Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology.

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study-GDAP1L1-to isolate highly functional live human neurons in vitro.

Adjuvant therapeutic decisions in elderly breast cancer patients: the role of chemotherapy in a retrospective analysis.

PURPOSE: Decisions on the type of adjuvant treatment in older breast cancer patients are challenging. Side effects of chemotherapy have to be weighed against life expectancy, comorbidities, functional status, and frailty on the basis of studies usually excluding patients over 69 years. To aid this decision, we analyzed a database of 6000 unselected patients and of those evaluated elderly primary breast cancer patients with hormone receptor-negative tumors from 1963 until 2003 in respect of survival data depending on adjuvant treatment. METHODS: A total of 131 elderly (i.e., >65 years) patients were observed retrospectively for a median of 72 months. Patients received breast-conserving therapy or mastectomy and adjuvant radiotherapy, chemotherapy, and endocrine therapy. Data were collected from a hospital-intern database. RESULTS: Median age at diagnosis was 72 years. Mostly, tumors were small (81 % T1, 17 % T2) but of unfavorable grading (40 % G2, 35 % G3). Lymph nodes were positive in 42 %. Mastectomy was performed in 65 %. While 42 % of patients received radiotherapy, only 10 % were treated with chemotherapy. Patients with G2 and G3 tumors (p = 0.027), younger women (p = 0.012), and patients with positive lymph node status (p < 0.0001) more likely received chemotherapy. Recurrence-free survival was longer in patients without chemotherapy (37 vs. 29 months, p = 0.234). Overall survival was non-significantly shorter in patients who received chemotherapy (59 vs. 81 months, p = 0.131). CONCLUSIONS: In this analysis, adjuvant chemotherapy was not associated with improved survival, presumably caused by an a priori poor prognosis of these patients. For an aging society more data are urgently needed to help selecting and personalizing adjuvant treatment within subgroups of breast cancer in older women.

Knowledge about post-anoxic somatosensory evoked potentials--present or not?

BACKGROUND AND PURPOSE: Median nerve somatosensory evoked potential (SEP) recordings play an important role in outcome algorithms in comatose patients after cardiopulmonary resuscitation. Knowledge of technical difficulties, clinical implications and uniform interpretation of SEP recordings is crucial. The aim of this study was to evaluate the skills of neurologists to interpret SEP recordings in post-anoxic patients. METHODS: Nationwide Dutch clinical neurophysiology examinations from 2007, 2008 and 2011, containing SEP related questions, were analysed. Participants were classified as neurology residents, neurologists with less than 10 years of experience, neurologists with more than 10 years of experience and clinical neurophysiologists. End-points were the knowledge of all participants about SEP recordings per year as well as improvement in knowledge over the years, as reflected by the test scores. RESULTS: A total of 194 participants completed the examination in 2007, 200 in 2008 and 263 in 2011. Between 2007 and 2008, all groups of respondents showed a significant increase in percentage of correct answers to SEP questions. Sixty-six participants completed all three examinations. The SEP score of this group improved in 2008 [75%, interquartile range (IQR) 50-75, P < 0.001] compared with 2007 (38%, IQR 38-50); there was no further improvement in 2011 (69%, IQR 54-77). CONCLUSION: Continuing education about technical knowledge, possible pitfalls and interpretation of SEP recordings remains of utmost importance.

The interplay of chronic stress and genetic traits discriminates between patients suffering from multisomatoform disorder with pain as the leading symptom and matched controls.

