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Domestication - the artificial selection of wild species to obtain variants with traits of human interest - was integral to the rise of complex societies. The oversupply of food was probably associated with the formalization of food preservation strategies through microbial fermentation. While considerable literature exists on the antiquity of fermented food, only few eukaryotic microbes have been studied so far for signs of domestication, less is known for bacteria. Here, we tested if cheese starter cultures harbour typical hallmarks of domestication by characterising over 100 community samples and over 100 individual strains isolated from historical and modern traditional Swiss cheese starter cultures. We find that cheese starter cultures have low genetic diversity both at the species and strain-level and maintained stable phenotypic traits. Molecular clock dating further suggests that the evolutionary origin of the bacteria approximately coincided with the first archaeological records of cheese making. Finally, we find evidence for ongoing genome decay and pseudogenization via transposon insertion related to a reduction of their niche breadth. Future work documenting the prevalence of these hallmarks across diverse fermented food systems and geographic regions will be key to unveiling the joint history of humanity with fermented food microbes., (© 2024. The Author(s).)

Metamaterials show elaborate mechanical behavior such as strain stiffening, which stems from their unit cell design. However, the stiffening response is typically programmed in the design step and cannot be adapted postmanufacturing. Here, we show hydrogel metamaterials with highly programmable strain-stiffening responses by exploiting the out-of-plane buckling of integrated pH-switchable hydrogel actuators. The stiffening upon reaching a certain extension stems from the initially buckled active hydrogel beams. At low strain, the beams do not contribute to the mechanical response under tension until they straighten with a resulting step-function increase in stiffness. In the hydrogel actuator design, the acrylic acid concentration hard-codes the configuration of the metamaterial and range of possible stiffening onsets, while the pH soft-codes the exact stiffening onset point after fabrication. The utilization of out-of-plane buckling to realize subsequent stiffening without the need to deform the passive structure extends the application of hydrogel actuators in mechanical metamaterials. Our concept of out-of-plane buckled active elements that stiffen only under tension enables strain-stiffening metamaterials with high programmability before and after fabrication.

Background: The "FIFA 11+" is an injury prevention program conceived for soccer athletes aged over 14. The use of FIFA 11+ Kids in soccer was associated with a reduction of the overall risk of injuries in children by 48%, and of 74% for serious injuries. However, to the best of our knowledge, a systematic review of the literature on the effects of FIFA 11+ Kids is still missing. Therefore, a systematic review was conducted to ascertain the benefits of the "FIFA 11+ KIDS" program in children who practice soccer., Methods: This systematic review was conducted according to the PRISMA recommendations and prospectively registered in PROSPERO. The electronic search was conducted in the following databases: Web of Science, PubMed, Medline via Ovid, EMBASE and SportDiscuss via EBSCO. Database searches were performed in January 2024. This review included studies that evaluated the effects of the "FIFA 11+ KIDS" program. Eligible studies had to describe program implementation and the mean age of the children., Results: A total of 11 articles were included in this systematic review from a pool of 8513 articles screened across various databases. These articles involved over 10,000 young participants from 8 countries, primarily aged 7-14 years, with the majority being soccer athletes. Study quality varied, with four categorized as high, four as good, and three as fair quality. Objectives varied across studies, with four focusing on FIFA 11+ Kids' efficacy in injury prevention, five examining its impact on performance and physical abilities, and two assessing its effects on children's focus and attention skills. Notably, injury prevention studies reported around a 50% reduction in overall injuries and nearly 60% in severe injuries, with a dose-response relationship observed with increased weekly sessions. Significant improvements were noted in physical and functional tests such as the Y balance, jump tests, and various soccer skills, along with positive effects on children's focus and attention, as indicated by 13-18% improvements in Attention Scale for Elementary School Children (ASESC) scores., Conclusion: The FIFA 11+ KIDS injury prevention program appears to be effective in reducing injuries in young football players. This can positively influence player and team overall performance and might support the long-term athlete development of these young athletes. These findings highlight the importance and necessity of injury prevention in young athletes., (© 2024. The Author(s).)

