Characterization of natural product inhibitors of quorum sensing in Pseudomonas aeruginosa reveals competitive inhibition of RhlR by ortho-vanillin.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. Ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR.

An Amino Acid Substitution in Elongation Factor EF-G1A Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa LasR-Null Mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

Tobramycin adaptation alters the antibiotic susceptibility of Pseudomonas aeruginosa quorum sensing-null mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum sensing system to increase resistance to the aminioglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism allowing the lasR-null mutants to persist under tobramycin selection. We hypothesized that the effects of inactivating lasR on tobramycin resistance might be dependent on the presence or absence of other gene mutations in that strain, a phenomenon known as epistasis. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. We show that the effects of ΔlasR on tobramycin resistance are strain dependent. The effects can be attributed to a point mutation in the gene encoding the translation elongation factor fusA1 (G61A nucleotide substitution), which confers a strong selective advantage to lasR-null PA14 under tobramycin selection. This fusA1 G61A mutation results in increased activity of the MexXY efflux pump and expression of the mexXY regulator ArmZ. The fusA1 mutation can also modulate ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results demonstrate the importance of epistatic gene interactions on antibiotic susceptibility of lasR-null mutants. These results support of the idea that gene interactions might play a significant role in the evolution of quorum sensing in P. aeruginosa.

Pubertal Suppression and Surgical Management of a Patient With 5-Alpha Reductase Deficiency.

Five-alpha reductase type 2 deficiency (5αRD2) is a rare cause of atypical genitalia in newborns. There are no definitive guidelines regarding management of children with this disorder. While many children are raised as female given the under-virilized appearance of their external genitalia at birth, these patients are historically counseled to undergo male puberty, resulting in a change in gender identity from female to male in more than half of post-pubertal patients. Here we report the first case of a patient with 5αRD2who identified as female from a very early age, strongly desired gender-affirming surgery, and elected to initiate puberty-blocking therapy prior to the onset of male puberty.

Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability.

BACKGROUND: Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs). RESULTS: Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G'). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-ß levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-ß was significantly greater for HMFs encapsulated in stiff hydrogels. CONCLUSIONS: Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn't support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Conjoint behavioral consultation: implementing a tiered home-school partnership model to promote school readiness.

An ecological perspective to school readiness focuses on child and family readiness by enhancing the developmental contexts and relationships within which children reside (e.g., home environment, parent-child relationship, home-school relationships). The Getting Ready intervention is an ecological, relationally based, tiered intervention providing both universal and intensive services to children and families to promote child and family school readiness. Intensive level consultation services were provided via Conjoint Behavioral Consultation (CBC; Sheridan & Kratochwill, 1992 , 2008 ). The purpose of this article is to describe the implementation and effects of CBC within the Getting Ready intervention to promote child and family school readiness. Keys to successful implementation of the CBC intervention and issues needing further investigation are discussed.

Functional analysis and intervention for chronic rumination.

We conducted a functional analysis and treatment evaluation of chronic rumination in a 19-year-old man with intellectual disabilities. Outcomes of the functional analysis suggested that rumination was maintained by automatic reinforcement. Results of the intervention evaluation suggested that (a) noncontingent access to food after meals reduced rumination more effectively than did noncontingent access to inedible stimuli, (b) a particular type of food was associated with lower levels of rumination than other types of food, and (c) both presession and continuous access to food reduced levels of rumination more effectively than did fixed-time access to food.

Review of sports performance research with youth, collegiate, and elite athletes.

This brief review summarizes translational and intervention research in the area of sports performance. We describe studies with youth, collegiate, and elite athletes; identify recent trends; and propose recommendations for future research.

Family involvement for children with disruptive behaviors: the role of parenting stress and motivational beliefs.

Children with disruptive behaviors are at risk for adverse outcomes. Family involvement is a significant predictor of positive child behavior outcomes; however, little research has investigated parent psychological variables that influence family involvement for children with disruptive behaviors. This study investigated the role of parental motivational beliefs (i.e., role construction and efficacy) as a potential mechanism by which parenting stress impacts family involvement for families of children with disruptive behaviors. Results indicated that parent role construction mediated the relation between parenting stress and all aspects of family involvement examined (i.e., home-based involvement, school-based involvement, and home-school communication). Parent efficacy mediated the relation between parenting stress and home-based involvement only. Parents of children with disruptive behaviors reporting stress may experience negative beliefs about their role and efficacy to support their child's education, which may thereby negatively influence their actual involvement. Therefore, parent motivational beliefs may serve as an important point for intervention to support involvement of families of children with disruptive behavior.

Diagnosis and management of primary insulin-like growth factor-I deficiency: current perspectives and clinical update.

