Post-synthetic benzylation of the mRNA 5' cap via enzymatic cascade reactions.

mRNAs are emerging modalities for vaccination and protein replacement therapy. Increasing the amount of protein produced by stabilizing the transcript or enhancing translation without eliciting a strong immune response are major steps towards overcoming the present limitations and improving their therapeutic potential. The 5' cap is a hallmark of mRNAs and non-natural modifications can alter the properties of the entire transcript selectively. Here, we developed a versatile enzymatic cascade for regioselective benzylation of various biomolecules and applied it for post-synthetic modification of mRNA at the 5' cap to demonstrate its potential. Starting from six synthetic methionine analogues bearing (hetero-)benzyl groups, S-adenosyl-l-methionine analogues are formed and utilized for N7G-cap modification of mRNAs. This post-synthetic enzymatic modification exclusively modifies mRNAs at the terminal N7G, producing mRNAs with functional 5' caps. It avoids the wrong orientation of the 5' cap-a problem in common co-transcriptional capping. In the case of the 4-chlorobenzyl group, protein production was increased to 139% during in vitro translation and to 128-150% in four different cell lines. This 5' cap modification did not activate cytosolic pathogen recognition receptors TLR3, TLR7 or TLR8 significantly more than control mRNAs, underlining its potential to contribute to the development of future mRNA therapeutics.

High-speed THz spectroscopic imaging at ten kilohertz pixel rate with amplitude and phase contrast.

By combining the advantages of the high-speed ASOPS technology and efficient THz generation, we have realized a high-speed laser-based spectroscopic THz imaging system with more than 10,000 pixels per second acquisition speed and an excellent signal-to-noise ratio of more than 100. Unlike THz line cameras or mm-wave intensity detectors, the present device allows for a much higher spatial resolution and attributes each imaging pixel with phase and amplitude information up to several THz while simultaneously maintaining a very high scanning speed unmatched by any other technique presented so far. The high-speed acquisition allows for samples to be scanned even at sample velocities of 5 m/s or higher while preserving the fundamental resolution limit of the THz radiation, which is on the order of 500 µm in the present case.

OSIRIS-REx Contamination Control Strategy and Implementation.

OSIRIS-REx will return pristine samples of carbonaceous asteroid Bennu. This article describes how pristine was defined based on expectations of Bennu and on a realistic understanding of what is achievable with a constrained schedule and budget, and how that definition flowed to requirements and implementation. To return a pristine sample, the OSIRIS-REx spacecraft sampling hardware was maintained at level 100 A/2 and <180 ng/cm2 of amino acids and hydrazine on the sampler head through precision cleaning, control of materials, and vigilance. Contamination is further characterized via witness material exposed to the spacecraft assembly and testing environment as well as in space. This characterization provided knowledge of the expected background and will be used in conjunction with archived spacecraft components for comparison with the samples when they are delivered to Earth for analysis. Most of all, the cleanliness of the OSIRIS-REx spacecraft was achieved through communication among scientists, engineers, managers, and technicians.

Non-motor symptoms and quality of life in dopa-responsive dystonia patients.

BACKGROUND: In patients with GTP-cyclohydrolase deficient dopa-responsive dystonia (DRD) the occurrence of associated non-motor symptoms (NMS) is to be expected. Earlier studies report conflicting results with regard to the nature and severity of NMS. The aim of our study was to investigate the prevalence of psychiatric disorders, sleep problems, fatigue and health-related quality of life (HR-QoL) in a Dutch DRD cohort. METHODS: Clinical characteristics, motor symptoms, type and severity of psychiatric co-morbidity, sleep problems, fatigue and HR-QoL were assessed in DRD patients with a confirmed GCH1 mutation and matched controls. RESULTS: Twenty-eight patients were included (18 adults and 10 children), from 10 families. Dystonia symptoms were well-controlled in all patients. According to the DSM IV patients significantly more often met the criteria for a lifetime psychiatric disorder than controls (61% vs. 29%, p < 0.05). In particular the frequencies of generalized anxiety and agoraphobia were higher in patients (both 29% vs. 4%, p < 0.05). Patients scored significantly higher on daytime sleepiness than controls (ESS, 11.2 vs 5.7, p < 0.05). Adult patients had significantly lower scores on the mental component of the HR-QoL (47 vs. 54, p < 0.05) than controls mainly associated with (worse) quality of sleep. CONCLUSION: NMS were highly prevalent in our cohort of DRD patients, despite adequate treatment of motor symptoms. Our findings support the accumulating evidence of an important non-motor phenotype in DRD, with possible involvement of serotonergic mechanisms. This highlights the need to address NMS and the underlying neurobiology in patients with DRD.