Since the first published genome sequence of Trypanosoma cruzi in 2005, there have been tremendous technological advances in genomics, reverse genetics, and assay development for this elusive pathogen. However, there is still an unmet need for new and better drugs to treat Chagas disease. Here, we introduce a T. cruzi assay strain that is useful for drug research and basic studies of host-pathogen interactions. T. cruzi STIB980 is a strain of discrete typing unit TcI that grows well in culture as axenic epimastigotes or intracellular amastigotes. We evaluated the optimal parameters for genetic transfection and constructed derivatives of T. cruzi STIB980 that express reporter genes for fluorescence- or bioluminescence-based drug efficacy testing, as well as a Cas9-expressing line for CRISPR/Cas9-mediated gene editing. The genome of T. cruzi STIB980 was sequenced by combining short-read Illumina with long-read Oxford Nanopore technologies. The latter served as the primary assembly and the former to correct mistakes. This resulted in a high-quality nuclear haplotype assembly of 28 Mb in 400 contigs, containing 10,043 open-reading frames with a median length of 1077 bp. We believe that T. cruzi STIB980 is a useful addition to the antichagasic toolbox and propose that it can serve as a DTU TcI reference strain for drug efficacy testing.

Programmable chemical circuits inspired by signaling networks in living cells are a promising approach for the development of adaptive and autonomous self-assembling molecular systems and material functions. Progress has been made at the molecular level, but connecting molecular control circuits to self-assembling larger elements such as colloids that enable real-space studies and access to functional materials is sparse and can suffer from kinetic traps, flocculation, or difficult system integration protocols. Herein, we report a toehold-mediated DNA strand displacement reaction network capable of autonomously directing two different microgels into transient and self-regulating co-assemblies. The microgels are functionalized with DNA and become elemental components of the network. The flexibility of the circuit design allows the installation of delay phases or accelerators by chaining additional circuit modules upstream or downstream of the core circuit. The design provides an adaptable and robust route to regulate other building blocks for advanced biomimetic functions.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

Background: Long-term cognitive impairment occurs in up to 60% of intensive care unit (ICU) survivors. Early use of functional and cognitive rehabilitation interventions, while patients are still in ICU, may reduce cognitive decline. We aim to describe the functional and cognitive interventions used during the ICU stay, the healthcare professionals providing interventions, and the potential impact on functional and cognitive rehabilitation., Method: In this integrative systematic review, we will include empirical qualitative, quantitative, mixed- and multiple-methods studies assessing the use of functional and cognitive rehabilitation provided in ICU. We will identify studies in relevant electronic databases from 2012 to 2022, which will be screened for eligibility by at least two reviewers. Literature reported as narrative reviews and editorials will be excluded. We will assess the impact of interventions evaluating a cognitive and functional function, quality of life, and all-cause mortality at 6-12 months after ICU discharge. The Revised Cochrane risk-of-bias Tool will be used for assessing risk of bias in clinical trials. For observational studies, we will use the National Institutes of Health Quality Assessment tool for Observational Cohort and Cross-Sectional Studies. Furthermore, we will use the critical appraisal skills programme for qualitative studies and the mixed methods appraisal tool for mixed methods studies. We will construct four matrices, including results describing which ICU patients and healthcare professionals were engaged in rehabilitation, which interventions were included in early rehabilitation in ICU, the potential impact on patient outcomes of rehabilitation interventions provided in ICU and a narrative synthesis of themes. A summary of the main results will be reported using modified GRADE methodology., Impact: This integrative review will inform the feasibility randomised clinical trial testing the development of a complex intervention targeting functional and cognitive rehabilitation for patients in ICU., (© 2023 The Authors. Acta Anaesthesiologica Scandinavica published by John Wiley & Sons Ltd on behalf of Acta Anaesthesiologica Scandinavica Foundation.)

The function of the aminotransferase Aat (GenBank Protein WP_159211138) from Pediococcus acidilactici FAM 18098 was studied in vivo . For this purpose, the gene was replaced with an erythromycin resistance gene using the temperature-sensitive Escherichia coli-Pediococcus shuttle plasmid pSET4T_Δ aat . The knockout was verified by PCR and genome sequencing. Subsequently, the differences between the metabolism of the knockout and of the wild-type strain were investigated by determining the free amino acids and organic acids in culture supernatants. It was found that the knockout mutant no longer synthesized 3-phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (HPLA). Additionally, the mutant strain no longer catabolized phenylalanine. Metabolic pathway analysis using the KEGG database indicate that P. acidilactici cannot synthesize α-ketoglutarate that is a predominant amino-group acceptor in many transamination reactions. To study the transfer of the amino group of phenylalanine, the wild-type strain was incubated with [ 15 N] phenylalanine. Mass spectrometry showed that during fermentation, [ 15 N] alanine was formed, indicating that pyruvic acid is an amino group acceptor in P. acidilactici . The present study shows that Aat plays a crucial role in PLA/HPLA biosynthesis and pyruvic acid is an amino acceptor in transamination reactions in P. acidilactici ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wenger, Bär, Portmann, Schmidt, Eugster, Weisskopf and Irmler.)

