may serve as a vital gateway for identifying and addressing concerns and promoting social-emotional learning, parent engagement, language development and school readiness.
Early Childhood Development; Developmental Screening; Early Intervention; Head Start Program; Preschool; Latino Families; Social-Emotional Development
To correlate MR 2D measurements of lateral ventricular width and 3D measures of lateral ventricular and supratentorial parenchymal volumes to postnatal outcomes in fetuses with ventriculomegaly (VM).
307 fetuses (mean gestational age 26.0 weeks, range 15.7-39.4 weeks) had MR volumetry after referral for VM. Fetuses were grouped into those with (N=114) or without (N=193) other CNS anomalies. Pregnancy outcome and postnatal neurodevelopmental outcomes up to age 3 were obtained. A subgroup analysis was performed excluding fetuses with other CNS anomalies. Logistic regression analysis was performed to assess which measure was most predictive of outcome.
There were 50 terminations and 2 stillbirths. There were 255 live births. 75 were lost to follow-up. Among 180 liveborn infants with follow-up, 140 had an abnormal and 40 had normal outcome. Atrial diameter (p<0.0001), frontal horn diameter (p<0.0001), and ventricular volume (p=0.04) were each predictive of live-birth, with each having 92% specificity at 60% sensitivity. Among fetuses without other CNS anomalies, 180/193 (93%) pregnancies resulted in live deliveries, with atrial diameter (p<0.0001), frontal horn diameter (p=0.003), and ventricular volume (p=0.008) associated with live birth, and with atrial diameter having highest specificity of >99% at 60% sensitivity. Parenchymal volume was not associated with normal or abnormal outcome (either livebirth vs. demise or normal vs. abnormal neurodevelopmental outcome). Among live-borns, there was no age-adjusted threshold for any of the measures that reliably distinguished between normal and abnormal neurodevelopmental outcome.
Ventricular volume and diameter, but not parenchymal volume, correlate with live birth in fetuses with VM. However, once live-born, neither 2D nor 3D measurements can distinguish a fetus that will go on to have a normal outcome.
central nervous system; fetus; MRI; ventriculomegaly; volumetry; neurodevelopment
NR4A orphan nuclear receptors are involved in multiple biological processes which are important in tumorigenesis such as cell proliferation, apoptosis, differentiation, and glucose utilization. The significance of NR4A family member NURR1 (NR4A2) in breast cancer etiology has not been elucidated. The purpose of this study was to ascertain the impact of NURR1 expression on breast transformation, tumor growth, and breast cancer patient survival.
We determined the expression of NURR1 in normal breast versus breast carcinoma in tissue microarrays (immunohistochemistry), tissue lysates (immunoblot), and at the mRNA level (publically available breast microarrays). In addition NURR1 expression was compared among breast cancer patients in cohorts based on p53 expression, estrogen receptor α expression, tumor grade, and lymph node metastases. Kaplan-Meier survival plots were used to determine the correlation between NURR1 expression and relapse free survival (RFS). Using shRNA-mediated silencing, we determined the effect of NURR1 expression on tumor growth in mouse xenografts.
Results from breast cancer tissue arrays demonstrate a higher NURR1 expression in the normal breast epithelium compared to breast carcinoma cells (p ≤ 0.05). Among cases of breast cancer, NURR1 expression in the primary tumors was inversely correlated with lymph node metastases (p ≤ 0.05) and p53 expression (p ≤ 0.05). Clinical stage and histological grade were not associated with variation in NURR1 expression. In gene microarrays, 4 of 5 datasets showed stronger mean expression of NURR1 in normal breast as compared to transformed breast. Additionally, NURR1 expression was strongly correlated with increase relapse free survival (HR = 0.7) in a cohort of all breast cancer patients, but showed no significant difference in survival when compared among patients whom have not been treated systemically (HR = 0.91). Paradoxically, NURR1 silenced breast xenografts showed significantly decreased growth in comparison to control, underscoring a biphasic role for NURR1 in breast cancer progression.
