Novel types of RiPP-modifying enzymes.

Novel discoveries in natural product biosynthesis reveal hidden bioactive compounds and expand our knowledge in enzymology. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of natural products featuring diverse non-canonical amino acids introduced by maturation enzymes as a class-defining characteristic. Underexplored RiPP sources, such as the human microbiome, the oceans, uncultured microorganisms, and plants are rich hunting grounds for novel enzymology. Unusual α- and ß-amino acids, peptide cleavages, lipidations, diverse macrocyclizations, and other features expand the range of chemical groups that are installed in RiPPs by often promiscuous enzymes. This review highlights the search for novelty in RiPP enzymology in the past two years, with respect to the discovery of new biochemical modifications but also towards novel applications.

Dollo Parsimony Overestimates Ancestral Gene Content Reconstructions.

Ancestral reconstruction is a widely used technique that has been applied to understand the evolutionary history of gain and loss of gene families. Ancestral gene content can be reconstructed via different phylogenetic methods, but many current and previous studies employ Dollo parsimony. We hypothesize that Dollo parsimony is not appropriate for ancestral gene content reconstruction inferences based on sequence homology, as Dollo parsimony is derived from the assumption that a complex character cannot be regained. This premise does not accurately model molecular sequence evolution, in which false orthology can result from sequence convergence or lateral gene transfer. The aim of this study is to test Dollo parsimony's suitability for ancestral gene content reconstruction and to compare its inferences with a maximum likelihood-based approach that allows a gene family to be gained more than once within a tree. We first compared the performance of the two approaches on a series of artificial data sets each of 5,000 genes that were simulated according to a spectrum of evolutionary rates without gene gain or loss, so that inferred deviations from the true gene count would arise only from errors in orthology inference and ancestral reconstruction. Next, we reconstructed protein domain evolution on a phylogeny representing known eukaryotic diversity. We observed that Dollo parsimony produced numerous ancestral gene content overestimations, especially at nodes closer to the root of the tree. These observations led us to the conclusion that, confirming our hypothesis, Dollo parsimony is not an appropriate method for ancestral reconstruction studies based on sequence homology.

The evolution of acute stroke care in Germany from 2019 to 2021: analysis of nation-wide administrative datasets.

BACKGROUND: The treatment of ischemic stroke (IS) has changed considerably in recent years. Particularly the advent of mechanical thrombectomy (MTE) has revolutionized the available treatment options. Most patients in developed countries have access to intravenous thrombolysis (IVT). However access to MTE remains restricted in some regions despite efforts to increase its availability. We performed an evaluation of national datasets to monitor improvements made in access to revascularization therapies for IS patients in Germany. METHODS: We analyzed national datasets on German Diagnosis-Related Groups and structured quality reports by extracting information of patients admitted with stroke with and without IVT and MTE for the period of 2019-2021. Data from 2016 and limited data for 2022 were also included for comparison. RESULTS: Admissions with ischemic stroke declined during the years of the COVID 19 pandemic by 4.5% from 227,258 in 2019 to 216,923 in 2021. IVT rates were stable with 16.3% being treated with IVT in 2019 and 2021. MTE rates continued to increase from 7.1 to 8.4% and the number of MTE centers increased by 14.8% in the same period. Over 87.3% of MTEs were performed in centers with a case volume exceeding 50 cases per year in 2021. The largest increase in the relative share of MTEs was seen in large MTE centers (n ≥ 200). Patient age for MTEs surpassed the age for IVTs in 2019 and the proportion of patients ≥ 80 years receiving MTE continued to increase. The proportion of regions in Germany with poor MTE rates (≤ 4.1%) decreased significantly from 2019 (12.3%) to 2021 (5.3%) (p < 0.001). CONCLUSIONS: We found strong evidence that while IVT rates reached a temporary ceiling effect, both the absolute number of and access to MTEs continued to increase in Germany. Regional disparities have become less significant and the majority of MTEs are performed in centers with medium or high case volumes.

Risk stratification of thyroid nodules: Assessing the suitability of ChatGPT for text-based analysis.