In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures' preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bettera, Dreier, Schmidt, Gatti, Berthoud and Bachmann.)

Background: Lung ultrasound (LUS) can be used to monitor critically ill patients with COVID-19, but the optimal number of examined lung zones is disputed., Methods: This was a prospective observational study. The objective was to investigate whether concise (6 zones) and extended (12 zones) LUS scoring protocols are clinically equivalent in critically ill ICU subjects with COVID-19. The primary outcome of this study was (statistical) agreement between concise and extended LUS score index evaluated in both supine and prone position. Agreement was determined using correlation coefficients and Bland-Altman plots to detect systematic differences between protocols. Secondary outcomes were difference between LUS score index in supine and prone position using similar methods., Results: We included 130 LUS examinations in 40 subjects (mean age 69.0 ± 8.5y, 75% male). Agreement between concise and extended LUS score index had no clinically relevant constant or proportional bias and limits of agreement were below the smallest detectable change. Across position changes, supine LUS score index was 8% higher than prone LUS score index and had limits above the smallest detectable change, indicating true LUS score index differences between protocols may occur due to the position change itself. Lastly, inter-rater and intra-rater agreement were very good., Conclusions: Concise LUS was equally informative as extended LUS for monitoring critically ill subjects with COVID-19 in supine or prone position. Clinicians can monitor patients undergoing position changes but must be wary that LUS score index alterations may result from the position change itself rather than disease progression or clinical improvement., Competing Interests: The authors have disclosed no conflicts of interest., (Copyright © 2023 by Daedalus Enterprises.)

Purpose: To examine whether lung ultrasound prior to prone positioning can predict the resulting gas-exchange response., Materials and Methods: This is a prospective observational study on critically-ill COVID-19 patients with a pilot and confirmation cohort. Lung ultrasound examinations were performed before prone positioning and gas-exchange parameters were recorded before and after position change., Results: A total of 79 patients, 36 in the pilot cohort and 43 in the confirmation cohort, were included. In the pilot cohort, a moderate correlation between pre-turn lung ultrasound score index (LUSI) and change in PaO2/FiO2 after prone positioning was found. These findings were corroborated and extended upon in the confirmation cohort. The confirmation cohort found that anterior LUSI had the strongest correlation with follow-up time-points 1, 6, 12, and 24 h after prone positioning, with strength of correlation gradually increasing up to 24 h. In a multivariate model anterior aeration loss (odds ratio 0.035; 95%CI 0.003-0.319 for anterior LUSI >50%) and higher pre-turn PaCO 2 (odds ratio 0.479 95% CI 0.235-0.979) were negatively predictive of a PaO2/FiO2 increase ≥20 mmHg., Conclusions: Anterior LUSI, in addition to other clinical parameters, may be used to aid COVID-19 respiratory strategy and a clinician's decision to prone., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Videos capture events that typically contain multiple sequential, and simultaneous, actions even in the span of only a few seconds. However, most large-scale datasets built to train models for action recognition in video only provide a single label per video. Consequently, models can be incorrectly penalized for classifying actions that exist in the videos but are not explicitly labeled and do not learn the full spectrum of information present in each video in training. Towards this goal, we present the Multi-Moments in Time dataset (M-MiT) which includes over two million action labels for over one million three second videos. This multi-label dataset introduces novel challenges on how to train and analyze models for multi-action detection. Here, we present baseline results for multi-action recognition using loss functions adapted for long tail multi-label learning, provide improved methods for visualizing and interpreting models trained for multi-label action detection and show the strength of transferring models trained on M-MiT to smaller datasets.