OBJECTIVE: Somatoform disorders and functional somatic syndromes (FSS) with symptoms that are not sufficiently explained by physical or technical examination are among the most challenging underlying causes. Many different somatoform disorders and FSS have overlapping symptoms, often with pain as the most prevalent one, leading to a high burden of disease. The concept of multisomatoform disorder (MSD) has been developed to acknowledge that fact. We analyzed a group of 151 patients and 149 matched controls to identify interactions of genetic and environmental factors with a possible influence on the development of MSD. DESIGN: In a retrospective case-control study, we performed a statistical analysis on 151 patients and 149 matched controls using logistic regression and a Classification and Regression Tree (CART) analysis. RESULTS: The logistic regression analysis of genes and environmental factors demonstrated significant differences in the results of the Trier Inventory of Chronic Stress (TICS) questionnaire, the single nucleotide polymorphism rs1800955 of the dopamine receptor D4 and the single nucleotide polymorphism rs4818 of the enzyme catechol-O-methyltransferase between patients with MSD and healthy controls. The resulting decision tree of the CART analysis determined that the TICS questionnaire was able to differentiate patients and controls most accurately, followed by certain genotypes of the 5-hydroxytryptamine receptor 2A and a single nucleotide polymorphism of the enzyme catechol-O-methyltransferase. CONCLUSIONS: The results of the statistical analysis identified a gene-environmental interaction possibly leading to MSD. The resulting identifiers could be used as a reference to inform diagnostic algorithms to easier identify patients suffering from MSD.

Early prediction of cardiac allograft vasculopathy and heart transplant failure.

Early risk-prediction is essential to prevent cardiac allograft vasculopathy (CAV) and graft failure in heart transplant patients. We developed multivariate models to identify patients likely to experience CAV, severe CAV, and failure due to CAV, at 1, 5 and 10 years. A cohort of 172 patients was followed prospectively for 6.7 ± 3.9 years. Logistic regression models were developed and cross-validated using bootstrap resampling. Predictive markers of atherothrombosis (myocardial fibrin deposition, and loss of vascular antithrombin and tissue plasminogen activator) and arterial endothelial activation (intercellular adhesion molecule-1 expression) were measured in serial biopsies obtained within 3 months posttransplant. Most markers were univariately associated with outcome. Multivariate models showed that loss of tissue plasminogen activator was the dominant and, in most cases, only predictor of long-term CAV (p < 0.001), severe CAV (p < 0.001), and graft failure due to CAV (p < 0.001). The models discriminated patients having adverse outcomes, had particularly high negative predictive values (graft failure due to CAV: 99%, 99% and 95% at 1, 5 and 10 years) and predicted event incidence and time to event. Early absence of atherothrombotic risk identifies a patient subgroup that rarely develops CAV or graft failure, implying that this low-risk subgroup could possibly be followed with fewer invasive procedures.

Distal muscle weakness and optic atrophy without central nervous system involvement in a patient with a homozygous missense mutation in the C19ORF12-gene.

Variants of the C19ORF12-gene have been described in patients with spastic paraplegia type 43 and in patients with mitochondrial membrane protein-associated neurodegeneration (MPAN), a subtype of neurodegeneration associated with brain iron accumulation (NBIA). In both subtypes optic atrophy and neuropathy have been frequently described. This case report describes a patient with bilateral optic atrophy and severe distal muscle weakness based on motor neuropathy without involvement of the central nervous system. Exome sequencing revealed a homozygous pathogenic missense variant (c.187G>C;p.Ala63Pro) of the C19ORF12-gene while iron deposits were absent on repeat MR-imaging of the brain, thus showing that peripheral neuropathy and optic neuropathy can be the sole manifestations of the C19ORF12-related disease spectrum whereby iron accumulation in the brain may be absent.

KIF1A variants are a frequent cause of autosomal dominant hereditary spastic paraplegia.

Variants in the KIF1A gene can cause autosomal recessive spastic paraplegia 30, autosomal recessive hereditary sensory neuropathy, or autosomal (de novo) dominant mental retardation type 9. More recently, variants in KIF1A have also been described in a few cases with autosomal dominant spastic paraplegia. Here, we describe 20 KIF1A variants in 24 patients from a clinical exome sequencing cohort of 347 individuals with a mostly 'pure' spastic paraplegia. In these patients, spastic paraplegia was slowly progressive and mostly pure, but with a highly variable disease onset (0-57 years). Segregation analyses showed a de novo occurrence in seven cases, and a dominant inheritance pattern in 11 families. The motor domain of KIF1A is a hotspot for disease causing variants in autosomal dominant spastic paraplegia, similar to mental retardation type 9 and recessive spastic paraplegia type 30. However, unlike these allelic disorders, dominant spastic paraplegia was also caused by loss-of-function variants outside this domain in six families. Finally, three missense variants were outside the motor domain and need further characterization. In conclusion, KIF1A variants are a frequent cause of autosomal dominant spastic paraplegia in our cohort (6-7%). The identification of KIF1A loss-of-function variants suggests haploinsufficiency as a possible mechanism in autosomal dominant spastic paraplegia.

