The Impact of Family Functioning on Help-Seeking Behavior and Symptom Severity in Obsessive-Compulsive Disorder.

ABSTRACT: Poor family functioning is associated with higher symptom severity in pediatric obsessive-compulsive disorder (OCD) and delayed help-seeking behavior in other forms of psychopathology. However, little is known about the impact of family functioning on help-seeking behavior and symptom severity in adults with OCD. The present study investigated the association between family functioning and both treatment delay and symptom severity in adults with obsessive-compulsive symptoms. Participants were 194 adults who self-identified as having OCD and completed an internet survey, including measures assessing family functioning, obsessive-compulsive symptom severity, help-seeking behavior, and depression symptom severity. Poorer family functioning was associated with higher obsessive-compulsive and depression symptom severity, after controlling for significant demographic variables. With respect to domains of family functioning, poorer general functioning, problem solving, communication skills, role functioning, affective involvement, and affective responsiveness were associated with higher obsessive-compulsive and depression symptom severity, after controlling for demographics. Poorer problem solving and communication were not significantly associated with treatment delay after controlling for demographics. Findings highlight the need for family intervention within the treatment framework for adult OCD and suggest targets ( e.g. , communication) to be addressed.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Structural and Functional Evaluation of Clinically Relevant Inhibitors of Steroidogenic Cytochrome P450 17A1.

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. (S)-seviteronel binds similarly to both observed CYP17A1 conformations. However, (S)-orteronel and (R)-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the (S)-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent (R)-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site.

Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.

The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

Evaluation of the toxicity of 2-aminoimidazole antibiofilm agents using both cellular and model organism systems.

Biofilm formation is a ubiquitous bacterial defense mechanism and has been shown to be a primary element in the antibiotic resistance of many human diseases, especially in the case of nosocomial infections. Recently, we have developed several compound libraries that are extremely effective at both dispersing preexisting biofilms and also inhibiting their initial formation. In addition to their antibiofilm properties, some of these molecules are able to resensitize resistant bacterial strains to previously ineffective antibiotics and are being assessed as adjuvants. In this study, we evaluated the toxic effects of three of our most effective 2-aminoimidazole compounds (dihydrosventrin, RA, and SPAR) using a rapid pipeline that combines a series of assays. A methylthiazolyldiphenyl-tetrazolium assay, using the HaCaT keratinocyte cell line was used to determine epidermal irritants and was combined with Caenorhabditis elegans fecundity assays that demonstrated the effects of environmental exposure to various concentrations of these molecules. In each case, the assays showed that the compounds did not exhibit toxicity until they reached well above their current biofilm dispersion/inhibition concentrations. The most effective antibiofilm compound also had significant effects when used in conjunction with several standard antibiotics against resistant bacteria. Consequently, it was further investigated using the C. elegans assay in combination with different antibiotics and was found to maintain the same low level of toxicity as when acting alone, bolstering its candidacy for further testing as an adjuvant.

Synthesis and biological evaluation of 2-aminoimidazole/carbamate hybrid anti-biofilm and anti-microbial agents.

The successful marriage of structural features from our 2-aminoimidazole and menthyl carbamate classes of anti-biofilm agents has resulted in the development of a novel hybrid scaffold of biofilm modulators. The compounds were evaluated against a panel of four bacterial strains for anti-biofilm and anti-microbial activity.

Synergistic effects between conventional antibiotics and 2-aminoimidazole-derived antibiofilm agents.

2-aminoimidazoles are an emerging class of small molecules that possess the ability to inhibit and disperse biofilms across bacterial order, class, and phylum. Herein, we report the synergistic effect between a 2-aminoimidazole/triazole conjugate and antibiotics toward dispersing preestablished biofilms, culminating with a 3-orders-of-magnitude increase of biofilm dispersion toward Staphylococcus aureus biofilms. Furthermore, we document that the 2-aminoimidazole/triazole conjugate will also resensitize multidrug-resistant strains of bacteria to the effects of conventional antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii.

Chemical synthesis and biological screening of 2-aminoimidazole-based bacterial and fungal antibiofilm agents.

A collection of 2-aminoimidazole/triazole amides has been synthesized and screened for antibiofilm activity. This class of small molecules was found to modulate the biofilm activity of Pseudomonas aeruginosa, a multidrug-resistant strain of Acinetobacter baumannii (MDRAB), a methicillin-resistant Staphylococcus aureus strain (MRSA), Escherichia coli, Rhodospirillum salexigens, Staphylococcus epidermidis, Vibrio vulnificus, and vancomycin-resistant Enterococcus faecium as well as the yeast Candida albicans and Cryptococcus neoformans. Furthermore, lead compounds were found to not lyse red blood cells at active concentrations.