Background: Phages are key drivers of genomic diversity in bacterial populations as they impose strong selective pressure on the evolution of bacterial defense mechanisms across closely related strains. The pan-immunity model suggests that such diversity is maintained because the effective immune system of a bacterial species is the one distributed across all strains present in the community. However, only few studies have analyzed the distribution of bacterial defense systems at the community-level, mostly focusing on CRISPR and comparing samples from complex environments. Here, we studied 2778 bacterial genomes and 188 metagenomes from cheese-associated communities, which are dominated by a few bacterial taxa and occur in relatively stable environments., Results: We corroborate previous laboratory findings that in cheese-associated communities nearly identical strains contain diverse and highly variable arsenals of innate and adaptive (i.e., CRISPR-Cas) immunity systems suggesting rapid turnover. CRISPR spacer abundance correlated with the abundance of matching target sequences across the metagenomes providing evidence that the identified defense repertoires are functional and under selection. While these characteristics align with the pan-immunity model, the detected CRISPR spacers only covered a subset of the phages previously identified in cheese, providing evidence that CRISPR does not enable complete immunity against all phages, and that the innate immune mechanisms may have complementary roles., Conclusions: Our findings show that the evolution of bacterial defense mechanisms is a highly dynamic process and highlight that experimentally tractable, low complexity communities such as those found in cheese, can help to understand ecological and molecular processes underlying phage-defense system relationships. These findings can have implications for the design of robust synthetic communities used in biotechnology and the food industry. Video Abstract., (© 2022. The Author(s).)

Purpose: Critically ill COVID-19 patients have an increased risk of developing pulmonary embolism (PE). Diagnosis of PE by point-of-care ultrasound (POCUS) might reduce the need for computed tomography pulmonary angiography (CTPA), while decreasing time-to-diagnosis., Materials & Methods: This prospective, observational study included adult ICU patients with COVID-19. Multi-organ (lungs, deep vein, cardiac) POCUS was performed within 24 h of CTPA, looking for subpleural consolidations, deep venous thrombosis (DVT), and right ventricular strain (RVS). We reported the scan time, and calculated diagnostic accuracy measures for these signs separately and in combination., Results: 70 consecutive patients were included. 23 patients (32.8%) had a PE. Median scan time was 14 min (IQR 11-17). Subpleural consolidations' diagnostic accuracy was: 42.9% (95%CI [34.1-52.0]). DVT's and RVS' diagnostic accuracy was: 75.6% (95%CI [67.1-82.9]) and 74.4% (95%CI [65.8-81.8]). Their sensitivity was: 24.0% (95%CI [9.4-45.1]), and 40.0% (95%CI [21.3-61.3]), while their specificity was: 88.8% (95%CI [80.8-94.3]), and: 83.0% (95%CI [74.2-89.8]), respectively. Multi-organ POCUS sensitivity was: 87.5% (95%CI [67.6-97.3]), and specificity was: 25% (95%CI [16.9-34.7])., Conclusions: Multi-organ rather than single-organ POCUS can be of aid in ruling out PE in critically ill COVID-19 and help select patients for CTPA. In addition, finding RVS can make PE more likely, while a DVT would preclude the need for a CTPA., Registration: www.trialregister.nl: NL8540., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest nor any financial disclosures., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this article, a novel approach for selective passivation of three-dimensional pyrolytic carbon microelectrodes via a facile electrochemical polymerization of a non-conductive polymer (polydopamine, PDA) onto the surface of carbon electrodes, followed by a selective laser ablation is elaborated. The 3D carbon electrodes consisting of 284 micropillars on a circular 2D carbon base layer were fabricated by pyrolysis of lithographically patterned negative photoresist SU-8. As a second step, dopamine was electropolymerized onto the electrode by cyclic voltammetry (CV) to provide an insulating layer at its surface. The CV parameters, such as the scan rate and the number of cycles, were investigated and optimized to achieve a reliable and uniform non-conductive coating on the surface of the 3D pyrolytic carbon electrode. Finally, the polydopamine was selectively removed only from the tips of the pillars, by using localized laser ablation. The selectively passivated electrodes were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy methods. Due to the surface being composed of highly biocompatible materials, such as pyrolytic carbon and polydopamine, these 3D electrodes are particularly suited for biological application, such as electrochemical monitoring of cells or retinal implants, where highly localized electrical stimulation of nerve cells is beneficial.

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics., (© 2021. The Author(s).)

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis ( LDL ) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either 'sensitive' or 'resistant' based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL , we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains' genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing.

