Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Simulator training in fetoscopic laser surgery for twin-twin transfusion syndrome: a pilot randomized controlled trial.

OBJECTIVE: To evaluate the effect of a newly developed training curriculum on the performance of fetoscopic laser surgery for twin-twin transfusion syndrome (TTTS) using an advanced high-fidelity simulator model. METHODS: Ten novices were randomized to receive verbal instructions and either skills training using the simulator (study group; n = 5) or no training (control group; n = 5). Both groups were evaluated with a pre-training and post-training test on the simulator. Performance was assessed by two independent observers and comprised a 52-item checklist for surgical performance (SP) score, measurement of procedure time and number of anastomoses missed. Eleven experts set the benchmark level of performance. Face validity and educational value of the simulator were assessed using a questionnaire. RESULTS: Both groups showed an improvement in SP score at the post-training test compared with the pre-training test. The simulator-trained group significantly outperformed the control group, with a median SP score of 28 (54%) in the pre-test and 46 (88%) in the post-test vs 25 (48%) and 36 (69%), respectively (P = 0.008). Procedure time decreased by 11 min (from 44 to 33 min) in the study group vs 1 min (from 39 to 38 min) in the control group (P = 0.69). There was no significant difference in the number of missed anastomoses at the post-training test between the two groups (1 vs 0). Subsequent feedback provided by the participants indicated that training on the simulator was perceived as a useful educational activity. CONCLUSIONS: Proficiency-based simulator training improves performance, indicated by SP score, for fetoscopic laser therapy. Despite the small sample size of this study, practice on a simulator is recommended before trainees carry out laser therapy for TTTS in pregnant women.

Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

[The course and development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannym mutatsiei.

AIM: To study the anamnesis, clinical state, electro-encephalographic and brain MRI characteristics in patients with Rett syndrome (МЕСР2) and epilepsy. MATERIAL AND METHODS: Eleven female patients, aged from 3 to 23 years, with Rett syndrome and MeCP2 mutations were studied. The study continued for 10 years (2006-2015). Assessment of neurological and mental status, night sleep video-EEG monitoring, MRI were performed. RESULTS AND CONCLUSION: Epilepsy was diagnosed in six cases (54.5%). Mean age at onset of epileptic seizures was 3 years 9 month. The following types of seizures were described: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus developed in one patient. Generalized seizures were identified in 56.25%, focal seizures in 43.75%. EEG changes were found in 9 patients (81.8%): slowing of the activity, episodes of periodic regional slowing, regional epileptiform activity and diffuse epileptiform activity, benign focal epileptiform discharges (BFED) of childhood, multiregional epileptiform activity. Five patients were treated with antiepileptic drugs. All of them had improved during treatment: a reduction of frequency of seizures was up to 50% in 4 cases (80%). One patient with resistant epilepsy was treated with the combination of drugs (levetiracetam, topiramate, zonisamide, benzodiazepine) that led to stopping of seizures during night sleep and decrease in the frequency of daytime seizures by 50%. Further research of epilepsy and efficacy of antiepileptic drugs in Rett syndrome is required.

[The course and the development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti techeniia i razvitiia épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannogo mutatsiei.

AIM: Studying data of anamnesis, clinical state, electro-encephalographic, brain MRI in patients with Rett syndrome (МЕСР2). MATERIAL AND METHODS: We studied 11 patients (female) from three to 23 years old with Rett syndrome and MeCP2 mutations. Observation continued 10 years (2006-2015). We analyzed the results of the neurological status, night sleep video-EEG monitoring, MRI. RESULTS AND CONCLUSION: Epilepsy diagnosed in six cases (54, 5%). The overage age of debut of epileptic seizures was 3 years 9 months. There are some types of seizures: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus evolved in one patient. Generalized seizures were 56, 25%, focal seizures - 43, 75%. EEG changing marked in nine patients (81, 8%): slowdown back activity, episodes of periodic regional slowdown, regional epileptiform activity, and diffuse epileptiform activity like benign focal epileptiform discharges (BFED). five patients took antiepileptic drugs. All of them had improved during treatment. There were reducing of frequency of the seizures up 50% - 4 cases (80%). one patients with resistant epilepsy was taken combination of drugs (levetirecetam, topiromat, zonisamide, benzodiazepine) with stopping of seizures in the night sleep and decreasing of frequency of daytime seizures to 50%. We believe there is very important of study epilepsy in patients with Rett syndrome and improvement of its treatment.

Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies.

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

Cryopreservation of fetal stem cells.

Fetal liver cells may be transplanted, in utero, to treat disorders affecting the hematopoietic system. Conventional immunological methods were employed to determine whether pooled cryopreserved cells could be a suitable basis for a tissue bank. We found that cryopreservation did not adversely change the population, viability or immunological capacity of human fetal liver cells and therefore, pooled and cryopreserved fetal stem cells may be suitable for transplantation.

Immunological capacity of human fetal liver cells.

The aim of this study was to evaluate immunological characteristics of human fetal liver (FL) cells, fresh and cryopreserved, 7-12 weeks post-conception. With monoclonal antibodies, HLA-associated determinants were demonstrated on FL. Although serological HLA determination of A, B, C and class II antigens was not possible, genomic HLA class II typing using RFLP technique or PCR amplification with sequence-specific primers was feasible. MLC induced only minor responses. Exposure to standard mitogens and polyclonal B cell activators did not stimulate DNA synthesis or antibody production. ABO antigens were expressed and determined. The apparent low immunological capacity of FL cells may reduce the risk of rejection and graft-versus-host disease when such cells are used in transplantation.

Effects of cryopreservation on subsets of fetal liver cells.

Human fetal livers from 6 to 13 weeks postconception were analysed before and after cryopreservation. The percentages of cell subsets, detected by MoAbs, did not change significantly after cryopreservation. Compared with BM, fetal liver contained significantly smaller subsets of cells identified by MoAbs, with two exceptions. Fetal liver contained a mean of 47% M5 positive cells versus 31% in BM, and there was no difference in the numbers of CD34+ cells. The colony-forming capacity was studied: 53 colonies grew from 10(5) cells from fresh fetal liver compared with 51 colonies from cryopreserved cells. For fresh BM the corresponding value was 88 per 10(5) cells. Incubation time for fetal stem cells was 17-18 days while the corresponding time for BM cells was 8-10 days.

In utero haematopoietic stem cell transplantation: current perspectives and future potential.

In utero transplantation (IUT) of haematopoietic cells is a new therapeutic option for families with increased risk of having a child with an inherited disorder. Immunological naiveté and the rapidly expanding haematopoietic system in the first trimester human fetus, make therapeutic intervention by IUT a real possibility for those disorders which can be diagnosed early in gestation. Fewer cells are required than in postnatal BMT and therapy can be offered before the pathological sequelae of a disorder become manifested. However, only a few cases of IUT have been performed in humans and it is imperative that consensus is reached quickly on issues such as cell numbers/cell types so that the benefits of this approach to treatment can be realised. This review presents the current status of IUT, the cases thus far recorded and offers a prospective view of developments in this rapidly expanding area.

Screening for fetal growth restriction: a mathematical model of the effect of time interval and ultrasound error.

OBJECTIVE: We estimated the effect of ultrasound error and time interval between examinations on the false-positive rate for detecting fetal growth restriction (FGR). METHODS: Using published growth curves for the fetal abdominal circumference and a coefficient of variation for ultrasound error of 5%, computer simulation was used to estimate false-positive rates in relation to the time interval between ultrasound examinations. Growth restriction was diagnosed when there was no apparent growth in fetal abdominal circumference between two consecutive examinations. In separate studies, the false-positive rate was plotted against gestational age at the first ultrasound examination. RESULTS: There was a dramatic increase in false-positive rates as the time interval between examinations was reduced. When the initial scan was performed at 32 weeks, the false-positive rate increased from 3.2% for an interval of 4 weeks to 30.8% for an interval of 1 week. At a 2-week interval, the error was 16.9%. There was a significant increase in the false-positive rate as the gestational age at the initial ultrasound was increased. At 28 weeks, the false-positive rate with a 2-week interval was 11.8%, increasing to 24.1% at 38 weeks. By varying the coefficient of variation of the ultrasound error, the false-positive rate increased from 0.8% at an error of 2% to 31.9% at an error of 10%. CONCLUSION: Ultrasound scanning at 2-week intervals is associated with false-positive rates for growth restriction in excess of 10%, increasing to much higher rates late in the third trimester. Improved screening performance should be attainable by increasing the interval between scans and reducing measurement errors.