NURR1 function presents a dichotomy in breast cancer etiology, in which NURR1 expression is associated with normal breast epithelial differentiation and efficacy of systemic cancer therapy, but silencing of which attenuates tumor growth. This provides a strong rationale for the potential implementation of NURR1 as a pharmacologic target and biomarker for therapeutic efficacy in breast cancer.
Breast cancer; NURR1; NR4A2; Orphan receptor
The identification of a hexanucleotide repeat expansion in the C9ORF72 gene as the cause of chromosome 9-linked frontotemporal dementia and motor neuron disease offers the opportunity for greater understanding of the relationship between these disorders and other clinical forms of frontotemporal lobar degeneration. In this study, we screened a cohort of 398 patients with frontotemporal dementia, progressive non-fluent aphasia, semantic dementia or mixture of these syndromes for mutations in the C9ORF72 gene. Motor neuron disease was present in 55 patients (14%). We identified 32 patients with C9ORF72 mutations, representing 8% of the cohort. The patients’ clinical phenotype at presentation varied: nine patients had frontotemporal dementia with motor neuron disease, 19 had frontotemporal dementia alone, one had mixed semantic dementia with frontal features and three had progressive non-fluent aphasia. There was, as expected, a significant association between C9ORF72 mutations and presence of motor neuron disease. Nevertheless, 46 patients, including 22 familial, had motor neuron disease but no mutation in C9ORF72. Thirty-eight per cent of the patients with C9ORF72 mutations presented with psychosis, with a further 28% exhibiting paranoid, deluded or irrational thinking, whereas <4% of non-mutation bearers presented similarly. The presence of psychosis dramatically increased the odds that patients carried the mutation. Mutation bearers showed a low incidence of motor stereotypies, and relatively high incidence of complex repetitive behaviours, largely linked to patients’ delusions. They also showed a lower incidence of acquired sweet food preference than patients without C9ORF72 mutations. Post-mortem pathology in five patients revealed transactive response DNA-binding protein 43 pathology, type A in one patient and type B in three. However, one patient had corticobasal degeneration pathology. The findings indicate that C9ORF72 mutations cause some but not all cases of frontotemporal dementia with motor neuron disease. Other mutations remain to be discovered. C9ORF72 mutations are associated with variable clinical presentations and pathology. Nevertheless, the findings highlight a powerful association between C9ORF72 mutations and psychosis and suggest that the behavioural characteristics of patients with C9ORF72 mutations are qualitatively distinct. Mutations in the C9ORF72 gene may be a major cause not only of frontotemporal dementia with motor neuron disease but also of late onset psychosis.
frontotemporal lobar degeneration; clinical characteristics; motor neuron disease; psychosis; neuropathology
This review article discusses recent work on the melatonin-mediated circadian regulation and integration of molecular, dietary and metabolic signaling mechanisms involved in human breast cancer growth and the consequences of circadian disruption by exposure to light-at-night (LAN). The antiproliferative effects of the circadian melatonin signal are mediated through a major mechanism involving the activation of MT1 melatonin receptors expressed in human breast cancer cell lines and xenografts. In estrogen receptor (ERα+) human breast cancer cells, melatonin suppresses both ERα mRNA expression and estrogen-induced transcriptional activity of the ERα via MT1-induced activation of Gαi2 signaling and reduction of cAMP levels. Melatonin also regulates the transactivation of additional members of the steroid hormone/nuclear receptor super-family, enzymes involved in estrogen metabolism, expression/activation of telomerase and the expression of core clock and clock-related genes. The anti-invasive/anti-metastatic actions of melatonin involve the blockade of p38 phosphorylation and the expression of matrix metalloproteinases. Melatonin also inhibits the growth of human breast cancer xenografts via another critical pathway involving MT1-mediated suppression of cAMP leading to blockade of linoleic acid (LA) uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Down-regulation of 13-HODE reduces the activation of growth factor pathways supporting cell proliferation and survival. Experimental evidence in rats and humans indicating that LAN-induced circadian disruption of the nocturnal melatonin signal activates human breast cancer growth, metabolism and signaling provides the strongest mechanistic support, thus far, for population and ecological studies demonstrating elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LAN.