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure-activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in-depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure-activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long-time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12'000 mutants showed no signs of resistance development to the synthetic derivatives., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

DNA mechanosensors offer unique properties for mechano-adaptive and self-reporting materials, such as programmable bond strength and geometrical strain response, tunable fluorescent strain sensing, interfacing to biological systems, and the ability to store mechanical information. However, the facile incorporation of advanced DNA motifs into polymer networks and achieving robustness in application settings remain difficult. Herein, we introduce one-component DNA mechanoprobes that can be easily polymerized into polymer hydrogels and even elastomers to allow strain-induced fluorescence sensing. The all-in-one mechanoprobe contains a DNA hairpin for programmable force sensing, an internal fluorophore-quencher pair as a reporter, and methacrylamide groups on both ends for rapid and facile photopolymerization into networks based on the nontoxic water-soluble monomer methoxy triethylene glycol acrylate (mTEGA). In addition to mechanosensing hydrogels, we utilize the low T g of p(mTEGA) to develop the first bulk elastomer materials with DNA force sensors, which show high elasticity and stronger mechanofluorescence. The system makes decisive steps forward for DNA-based mechanoprobes by overcoming the classical multicomponent design of such probes, allowing photopolymerization useful for the design of complex objects or even 3D printing and demonstrating that such motifs may even be useful in dry bulk materials.

This paper demonstrates an improved method to accurately extract the surface morphology of black silicon (BSi). The method is based on an automated Xe + plasma focused ion beam (PFIB) and scanning electron microscope (SEM) tomography technique. A comprehensive new sample preparation method is described and shown to minimize the PFIB artifacts induced by both the top surface sample-PFIB interaction and the non-uniform material density. An optimized post-image processing procedure is also described that ensures the accuracy of the reconstructed 3D surface model. The application of these new methods is demonstrated by applying them to extract the surface topography of BSi formed by reactive ion etching (RIE) consisting of 2 µm tall needles. An area of 320 µm 2 is investigated with a controlled slice thickness of 10 nm. The reconstructed 3D model allows the extraction of critical roughness characteristics, such as height distribution, correlation length, and surface enhancement ratio. Furthermore, it is demonstrated that the particular surface studied contains regions in which under-etching has resulted in overhanging structures, which would not have been identified with other surface topography techniques. Such overhanging structures can be present in a broad range of BSi surfaces, including BSi surfaces formed by RIE and metal catalyst chemical etching (MCCE). Without proper measurement, the un-detected overhangs would result in the underestimation of many critical surface characteristics, such as absolute surface area, electrochemical reactivity and light-trapping., (Copyright © 2020 Elsevier B.V. All rights reserved.)

BACKGROUND Primary ciliary dyskinesia (PCD) is a disease characterized by motor ciliary dysfunction, which leads to the accumulation of secretions in the lower airways and, consequently, to atelectasis and repeated infections. During the neonatal period, diagnosis can be difficult because the symptoms are frequently associated with other respiratory diseases common in neonates. The laterality defects should warn the clinician of the need for further investigation using clinical criteria, but the confirmation depends on a genetic test. CASE REPORT The objective of this report is to present a case of PCD manifesting in the neonatal period that was diagnosed due to respiratory failure associated with recurrent atelectasis and situs inversus totalis. CONCLUSIONS This disease is not well known by neonatologists, but early diagnosis decreases morbidity and improves patient quality of life.

During cheese ripening, the bacterial strain Pediococcus acidilactici FAM18098 produces the non-proteinogenic amino acid, α-aminobutyrate (AABA). The metabolic processes that lead to the biosynthesis of this compound are unknown. In this study, 10 P. acidilactici, including FAM18098 and nine Pediococcus pentosaceus strains, were screened for their ability to produce AABA. All P. acidilactici strains produced AABA, whereas the P. pentosaceus strains did not. The genomes of the pediococcal strains were sequenced and searched for genes encoding aminotransferases to test the hypothesis that AABA could result from the transamination of α-ketobutyrate. A GenBank and KEGG database search revealed the presence of a species-specific aminotransferase in P. acidilactici. The gene was cloned and its gene product was produced as a His-tagged fusion protein in Escherichia coli to determine the substrate specificity of this enzyme. The purified recombinant protein showed aminotransferase activity at pH 5.5. It catalyzed the transfer of the amino group from leucine, methionine, AABA, alanine, cysteine, and phenylalanine to the amino group acceptor α-ketoglutarate. Αlpha-ketobutyrate could replace α-ketoglutarate as an amino group acceptor. In this case, AABA was produced at significantly higher levels than glutamate. The results of this study show that P. acidilactici possesses a novel aminotransferase that might play a role in cheese biochemistry and has the potential to be used in biotechnological processes for the production of AABA.