Cytokines in fetal blood and amniotic fluid in Rh-immunized pregnancies.

OBJECTIVE: To determine fetal serum and amniotic fluid (AF) levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin, and to explore the relationship between cytokines and hemoglobin concentration, white blood cell count (WBC), and platelet count in fetuses affected by Rh immunization. METHODS: Thirty-four consecutive Rh-immunized patients in gestational weeks 19-33 were included. All patients were investigated by funipuncture and 13 by amniocentesis. The levels of IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin were estimated using commercially available immunoassays. RESULTS: There was a significant correlation between erythropoietin concentrations in fetal serum and AF (r = 0.54, P < .05), whereas none of the other cytokines showed a positive correlation between these two compartments. Fetal serum contained higher concentrations of IL-3, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin compared with AF. In contrast, the IL-6 level was significantly higher in AF compared with fetal serum (P = .002). Erythropoietin and IL-3 levels were both negatively correlated with fetal hemoglobin concentrations (r = -0.75, P = .02, and r = -0.67, P = .045). The fetal WBC correlated significantly with the fetal serum concentration of granulocyte-macrophage colony-stimulating factor (r = 0.38, P = .04). CONCLUSION: Human fetuses with anemia due to erythrocyte immunization exhibit an increased production of erythropoietin and IL-3. Other studied cytokines (such as stem cell factor, granulocyte-macrophage colony-stimulating factor, and IL-6) did not correlate with the degree of fetal anemia. Among the studied cytokines, only erythropoietin showed a positive correlation between fetal serum and AF.

The relationships between personality traits and plasma gastrin, cholecystokinin, somatostatin, insulin, and oxytocin levels in healthy women.

In earlier studies performed on a group of women with gastrointestinal symptoms, significant positive correlations between the gastrointestinal hormone gastrin and anxiety, and a negative correlation with socialization were obtained. These and other relationships were tested on 33 healthy women. A comprehensive and concise statistical model was used for the analysis of correlations between, on one hand, the levels of oxytocin and the gastrointestinal hormones gastrin, cholecystokinin, somatostatin and insulin, and, on the other hand, personality traits. Almost all explained variance of the hormone levels could be referred to three personality trait factors, Anxiety, Aggressive non-conformity, and Detachment. The statistical explanation of the gastrin level variance was most successful, the three personality trait factors explaining 48% of this variance. Gastrin "increased" Anxiety while reducing Aggressive non-conformity and Detachment. A similar pattern for insulin was also reliable. Considering general trends, the negative correlations between all hormones and Detachment are interesting. Present data suggest that there is a psychoendocrinological antithesis to the fight-flight individual, characterized by high activity in the sympathoadrenal system: these contrasting persons, with high levels of the gastrointestinal hormones gastrin and insulin, tend to be warm and caring and non-aggressive--but often not free from anxiety. We do not think that the demonstrated associations between hormone levels and personality traits implicate a direct causal relationship. They rather may mirror the activity of centrally acting or hypothalamic control systems which influence both behavioural and endocrine profiles.

Influence of ingesting a solution of branched-chain amino acids on plasma and muscle concentrations of amino acids during prolonged submaximal exercise.