With the availability of recombinant human (rh) IGF-I as a new therapeutic agent, the criteria for diagnosis and strategies for management of growth deficiencies continue to evolve. This supplement provides a clinical update on molecular, therapeutic, and metabolic aspects of the management of short stature associated with insulin-like growth factor-I deficiency (IGFD). Several distinct, single-gene defects associated with primary IGFD now have been identified. The first section of this supplement focuses on selecting certain patients for specific genetic testing of the GH/IGF-I axis, based on previously obtained clinical and biochemical assessments. Management of short stature in children responding poorly to rhGH and definitions of a good and poor response are discussed in the next section. In addition, the authors further address different methods to help practicing clinicians predict and assess GH response, review the effect of rhGH on final adult height, and discuss the role IGF-I may have in the therapeutic approach to short stature. Finally, the metabolic aspects related to the treatment of short stature are discussed in the third part of this supplement.

HESX1 mutations are an uncommon cause of septooptic dysplasia and hypopituitarism.

CONTEXT: Mutations in the transcription factor HESX1 have previously been described in association with septooptic dysplasia (SOD) as well as isolated defects of the hypothalamic-pituitary axis. OBJECTIVE: Given that previous screening was carried out by SSCP detection alone and limited to coding regions, we performed an in-depth genetic analysis of HESX1 to establish the true contribution of HESX1 genetic defects to the etiology of hypopituitarism. DESIGN: Nonfamilial patients (724) with either SOD (n = 314) or isolated pituitary dysfunction, optic nerve hypoplasia, or midline neurological abnormalities (n = 410) originally screened by SSCP were rescreened by heteroduplex detection for mutations in the coding and regulatory regions of HESX1. In addition, direct sequencing of HESX1 was performed in 126 patients with familial hypopituitarism from 66 unrelated families and in 11 patients born to consanguineous parents. PATIENTS: All patients studied had at least one of the three classical features associated with SOD (optic nerve hypoplasia, hypopituitarism, midline forebrain defects). RESULTS: Novel sequence changes identified included a functionally significant heterozygous mutation at a highly conserved residue (E149K) in a patient with isolated GH deficiency and digital abnormalities. The overall incidence of coding region mutations within the cohort was less than 1%. CONCLUSIONS: Mutations within HESX1 are a rare cause of SOD and hypopituitarism. However, the large number of familial patients with SOD in whom no mutations were identified is suggestive of an etiological role for other genetic factors. Furthermore, we have found that within our cohort SOD is associated with a reduced maternal age compared with isolated defects of the hypothalamopituitary axis.

Evaluation of two assessment tools in predicting driving ability of senior drivers.

OBJECTIVE: To evaluate Motor Free Visual Perceptual Test (MVPT) and Clock Drawing Task (clock test) as quick assessment tools in predicting driving capability of senior drivers for an on-road driving test. DESIGN: Senior drivers (> or = 55 yrs) referred for evaluation and recommendation for license renewal were given the MVPT, clock test, and an on-road driving test. Receiving operating characteristic (ROC) analysis and stepwise multivariate logistic regression (SMLR) were used to develop a probability model to differentiate between capable and incapable senior drivers. RESULTS: Data for 232 seniors who had completed all written tests and the on-road driving test were analyzed. Of the 232 seniors, 131 (56%) were classified as capable and 101 (44%) as incapable drivers on the road test. Mean scores for capable and incapable drivers were MVPT 32.0 +/- 4.0 vs. 28.4 +/- 4.6 and mean clock test score 3.5 +/- 0.8 vs. 2.7 +/- 1.2, and mean processing time was 7.1 + 6.5 vs. 10.6 + 5.5. The means of the three measurements were significantly different between the two groups (P value <0.001). ROC curve analysis revealed an optimal cut point of > or = 32 for MVPT score with 60% sensitivity and 83% specificity. The optimal cut point for clock test scores is > or = 3 with 70% sensitivity and 65% specificity. The optimal cut point for processing times is < or = 6.27 secs with 60% sensitivity and 80% specificity. SMLR showed that the most significant predictor of seniors' driving capabilities are the MVPT test scores and clock test scores. CONCLUSION: MVPT and clock test tools are significant predictors of driving capability on an on-road driving test.

Novel mutations within the POU1F1 gene associated with variable combined pituitary hormone deficiency.