[Preferred Medical Specialties of Medical Students in Contrast to the Need for General Practitioners in Saxony]. / Facharztpräferenzen von Medizinstudierenden im Kontrast zum Bedarf an Allgemeinmedizinern in Sachsen.

Aim of the study: Especially in the rural areas of Germany, there are not enough general practitioners (GPs) for primary care. Preferred medical specialties of medical students can help get an estimate of the number of future GPs. This study compares this estimate to the current need for GPs in Saxony. Methods: 587 medical students at the second, sixth and tenth semester were invited to take part in an anonymous cross-sectional study regarding their specialty preferences at the Technical University Dresden. Based on the data of the medical requirements for Saxony, 4 model calculations were generated for comparison of the estimated numbers of future GPs and the current need for GPs. Results: The most commonly preferred medical specialties were surgery (19.1%), internal medicine (12.9%), pediatrics (11.6%) and general practice (9.9%). A significant increase in specialist preference for GP was observed from the sixth (4.9%) to the tenth semester (14.0%). The model calculations show that approximately 29% to 111% of the open positions for GPs could be filled by the potential new GPs from Dresden. Conclusion: Currently, medical students planning to become GPs cannot meet the corresponding need for GPs. Future studies should include the points of view of students, continuing education assistants, GPs and patients.

Psychiatric co-morbidity is highly prevalent in idiopathic cervical dystonia and significantly influences health-related quality of life: Results of a controlled study.

INTRODUCTION: The aim of this study was to systematically investigate the prevalence of psychiatric disorders and factors influencing health-related quality of life (HR-QoL) in cervical dystonia (CD) patients, in the context of objective dystonia motor severity. METHODS: We studied 50 CD patients and 50 matched healthy controls. Psychiatric assessment included the MINI-PLUS interview and quantitative questionnaires. Dystonia motor severity (based on video evaluation), pain and disability were determined with the TWSTRS rating scale. In addition, severity of tremor and jerks was evaluated with the 7-point CGI-S scale. HR-QoL was determined with the RAND-36 item Health Survey and predictors of HR-QoL were assessed using multiple regression analysis. RESULTS: In CD patients, the MINI-PLUS revealed a significantly higher prevalence of psychiatric disorders (64% vs. 28%, p = 0.001), with substantially more depression (32% vs. 14%) and anxiety disorders (42% vs. 8%). This was confirmed by the quantitative rating scales. Disease characteristics did not differ between patients with and without a psychiatric diagnosis. HR-QoL in dystonia patients was significantly lowered. The most important predictors of HR-QoL appeared severity of depressive symptoms, pain and disability, but not severity of motor symptoms. CONCLUSION: Psychiatric co-morbidity is highly prevalent and is an important predictor of HR-QoL in CD patients, rather than dystonia motor severity. Our findings support the theory of a shared neurobiology for motor and non-motor features and highlight the need for systematic research into psychiatric disorders in dystonia. Adequate treatment of psychiatric symptoms could significantly contribute to better overall quality of life of CD patients.

Frequency comb metrology with an optical parametric oscillator.