Fascioliasis is a zoonotic infection of humans and, more commonly, ruminants. It is caused by 2 liver fluke species, Fasciola hepatica and Fasciola gigantica, which differ in size. The traditional morphological methods used to distinguish the 2 species can be unreliable, particularly in the presence of hybrids between the 2 species. The development of advanced molecular methods has allowed for more definitive identification of Fasciola species, including their hybrids. Hybrids are of concern, as it is thought that they could acquire advantageous traits such as increased pathogenicity and host range. In 2013, we collected flukes from Fasciola-positive cattle, sheep, and goats slaughtered in 4 Chadian abattoirs. DNA from 27 flukes was extracted, amplified, and analyzed to identify species using the ITS1+2 locus. Twenty-six of the 27 flukes were identified as F. gigantica, while the remaining fluke showed heterozygosity at all variable sites that distinguish F. hepatica and F. gigantica. Cloning and sequencing of both alleles confirmed the presence of 1 F. hepatica and 1 F. gigantica allele. To our knowledge, this is the first unambiguous, molecular demonstration of the presence of such a hybrid in a bovine in sub-Saharan Africa., (© American Society of Parasitologists 2020.)

Background: Vestibular schwannoma (VS) is the most common benign tumor originating in the cerebellopontine angle. In most cases, tumors tend to grow and deserve proper treatment. Sometimes they stabilize, and rarely they decrease in size spontaneously., Methods: We evaluated retrospectively the images of patients with spontaneous tumor regression. We describe the common neuroimage findings of patients with spontaneous tumoral regression., Results: Four patients with diagnosis of VS were followed with magnetic resonance imaging (MRI). There were some relevant features in MRI: a heterogeneous contrast enhancement in the outer layer of the tumor and presence of a cerebrospinal fluid column between the tumor and the entrance of the internal auditory canal. The percentage of tumor diameter reduction ranged from 20% to 40%., Conclusions: Some MRI features may demonstrate a spontaneous involution of VS and may be closely followed in asymptomatic or oligosymptomatic patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Antimalarial peroxides such as the phytochemical artemisinin or the synthetic ozonides arterolane and artefenomel undergo reductive cleavage of the pharmacophoric peroxide bond by ferrous heme, released by parasite hemoglobin digestion. The generated carbon-centered radicals alkylate heme in an intramolecular reaction and proteins in an intermolecular reaction. Here, we determine the proteinaceous alkylation signatures of artemisinin and synthetic ozonides in Plasmodium falciparum using alkyne click chemistry probes to identify target proteins by affinity purification and mass spectrometry-based proteomics. Using stringent controls and purification procedures, we identified 25 P. falciparum proteins that were alkylated by the antimalarial peroxides in a peroxide-dependent manner, but the alkylation patterns were more random than we had anticipated. Moreover, there was little overlap in the alkylation signatures identified in this work and those disclosed in previous studies. Our findings suggest that alkylation of parasite proteins by antimalarial peroxides is likely to be a nonspecific, stochastic process.