CONTEXT: Mutations within the gene encoding the pituitary-specific transcription factor POU1F1 are associated with combined pituitary hormone deficiency (CPHD). Most of the affected individuals manifest GH, prolactin, and TSH deficiency. OBJECTIVE: We have now screened 129 individuals with CPHD and isolated GH deficiency for mutations within POU1F1. RESULTS: Causative mutations were identified in 10 of 129 individuals (7.8%). Of these, five patients harbored the dominant negative R271W mutation, which is a well-recognized mutational hot spot. We have also identified a second frequently occurring mutation, E230K, which appears to be common in Maltese patients. Additionally, we describe two novel mutations within POU1F1, an insertion of a single base pair (ins778A) and a missense mutation (R172Q). Functional studies have revealed that POU1F1 (E230K) is associated with a reduction in transactivation, although DNA-binding affinity is similar to the wild-type protein. On the other hand, POU1F1 (R172Q) is associated with a reduction in DNA binding and transactivation, whereas POU1F1 (ins778A) is associated with loss of DNA binding and a reduction in transactivation. CONCLUSIONS: Our data suggest that the phenotype associated with POU1F1 mutations may be more variable, with the occasional preservation of TSH secretion. Additionally, our data revealed POU1F1 mutations in three patients who were diagnosed as having ACTH deficiency but who, on further evaluation, were found to have normal cortisol secretion. Hence, elucidation of the genotype led to further evaluation of the phenotype, with the cessation of cortisol replacement that had been commenced unnecessarily. These data reflect the importance of mutational analysis in patients with CPHD.

Mutations within the transcription factor PROP1 are rare in a cohort of patients with sporadic combined pituitary hormone deficiency (CPHD).

OBJECTIVE: Mutations within the pituitary-specific paired-like homeobox gene PROP1 have been described in 50-100% of patients with familial combined pituitary hormone deficiency (CPHD). We screened a cohort of sporadic (n = 189) and familial (n = 44) patients with hypopituitarism (153 CPHD and 80 isolated hormone deficiencies) for mutations within the coding sequence of PROP1. DESIGN AND PATIENTS: Patients with congenital hypopituitarism were recruited from the London Centre for Paediatric Endocrinology as well as several national and international centres. The pituitary phenotype ranged from isolated growth hormone deficiency (IGHD) to panhypopituitarism. Clinical data, including endocrine and neuro-radiological studies were obtained from patient records, and DNA was collected and screened for mutations within PROP1 using PCR and single-stranded conformation polymorphism (SSCP) analysis. Positive results on SSCP were sequenced directly. RESULTS: The prevalence of PROP1 mutations in unselected sporadic cases of hypopituitarism was lower (1.1%) than in familial cases (29.5%). PROP1 mutations can be associated with a highly variable phenotype, and both pituitary hypoplasia and pituitary hyperplasia. We describe the waxing and waning of a pituitary mass over 20 months in association with a PROP1 mutation that is predicted to lead to complete loss of function. Additionally, we have identified a possible founder mutation in CPHD patients from the Indian subcontinent. CONCLUSIONS: PROP1 mutations are rare in sporadic cases of CPHD, although the prevalence rises if there is a positive family history or if the patients are carefully selected with respect to the endocrine and neuroradiological phenotype. There is considerable phenotypic variability in families with the same mutation, indicating the role of other genetic or environmental factors on phenotypic expression. Finally, the pituitary enlargement that is observed in patients with PROP1 mutations can wax and wane in size before eventual involution.

Over- and underdosage of SOX3 is associated with infundibular hypoplasia and hypopituitarism.

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

A homozygous mutation in HESX1 is associated with evolving hypopituitarism due to impaired repressor-corepressor interaction.

The paired-like homeobox gene expressed in embryonic stem cells Hesx1/HESX1 encodes a developmental repressor and is expressed in early development in a region fated to form the forebrain, with subsequent localization to Rathke's pouch, the primordium of the anterior pituitary gland. Mutations within the gene have been associated with septo-optic dysplasia, a constellation of phenotypes including eye, forebrain, and pituitary abnormalities, or milder degrees of hypopituitarism. We identified a novel homozygous nonconservative missense mutation (I26T) in the critical Engrailed homology repressor domain (eh1) of HESX1, the first, to our knowledge, to be described in humans, in a girl with evolving combined pituitary hormone deficiency born to consanguineous parents. Neuroimaging revealed a thin pituitary stalk with anterior pituitary hypoplasia and an ectopic posterior pituitary, but no midline or optic nerve abnormalities. This I26T mutation did not affect the DNA-binding ability of HESX1 but led to an impaired ability to recruit the mammalian Groucho homolog/Transducin-like enhancer of split-1 (Gro/TLE1), a crucial corepressor for HESX1, thereby leading to partial loss of repression. Thus, the novel pituitary phenotype highlighted here appears to be a specific consequence of the inability of HESX1 to recruit Groucho-related corepressors, suggesting that other molecular mechanisms govern HESX1 function in the forebrain.

The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.