We report on the first demonstration of absolute frequency comb metrology with an optical parametric oscillator (OPO) frequency comb. The synchronously-pumped OPO operated in the 1.5-µm spectral region and was referenced to an H-maser atomic clock. Using different techniques, we thoroughly characterized the frequency noise power spectral density (PSD) of the repetition rate frep, of the carrier-envelope offset frequency fCEO, and of an optical comb line νN. The comb mode optical linewidth at 1557 nm was determined to be ~70 kHz for an observation time of 1 s from the measured frequency noise PSD, and was limited by the stability of the microwave frequency standard available for the stabilization of the comb repetition rate. We achieved a tight lock of the carrier envelope offset frequency with only ~300 mrad residual integrated phase noise, which makes its contribution to the optical linewidth negligible. The OPO comb was used to measure the absolute optical frequency of a near-infrared laser whose second-harmonic component was locked to the F = 2→3 transition of the 87Rb D2 line at 780 nm, leading to a measured transition frequency of νRb = 384,228,115,346 ± 16 kHz. We performed the same measurement with a commercial fiber-laser comb operating in the 1.5-µm region. Both the OPO comb and the commercial fiber comb achieved similar performance. The measurement accuracy was limited by interferometric noise in the fibered setup of the Rb-stabilized laser.

Serotonergic perturbations in dystonia disorders-a systematic review.

Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent muscle contractions. Emerging data describe high prevalences of non-motor symptoms, including psychiatric co-morbidity, as part of the phenotype of dystonia. Basal ganglia serotonin and serotonin-dopamine interactions gain attention, as imbalances are known to be involved in extrapyramidal movement and psychiatric disorders. We systematically reviewed the literature for human and animal studies relating to serotonin and its role in dystonia. An association between dystonia and the serotonergic system was reported with decreased levels of 5-hydroxyindolacetic acid, the main metabolite of serotonin. A relation between dystonia and drugs affecting the serotonergic system was described in 89 cases in 49 papers. Psychiatric co-morbidity was frequently described, but likely underestimated as it was not systematically examined. Currently, there are no good (pharmaco)therapeutic options for most forms of dystonia or associated non-motor symptoms. Further research using selective serotonergic drugs in appropriate models of dystonia is required to establish the role of the serotonergic system in dystonia and to guide us to new therapeutic strategies.

Damaging biting behaviors in intensively kept rearing gilts: the effect of jute sacks and relations with production characteristics.

Pigs may display biting behavior directed at pen mates, resulting in body damage such as tail wounds. We assessed the suitability of jute sacks (hung vertically at wall) to reduce biting behaviors and tail wounds in rearing gilts. Additionally, we assessed several characteristics of different types of tail biters. Tail docked rearing gilts originated from 72 litters, which were kept in partly slatted pens with jute sacks (J) or barren control pens (CON; 36 litters per treatment). Tail and ear damage were observed at weaning (4 wk) and during the weaner and rearing phase (17 J and 19 CON pens). Sow (dam) damage was also considered. Biting behaviors (tail, ear, and other) were observed during the weaner and rearing phase. Weight was recorded at birth, weaning, and end of the weaner phase and ADG was calculated from birth till weaning and from weaning till 9 wk. Furthermore, estimated breeding values for litter size, litter birth weight, back fat, and growth between birth and ∼ 105 kg, and ∼ 25 to 105 kg were determined. Jute sacks reduced tail and ear damage at weaning (both P < 0.001) and tended to reduce sow tail damage (P = 0.09). Jute sacks also reduced tail damage post-weaning (P < 0.001 to 0.03), with a 5-fold reduction of (mild) tail wounds at the age of 13 wk. Biting behaviors directed at pen mates were up to 50% lower in J pens (P < 0.001 to 0.04). Higher genotypic litter size, litter birth weight, growth, and lower back fat seemed associated with higher levels of ear or tail-biting behavior (P = 0.002 to 0.09), albeit dependent on treatment and phase. Higher phenotypic litter sizes were associated with higher levels of biting behaviors (P = 0.004-0.08). High-tail-biters and Medium-tail-biters (the latter less pronounced) stemmed from larger litters (P = 0.01 to 0.05), were heavier (P = 0.03 to 0.04), grew faster (P = 0.05 to 0.08), and displayed higher levels of all kinds of biting behavior directed to pen mates and the jute sack (P < 0.001 to 0.10) compared with non-tail-biters, the effect size dependent on treatment and phase of life. To conclude, jute sacks may profoundly reduce damaging behaviors and tail wounds in rearing gilts, probably because they partly meet the behavioral need of pigs for rooting and chewing. Furthermore, (tail) biting is associated with production level of the gilts (phenotypically and genetically), which suggests a role for breeding programs and additional research focusing on metabolic demands of (tail) biting pigs.