Melatonin; Breast Cancer; Diet; Metabolism; Molecular Signaling; Circadian; Disruption
This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells.
melatonin; clock genes; circadian; serum shock; breast cancer
Biotroph pathogens establish intimate interactions with their hosts that are conditioned by the successful secretion of effectors in infected tissues and subsequent manipulation of host physiology. The identification of early-expressed pathogen effectors and early-modulated host functions is currently a major goal to understand the molecular basis of biotrophy. Here, we report the 454-pyrosequencing transcriptome analysis of early stages of poplar leaf colonization by the rust fungus Melampsora larici-populina. Among the 841,301 reads considered for analysis, 616,879 and 649 were successfully mapped to Populus trichocarpa and M. larici-populina genome sequences, respectively. From a methodological aspect, these results indicate that this single approach is not appropriate to saturate poplar transcriptome and to follow transcript accumulation of the pathogen. We identified 19 pathogen transcripts encoding early-expressed small-secreted proteins representing candidate effectors of interest for forthcoming studies. Poplar RNA-Seq data were validated by oligoarrays and quantitatively analysed, which revealed a highly stable transcriptome with a single transcript encoding a sulfate transporter (herein named PtSultr3;5, POPTR_0006s16150) showing a dramatic increase upon colonization by either virulent or avirulent M. larici-populina strains. Perspectives connecting host sulfate transport and biotrophic lifestyle are discussed.
Human immunodeficiency virus (HIV)-infected persons are at risk for severe influenza infections. Although vaccination against the H1N1 pandemic influenza strain is recommended, currently, there are no data on the durability of post-vaccination antibody responses in this population.
HIV-infected and HIV-uninfected adults (18–50 years old) received a single dose of monovalent 2009 influenza A (H1N1) vaccine (strain A/California/7/2009H1N1). Antibody levels to the 2009 H1N1 pandemic strain were determined at day 0, day 28, and 6 months by hemagglutination-inhibition assay. A seroprotective response was a post-vaccination titer of ≥1:40 among those with a pre-vaccination level of ≤1:10. Geometric mean titers (GMT) and factors associated with higher levels were also evaluated.
We studied 127 participants with a median age of 35 (interquartile range (IQR) 28, 42) years. Among the HIV-infected arm (n=63), the median CD4 count was 595 (IQR 476, 819) cells/mm3 and 83% were receiving HAART. Thirty-five percent of all participants had a pre-vaccination level of >1:10. HIV-infected compared to HIV-uninfected adults were less likely to generate a seroprotective response at day 28 (54% vs. 75%, adjusted OR 0.23, p=0.021) or have a durable response at 6 months post-vaccination (28% vs. 56%, adjusted OR 0.19, p=0.005). Additionally, although pre-vaccination GMT were similar in both arms (median 7 vs. 8, p=0.11), the GMT at 6 months was significantly lower among HIV-infected versus HIV-uninfected adults (median 20 vs. 113, p=0.003). Among HIV-infected persons, younger age (p=0.035) and receipt of HAART (p=0.028) were associated with higher GMTs at 6 months.
Despite vaccination, most HIV-infected adults do not have durable seroprotective antibody responses to the 2009 influenza A (H1N1) virus, and hence may remain vulnerable to infection. In addition to HAART use, more immunogenic vaccines are likely needed for improving protection against influenza in this population.
