CpG-ODNs activate dendritic cells (DCs) to produce interferon alpha (IFNα) and beta (IFNβ). Previous studies demonstrated that Toll-like receptor 9 (TLR9) deficient DCs exhibited a residual IFNα ...response to CpG-A, indicating that yet-unidentified molecules are also involved in induction of IFNα by CpG-A. Here, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) but not Ku70 deficient BMDCs showed defective IFNα and IFNβ responses to CpG-A or CpG-B. Loss of both DNA-PKcs and TLR9 further reduced the IFNα response to CpG-A. These DNA-PKcs and TLR9 effects were mediated by their downstream Akt/mTORC1 pathway and downstream events IRAK1 and IKKα. Loss of DNA-PKcs, TLR9, MyD88 or IRAK4 impaired phosphorylation of Akt(S473), S6K, S6, IRAK1, or IKKα in BMDCs in response to CpG-ODNs. The residual IFNα and IFNβ in DNA-PKcs-deficient BMDCs were partially responsible for the induction of IL-6 and IL-12 by CpG-ODNs and their stimulatory effect was blocked by IFNAR1 neutralizing antibodies. Further analysis indicated that CpG-ODN associated with DNA-PKcs and Ku70, and induced DNA-PKcs's interaction with TRAF3. Intriguingly, DNA-PKcs but not Ku70 expression level was reduced in TLR9-deficient BMDCs. Taken together, our data suggest that DNA-PKcs is an important mediator in the type I IFN response to CpG-ODNs in TLR9-dependent or -independent fashions.
Shared Decision Making (SDM) as means to the involvement of patients in medical decision making is increasingly demanded by treatment guidelines and legislation. Also, matching of patients' ...preferences to treatments has been shown to be effective regarding symptom reduction. Despite promising results for patients with substance use disorders (SUD) no systematic evaluation of the literature has been provided. The aim is therefore to give a systematic overview of the literature of patient preferences and SDM in the treatment of patients with SUD.
An electronic literature search of the databases Medline, Embase, Psyndex and Clinical Trials Register was performed. Variations of the search terms substance use disorders, patient preferences and SDM were used. For data synthesis the populations, interventions and outcomes were summarized and described according to the PRISMA statement. Methodological quality of the included articles was assessed with the Mixed Methods Appraisal Tool.
N = 25 trials were included in this review. These were conducted between 1986 and 2014 with altogether n = 8.729 patients. Two studies found that patients with SUD preferred to be actively involved in treatment decisions. Treatment preferences were assessed in n = 18 studies, where the majority of patients preferred outpatient compared with inpatient treatment. Matching patients to preferences resulted in a reduction on substance use (n = 3 studies), but the majority of studies found no significant effect. Interventions for SDM differed across patient populations and optional therapeutic techniques.
Patients with substance use disorders should be involved in medical treatment decisions, as patients with other health conditions. A suitable approach is Shared Decision Making, emphasizing the patients' preferences. However, due to the heterogeneity of the included studies, results should be interpreted with caution. Further research is needed regarding SDM interventions in patient populations with substance use disorders.
We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction ...with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.
The caspase family of cysteine proteases are highly sought‐after drug targets owing to their essential roles in apoptosis, proliferation, and inflammation pathways. High‐throughput screening efforts ...to discover inhibitors have gained little traction. Fragment‐based screening has emerged as a powerful approach for the discovery of innovative drug leads. This method has become a central facet of drug discovery campaigns in the pharmaceutical industry and academia. A fragment‐based drug discovery campaign against human caspase‐7 resulted in the discovery of a novel series of allosteric inhibitors. An X‐ray crystal structure of caspase‐7 bound to a fragment hit and a thorough kinetic characterization of a zymogenic form of the enzyme were used to investigate the allosteric mechanism of inhibition. This work further advances our understanding of the mechanisms of allosteric control of this class of pharmaceutically relevant enzymes, and provides a new path forward for drug discovery efforts.
A biophysical fragment‐based screen was employed to discover a new series of allosteric caspase inhibitors. A crystal structure of the lead fragment bound to caspase‐7 revealed significant distortion of the substrate‐binding loops and movement of the catalytic cysteine/histidine dyad.
Central to the pharmacological use of glucocorticoids (GCs) is the availability of the glucocorticoid receptor alpha (GRα). However, chronic GC therapy often results in the ligand-mediated ...downregulation of the GRα, and the subsequent development of an acquired GC resistance. While studies have demonstrated the dimerization-dependent downregulation of GRα, as well as the molecular mechanisms through which ligand-mediated downregulation occurs, little is known regarding the relative contribution of these molecular mechanisms to the cumulative ligand-mediated downregulation of the receptor, especially within an endogenous system. Thus, to probe this, the current study evaluates the conformational-dependent regulation of GRα protein using mouse embryonic fibroblast (MEF) cells containing either wild type GRα (MEFwt) or the dimerization deficient GRα mutant (MEFdim) and inhibitors of transcription, translation, and proteasomal degradation. Results show that the promotion of GRα dimerization increases the downregulation of the receptor via two main mechanisms, proteasomal degradation of the receptor protein, and downregulation of GRwt mRNA transcripts. In contrast, when receptor dimerization is restricted these two mechanisms play a lesser role and results suggest that stabilization of GRα protein by non-coding RNAs may potentially be the major regulatory mechanism. Together, these findings clarify the relative contribution of the molecular mechanisms involved in ligand-mediated downregulation of GRα and provides pharmacological targets for the development of GRα ligands with a more favourable therapeutic index.