[Sexual risk behaviour of nursing students. A cross-sectional study at nursing schools in Saxony-Anhalt]. / Das sexuelle Risikoverhalten von Auszubildenden der Gesundheits- und Krankenpflege. Eine Querschnittstudie an Krankenpflegeschulen in Sachsen-Anhalt.

AIM: This work describes the sexual risk behaviour of nursing students. METHODS: 22 of 23 state-approved nursing schools in Saxony-Anhalt, Germany, participated in this cross-sectional study (study period 7/2005-5/2006). Participation was voluntarily and anonymous. RESULTS: More male than female students (59.6% vs. 44.8%) used condoms to prevent STIs. More female students (58.9% vs. 43.4%) reported constant partnership with just one partner as an STI prevention method (chi² test, p<0.005). 29.6% of the nursing students reported >1 sexual partner in the past 12 months. 3.9% of the students demanded an HIV test from the partner, 69.3% had never tested themselves for HIV. 10.2% used no prevention against STIs. CONCLUSIONS: Nursing students should be sensitised for sexual risk behaviour and STIs. This should be included in the content of teaching throughout the education, not least since nursing students are multiplicators and have an influence on health behaviour of the patients.

[Inappropriately rough play behaviour and predatory attacks against people by a tomcat. A case report]. / Unangemessen grobes Spielverhalten und jagdliche Attacken eines Katers gegenüber Menschen. Ein Fallbericht.

The owner of a 6-months-old tomcat came to seek help because the cat had attacked her face on a near-daily basis. Through a detailed behavioural history, the cat's behaviour was diagnosed as human-directed predatory attack behaviour, play-related aggression and reduced motor as well as emotional self-control. Within a few weeks, behavioural therapy produced a significant improvement. After 5 months of therapy, the cat showed neither predatory attacks nor inappropriately rough or aggressive behaviour in play towards its owner or other humans.

Maternal serum cholesterol levels are elevated from the 1st trimester of pregnancy: a cross-sectional study.

Cholesterol is monitored in the non-pregnant adult population, where normal values are established. Although reported to be elevated in pregnancy, cholesterol is neither routinely measured nor treated. We aimed to investigate cholesterol levels throughout pregnancy and to establish reference values for cholesterol in healthy pregnant women. This was a cross-sectional analysis of serum cholesterol in healthy women with an uncomplicated singleton pregnancy. Pregnant women attending for antenatal care were recruited and cholesterol levels assayed at 12, 20, 28 and 36 weeks' gestation and on day 1-3 postpartum. A total of 222 women were recruited. The majority (95%) were white Irish, with a median age of 31 years (range 16-46). Median BMI was 25.9 kg/m2 (range 18-40) and 16% were smokers. Cholesterol levels were elevated in all trimesters of pregnancy, with median values from 1st trimester raised outside the non-pregnant adult range. High-density lipoprotein (HDL) levels ranged from 0.9 to 3.7 mmol/l and low-density lipoprotein (LDL) levels ranged from 1.3 to 6.1 mmol/l. Fasting, smoking and obesity did not have any significant effects on results. Total and LDL-cholesterol levels were raised throughout pregnancy. Levels were above non-pregnant adult ranges as early as the 1st trimester. The implications of this on fetus and mother are undetermined and deserve further investigation.

Reference standard for serum bile acids in pregnancy.