Immunological effects of stress in psoriasis.

BACKGROUND: Psychological stress causes phenotypic changes in circulating lymphocytes and is regarded as an important trigger of the Th1-polarized inflammatory skin disease psoriasis. OBJECTIVE: To study the effects of psychological stress on immunological parameters, i.e. membrane molecules relevant to the pathophysiology of psoriasis, especially cutaneous lymphocyte-associated antigens (CLA) involved in T and natural killer (NK) cells homing in on the skin. METHODS: The severity of psoriasis was assessed in patients using the Psoriasis Area and Severity Index. Patients with psoriasis (n=15) and healthy volunteers (n=15) were exposed to brief psychological stress in the laboratory. In vitro analyses were conducted 1 h before, immediately following and 1 h after stress exposure. Peripheral T- and NK-cell subsets including CD8+ T lymphocytes, CLA+ lymphocytes and lymphocyte function-associated antigen type 1 (LFA-1)+ lymphocytes were analysed by flow cytometry. RESULTS: We found a significant stress-induced increase of CD3+ T lymphocytes in patients with psoriasis only. Analyses of T-cell subsets revealed that this increase was observable for cytotoxic CD8+ T lymphocytes and CLA+ CD3+ lymphocytes. The total number of circulating NK cells (CD16+, CD56+) increased immediately after stress in both groups whereas only patients with psoriasis showed a significant increase in CLA+ NK cells. CONCLUSIONS: A higher stress-induced increase of CLA+ T and CLA+ NK cells in the circulation of patients with psoriasis might point to an increased ability of T and NK cells in the presence of psoriasis to home in on the skin during mental stress. Further studies are needed to verify these relationships in more detail and to investigate the time point at which these cells accumulate within lesional skin, and whether or not psychotherapy improves the quality of life of patients with psoriasis and influences stress-dependent parameters.

The HER2 phenotype of circulating tumor cells in HER2-positive early breast cancer: A translational research project of a prospective randomized phase III trial.

BACKGROUND: HER2 is one of the predominant therapeutic targets in breast cancer. The metastatic selection process may lead to discrepancies between the HER2 status of the primary tumor and circulating tumor cells (CTCs). This study analyzed the HER2 status of CTCs in patients with HER2-positive primary breast cancer at the time of diagnosis. Aim of the study was to assess potential discordance of HER2 status between primary tumor and CTCs, as this may have important implications for the use of HER2-targeted therapy. METHODS: The number and HER2 status of CTCs out of 30ml peripheral blood were assessed in 642 patients using the CellSearch System (Janssen Diagnostics, USA). The cutoff for CTC positivity was the presence of at least 1 CTC, and the cutoff for HER2 positivity of CTCs was the presence of at least 1 CTC with a strong HER2 staining. RESULTS: 258 (40.2%) of the 642 patients were positive for CTCs (median 2; range 1-1,689). 149 (57.8%) of these 258 patients had at least 1 CTC with strong HER2 staining. The presence of HER2-positive CTCs was not associated with tumor size (p = 0.335), histopathological grading (p = 0.976), hormone receptor status (ER: p = 0.626, PR: p = 0.263) or axillary lymph node involvement (p = 0.430). Overall, 83 (32.2%) of the CTC-positive patients exclusively had CTCs with strong HER2 staining, whereas 31 (12.0%) had only CTCs with negative HER2 staining. Within-sample variation in the HER2 status of CTCs was found in 86 (57.8%) of the 149 patients with more than 1 CTC. CONCLUSION: This study demonstrated that discordance between the HER2 expression of CTCs and that of the primary tumor frequently occurs in early breast cancer. Future follow-up evaluation will assess whether this discrepancy may contribute to trastuzumab resistance.

Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation.

We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.

First on-line survey of an international multidisciplinary working group (MightyMedic) on current practice in diagnosis, therapy and follow-up of dyslipidemias.

The MightyMedic (Multidisciplinary International Group for Hemapheresis TherapY and MEtabolic DIsturbances Contrast) Working Group has been founded in 2013. The leading idea was to establish an international network of interdisciplinary nature aimed at working to cross national borders research projects, clinical trials, educational initiatives (meetings, workshops, summer schools) in the field of metabolic diseases, namely hyperlipidemias, and diabetes, preventive cardiology, and atherosclerosis. Therapeutic apheresis, its indications and techniques, is a parallel field of investigation. The first on-line survey of the Group has been completed in the first half of 2014. The survey included # 24 Centers in Italy, Germany, Greece, UK, Sweden, Japan and USA. Relevant data have been collected on current practice in diagnosis, therapy and follow-up of dyslipidemias. 240 subjects with hyperlipidemia and treated with lipoprotein apheresis have been reported in the survey, but a large percentage of patients (35%) who could benefit from this therapeutic option are still treated by conventional drug approach. Genetic molecular diagnosis is performed in only 33% of patients while Lipoprotein(a) (Lp(a)) is included in cardiovascular disease risk assessment in 71% of participating Centers. New detailed investigations and prospective multicenter studies are needed to evaluate changes induced by the impact of updated indications and strategies, as well as new treatment options, targeting standardization of therapeutic and diagnostic approaches.

Diet, nutrition intake, and metabolism in populations at high and low risk for colon cancer. Metabolism of bile acids.

High levels of fecal bile acids have been associated with populations at high risk for developing colon cancer. In this study, 168 subjects were drawn from populations that show low and high mortality from colon cancer [pure vegetarians, lacto-ovo vegetarians, and nonvegetarians Seventh-day Adventists (SDA) and demographically comparable group from the general population]. Lyophilized aliquots of 3-day stool samples were examined for levels of primary (cholic and chenodeoxycholic acids) and secondary (deoxycholic and lithocholic acids) bile acids. Total bile acids (mg/g lyophilized stools) were statistically different among dietary groups: SDA pure vegetarians 2.16 +/- 0.32, SDA lacto-ovo vegetarians 3.66 +/- 0.41, SDA nonvegetarians 4.39 +/- 0.44, general population nonvegetarians 6.04 +/- 0.75; but were similar when stool weights, body weights, and fat intake were taken into account. The most striking difference was evident in the ratio of secondary to primary bile acids: when compared to SDA pure vegetarians, both SDA lacto-ovo vegetarians and SDA nonvegetarians had twice the ratio while values for general population nonvegetarians were five to six times. The data indicate that these differences in excretion patterns among dietary groups reveal distinctly characteristic metabolic features associated with diet and lifestyle.

Reflex sympathetic dystrophy of the face. Report of two cases and a review of the literature.

Reflex sympathetic dystrophy of the face is an infrequently reported cause of chronic facial pain. We report the cases of two patients who were diagnosed and treated by sympathetic blockade of the stellate ganglion. Pathophysiologic mechanisms and rationale for treatment are discussed.

Myofascial pain may present clinically as occipital neuralgia.

Three case presentations illustrate that the clinical signs and symptoms of occipital neuralgia may be produced by myofascial pain. Assessment of myofascial trigger points is needed before making a diagnosis of occipital neuralgia. Myofascial trigger points can be effectively treated with minimally invasive procedures, thereby avoiding irreversible surgical interventions.

Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE).

ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.

Molecular identification and differentiation of Staphylococcus species and strains of cheese origin.