PURPOSE: Accurate risk stratification of thyroid nodules is essential for optimal patient management. This study aimed to assess the suitability of ChatGPT for risk stratification of thyroid nodules using a text-based evaluation. METHODS: A dataset was compiled comprising 50 anonymized clinical reports and associated risk assessments for thyroid nodules. The Chat Generative Pre-trained Transformer (ChatGPT) was used to classify sonographic patterns in accordance with the Thyroid Imaging Reporting and Data System (TI-RADS). The model's performance was assessed using various criteria, including sensitivity, specificity, and accuracy. A comparative analysis was conducted, evaluating the model against investigator-based risk stratification as well as histology. RESULTS: With an overall agreement rate of 42 % in comparison with examiner-based evaluation (TI-RADS 1-5), the results show that ChatGPT has moderate potential for predicting the risk of malignancy in thyroid nodules using text-based reports. The chatbot model achieved a sensitivity of 86.7 %, a specificity of 10.7 %, and an overall accuracy of 68 % when distinguishing between low-risk (TI-RADS 2 and 3) and high-risk (TI-RADS 4 and 5) categories. Interrater reliability was calculated with a Cohen's kappa of 0.686. CONCLUSION: This study highlights the potential of ChatGPT in assisting clinicians with risk stratification of thyroid nodules. The results suggest that ChatGPT can facilitate personalized treatment decisions, although the agreement rate is still low. Further research and validation studies are necessary to establish the clinical applicability and generalizability of ChatGPT in routine practice. The integration of ChatGPT into clinical workflows has the potential to enhance thyroid nodule risk assessment and improve patient care.

The protist cultural renaissance.

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

The primacy of temporal dynamics in driving spatial self-organization of soil iron redox patterns.

This study investigates mechanisms that generate regularly spaced iron-rich bands in upland soils. These striking features appear in soils worldwide, but beyond a generalized association with changing redox, their genesis is yet to be explained. Upland soils exhibit significant redox fluctuations driven by rainfall, groundwater changes, or irrigation. Pattern formation in such systems provides an opportunity to investigate the temporal aspects of spatial self-organization, which have been heretofore understudied. By comparing multiple alternative mechanisms, we found that regular iron banding in upland soils is explained by coupling two sets of scale-dependent feedbacks, the general principle of Turing morphogenesis. First, clay dispersion and coagulation in iron redox fluctuations amplify soil Fe(III) aggregation and crystal growth to a level that negatively affects root growth. Second, the activation of this negative root response to highly crystalline Fe(III) leads to the formation of rhythmic iron bands. In forming iron bands, environmental variability plays a critical role. It creates alternating anoxic and oxic conditions for required pattern-forming processes to occur in distinctly separated times and determines durations of anoxic and oxic episodes, thereby controlling relative rates of processes accompanying oxidation and reduction reactions. As Turing morphogenesis requires ratios of certain process rates to be within a specific range, environmental variability thus modifies the likelihood that pattern formation will occur. Projected changes of climatic regime could significantly alter many spatially self-organized systems, as well as the ecological functioning associated with the striking patterns they present. This temporal dimension of pattern formation merits close attention in the future.

Legacies of Pre-1960s Municipal Waste Incineration in the Pb of City Soils.

A 1937 street map of Durham, North Carolina, located four city-run waste incinerators that we recognized to be sites of contemporary city parks. We obtained city permission to sample three park's soils, developed a sampling design for geospatial mapping of hypothetical incinerator-ash contamination of park soils, and queried online Durham newspapers to understand histories of incinerator operations, ash disposal, and incinerator-to-park conversions. In 2021-2022, seven decades after parks were created, two parks had soil-Pb > 400 mgPb/kg, EPA's threshold for safe soil in play areas. At Walltown Park, six of 97 surface samples ranged from 416 to 1338 mg Pb/kg within meters of a basketball court and a park path. East Durham Park had a hectare-sized area where 12 samples averaged 1294 mgPb/kg (median 1335 mg/kg). Engineering surveys of United States and Canadian cities in 1941 and 1958 suggest that half incinerated solid wastes. Many records describe how incinerator ash was dumped with little regard for health or environmental hazards. Legacy soil contaminations of incinerator ash can be identified, as we have done in Durham, from historical records of city-waste incinerator operations, online access to newspaper archives that describe incinerator-to-park conversions, and a XRF to screen for soil-Pb contamination.

Inpatient care of neuromyelitis optica spectrum disorder in Germany: Nationwide analysis from 2010 to 2021.

