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Custom proteases find applications as therapeutics, in research and in biotechnological applications. Here, the authors establish a protease selection system based on bacterial alpha-2-macroglobulin protease inhibitors and evolve staphylococcal proteases for increased activity and altered specificity.

Abstract Cardiac alterations are frequently observed after acute neurological disorders. Takotsubo syndrome (TTS) represents an acute heart failure syndrome and is increasingly recognized as part of the spectrum of cardiac complications observed after neurological disorders. A systematic investigation of TTS patients with neurological disorders has not been conducted yet. The aim of the study was to expand insights regarding neurological disease entities triggering TTS and to investigate the clinical profile and outcomes of TTS patients after primary neurological disorders. The International Takotsubo Registry is an observational multicenter collaborative effort of 45 centers in 14 countries (ClinicalTrials.gov, identifier NCT01947621). All patients in the registry fulfilled International Takotsubo Diagnostic Criteria. For the present study, patients were included if complete information on acute neurological disorders were available. 2402 patients in whom complete information on acute neurological status were available were analyzed. In 161 patients (6.7%) an acute neurological disorder was identified as the preceding triggering factor. The most common neurological disorders were seizures, intracranial hemorrhage, and ischemic stroke. Time from neurological symptoms to TTS diagnosis was ≤ 2 days in 87.3% of cases. TTS patients with neurological disorders were younger, had a lower female predominance, fewer cardiac symptoms, lower left ventricular ejection fraction, and higher levels of cardiac biomarkers. TTS patients with neurological disorders had a 3.2-fold increased odds of in-hospital mortality compared to TTS patients without neurological disorders. In this large-scale study, 1 out of 15 TTS patients had an acute neurological condition as the underlying triggering factor. Our data emphasize that a wide spectrum of neurological diseases ranging from benign to life-threatening encompass TTS. The high rates of adverse events highlight the need for clinical awareness.

PURPOSE:There is a strong association between severe hyperhomocysteinemia and myopia. Thus we studied the hypothesis that even moderately increased levels of homocysteine (Hcy) might be a potentially treatable risk factor for myopia. METHODS:The Gutenberg Health Study (GHS) is a population-based, prospective, observational cohort study in Germany, including 15,010 participants aged between 35 and 74 at recruitment. The baseline examination was conducted from 2007-2012. Refraction was measured using autorefraction (HARK 599, Carl Zeiss AG, Jena, Germany). Hcy was measured by an immunoassay. We included only phakic participants without a history of corneal surgery or corneal laser treatment. We used linear regression models to evaluate the potential association between Hcy and refraction at baseline, and between Hcy and change in refraction between baseline and 5-year-follow-up examination. We used generalized estimating equation models to account for the correlation between fellow eyes. RESULTS:We included 13,749 participants, categorized as having no myopia (spherical equivalent > -0.75 D, 65.2%), low myopia (-0.75 D--2.75 D, 21.5%), moderate myopia (-3.00 D- 5.75 D, 9.8%) and high myopia (≤ -6 D, 3.5%). Median Hcy levels were similar in all groups (μmol/l). We observed no association of Hcy with refraction or 5-year change in refraction in the models adjusted for age, sex and socioeconomic status. CONCLUSION:We found no evidence for an association of Hcy levels and refractive error.

Abstract Takotsubo syndrome (TTS) is characterized by acute left ventricular dysfunction, with a hospital-mortality rate similar to acute coronary syndrome (ACS). However, the aetiology of TTS is still unknown. In the present study, a multivariate pattern analysis using machine learning with multimodal magnetic resonance imaging (MRI) data of the human brain of TTS patients and age- and gender-matched healthy control subjects was performed. We found consistent structural and functional alterations in TTS patients compared to the control group. In particular, anatomical and neurophysiological measures from brain regions constituting the emotional-autonomic control system contributed to a prediction accuracy of more than 82%. Thus, our findings demonstrate homogeneous neuronal alterations in TTS patients and substantiate the importance of the concept of a brain-heart interaction in TTS.