Introduction: Chordoma is a malignant tumor that usually involves the axial skeleton. Intradural chordomas are even rarer and 37 cases have been reported to the best of our knowledge. We present a case of a patient with an atypical metastatic diffuse intradural spinal involvement., Case Description: We present a 33-year-old woman previously submitted to five brain surgeries to treat a posterior fossa intradural chordoma since December 2012. Currently, she presented almost with right and left hemiplegia (grade 2 bilaterally) and also left oculomotor, adbucent and facial nerve paresis. We performed neuroaxis magnetic resonance MR which disclosed stability of posterior fossa tumor which was previously irradiated. However, there were new intradural lesions at the level of C3, T11/T12 and L4/L5/S1 vertebrae., Discussion: With the advent of contemponaeous surgery, radiotherapy options and even available chemotherapy to treat Chordomas (Imatinib), patients may experience enlarged survival and thus face complications such as drop metastases along neuroaxis. Our case illustrates a late (6 years) follow-up presentation of an initial posterior fossa intradural chordoma. It suggests that whole neuraxis involvement may be the final presentation of all patients harbouring chordomas and surviving after adequate initial treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cleft lip and palate is a congenital malformation that requires a multidisciplinary treatment that evolves pediatrician, obstetrics, fetal medicine, genetics, plastic surgery, orthodontics, speech therapist, nursery, and psychology. Actually, the authors believe that it could be possible to ad protocols to use stem cells.The intrauterine diagnosis leads to preborn parental orientation and better parental collaboration to accept a precocious multidisciplinary treatment. After birth the authors' protocol is: orthodontic devices, phonoaudiology, and surgical procedures.The authors' cleft lip and palate reconstructive surgery protocol demands several steps and begins at 4 to 6-month old with rhinocheiloplasty and soft palate closure at the same moment. The treatment sequence involves the hard palate surgery (8-18 months after the first surgical step), alveoloplasty (after 10 years old), and secondary rhinoplasty (after 14 years old).New ideas to use stem cells and blood from the umbilical cord and also blood from placenta are discussed to improve final surgical results. Maternal stem cells are easy to collect, there are no damage to the patient and mother, it is autologous and it could be very useful in the authors' protocol.Nine patients with clef lip and palate were operated and had stem cells from umbilical cord blood and placenta blood injected into the bone and soft tissue during the primary procedure (rhinocheiloplasty).The stem cells activity into soft tissue and bone were evaluated. Preliminary results have shown no adverse results and improvement at the inflammatory response. A treatment protocol with stem cells was developed. It had a long time follow-up of 10 years.

One in four asylum applicants in Europe are children, and 23% of whom are unaccompanied and may be at increased risk of mental illness. This study contributes to the limited evidence base by comparing the incidence of psychiatric disorders among unaccompanied and accompanied refugee children. We linked a cohort of refugee children who obtained right of residency in Denmark between 01 January 1993 and 31 December 2010 to the Danish Psychiatric Central Register, and calculated incidence rates per 100,000 person years and incidence rate ratios of overall psychiatric disorder, psychotic disorders, affective disorders, and neurotic disorders for accompanied and unaccompanied minors using Poisson regression. We adjusted the analyses for sex, age at residency, and age at arrival (aIRR). Stratified analyses were conducted by nationality. Unaccompanied minors had significantly higher rates of any psychiatric disorder (aIRR: 1.38, 95% CI 1.14-1.68) and neurotic disorders (aIRR: 1.67, 95% CI 1.32-2.13) than accompanied minors. Among children from Afghanistan, unaccompanied minors had significantly higher rates of any psychiatric disorder (aIRR: 2.23, 95% CI 1.26-3.93) and neurotic disorders (aIRR: 3.50, 95% CI 1.72-7.11). Among children from Iraq, unaccompanied minors had higher rates of any psychiatric disorder (aIRR: 2.02, 95% CI 1.18-3.45), affective disorders (aIRR: 6.04, 95% CI 2.17-16.8), and neurotic disorders (aIRR: 3.04, 95% CI 1.62-5.70). Unaccompanied children were found to experience a higher incidence of any psychiatric disorder and neurotic disorders. Strategies are needed to address the specific mental health and social needs of unaccompanied minors.

Trypanosoma brucei comprise the causative agents of sleeping sickness, T. b. gambiense and T. b. rhodesiense, as well as the livestock-pathogenic T. b. brucei. The parasites are transmitted by the tsetse fly and occur exclusively in sub-Saharan Africa. T. brucei are not only lethal pathogens but have also become model organisms for molecular parasitology. We focus here on membrane transport proteins of T. brucei, their contribution to homeostasis and metabolism in the context of a parasitic lifestyle, and their pharmacological role as potential drug targets or routes of drug entry. Transporters and channels in the plasma membrane are attractive drug targets as they are accessible from the outside. Alternatively, they can be exploited to selectively deliver harmful substances into the trypanosome's interior. Both approaches require the targeted transporter to be essential: in the first case to kill the trypanosome, in the second case to prevent drug resistance due to loss of the transporter. By combining functional and phylogenetic analyses, we were mining the T. brucei predicted proteome for transporters of pharmacological significance. Here, we review recent progress in the identification of transporters of lipid precursors, amino acid permeases and ion channels in T. brucei., (© 2017 Federation of European Biochemical Societies.)