OBJECTIVE: Obstetric cholestasis (OC) is a liver disorder characterised by pruritus and elevated serum bile acids (SBA) that affects one in 200 pregnant women. It is associated with adverse perinatal outcomes such as premature delivery and stillbirth. Mild OC is defined as SBA levels of 10-39 µmol/l, and severe OC is defined by levels >40 µmol/l. SBA levels in normal pregnancy have not been investigated. We aimed to establish reference values for SBA in healthy pregnant women across different trimesters of pregnancy. DESIGN: Cross-sectional analysis of SBA levels. SETTING: A large tertiary referral university teaching maternity hospital. POPULATION: Healthy pregnant women with a singleton pregnancy and a body mass index (BMI) < 40, excluding women with significant alcohol intake, history of liver disease, prior cholecystectomy and OC. METHODS: Cross-sectional analysis of SBA levels at 12, 20, 28 and 36 weeks of gestation, and on days 1-3 postpartum. MAIN OUTCOME MEASURES: SBA levels in µmol/l. RESULTS: A total of 219 women attending for antenatal care were recruited, and SBA levels were assayed at 12, 20, 28 and 36 weeks of gestation, and up to 72 hours postpartum (n = 44-49 cases at each stage). The majority were white European women, with a median age of 30 years (range 17-46 years) and median BMI of 25 (range 18-38). Values of SBA ranged from 0.3 to 9.8 µmol/l in 216 women, with only three measurements outside this range. There were no significant changes throughout pregnancy. CONCLUSIONS: SBA values in uncomplicated pregnancies are consistent, regardless of gestation, and are not elevated in pregnancy. The current reference values for the diagnosis of OC appear to be appropriate.

Blood-brain barrier P-glycoprotein function in neurodegenerative disease.

Protection of the brain is strengthened by active transport and ABC transporters. P-glycoprotein (P-gp) at the blood-brain barrier (BBB) functions as an active efflux pump by extruding a substrate from the brain, which is important for maintaining loco-regional homeostasis in the brain and protection against toxic compounds. Importantly, dysfunctional BBB P-gp transport is postulated as an important factor contributing to accumulation of aggregated protein in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Furthermore, P-gp is a major factor in mediating resistance to brain entry of numerous exogenous compounds, including toxins that can be involved in PD pathogenesis. This review highlights the role of altered P-gp function in the pathogenesis and progression of neurodegenerative disease. Also the implications of alterations in P-gp function for the treatment of these diseases are discussed.

Subharmonic resonant optical excitation of confined acoustic modes in a free-standing semiconductor membrane at GHz frequencies with a high-repetition-rate femtosecond laser.

We propose subharmonic resonant optical excitation with femtosecond lasers as a new method for the characterization of phononic and nanomechanical systems in the gigahertz to terahertz frequency range. This method is applied for the investigation of confined acoustic modes in a free-standing semiconductor membrane. By tuning the repetition rate of a femtosecond laser through a subharmonic of a mechanical resonance we amplify the mechanical amplitude, directly measure the linewidth with megahertz resolution, infer the lifetime of the coherently excited vibrational states, accurately determine the system's quality factor, and determine the amplitude of the mechanical motion with femtometer resolution.

11C-verapamil to assess P-gp function in human brain during aging, depression and neurodegenerative disease.

P-glycoprotein (P-gp) at the blood-brain barrier (BBB) functions as an active efflux pump by extruding a wide range of substrates from the brain. This is important for maintaining loco-regional homeostasis and for protecting the brain against blood-borne toxic substances. Altered P-gp function seems to be involved in the pathophysiology of neurodegenerative disease and various neurological and psychiatric disorders. Positron emission tomography (PET) with the radiotracer (11)C-verapamil (VPM-PET) is a validated technique allowing measurement of P-gp function at the human BBB. In this review, we highlight changes of P-gp function, as measured with VPM-PET, in aging and in the pathogenesis and progression of neurodegenerative disease, as well as their role in depressive disorders.

Terahertz emission from lateral photo-Dember currents.

The photo-Dember effect is a source of impulsive THz emission following femtosecond pulsed optical excitation. This emission results from the ultrafast spatial separation of electron-hole pairs in strong carrier gradients due to their different diffusion coefficients. The associated time dependent polarization is oriented perpendicular to the excited surface which is inaptly for efficient out coupling of THz radiation. We propose a scheme for generating strong carrier gradients parallel to the excited surface. The resulting photo-Dember currents are oriented in the same direction and emit THz radiation into the favorable direction perpendicular to the surface. This effect is demonstrated for GaAs and In(0.53)Ga(0.47)As. Surprisingly the photo-Dember THz emitters provide higher bandwidth than photoconductive emitters. Multiplexing of phase coherent photo-Dember currents by periodically tailoring the photoexcited spatial carrier distribution gives rise to a strongly enhanced THz emission, which reaches electric field amplitudes comparable to a high-efficiency externally biased photoconductive emitter.

