Basal signaling activity of mu opioid receptor in mouse brain: role in narcotic dependence

Narcotic analgesics cause addiction by poorly understood mechanisms, involving mu opoid receptor (MOR). Previous cell culture studies have demonstrated significant basal, spontaneous MOR signaling activity, but its relevance to narcotic addiction remained unclear. In this study, we tested basal MOR-signaling activity in brain tissue from untreated and morphine-pretreated mice, in comparison to antagonist-induced withdrawal in morphine-dependent mice. Using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding and adenylyl cyclase activity assay in brain homogenates, we demonstrated that morphine pretreatment of mice enhanced basal MOR signaling in mouse brain homogenates and, moreover, caused persistent changes in the effects of naloxone and naltrexone, antagonists that elicit severe withdrawal in dependent subjects. Naloxone and naltrexone suppressed basal [(35)S]GTP gamma S binding (acting as "inverse agonists") only after morphine pretreatment, but not in drug-naive animals. Moreover, naloxone and naltrexone stimulated adenylyl cyclase activity in striatum homogenates only after morphine pretreatment, by reversing the inhibitory effects of basal MOR activity. After cessation of morphine treatment, the time course of inverse naloxone effects on basal MOR signaling was similar to the time course of naltrexone-stimulated narcotic withdrawal over several days. The neutral antagonist 6 beta-naltrexol blocked MOR activation without affecting basal signaling (G protein coupling and adenylyl cyclase regulation) and also elicited substantially less severe withdrawal. These results demonstrate long-lasting regulation of basal MOR signaling as a potential factor in narcotic dependence.     

Current perspectives on behavioural and cellular mechanisms of illness anorexia

Here we review our current understanding of the integration of immune, neural, metabolic and endocrine signals involved in the generation of anorexia during acute infection, with the focus on anorexia elicited by peripheral administration of bacterial lipopolysaccharide (LPS). We chose to limit this review to peripheral LPS-anorexia because the mechanisms underlying this response may also be valid for anorexia during other types of acute or chronic infections, with slight differences in the duration of anorexia, levels of circulating concentrations of pro-inflammatory cytokines and hypermetabolism. Evidence so far indicates that LPS-anorexia is a complex response beneficial to host defence that involves both peripheral and central action of pro-inflammatory cytokines, other immune factors, such as prostanoids, and neurotransmitters, such as serotonin. One interesting characteristic of LPS-anorexia is its sexual differentiation, an aspect mainly mediated by the gonadal hormone estradiol. Understanding the behavioural and molecular mechanisms of LPS-anorexia may even provide useful leads for identifying mechanisms of eating disorders in humans.     

Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology

CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amplicon 3, 77%; and amplicon 4, 69%. A new emPCR condition, improving the standard titanium assay, presents a robust solution to sequence amplicons with a GC content of up to 77%. Moreover, this assay was subsequently tested on a larger independent cohort of 23 AML patients. For each patient, a median of 737 reads was generated (coverage range, 397-fold to 1194-fold) and therefore allowed a robust detection of insertions, deletions, and point mutations. In conclusion, next-generation amplicon sequencing enables the highly sensitive detection of molecular mutations and is a feasible assay for routine assessment of GC-rich content amplicons.     

Association of costs with somatic symptom severity in patients with medically unexplained symptoms

Objective

To analyse the association of direct and indirect costs in patients with medically unexplained symptoms (MUS) with somatic symptom severity (SSS).

Methods

A cross-sectional cost analysis for retrospective 6 months was conducted in 294 primary care patients with MUS. Health care utilisation and loss of productivity were measured by questionnaires. SSS was measured using the “Patient Health Questionnaire 15” (PHQ-15). Direct and indirect costs and the association of costs with SSS were analysed via multiple linear regression analysis.

Results

Patients with MUS had average 6-month direct costs of 1098 EUR and indirect costs of 7645 EUR. For direct costs, outpatient physician visits were the most expensive single cost category (36%), followed by pharmaceuticals (25%) and hospital stays (19%). Indirect costs were predominantly caused by productivity reduction at work (56%) followed by early retirement (29%) and acute sickness absence (14%). As compared to mild SSS, moderate SSS was not significantly associated with direct, but with indirect costs (+ 2948 EUR; p < .001); severe SSS was associated with increased direct cost (+ 658 EUR; p = .001) and increased indirect costs (+ 4630 EUR; p < .001). Age was positively associated with direct cost (+ 15 EUR for each additional year; p = .015) as well as indirect cost (+ 104 EUR for each additional year; p < .001).

Conclusions

MUS are associated with relevant direct and even much higher indirect costs that strongly depend on SSS.     