Modulating the development of E. coli biofilms with 2-aminoimidazoles.

The synthesis of a 20 member 2-aminoimidazole/triazole pilot library is reported. Each member of the library was screened for its ability to inhibit or promote biofilm development of either Escherichia coli and Acinetobacter baumannii. From this screen, E. coli-selective 2-aminoimidazoles were discovered, with the best inhibitor inhibiting biofilm development with an IC(50) of 13µM. The most potent promoter of E. coli biofilm formation promoted biofilm development by 321% at 400µM.

Synthesis and bacterial biofilm inhibition studies of ethyl N-(2-phenethyl) carbamate derivatives.

An 88 member library based upon the marine bacterial metabolite ethyl N-(2-phenethyl) carbamate was evaluated for bacterial biofilm inhibition against a panel of medically relevant strains. These studies culminated in the discovery of a new class of molecules capable of inhibiting the formation of S. aureus biofilms with low micromolar IC(50) values.

A nitroenolate approach to the synthesis of 4,5-disubstituted-2-aminoimidazoles. Pilot library assembly and screening for antibiotic and antibiofilm activity.

A library of 4,5-disubstituted-2-aminoimidazoles was synthesized using a nitroenolate route and then screened for antibiofilm and antimicrobial activity. These compounds displayed notable biofilm dispersal and planktonic microbicidal activity against various Gram-positive and Gram-negative bacteria.

The chemical synthesis and antibiotic activity of a diverse library of 2-aminobenzimidazole small molecules against MRSA and multidrug-resistant A. baumannii.

Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein is described the development of a class of novel 2-aminobenzimidazoles with antibiotic activity. These active 2-aminobenzimidazoles retain their antibiotic activity against several strains of multidrug-resistant Staphylococcus aureus and Acinetobacter baumannii when compared to susceptible strains.

A 2-aminobenzimidazole that inhibits and disperses gram-positive biofilms through a zinc-dependent mechanism.

A number of 2-aminobenzimidazole derivatives were synthesized and screened for their ability to inhibit and disperse bacterial biofilms. From these compounds, a lead 2-aminobenzimidazole was identified that both inhibited and dispersed MRSA, vancomycin-resistant Enterococcus faecium, and Staphylococcus epidermidis biofilms. Mechanistic studies showed that the activity is Zn(II)-dependent, potentially via a direct zinc-chelating mechanism.

Tandem dispersion and killing of bacteria from a biofilm.

The combined effects of biofilm dispersion with a 2-aminoimidazole-triazole conjugate and bactericidal activity with a photodynamic inactivation agent suggest a novel combination therapy for treating diverse microbial infections.

Inhibition of Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa biofilm formation with a class of TAGE-triazole conjugates.

A chemically diverse library of TAGE-triazole conjugates was synthesized utilizing click chemistry on the TAGE scaffold. This library of small molecules was screened for anti-biofilm activity and found to possess the ability of inhibiting biofilm formation against Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa. One such compound in this library demonstrated the most potent inhibitory effect against Staphylococcus aureus biofilm formation that has been displayed by any 2-aminoimidazole derivative.

Target Deconvolution of a Multikinase Inhibitor with Antimetastatic Properties Identifies TAOK3 as a Key Contributor to a Cancer Stem Cell-Like Phenotype.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Concentration of Listeria monocytogenes in skim milk and soft cheese through microplate immunocapture.

Microplate immunocapture is an inexpensive method for the concentration of foodborne pathogens using an antibody-coated microplate. The objective of this study was to determine the efficacy of microplate immunocapture as an alternative to traditional enrichment for concentrating Listeria monocytogenes to levels detectable with selective plating or real-time PCR. L. monocytogenes isolates serologically characterized as Type 1 (1/2a) and Type 4 (untypeable) were grown overnight and diluted to 100 to 106 colony-forming units (CFU)/mL. The isolates were used to optimize microplate immunocapture in tryptic soy broth with 0.6% yeast extract (TSBYE), skim milk, and queso fresco samples. Following microplate immunocapture, the bacteria were streaked onto polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) agar, followed by incubation at 37 °C for 24 ±â€¯2 h. The bacteria also underwent real-time polymerase chain reaction (PCR). The optimized microplate immunocapture method was tested in triplicate for its ability to capture L. monocytogenes in broth and food samples. Overall recovery rates for L. monocytogenes in food samples at cell populations of 100, 102, and 104 CFU/25 g using microplate immunocapture with real-time PCR were 88.9%, 94.4%, and 100%, respectively. Recovery in these matrices using microplate immunocapture with selective plating was comparatively lower, at 0%, 44.4%, and 100%, respectively. Conventional culture method showed 100% detection at each inoculation level. Microplate immunocapture combined with real-time PCR shows high potential to reduce the time required for detection, with concentration of L. monocytogenes to detectable levels within 1-4 h. The incorporation of a short enrichment step may improve recovery rates at low cell levels.