•Dexamethasone downregulates GRα in a dose- and dimerization dependent manner.•GRα dimerization required for downregulation of both receptor mRNA and protein.•Dimerised GRα downregulated mainly via proteasome.•Downregulation of dimerization deficient GRα not via known molecular mechanisms.•Upon transcriptional inhibition, downregulation of GRdim equals GRwt.
In this article, we consider the gender gap in political participation by analyzing recent survey data about German adolescents. Differentiating between institutional, non-institutional, and ...expressive participation, we show that, even in Germany where there is strong gender equality, type-specific gender differences persist Testing for resource, socialization, and attitudinal explanations, in multivariate regression analyses, we identify socialization in civic forms of participation together with the lower confidence of women in their personal and political skills as major drivers for the sexual differences in political engagement, especially so for institutionalized forms of participation.
OBJECTIVE:
To create and validate a questionnaire for assessing symptom severity and symptom impact on health-related quality of life for women with leiomyomata.
METHODS:
The questionnaire was ...derived from focus groups of women with leiomyomata. Content validity was established through cognitive debriefings of women with leiomyomata and review by expert clinicians. Patients for the validation study were recruited from five gynecologists’ offices, an interventional radiology department, and a University campus. Instruments used for validation were the Short Form-36, Menorrhagia Questionnaire, the Revicki-Wu Sexual Function Scale, and a physician and a patient assessment of severity. Item and exploratory factor analysis were performed to assess the subscale structure of the questionnaire. Psychometric evaluation was conducted to assess reliability and validity. Test-retest was performed on a random subset of the sample within 2 weeks of the initial visit.
RESULTS:
A total of 110 patients with confirmed leiomyomata and 29 normal subjects participated in the validation. The final questionnaire consists of eight symptom questions and 29 health-related quality of life questions with six subscales. Subscale Cronbach’s α ranged from 0.83 to 0.95, with the overall health-related quality of life score α = 0.97. The Uterine Fibroid Symptom and Quality of Life (UFS-QOL) questionnaire subscales discriminated not only from normal controls but also among leiomyomata patients with varying degrees of symptom severity. Test-retest reliability was good with intraclass correlation coefficients of 0.76–0.93.
CONCLUSION:
The UFS-QOL appears to be a useful new tool for detecting differences in symptom severity and health-related quality of life among patients with uterine leiomyomata. Additional study is underway to determine the responsiveness of the UFS-QOL to therapies for leiomyomata.
Across species, sleep is characterized by a complex architecture. Sleep deprivation is a classic method to study the consequences of sleep loss, which include alterations in the activity of sleep ...circuits and detrimental consequences on well being. Automating the observation and manipulation of sleep is advantageous to study its regulation and functions. Caenorhabditis elegans shows sleep behavior similar to other animals that have a nervous system. However, a method for real-time automatic sleep detection that allows sleep-specific manipulations has not been established for this model animal. Also, our understanding of how sleep deprivation affects sleep neurons in this system is incomplete. Here we describe a system for real-time automatic sleep detection of C. elegans grown in microfluidic devices based on a frame-subtraction algorithm using a dynamic threshold. As proof of principle for this setup, we used automated mechanical stimulation to perturb sleep behavior and followed the activity of the sleep-active RIS neuron. We show that our system can automatically detect sleep bouts and deprive worms of sleep. We found that mechanical stimulation generally leads to the activation of the sleep-active RIS neuron, and this stimulation-induced RIS depolarization is most prominent during sleep deprivation.
Terahertz (THz) spectroscopy is a powerful tool for unambiguously extracting complex-valued material properties (e.g., refractive index, conductivity, etc.) from a wide range of samples, with ...applications ranging from materials science to biology. However, extracting complex refractive indices from THz time-domain spectroscopy data can prove challenging, especially for multilayer samples. These challenges arise from the large number of transmission-reflection paths the THz pulse can take through the sample layers, leading to unwieldy strings of Fresnel coefficients. This issue has often been addressed using various approximations. However, these approximations are only applicable to specific classes of samples and can give erroneous results when misapplied. An alternative to this approach is to programmatically model all possible paths through the sample. The many paths through the sample layers can be modeled as a tree that branches at every point where the paths diverge, i.e., whenever the pulse can either be transmitted or reflected. This tree can then be used to generate expressions relating the unknown refractive index to the observed time domain data. Here, we provide a freely available open-source package implementing this method as both a MATLAB library and a corresponding graphical user interface, which can also be run without a MATLAB license (https://github.com/YaleTHz/nelly). We have tested this method for a range of samples and compared the results to commonly used approximations to demonstrate its accuracy and wide applicability. Our method consistently gives better agreement than common approximations.
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