influenza; pandemic 2009 H1N1; vaccine responses; HIV; durability; long-term immunity
Microbial resistance has reached alarming levels, threatening to outpace the ability to counter with more potent antimicrobial agents. In particular, methicillin-resistant Staphylococcus aureus (MRSA) has become a leading cause of skin and soft-tissue infections and PVL-positive strains have been associated with necrotizing pneumonia. Increasing reports of growing resistance to glycopeptides have been noted, further limiting the efficacy of standard antibiotics, such as vancomycin. Ceftaroline is a novel fifth-generation cephalosporin, which exhibits broad-spectrum activity against Gram-positive bacteria, including MRSA and extensively-resistant strains, such as vancomycin-intermediate S. aureus (VISA), heteroresistant VISA (hVISA), and vancomycin-resistant S. aureus (VRSA). In addition to being an exciting new agent in the anti-MRSA armamentarium, ceftaroline provides efficacy against many respiratory pathogens including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Ceftaroline (600 mg intravenously every 12 hours) has been shown effective in phase III studies in the treatment of complicated skin and soft tissue infections and community-acquired pneumonia. To date, this unique antibiotic exhibits a low propensity for inducing resistance and has a good safety profile, although further post-marketing data and clinical experience are needed. In summary, ceftaroline provides an additional option for the management of complex multidrug resistant infections, including MRSA.
Ceftaroline; antibiotic; cephalosporin; methicillin-resistant Staphylococcus aureus; MRSA; multidrug resistant organisms
Background. Limited data exist on the immunogenicity of the 2009 influenza A (H1N1) vaccine among immunocompromised persons, including those with human immunodeficiency virus (HIV) infection.
Methods. We compared the immunogenicity and tolerability of a single dose of the monovalent 2009 influenza A (H1N1) vaccine (strain A/California/7/2009H1N1) between HIV-infected and HIV-uninfected adults 18–50 years of age. The primary end point was an antibody titer of ≥1:40 at day 28 after vaccination in those with a prevaccination level of ≤1:10, as measured by hemagglutination-inhibition assay. Geometric mean titers, influenza-like illnesses, and tolerability were also evaluated.
Results. One hundred thirty-one participants were evaluated (65 HIV-infected and 66 HIV-uninfected patients), with a median age of 35 years (interquartile range, 27–42 years). HIV-infected persons had a median CD4 cell count of 581 cells/mm3 (interquartile range, 476–814 cells/mm3) , and 82% were receiving antiretroviral medications. At baseline, 35 patients (27%) had antibody titers of >1:10. HIV-infected patients (29 [56%] of 52), compared with HIV-uninfected persons (35 [80%] of 44), were significantly less likely to develop an antibody response (odds ratio, .20; P = .003). Changes in the median geometric mean titer from baseline to day 28 were also significantly lower in HIV-infected patients than in HIV-uninfected persons (75 vs 153; P = .001). Five influenza-like illnesses occurred (2 cases in HIV-infected persons), but none was attributable to the 2009 influenza H1N1 virus. The vaccine was well tolerated in both groups.
Conclusions. Despite high CD4 cell counts and receipt of antiretroviral medications, HIV-infected adults generated significantly poorer antibody responses, compared with HIV-uninfected persons. Future studies evaluating a 2-dose series or more-immunogenic influenza A (H1N1) vaccines among HIV-infected adults are needed (ClinicalTrials.gov NCT00996970).
leptospirosis; hemoptysis; bacteria; Hawaii; severe pulmonary hemorrhagic syndrome; SPHS; zoonosis; travel
Poplars are extensively cultivated worldwide, and their susceptibility to the leaf rust fungus Melampsora larici-populina leads to considerable damages in plantations. Despite a good knowledge of the poplar rust life cycle, and particularly the epidemics on poplar, the perennial status of the plant host and the obligate biotrophic lifestyle of the rust fungus are bottlenecks for molecular investigations. Following the completion of both M. larici-populina and Populus trichocarpa genome sequences, gene families involved in poplar resistance or in rust fungus virulence were investigated, allowing the identification of key genetic determinants likely controlling the outcome of the interaction. Specific expansions of resistance and defense-related genes in poplar indicate probable innovations in perennial species in relation with host-pathogen interactions. The genome of M. Larici-populina contains a strikingly high number of genes encoding small secreted proteins (SSPs) representing hundreds of candidate effectors. Transcriptome analyses of interacting partners in compatible and incompatible interactions revealed conserved set of genes involved in poplar defense reactions as well as timely regulated expression of SSP transcripts during host tissues colonisation. Ongoing functional studies of selected candidate effectors will be achieved mainly on the basis of recombinant protein purification and subsequent characterisation.
Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is challenging, particularly in developing countries. Highly sensitive diagnostic methods are costly, while less expensive methods often lack sensitivity or specificity. Cost-effectiveness comparisons of the various diagnostic options have not been presented.
Methods and Findings
We compared cost-effectiveness, as measured by cost per life-years gained and proportion of patients successfully diagnosed and treated, of 33 PCP diagnostic options, involving combinations of specimen collection methods [oral washes, induced and expectorated sputum, and bronchoalveolar lavage (BAL)] and laboratory diagnostic procedures [various staining procedures or polymerase chain reactions (PCR)], or clinical diagnosis with chest x-ray alone. Our analyses were conducted from the perspective of the government payer among ambulatory, HIV-infected patients with symptoms of pneumonia presenting to HIV clinics and hospitals in South Africa. Costing data were obtained from the National Institutes of Communicable Diseases in South Africa. At 50% disease prevalence, diagnostic procedures involving expectorated sputum with any PCR method, or induced sputum with nested or real-time PCR, were all highly cost-effective, successfully treating 77–90% of patients at $26–51 per life-year gained. Procedures using BAL specimens were significantly more expensive without added benefit, successfully treating 68–90% of patients at costs of $189–232 per life-year gained. A relatively cost-effective diagnostic procedure that did not require PCR was Toluidine Blue O staining of induced sputum ($25 per life-year gained, successfully treating 68% of patients). Diagnosis using chest x-rays alone resulted in successful treatment of 77% of patients, though cost-effectiveness was reduced ($109 per life-year gained) compared with several molecular diagnostic options.
For diagnosis of PCP, use of PCR technologies, when combined with less-invasive patient specimens such as expectorated or induced sputum, represent more cost-effective options than any diagnostic procedure using BAL, or chest x-ray alone.
Bilateral perisylvian polymicrogyria (BPP) is a well-recognized malformation of cortical development commonly associated with epilepsy, cognitive impairment, and oromotor apraxia. Reports have suggested the association of BPP with arthrogryposis multiplex congenita. We sought to investigate the clinical, electrophysiological, and neuroradiological features of this combined syndrome to determine if there are unique features that distinguish BPP with arthrogryposis from BPP alone.
Cases of BPP with congenital arthrogryposis were identified from a large research database of individuals with polymicrogyria. Clinical features (including oromotor function, seizures, and joint contractures), MR brain imaging, and results of neuromuscular testing were reviewed.
Ten cases of BPP with congenital arthrogryposis were identified. Most cases had some degree of oromotor apraxia. Only a few had seizures, but a majority of cases were still young children. Electrophysiological studies provided evidence for lower motor neuron or peripheral nervous system involvement. On brain imaging, bilateral polymicrogyria (PMG) centered along the Sylvian fissures was seen, with variable extension frontally or parietally; no other cortical malformations were present. We did not identify obvious neuroimaging features that distinguish this syndrome from that of BPP without arthrogryposis.
The clinical and neuroimaging features of the syndrome of BPP with congenital arthrogryposis appear similar to those seen in cases of isolated BPP without joint contractures, but electrophysiological studies often demonstrate coexistent lower motor neuron or peripheral nervous system pathology. These findings suggest that BPP with arthrogryposis may have a genetic etiology with effects at two levels of the neuraxis.