Diabetes plays an important role in the complex relationship between chronic kidney disease (CKD) and cardiovascular disease. This retrospective observational study compared the influence of estimated glomerular filtration rate (eGFR) and proteinuria on the risk of major adverse cardiovascular event (MACE; myocardial infarction or stroke) in CKD patients with and without diabetes. Data were from a linked database of UK electronic health records. Individuals with CKD and no prior MACE were classified as type 1 diabetes (T1DM; n = 164), type 2 diabetes (T2DM; n = 9,711), and non-diabetes (non-DM; n = 75,789). Monthly updated time-dependent Cox proportional hazard models were constructed to calculate adjusted hazard ratios (aHRs) for progression to MACE from first record of abnormal eGFR or proteinuria (index date). In non-DM, aHRs (95% CIs) by baseline eGFR category (referent G2) were G1: 0.70 (0.55-0.90), G3a: 1.28 (1.20-1.35), G3b: 1.64 (1.52-1.76), G4: 2.19 (1.98-2.43), and G5: 3.12 (2.44-3.99), and by proteinuria category (referent A1) were A2: 1.13 (1.00-1.28), A2/3 (severity indeterminable): 1.58 (1.28-1.95), and A3: 1.64 (1.38-1.95). In T2DM, aHRs were G1: 0.98 (0.72-1.32), G3a: 1.18 (1.03-1.34), G3b: 1.31 (1.12-1.54), G4: 1.87 (1.53-2.29), G5: 2.87 (1.82-4.52), A2: 1.22 (1.04-1.42), A2/3: 1.45 (1.17-1.79), and A3: 1.82 (1.53-2.16). Low numbers in T1DM precluded analysis. Modelling T2DM and non-DM together, aHRs were, respectively, G1: 3.23 (2.38-4.40) and 0.70 (0.55-0.89); G2: 3.18 (2.73-3.70) and 1.00 (referent); G3a: 3.65 (3.13-4.25) and 1.28 (1.21-1.36); G3b: 4.01 (3.40-4.74) and 1.65 (1.54-1.77); G4: 5.78 (4.70-7.10) and 2.21 (2.00-2.45); G5: 9.00 (5.71-14.18) and 3.14 (2.46-4.00). In conclusion, reduced eGFR and proteinuria were independently associated with increased risk of MACE regardless of diabetes status. However, the risk of MACE in the same eGFR state was 4.6-2.4 times higher in T2DM than in non-DM.

In this mini-review, I summarize and interpret the current status of sex/gender differences in terms of brain anatomy, brain function, behavior, and cognition. Based on this review and the reported findings, I conclude that most of these sex/gender differences are not large enough to support the assumption of sexual dimorphism in terms of brain anatomy, brain function, cognition, and behavior. Instead, I suggest that many brain and cognitive features are modulated by environment, culture, and practice (and several other influences). These influences interact with the menstrual cycle, the general hormone level, and current gender stereotypes in a way that has not yet been fully understood.

Language in its highest complexity is a unique human faculty with simultaneous translation being among the most demanding language task involving both linguistic and executive functions. In this context, bilingually grown up individuals as well as simultaneous interpreters (SIs) represent appropriate groups for studying expertise-related neural adaptations in the human brain. The present study was performed to examine if a domain-specific neural network activation pattern, constituted by brain regions involved in speech processing as well as cognitive control mechanisms can be detected during a task-free resting state condition. To investigate this, electroencephalographic (EEG) data were recorded from 16 SIs and 16 age and gender-matched multilingual control subjects. Graph-theoretical network analyses revealed interhemispheric hyperconnectivity between the ventral part of the prefrontal cortex (pars opercularis and pars triangularis) and the dorsolateral prefrontal cortex (DLPFC) in language experts compared to multilingual controls in the alpha frequency range. This finding suggests that the high cognitive demands placed on simultaneous interpreting lead to an increased neural communication between prefrontal brain regions essentially engaged in supporting executive control-a neural fingerprint that is even detectable during rest.

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements.

The problem size effect is a well-established finding in arithmetic problem solving and is characterized by worse performance in problems with larger compared to smaller operand size. Solving small and large arithmetic problems has also been shown to involve different cognitive processes and distinct electroencephalography (EEG) oscillations over the left posterior parietal cortex (LPPC). In this study, we aimed to provide further evidence for these dissociations by using transcranial direct current stimulation (tDCS). Participants underwent anodal (30min, 1.5 mA, LPPC) and sham tDCS. After the stimulation, we recorded their neural activity using EEG while the participants solved small and large arithmetic problems. We found that the tDCS effects on performance and oscillatory activity critically depended on the problem size. While anodal tDCS improved response latencies in large arithmetic problems, it decreased solution rates in small arithmetic problems. Likewise, the lower-alpha desynchronization in large problems increased, whereas the theta synchronization in small problems decreased. These findings reveal that the LPPC is differentially involved in solving small and large arithmetic problems and demonstrate that the effects of brain stimulation strikingly differ depending on the involved neuro-cognitive processes.