Background: Despite tremendous development in the treatment of neuromyelitis optica spectrum disorder (NMOSD), less is known about the characteristics of hospitalized patients and inpatient care utilization. Objective: To investigate the development of inpatient NMOSD case numbers and implemented immunotherapies in the last decade in Germany. Methods: We conducted a nationwide retrospective study using an administrative database of all hospitalized NMOSD patients between 2010 and 2021. We evaluated yearly data on case numbers, demographics, treatment regimens, and seasonal variations of apheresis therapy as a surrogate marker of severe relapse incidence. Results: During the observational period case number of inpatients substantially increased (2010:n = 463, 2021:n = 992). The mean age was 48.1 ± 2.5 years (74% females). The pooled yearly rate of plasmapheresis/immunoadsorption was 14% (95% CI [13-15%]), without seasonal variations. Its application peaked in 2013 (18%, 95% CI [15-21%]) with decreasing trend since. Predominant immunotherapy was rituximab (40%, 95% CI [34-45%]), followed by tocilizumab (4%, 95% CI [3-5%]) since 2013 and eculizumab (4%, 95% CI [3-5%]) since 2020. Inpatient mortality ranged between 0% and 1% per year. Conclusions: Inpatient case numbers of NMOSD substantially increased during the past decade, probably reflecting improving disease awareness. In parallel with the administration of highly effective therapies rate of apheresis therapies decreased. A stable apheresis rate over the year makes seasonal variations of the steroid-refractive relapses unlikely.

Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous ß-amino acid dienophiles.

The tetrazine ligation is an inverse electron-demand Diels-Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M-1 s-1 and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

Software- and TDM-Guided Dosing of Meropenem Promises High Rates of Target Attainment in Critically Ill Patients.

Various studies have reported insufficient beta-lactam concentrations in critically ill patients. The optimal dosing strategy for beta-lactams in critically ill patients, particularly in septic patients, is an ongoing matter of discussion. This retrospective study aimed to evaluate the success of software-guided empiric meropenem dosing (CADDy, Calculator to Approximate Drug-Dosing in Dialysis) with subsequent routine meropenem measurements and expert clinical pharmacological interpretations. Adequate therapeutic drug exposure was defined as concentrations of 8-16 mg/L, whereas concentrations of 16-24 mg/L were defined as moderately high and concentrations >24 mg/L as potentially harmful. A total of 91 patients received meropenem as a continuous infusion (229 serum concentrations), of whom 60% achieved 8-16 mg/L, 23% achieved 16-24 mg/L, and 10% achieved unnecessarily high and potentially harmful meropenem concentrations >24 mg/L in the first 48 h using the dosing software. No patient showed concentrations <2 mg/L using the dosing software in the first 48 h. With a subsequent TDM-guided dose adjustment, therapeutic drug exposure was significantly (p ≤ 0.05) enhanced to 70%. No patient had meropenem concentrations >24 mg/L with TDM-guided dose adjustments. The combined use of dosing software and consecutive TDM promised a high rate of adequate therapeutic drug exposures of meropenem in patients with sepsis and septic shock.

First steps in the development of next-generation chirped volume Bragg gratings by means of fs laser inscription in fused silica.

The first steps towards the development and characterization of next-generation chirped volume Bragg gratings (CVBGs) by means of fs laser inscription were made. Based on the phase mask inscription technique we realized CVBGs in fused silica with a 3 × 3 mm2 aperture and a length of almost 12 mm with a chirp rate of ∼190 ps/nm around a central wavelength of 1030.5 nm. Strong mechanical stresses induced serious polarization and phase distortions of the radiation. We show a possible approach to solution of this problem. The change in the linear absorption coefficient associated with local modification of fused silica is quite small, enabling utilization of this type of gratings in high average power lasers.

Cortical blindness as a sign of delayed post-hypoxic encephalopathy: a case report.

We present a case of a 67-year-old female patient, who presented with acute cortical blindness five days after a successful resuscitation from cardiac arrest. The magnetic resonance tomography revealed a mild FLAIR signal increase of the bilateral occipital cortex. A lumbar puncture revealed considerably elevated tau protein levels, in the presence of normal phospho-tau, as a marker of brain injury, whilst neuron-specific enolase levels were normal. The diagnosis of delayed post-hypoxic encephalopathy was set. We hereby describe a rare clinical manifestation after initially successful resuscitation and encourage the studying of tau protein as a potential marker of this disease entity.

Regulatory and coding sequences of TRNP1 co-evolve with brain size and cortical folding in mammals.