On two occasions, seven male endurance-trained cyclists performed sustained exhaustive exercise with reduced muscle glycogen stores. During exercise, the subjects were supplied in random order with an aqueous solution of branched-chain amino acids (BCAA) or flavored water (placebo). Ingestion of BCAA caused the concentration of these amino acids to increase by 135% in the plasma and by 57% in muscle tissue during exercise, whereas in the placebo trial there was no change or a slight decrease in the concentration in plasma and a decrease of 18% in the muscle. The plasma concentration of alanine increased by 48% during exercise when BCAA were ingested, and the increase in the muscle concentration of alanine during exercise was larger (70% versus 31% in the placebo trial), suggesting an increased rate of alanine production. Also, the plasma concentration of arginine increased by 14% during exercise when BCAA were ingested, whereas there was no change during exercise in the placebo trial. There was a smaller decrease in the muscle glutamate concentration during exercise in the BCAA trial (32% versus 47% in the placebo trial; p < 0.05), but, for the remaining amino acids, there was no difference between the BCAA and placebo trials. There was a significant decrease in the muscle glycogen concentration during exercise in the placebo trial, whereas only a small decrease was found in the BCAA trial (28 and 9 mmol/kg wet wt [p < 0.05] in the placebo and BCAA trial, respectively). This might indicate that an increased supply of BCAA has a sparing effect on muscle glycogen degradation during exercise.

New device for hand-assisted laparoscopic surgery.

Laparoscopic surgery has undergone a rapid evolution since the first laparoscopic cholecystectomy of Erich Mühe in 1985. Many surgeons felt that further technological success would be related not only to increasing experience and skill of surgeons, but also technological advances which would enable surgeons to perform increasingly more difficult and complex tasks. Progress has been rapid for some, but broad acceptance by surgeons has been slow.

[Twin-twin transfusion syndrome--front line in fetal medicine]. / Tvilling--tvillingtransfusionssyndrom--frontlinje i fostermedicin.

Twin-twin transfusion develops in 10-20% of monochorionic twin pregnancies. As far as we know the underlying disturbance is an unbalanced anastomosis between the two fetuses on the placental surface. Without treatment mortality is as high as 80% if diagnosed before viability. One of this article's authors spent two months in Hamburg at the 'Allgemeine Krankenhaus', Barmbeck in Germany and describes the technique used there to coagulate these anastomoses via fetoscope. The outcome seems very promising with overall survival of 80% using this method. In the article pathogenesis and alternative treatments are also discussed.

[Twin-twin transfusion syndrome--front line in fetal medicine]. / Tvilling-tvillingtransfusionssyndrom--frontlinje i fostermedicin.

Twin-twin transfusion develops in 10-20% of monochorionic twin pregnancies. As far as we know the underlying disturbance is an unbalanced anastomosis between the two fetuses on the placental surface. Without treatment mortality is as high as 80% if diagnosed before viability. One of this article's authors spent two months in Hamburg at the "Allgemeine Krankenhaus", Barmbeck in Germany and describes the technique used there to coagulate these anastomoses via fetoscope. The outcome seems very promising with overall survival of 80% using this method. In the article pathogenesis and alternative treatments are also discussed.

Random nanolasing in the Anderson localized regime.

The development of nanoscale optical devices for classical and quantum photonics is affected by unavoidable fabrication imperfections that often impose performance limitations. However, disorder may also enable new functionalities, for example in random lasers, where lasing relies on random multiple scattering. The applicability of random lasers has been limited due to multidirectional emission, lack of tunability, and strong mode competition with chaotic fluctuations due to a weak mode confinement. The regime of Anderson localization of light has been proposed for obtaining stable multimode random lasing, and initial work concerned macroscopic one-dimensional layered media. Here, we demonstrate on-chip random nanolasers where the cavity feedback is provided by the intrinsic disorder. The strong confinement achieved by Anderson localization reduces the spatial overlap between lasing modes, thus preventing mode competition and improving stability. This enables highly efficient, stable and broadband wavelength-controlled lasers with very small mode volumes. Furthermore, the complex interplay between gain, dispersion-controlled slow light, and disorder is demonstrated experimentally for a non-conservative random medium. The statistical analysis shows a way towards optimizing random-lasing performance by reducing the localization length, a universal parameter.