There is no doubt that good bimanual performance is very important for skilled handball playing. The control of the non-dominant hand is especially demanding since efficient catching and throwing needs both hands.We investigated training-induced structural neuroplasticity in professional handball players using several structural neuroimaging techniques and analytic approaches and also provide a review of the literature about sport-induced structural neuroplastic alterations. Structural brain adaptations were expected in regions relevant for motor and somatosensory processing such as the grey matter (GM) of the primary/secondary motor (MI/supplementary motor area, SMA) and somatosensory cortex (SI/SII), basal ganglia, thalamus, and cerebellum and in the white matter (WM) of the corticospinal tract (CST) and corpus callosum, stronger in brain regions controlling the non-dominant left hand.Increased GM volume in handball players compared with control subjects were found in the right MI/SI, bilateral SMA/cingulate motor area, and left intraparietal sulcus. Fractional anisotropy (FA) and axial diffusivity were increased within the right CST in handball players compared with control women. Age of handball training commencement correlated inversely with GM volume in the right and left MI/SI and years of handball training experience correlated inversely with radial diffusivity in the right CST. Subcortical structures tended to be larger in handball players. The anatomical measures of the brain regions associated with handball playing were positively correlated in handball players, but not interrelated in control women.Training-induced structural alterations were found in the somatosensory-motor network of handball players, more pronounced in the right hemisphere controlling the non-dominant left hand. Correlations between handball training-related measures and anatomical differences suggest neuroplastic adaptations rather than a genetic predisposition for a ball playing affinity. Investigations of neuroplasticity specifically in sportsmen might help to understand the neural mechanisms of expertise in general.

BackgroundIn accordance with the Theory of Structural Dissociation of the Personality (TSDP), studies of dissociative identity disorder (DID) have documented that two prototypical dissociative subsystems of the personality, the "Emotional Part" (EP) and the "Apparently Normal Part" (ANP), have different biopsychosocial reactions to supraliminal and subliminal trauma-related cues and that these reactions cannot be mimicked by fantasy prone healthy controls nor by actors.MethodsArterial spin labeling perfusion MRI was used to test the hypotheses that ANP and EP in DID have different perfusion patterns in response to rest instructions, and that perfusion is different in actors who were instructed to simulate ANP and EP. In a follow-up study, regional cerebral blood flow of DID patients was compared with the activation pattern of healthy non-simulating controls.ResultsCompared to EP, ANP showed elevated perfusion in bilateral thalamus. Compared to ANP, EP had increased perfusion in the dorsomedial prefrontal cortex, primary somatosensory cortex, and motor-related areas. Perfusion patterns for simulated ANP and EP were different. Fitting their reported role-play strategies, the actors activated brain structures involved in visual mental imagery and empathizing feelings. The follow-up study demonstrated elevated perfusion in the left temporal lobe in DID patients, whereas non-simulating healthy controls had increased activity in areas which mediate the mental construction of past and future episodic events.ConclusionDID involves dissociative part-dependent resting-state differences. Compared to ANP, EP activated brain structures involved in self-referencing and sensorimotor actions more. Actors had different perfusion patterns compared to genuine ANP and EP. Comparisons of neural activity for individuals with DID and non-DID simulating controls suggest that the resting-state features of ANP and EP in DID are not due to imagination. The findings are consistent with TSDP and inconsistent with the idea that DID is caused by suggestion, fantasy proneness, and role-playing.

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.

Graph theory deterministically models networks as sets of vertices, which are linked by connections. Such mathematical representation of networks, called graphs are increasingly used in neuroscience to model functional brain networks. It was shown that many forms of structural and functional brain networks have small-world characteristics, thus, constitute networks of dense local and highly effective distal information processing. Motivated by a previous small-world connectivity analysis of resting EEG-data we explored implications of a commonly used analysis approach. This common course of analysis is to compare small-world characteristics between two groups using classical inferential statistics. This however, becomes problematic when using measures of inter-subject correlations, as it is the case in commonly used brain imaging methods such as structural and diffusion tensor imaging with the exception of fibre tracking. Since for each voxel, or region there is only one data point, a measure of connectivity can only be computed for a group. To empirically determine an adequate small-world network threshold and to generate the necessary distribution of measures for classical inferential statistics, samples are generated by thresholding the networks on the group level over a range of thresholds. We believe that there are mainly two problems with this approach. First, the number of thresholded networks is arbitrary. Second, the obtained thresholded networks are not independent samples. Both issues become problematic when using commonly applied parametric statistical tests. Here, we demonstrate potential consequences of the number of thresholds and non-independency of samples in two examples (using artificial data and EEG data). Consequently alternative approaches are presented, which overcome these methodological issues.