Amplified Ribosomal-DNA Restriction Analysis (ARDRA) was used to differentiate among 12 species and 4 subspecies of the genus Staphylococcus. With a universal primer pair a 2.4 kbp PCR-product was amplified, including the 16S rDNA, the 16S-23S rDNA interspacer region, and about 500 bp of the 23S rDNA. Species-specific restriction patterns were found using the restriction enzymes HindIII and XmnI separately. Cheese related staphylococci were clearly differentiated. ARDRA results were in good agreement with results of partial sequencing of the 16S rDNA. ARDRA could fully replace the biochemical identification with ID32 Staph (BioMerieux) which was less reliable when staphylococci of cheese origin were analysed. Genomic restriction digests of cheese-related S. equorum strains by SmaI and SacI gave unique strain-specific restriction patterns which can be used to identify starter staphylococci in a complex microbial environment such as the surface of Red-Smear cheeses.

Heparin-mediated extracorporeal LDL/fibrinogen precipitation--H.E.L.P.--in coronary and cerebral ischemia.

Cerebral and myocardial infarctions share common aspects of pathobiochemistry. The central problem is the oxygen supply of the infarcted region. To maintain this supply, H.E.L.P.-apheresis (Heparin-mediated Extracorporeal LDL/Fibrinogen Precipitation) has already proven beneficial in the prevention and therapy of myocardial infarction. Since H.E.L.P.-apheresis can lower significantly plasma viscosity and erythrocyte aggregation without reducing the oxygen transport capacity, patients with cerebral infarction (stroke) may also benefit from our experiences in myocardial ischemia. The system is designed to remove selectively plasma fibrinogen, LDL-cholesterol and lipoprotein(a) from blood circulation, simultaneously. The removal of the plasma compounds is achieved by extracorporeal precipitation with heparin at low pH. Excess heparin is completely removed by an adsorber before the plasma is given back to the patient. H.E.L.P.-apheresis has proved to be safe in patients with coronary heart disease and allows a controlled reduction of thrombogenic plasma compounds. It is therefore hoped to be effective also in patients with acute ischemic stroke.

TQM as a managerial innovation: research issues and implications.

TQM is a managerial innovation that is likely to have profound consequences on the delivery of health services. As an innovation, it is important that attention be given to the fundamental research issues associated with implementation and impact. Using a variance and process perspective, selected research issues are identified within organizations and among work units within these organizations. The variance perspective at both the organizational and work unit level considers the explanation of impact and adoption at a particular point in time. The process perspective considers the particular steps or events in the overall adoption process. The managerial implications for each perspective are discussed.

Facilities planning: opening and activation.

Making your facility work is the real objective. Getting it designed and built are only the means to this end. The authors of this article examine the transition that occurs in the later stages of construction as an institution prepares to occupy and use a new facility. Practical guidelines are presented for equipping the project, training personnel, and accomplishing the move-in.

Evaluation of two different analytical methods for circulating tumor cell detection in peripheral blood of patients with primary breast cancer.

BACKGROUND: Evidence is accumulating that circulating tumor cells (CTC) out of peripheral blood can serve as prognostic marker not only in metastatic but also in early breast cancer (BC). Various methods are available to detect CTC. Comparisons between the different techniques, however, are rare. MATERIAL AND METHODS: We evaluate two different methods for CTC enrichment and detection in primary BC patients: the FDA-approved CellSearch System (CSS; Veridex, Warren, USA) and a manual immunocytochemistry (MICC). The cut-off value for positivity was ≥1 CTC. RESULTS: The two different nonoverlapping patient cohorts evaluated with one or the other method were well balanced regarding common clinical parameters. Before adjuvant CHT 21.1% (416 out of 1972) and 20.6% (247 out of 1198) of the patients were CTC-positive, while after CHT 22.5% (359 out of 1598) and 16.6% (177 out of 1066) of the patients were CTC-positive using CSS or MICC, respectively. CTC positivity rate before CHT was thus similar and not significantly different (P = 0.749), while CTC positivity rate immediately after CHT was significantly lower using MICC compared to CSS (P < 0.001). CONCLUSION: Using CSS or MICC for CTC detection, we found comparable prevalence of CTC before but not after adjuvant CHT.