Cortical malformation; dysplasia; perisylvian; polymicrogyria; contractures; arthrogryposis
As the mainstay treatment for advanced prostate cancer, androgen-deprivation therapy (ADT) targets the action of androgen receptor (AR) by reducing androgen level and/or by using anti-androgen to compete with androgens for binding to AR. Albeit effective in extending survival, ADT is associated with dose-limiting toxicity and the development of castration-resistant prostate cancer (CRPC) after prolonged use. Since CRPC is lethal and incurable, developing effective strategies to enhance the efficacy of ADT and circumvent resistance becomes an urgent task. Continuous AR signaling constitutes one major mechanism underlying the development of CRPC. The present study showed that methylseleninic acid (MSA), an agent that effectively reduces AR abundance, could enhance the cancer-killing efficacy of the anti-androgen bicalutamide in both androgen-dependent and castration-resistant prostate cancer cells. We found that combination of MSA and bicalutamide produced a robust downregulation of prostate-specific antigen and a recently identified AR target, telomerase and its catalytic subunit, telomere reverse transcriptase (hTERT). The downregulation of hTERT occurs mainly at the transcriptional level, and reduced AR occupancy of the promoter contributes to the downregulation. Furthermore, apoptosis induction by the two agents is significantly mitigated by restoration of hTERT. Our findings thus indicate that MSA in combination with anti-androgen could represent a viable approach to improve the therapeutic outcome of ADT. Given the critical role of hTERT/telomerase downregulation in mediating the combination effect and the fact that hTERT/telomerase could be measured in blood and urine, hTERT/telomerase could serve as an ideal tumor-specific biomarker to monitor the efficacy of the combination therapy non-invasively.
anti-androgen; methylseleninic acid; androgen receptor; telomerase; prostate cancer
Plant inducible immunity includes the accumulation of a set of defense proteins during infection called pathogenesis-related (PR) proteins, which are grouped into families termed PR-1 to PR-17. The PR-5 family is composed of thaumatin-like proteins (TLPs), which are responsive to biotic and abiotic stress and are widely studied in plants. TLPs were also recently discovered in fungi and animals. In the poplar genome, TLPs are over-represented compared with annual species and their transcripts strongly accumulate during stress conditions.
Our analysis of the poplar TLP family suggests that the expansion of this gene family was followed by diversification, as differences in expression patterns and predicted properties correlate with phylogeny. In particular, we identified a clade of poplar TLPs that cluster to a single 350 kb locus of chromosome I and that are up-regulated by poplar leaf rust infection. A wider phylogenetic analysis of eukaryote TLPs - including plant, animal and fungi sequences - shows that TLP gene content and diversity increased markedly during land plant evolution. Mapping the reported functions of characterized TLPs to the eukaryote phylogenetic tree showed that antifungal or glycan-lytic properties are widespread across eukaryote phylogeny, suggesting that these properties are shared by most TLPs and are likely associated with the presence of a conserved acidic cleft in their 3D structure. Also, we established an exhaustive catalog of TLPs with atypical architectures such as small-TLPs, TLP-kinases and small-TLP-kinases, which have potentially developed alternative functions (such as putative receptor kinases for pathogen sensing and signaling).
Our study, based on the most recent plant genome sequences, provides evidence for TLP gene family diversification during land plant evolution. We have shown that the diverse functions described for TLPs are not restricted to specific clades but seem to be universal among eukaryotes, with some exceptions likely attributable to atypical protein structures. In the perennial plant model Populus, we unravelled the TLPs likely involved in leaf rust resistance, which will provide the foundation for further functional investigations.
Heterokont algae form a monophyletic group within the stramenopile branch of the tree of life. These organisms display wide morphological diversity, ranging from minute unicells to massive, bladed forms. Surprisingly, chloroplast genome sequences are available only for diatoms, representing two (Coscinodiscophyceae and Bacillariophyceae) of approximately 18 classes of algae that comprise this taxonomic cluster.
A universal challenge to chloroplast genome sequencing studies is the retrieval of highly purified DNA in quantities sufficient for analytical processing. To circumvent this problem, we have developed a simplified method for sequencing chloroplast genomes, using fosmids selected from a total cellular DNA library. The technique has been used to sequence chloroplast DNA of two Heterosigma akashiwo strains. This raphidophyte has served as a model system for studies of stramenopile chloroplast biogenesis and evolution.