High-speed asynchronous optical sampling with sub-50fs time resolution.

We report an ultrafast time-domain spectroscopy system based on high-speed asynchronous optical sampling operating without mechanical scanner. The system uses two 1 GHz femtosecond oscillators that are offset-stabilized using high-bandwidth feedback electronics operating at the tenth repetition rate harmonics. Definition of the offset frequency, i.e. the time-delay scan rate, in the range of a few kilohertz is accomplished using direct-digital-synthesis electronics for the first time. The time-resolution of the system over the full available 1 ns time-delay window is determined by the laser pulse duration and is 45 fs. This represents a three-fold improvement compared to previous approaches where timing jitter was the limiting factor. Two showcase experiments are presented to verify the high time-resolution and sensitivity of the system.

[11C]-PK11195 PET: quantification of neuroinflammation and a monitor of anti-inflammatory treatment in Parkinson's disease?

UNLABELLED: [(11)C]-PK11195 PET has been used for in vivo brain imaging of microglia activation in Parkinson's disease (PD) patients. COX-2 inhibition has been shown to reduce neuroinflammation and neurodegeneration in animal models of PD. This pilot study assessed the use of [(11)C]-PK11195 PET to quantify neuroinflammation and evaluate the ability of COX-2 inhibition to reduce neuroinflammation in PD patients. METHODS: Fourteen PD patients and eight healthy, age matched controls underwent a [(11)C]-PK11195 PET and MRI scan. Five PD patients were scanned before and after one month of celecoxib treatment 200 mg/day. Arterial plasma sampling and metabolite analysis were performed to create plasma input curves. A 2-compartment model and Logan analysis were applied and parametric DV images were compared using t-test in SPM2. In addition a simplified reference region model (SRTM) was applied, with both the cerebellum and a reference region derived from cluster analysis. RESULTS: Using the cluster analysis, PD patients showed higher contralateral putamen BP and midbrain BP compared to controls, although considerable overlap was seen and differences were not statistically significant. Unexpectedly, BP and DV after celecoxib were slightly higher. Cerebellum as reference region resulted in lower BP values and k(3)/k(4) gave 10-fold higher BP values. Linearization of the data did not show differences between PD patients and controls. CONCLUSIONS: In current practice, [(11)C]-PK11195 seems an unsuitable tracer for accurate or reliable quantification of neuroinflammation. Refinement of [(11)C]-PK11195 uptake analysis and, more importantly, further development of better tracers is necessary to enable accurate measurement of neuroinflammation and effects of anti-inflammatory treatment in patients.

Identificação de portadores de Staphylococcus enterotoxigênicos e avaliação da sensibilidade a antimicrobianos / Identification of enterotoxigenic Staphylococcus carriers and antimicrobial susceptibility evaluation

This study identified Staphylococcus aureus carriers among students of nutrition course, evaluating the capacity of producing staphylococcal enterotoxins (VIDAS Kit) and also the antimicrobial susceptibility (Kirby-Bauer method) of the isolates. Among 173 students sampled, 62 (35.8%) were detected as carriers of S. aureus, being 38 (61.3%) in nostrils; nine (14.5%) on the hands, and 15 (24.2%) in both sites. The production of staphylococcal toxins was detected in 71.4% of the positive-coagulase pools and in 7.1% of the negative-coagulase pools. Even though considerable resistance had been observed with azithromycin (38%), erithromycin (36%), and tetracycline (14%); cephalothin, cefoperazone, ceftriaxone, ciprofloxacin, imipenem, and oxacillin were effective to inhibit the microorganism growth. The high score of healthy carriers was alarming; however, many of the isolated Staphylococcus showed sensitive to most of the antimicrobial tested. The production of toxins was also relevant, mainly by strains that the current Brazilian legislation consider harmless.