Brain size and cortical folding have increased and decreased recurrently during mammalian evolution. Identifying genetic elements whose sequence or functional properties co-evolve with these traits can provide unique information on evolutionary and developmental mechanisms. A good candidate for such a comparative approach is TRNP1, as it controls proliferation of neural progenitors in mice and ferrets. Here, we investigate the contribution of both regulatory and coding sequences of TRNP1 to brain size and cortical folding in over 30 mammals. We find that the rate of TRNP1 protein evolution (ω) significantly correlates with brain size, slightly less with cortical folding and much less with body size. This brain correlation is stronger than for >95% of random control proteins. This co-evolution is likely affecting TRNP1 activity, as we find that TRNP1 from species with larger brains and more cortical folding induce higher proliferation rates in neural stem cells. Furthermore, we compare the activity of putative cis-regulatory elements (CREs) of TRNP1 in a massively parallel reporter assay and identify one CRE that likely co-evolves with cortical folding in Old World monkeys and apes. Our analyses indicate that coding and regulatory changes that increased TRNP1 activity were positively selected either as a cause or a consequence of increases in brain size and cortical folding. They also provide an example how phylogenetic approaches can inform biological mechanisms, especially when combined with molecular phenotypes across several species.

Taxonomic composition, community structure and molecular novelty of microeukaryotes in a temperate oligomesotrophic lake as revealed by metabarcoding.

Microbial eukaryotes are diverse and ecologically important organisms, yet sampling constraints have hindered the understanding of their distribution and diversity in freshwater ecosystems. Metabarcoding has provided a powerful complement to traditional limnological studies, revealing an unprecedented diversity of protists in freshwater environments. Here, we aim to expand our knowledge of the ecology and diversity of protists in lacustrine ecosystems by targeting the V4 hypervariable region of the 18S rRNA gene in water column, sediment and biofilm samples collected from Sanabria Lake (Spain) and surrounding freshwater ecosystems. Sanabria is a temperate lake, which are relatively understudied by metabarcoding in comparison to alpine and polar lakes. The phylogenetic diversity of microbial eukaryotes detected in Sanabria spans all currently recognized eukaryotic supergroups, with Stramenopiles being the most abundant and diverse supergroup in all sampling sites. Parasitic microeukaryotes account for 21% of the total protist ASVs identified in our study and were dominated by Chytridiomycota, both in terms of richness and abundance, in all sampling sites. Sediments, biofilms and water column samples harbour distinct microbial communities. Phylogenetic placement of poorly assigned and abundant ASVs indicates molecular novelty inside Rhodophyta, Bigyra, early-branching Nucletmycea and Apusomonadida. In addition, we report the first freshwater incidence of the previously exclusively marine genera Abeoforma and Sphaeroforma. Our results contribute to a deeper understanding of microeukaryotic communities in freshwater ecosystems, and provide the first molecular reference for future biomonitoring surveys in Sanabria Lake.

[Patient Preferences for Exercise Therapy in People with Severe Mental Illness in Germany]. / Der subjektive Stellenwert von Bewegungs- und Sporttherapie bei Menschen mit schwerer psychischer Erkrankung in Deutschland.

PURPOSE: Assessing the experience with and the attitudes towards exercise therapy in persons with severe mental illness (SMI). Furthermore, potential variables of high preference towards exercise therapy are investigated. METHODS: Cross-sectional observational study of SMI patients aged between 18 and 65 years (n=385). Patients were interviewed by trained staff using standardised instruments. Potential variables were analysed using a hierarchic binary logistic regression model. RESULTS: 84,4% of SMI patients had a high preference for exercise therapy; of these, 44,1% exercised regularly. Among patients with severe mental illness especially a higher value in the GAF-assessment (p=0,041) and living in a metropolitan area (p=0,011) predict a high preference for exercise therapy. CONCLUSION: Most of the patients with severe mental illness interviewed in this study place a surprisingly high value on sports and exercise therapy. Due to the increasing evidence with regard to positive effects of these therapies, it may be an excellent starting point to expand sports and exercise therapy as part of a comprehensive treatment plan. At the same time, strategies for everyday transfer need to be implemented more rigorously.

Prognostic markers of post-stroke depression (PROMoSD): study protocol of a prospective single-center observational study on raphe hypoechogenicity as a predictor of post-stroke depression.