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements.

Worldwide, methicillin-resistant Staphylococcus aureus (MRSA) pose an increased risk for healthcare- and community-associated infections. Since the first report of MRSA in England in 1961, several distinct clones or strains have emerged. Changes within the MRSA population of whole countries, small regions or of single hospitals have been observed with some clones replacing others. In this study, the clonal replacement of MRSA isolates in a South-eastern German tertiary care hospital between 2000 and 2010 is described based on microarray analyses of 778 isolates and at least 50 MRSA per year. Within these eleven years, four common epidemic strains, CC22-MRSA-IV, CC45-MRSA-IV, CC5/ST228-MRSA-I (including a variant with a truncated SCCmec element) and CC5-MRSA-II were identified. The PVL-negative CC22-MRSA-IV strain (Barnim Epidemic Strain, UK-EMRSA-15) was detected for the first time in 2001 and its abundance increased since then to 58.6% in 2010. CC5-MRSA-II increased from 2% (2000) to about 30% (2003), and since then it fluctuates between 23 and 37% of isolates. CC5/ST228-MRSA-I decreased from about the half of tested isolates (2000) to 2.3% (2010). A similar trend was observed for CC45-MRSA-IV, which decreased drastically down to 3.4% in 2010 after reaching a maximum of 62.0% in 2002. Seventeen other PVL-negative MRSA strains were identified sporadically with no significant trend being observed. Seven PVL-positive MRSA strains were found, but they remained rare during the study period accounting together for 2.7% of isolates.

BackgroundExposure to bright light such as sunlight elicits a sneeze or prickling sensation in about one of every four individuals. This study presents the first scientific examination of this phenomenon, called 'the photic sneeze reflex'.Methodology and principal findingsIn the present experiment, 'photic sneezers' and controls were exposed to a standard checkerboard stimulus (block 1) and bright flashing lights (block 2) while their EEG (electro-encephalogram) was recorded. Remarkably, we found a generally enhanced excitability of the visual cortex (mainly in the cuneus) to visual stimuli in 'photic sneezers' compared with control subjects. In addition, a stronger prickling sensation in the nose of photic sneezers was found to be associated with activation in the insula and stronger activation in the secondary somatosensory cortex.ConclusionWe propose that the photic sneeze phenomenon might be the consequence of higher sensitivity to visual stimuli in the visual cortex and of co-activation of somatosensory areas. The 'photic sneeze reflex' is therefore not a classical reflex that occurs only at a brainstem or spinal cord level but, in stark contrast to many theories, involves also specific cortical areas.

BACKGROUND: Several recent studies have shown practice-dependent structural alterations in humans. Cross-sectional studies of intensive practice of specific tasks suggest associated long-term structural adaptations. Playing golf at a high level of performance is one of the most demanding sporting activities. In this study, we report the relationship between a particular level of proficiency in playing golf (indicated by golf handicap level) and specific neuroanatomical features. PRINCIPAL FINDINGS: Using voxel-based morphometry (VBM) of grey (GM) and white matter (WM) volumes and fractional anisotropy (FA) measures of the fibre tracts, we identified differences between skilled (professional golfers and golfers with an handicap from 1-14) and less-skilled golfers (golfers with an handicap from 15-36 and non-golfer). Larger GM volumes were found in skilled golfers in a fronto-parietal network including premotor and parietal areas. Skilled golfers revealed smaller WM volume and FA values in the vicinity of the corticospinal tract at the level of the internal and external capsule and in the parietal operculum. However, there was no structural difference within the skilled and less-skilled golfer group. CONCLUSION: There is no linear relationship between the anatomical findings and handicap level, amount of practice, and practice hours per year. There was however a strong difference between highly-practiced golfers (at least 800-3,000 hours) and those who have practised less or non-golfers without any golfing practise, thus indicating a step-wise structural and not a linear change.