Assessing health-related quality of life in NeuroAIDS: some psychometric properties of the Neurological Quality of Life Questionnaire (NeuroQOL).

Several studies were undertaken to assess the psychometric properties (reliability and initial convergent and discriminant construct validity) of the Neurological Quality of Life Questionnaire (NeuroQOL). The NeuroQOL contains 114 items answered in self report Likert format, with higher scores reflecting better quality of life. Study one compared the questionnaire with existing quality of life measures (Symptom Distress Scale, Sickness Impact Profile) and a significant (p<0.05) correlation was found. Studies two through five evaluated the relationship between the NeuroQOL and disease stage, psychological, neuropsychological and neurological measures, and a significant correlation was also found with each domain. The internal consistency reliability (alpha=0.96), split half reliability (r(12)=0.97), and test-retest reliability (coefficients were 0.78 for 6 months and 0.67 for one year intervals between test and retest) were all found to be high and adequately stable. Overall, these results indicate acceptable reliability and initial construct validity for the NeuroQOL.

Identification of novel small molecule Beclin 1 mimetics activating autophagy.

Anti-apoptotic proteins Bcl-2 and Bcl-xL could block autophagy by binding to Beclin 1 protein, an essential inducer of autophagy. Compounds mimicking Beclin 1 might be able to disrupt Bcl-xL/2-Beclin 1 interaction, free out Beclin 1, and thus trigger autophagy. In order to identify small molecule Beclin 1 mimetics, a fluorescence polarization-based high-throughput screening of 50,316 compounds was carried out with a Z' score of 0.82 ± 0.05, and an outcome of 58 hits. After the structure analysis, three acridine analogues were unveiled and confirmed using the fluorescence polarization assay and the surface plasmon resonance assay. Moreover, a set of 17 additional acridine analogues was prepared and tested. Compound 7 showed selectivity for Bcl-xL (KD = 6.5 µM) over Bcl-2 (KD = 160 µM) protein, and potent cytotoxicity (nanomolar scale) in PC-3, PC-3a and DU145 prostate cancer cells. Furthermore, induction of autophagy was also demonstrated in PC-3 and PC-3a cells treated with some acridine compounds by LC3 conversion immunoblotting and LC3 fluorescence microscopy. These Beclin 1 mimetics will be invaluable tools for developing novel autophagy inducers, better understanding the roles of autophagy in cancer, and will contribute to cancer therapy.

Cognitive sequelae of subthalamic nucleus deep brain stimulation in Parkinson's disease: a meta-analysis.

BACKGROUND: Deep brain stimulation of the subthalamic nucleus (STN DBS) is an increasingly common treatment for Parkinson's disease. Qualitative reviews have concluded that diminished verbal fluency is common after STN DBS, but that changes in global cognitive abilities, attention, executive functions, and memory are only inconsistently observed and, when present, often nominal or transient. We did a quantitative meta-analysis to improve understanding of the variability and clinical significance of cognitive dysfunction after STN DBS. METHODS: We searched MedLine, PsycLIT, and ISI Web of Science electronic databases for articles published between 1990 and 2006, and extracted information about number of patients, exclusion criteria, confirmation of target by microelectrode recording, verification of electrode placement via radiographic means, stimulation parameters, assessment time points, assessment measures, whether patients were on levodopa or dopaminomimetics, and summary statistics needed for computation of effect sizes. We used the random-effects meta-analytical model to assess continuous outcomes before and after STN DBS. FINDINGS: Of 40 neuropsychological studies identified, 28 cohort studies (including 612 patients) were eligible for inclusion in the meta-analysis. After adjusting for heterogeneity of variance in study effect sizes, the random effects meta-analysis revealed significant, albeit small, declines in executive functions and verbal learning and memory. Moderate declines were only reported in semantic (Cohen's d 0.73) and phonemic verbal fluency (0.51). Changes in verbal fluency were not related to patient age, disease duration, stimulation parameters, or change in dopaminomimetic dose after surgery. INTERPRETATION: STN DBS, in selected patients, seems relatively safe from a cognitive standpoint. However, difficulty in identification of factors underlying changes in verbal fluency draws attention to the need for uniform and detailed reporting of patient selection, demographic, disease, treatment, surgical, stimulation, and clinical outcome parameters.