A laboratory method for parallel determination of platelet aggregation and thrombin generation

Optimal use of combinations of antiaggregating and antithrombotic drugs in vivo requires improved methods for testing of possible synergistic drug effects. Therefore, we developed an in vitro model that allows parallel evaluation of the influence of drugs on the time course of platelet aggregation inhibition and on thrombin generation inhibition. Platelet rich plasma samples were incubated with different amounts of antiaggregating and/or antithrombotic agents and the effects on thrombin generation, lag phase until the onset of platelet aggregation, and on inhibition of platelet aggregation were detected. Plasma activation was performed by addition of high (29 nM final concentration, "high coagulant challenge") or low (5 pM final concentration, "low coagulant challenge") amounts of tissue factor. Thus, platelet activation is initiated in our model by endogenously generated thrombin and not by exogenously added agonists, as usual in conventional evaluation of platelet aggregation. The combination of glycoprotein IIb/IIIa inhibitors (eptifibatide or abciximab) and anticoagulants (unfractionated heparin, low molecular weight heparin, or recombinant hirudin) used in this study exhibited an additive effect on prolongation of the lag phase until the onset of platelet aggregation. Under high but not under low coagulant challenge the combination of eptifibatide and anticoagulants had a synergistic inhibitory effect on platelet aggregation. Combination of abciximab and anticoagulants had no additive inhibitory effects on platelet aggregation under both high and low coagulant challenge. Under low coagulant challenge combination of eptifibatide but not abciximab with low molecular weight heparin significantly reduced the thrombin generation. We suggest that our laboratory method might be useful for pre-clinical testing of in vitro effects of glycoprotein IIb/IIIa inhibitors and anticoagulants.     

Novel Method for Three-Dimensional Facial Expression Recognition Using Self-Normalizing Neural Networks and Mobile Devices

Introduction To date, most ways to perform facial expression recognition rely on two-dimensional images, advanced approaches with three-dimensional data exist. These however demand stationary apparatuses and thus lack portability and possibilities to scale deployment. As human emotions, intent and even diseases may condense in distinct facial expressions or changes therein, the need for a portable yet capable solution is signified. Due to the superior informative value of three-dimensional data on facial morphology and because certain syndromes find expression in specific facial dysmorphisms, a solution should allow portable acquisition of true three-dimensional facial scans in real time. In this study we present a novel solution for the three-dimensional acquisition of facial geometry data and the recognition of facial expressions from it. The new technology presented here only requires the use of a smartphone or tablet with an integrated TrueDepth camera and enables real-time acquisition of the geometry and its categorization into distinct facial expressions. Material and Methods Our approach consisted of two parts: First, training data was acquired by asking a collective of 226 medical students to adopt defined facial expressions while their current facial morphology was captured by our specially developed app running on iPads, placed in front of the students. In total, the list of the facial expressions to be shown by the participants consisted of “disappointed”, “stressed”, “happy”, “sad” and “surprised”. Second, the data were used to train a self-normalizing neural network. A set of all factors describing the current facial expression at a time is referred to as “snapshot”. Results In total, over half a million snapshots were recorded in the study. Ultimately, the network achieved an overall accuracy of 80.54% after 400 epochs of training. In test, an overall accuracy of 81.15% was determined. Recall values differed by the category of a snapshot and ranged from 74.79% for “stressed” to 87.61% for “happy”. Precision showed similar results, whereas “sad” achieved the lowest value at 77.48% and “surprised” the highest at 86.87%. Conclusions With the present work it can be demonstrated that respectable results can be achieved even when using data sets with some challenges. Through various measures, already incorporated into an optimized version of our app, it is to be expected that the training results can be significantly improved and made more precise in the future. Currently a follow-up study with the new version of our app that encompasses the suggested alterations and adaptions, is being conducted. We aim to build a large and open database of facial scans not only for facial expression recognition but to perform disease recognition and to monitor diseases’ treatment progresses.     

Update Breast Cancer 2022 Part 4 – Advanced-Stage Breast Cancer

For the treatment of patients with advanced HER2-negative hormone receptor-positive breast cancer, several substances have been introduced into practice in recent years. In addition, other drugs are under development. A number of studies have been published over the past year which have shown either an advantage for progression-free survival or for overall survival. This review summarizes the latest results, which have been published at current congresses or in specialist journals, and classifies them in the clinical treatment context. In particular, the importance of therapy with CDK4/6 inhibitors – trastuzumab deruxtecan, sacituzumab govitecan and capivasertib – is discussed. For trastuzumab deruxtecan, an overall survival benefit in HER2-negative breast cancer with low HER2 expression (HER2-low expression) was reported in the Destiny-Breast-04 study. Similarly, there was an overall survival benefit in the FAKTION study with capivasertib. The lack of overall survival benefit for palbociclib in the first line of therapy raises the question of clinical classification.Key words: advanced breast cancer, chemotherapy, endocrine therapy, antibody drug conjugates     

Update Breast Cancer 2022 Part 5 – Early Stage Breast Cancer

The treatment of patients with early stage breast cancer has changed in recent years due to the introduction of pembrolizumab, olaparib, and abemaciclib. These and other drugs with the same class of active ingredient are currently in trial for various indications. This review article summarizes the latest results that have either been presented at major conferences such as the ESMO 2022 or published recently in international journals. This includes reports on newly discovered breast cancer genes, atezolizumab in neoadjuvant therapy in HER2-positive patients, long-term data from the APHINITY study, and on how preoperative peritumoral application of local anesthetics can influence the prognosis. We also present solid data on dynamic Ki-67 from the ADAPT studies.Key words: breast cancer, surgery, chemotherapy, therapy standard