INTRODUCTION: Post-stroke depression (PSD) is an important and frequent non-motor complication after a stroke. As valid prediction of PSD occurrence is still not possible, the unselective use of preventive therapy in stroke patients has risen a questionable risk-to-benefit consideration. Therefore, there is a need to increase the prediction probability of PSD to identify patients at very high risk of a depressive complication who might benefit from preventive therapy. In this context, a brainstem raphe hypoechogenicity (BRH) in transcranial sonography (TCS) has previously been associated with depressive symptoms in a broad spectrum of diseases. BRH might therefore represent a valid maker of vulnerability for depressive symptoms that could be of interest in the risk assessment of PSD occurrence. METHODS: In the prognostic markers of post-stroke depression (PROMoSD) study, a prospective, observational, single-center, investigator-initiated study, we aim to include 100 patients with acute ischemic stroke (AIS). Besides data on clinical characteristics and baseline psychiatric assessment, we conduct a TCS examination to identify patients with BRH. The primary outcome is the incidence of PSD three months after inclusion, determined by a blinded investigator according to the fifth version of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) criteria. PERSPECTIVE: The results of PROMoSD will answer the question of whether screening of BRH after AIS improves the prediction of PSD occurrence. A positive result of this study could have direct consequences on psychiatric support after AIS by streamlining diagnostic and therapeutic algorithms. Trial registration ClinicalTrials.gov identifier no. NCT05580198.

In vivo PDX CRISPR/Cas9 screens reveal mutual therapeutic targets to overcome heterogeneous acquired chemo-resistance.

Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

Cross-sectional analysis of clinical aspects in patients with long-COVID and post-COVID syndrome.

Objective: Regarding pathogenesis, clinical manifestations, at-risk individuals, and diagnostic methods for stratifying patients for therapeutic approaches, our understanding of post-COVID syndrome is limited. Here, we set out to assess sociodemographic and clinical aspects in patients with the long-COVID and post-COVID syndrome. Methods: We performed a cross-sectional analysis of patients presenting at our specialized university hospital outpatient clinic. We assessed patients' clinical presentation, fatigue, symptoms of depression and anxiety, and impairment of smell. Results: A total of 101 patients were included (73.3% female), of whom 78.2% had a mild course of COVID-19. At presentation, 93.1% suffered from fatigue, 82.2% from impaired concentration, and 79.2% from impaired memory, 53.5% had impaired sleep. The most common secondary diagnosis found in our cohort was thyroid disease. Fatigue analysis showed that 81.3% of female and 58.8% of male patients had severe combined fatigue. Female gender was an independent risk factor for severe fatigue (severe cognitive fatigue OR = 8.045, p = 0.010; severe motor fatigue OR = 7.698, p = 0.013). Males suffered from more depressive symptoms, which correlated positively with the duration of symptom onset. 70.3% of patients with anamnestic smell impairment had hyposmia, and 18.9% were anosmic. Interpretation: Most long-COVID patients suffered from severe fatigue, with the female sex as an independent risk factor. Fatigue was not associated with symptoms of depression or anxiety. Patients with long-COVID symptoms should receive an interdisciplinary diagnostic and therapeutic approach depending on the clinical presentation.

Plasma Transfusion in Septic Shock-A Secondary Analysis of a Retrospective Single-Center Cohort.

In sepsis, both beneficial and detrimental effects of fresh frozen plasma (FFP) transfusion have been reported. The aim of this study was to analyze the indication for and effect of FFP transfusion in patients with septic shock. We performed a secondary analysis of a retrospective single-center cohort of all patients treated for septic shock at the interdisciplinary surgical intensive care unit (ICU) of the Heidelberg University Hospital. Septic shock was defined according to sepsis-3 criteria. To assess the effects of FFP administration in the early phase of septic shock, we compared patients with and without FFP transfusion during the first 48 h of septic shock. Patients who died during the first 48 h of septic shock were excluded from the analysis. Primary endpoints were 30- and 90-day mortality. A total of 261 patients were identified, of which 100 (38.3%) received FFP transfusion within the first 48 h after septic shock onset. The unmatched analysis showed a trend toward higher 30- and 90-d mortality in the FFP group (30 d: +7% p = 0.261; 90 d: +11.9% p = 0.061). In the propensity-matched analysis, 30- and 90-day mortality were similar between groups. Plasma administration did not influence fluid or vasopressor need, lactate levels, ICU stay, or days on a ventilator. We found no significant harm or associated benefit of FFP use in the early phase of septic shock. Finally, plasma should only be used in patients with a strong indication according to current recommendations, as a conclusive evaluation of the risk-benefit ratio for plasma transfusion in septic shock cannot be made based on the current data.