H. akashiwo strain CCMP452 (West Atlantic) chloroplast DNA is 160,149 bp in size with a 21,822-bp inverted repeat, whereas NIES293 (West Pacific) chloroplast DNA is 159,370 bp in size and has an inverted repeat of 21,665 bp. The fosmid cloning technique reveals that both strains contain an isomeric chloroplast DNA population resulting from an inversion of their single copy domains. Both strains contain multiple small inverted and tandem repeats, non-randomly distributed within the genomes. Although both CCMP452 and NIES293 chloroplast DNAs contains 197 genes, multiple nucleotide polymorphisms are present in both coding and intergenic regions. Several protein-coding genes contain large, in-frame inserts relative to orthologous genes in other plastids. These inserts are maintained in mRNA products. Two genes of interest in H. akashiwo, not previously reported in any chloroplast genome, include tyrC, a tyrosine recombinase, which we hypothesize may be a result of a lateral gene transfer event, and an unidentified 456 amino acid protein, which we hypothesize serves as a G-protein-coupled receptor. The H. akashiwo chloroplast genomes share little synteny with other algal chloroplast genomes sequenced to date.
The fosmid cloning technique eliminates chloroplast isolation, does not require chloroplast DNA purification, and reduces sequencing processing time. Application of this method has provided new insights into chloroplast genome architecture, gene content and evolution within the stramenopile cluster.
Maintenance of homeostasis requires that an organism perceive selected physical and chemical signals within an informationally dense environment. Functionally, an organism uses a variety of signal transduction arrays to amplify and convert these perceived signals into appropriate gene transcriptional responses. These changes in gene expression serve to modify selective metabolic processes and thus optimize reproductive success. Here we analyze a chloroplast-encoded His-to-Asp signal transduction circuit in the stramenopile Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor]. The presence, structure and putative function of this protein pair are discussed in the context of their evolutionary homologues.
Bioinformatic analysis of the Heterosigma akashiwo chloroplast genome sequence revealed the presence of a single two-component His-to-Asp (designated Tsg1/Trg1) pair in this stramenopile (golden-brown alga). These data represent the first documentation of a His-to-Asp array in stramenopiles and counter previous reports suggesting that such regulatory proteins are lacking in this taxonomic cluster. Comparison of the 43 kDa H. akashiwo Tsg1 with bacterial sensor kinases showed that the algal protein exhibits a moderately maintained PAS motif in the sensor kinase domain as well as highly conserved H, N, G1 and F motifs within the histidine kinase ATP binding site. Molecular modelling of the 27 kDa H. akashiwo Trg1 regulator protein was consistent with a winged helix-turn-helix identity – a class of proteins that is known to impact gene expression at the level of transcription. The occurrence of Trg1 protein in actively growing H. akashiwo cells was verified by Western analysis. The presence of a PhoB-like RNA polymerase loop in Trg1 and its homologues in the red-algal lineage support the hypothesis that Trg1 and its homologues interact with a sigma 70 (σ70) subunit (encoded by rpoD) of a eubacterial type polymerase. Sequence analysis of H. akashiwo rpoD showed this nuclear-encoded gene has a well-defined 4.2 domain, a region that augments RNA polymerase interaction with transcriptional regulatory proteins and also serves in -35 promoter recognition. The presence/loss of the His-to-Asp pairs in primary and secondary chloroplast lineages is assessed.
His-to-Asp signal transduction components are found in most rhodophytic chloroplasts, as well as in their putative cyanobacterial progenitors. The evolutionary conservation of these proteins argues that they are important for the maintenance of chloroplast homeostasis. Our data suggest that chloroplast gene transcription may be impacted by the interaction of the His-to-Asp regulator protein (which is less frequently lost than the sensor protein) with the RNA polymerase σ70 subunit.
To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10−6. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.
cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle Tuber borchii. Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.
The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.
The virulent cos-type Streptococcus thermophilus phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative ori of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between orf36 and orf37. As the replication ability of the putative ori and flanking genes could not be established, its ability to confer phage resistance was tested. When ori is cloned on a high-copy-number plasmid, it provides protection to S. thermophilus strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the cro-like gene (orf29) and the ori. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the cro-ori region in the three S. thermophilus cos-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of orf29 was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the S. thermophilus operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the pts promoter in Lactococcus lactis and in Escherichia coli.
The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.