A 4-year-old girl was admitted to the emergency department after having been buried beneath a wall. A computed tomography scan revealed anterior grade V L5-S1 spondylolisthesis, and magnetic resonance imaging showed a traumatic rupture of the fibrous annulus of the L5-S1 intervertebral disc and lesion of the anterior longitudinal and yellow ligaments. The patient underwent anterior and posterior fixation. Four months later she was able to walk independently, despite a persistent left foot drop. Additionally, we conducted a literature review on lumbosacral spondyloptosis in the pediatric population published between 1990 and 2017. We found 16 cases, 86.6% of which were male, with a mean patient age of 16 ± 5.05 years. Most patients underwent spine instrumentation. Based on the data reviewed, the neurological status at admission might be a valid predictor of outcome. Pedicle screws are a safe and reliable procedure for stable fixation of the spine in these cases. The removal of screws is discouraged., (© 2018 S. Karger AG, Basel.)

Suramin is one of the first drugs developed in a medicinal chemistry program (Bayer, 1916), and it is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense. Cellular uptake of suramin occurs by endocytosis, and reverse genetic studies with T. b. brucei have linked downregulation of the endocytic pathway to suramin resistance. Here we show that forward selection for suramin resistance in T. brucei spp. cultures is fast, highly reproducible and linked to antigenic variation. Bloodstream-form trypanosomes are covered by a dense coat of variant surface glycoprotein (VSG), which protects them from their mammalian hosts' immune defenses. Each T. brucei genome contains over 2000 different VSG genes, but only one is expressed at a time. An expression switch to one particular VSG, termed VSG Sur , correlated with suramin resistance. Reintroduction of the originally expressed VSG gene in resistant T. brucei restored suramin susceptibility. This is the first report of a link between antigenic variation and drug resistance in African trypanosomes., (© 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.

Being the principal causative agent of bacterial pneumonia, otitis media, meningitis and septicemia, the bacterium Streptococcus pneumoniae is a major global health problem. To highlight the molecular basis of this problem, we have portrayed essential biological processes of the pneumococcal life cycle in eight watercolor paintings. The paintings are done to a consistent nanometer scale based on currently available data from structural biology and proteomics. In this review article, the paintings are used to provide a visual review of protein synthesis, carbohydrate metabolism, cell wall synthesis, cell division, teichoic acid synthesis, virulence, transformation and pilus synthesis based on the available scientific literature within the field of pneumococcal biology. Visualization of the molecular details of these processes reveals several scientific questions about how molecular components of the pneumococcal cell are organized to allow biological function to take place. By the presentation of this visual review, we intend to stimulate scientific discussion, aid in the generation of scientific hypotheses and increase public awareness. A narrated video describing the biological processes in the context of a whole-cell illustration accompany this article., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Reconstructive surgery to craniofacial deformities caused by tumor ressections, traumas or congenital malformation are frequent in medicine practice. It aims to provide the patients with better quality of life and functional improvement of speech, breathing, chewing, and swallowing. Many are the techniques described in the literature to recover bone defects. This study evaluated a vascularized galeal and periosteum flap in rabbits, which could possibly substitute the bone graft in reconstructive surgery, especially for facial defects. It involved rabbits, divided into 12 groups, submitted to a surgical procedure to construct the galea and periosteum cranial flap filled with fragments of cranial bone, platelet-rich plasma, mesenchimal stem cells, and hyaluronic acid. The evaluation methods included image examinations and histological analysis.The results demonstrated bone formation with the use of platelet-rich plasma, mesenchimal stem cells, and bone fragments. The use of several enrichment materials of osseous cellular stimulation improved the quality and bone tissue organization. The more enrichment factor used, the better the tissue quality result was.Much research should be done to improve the methods and to analyze if results in human have the same bone formation as it happened in rabbits.

Purpose: To evaluate three different kinds of neurorrhaphy of the peroneal nerve., Methods: Eigthy rats were divided into 5 groups. Control: nerve had no intervention. End-to-end (EE): nerve was cut and elongated with a nerve graft with two end-to-end neurorrhaphies. End-to-side (ES): nerve was cut and sutured to the graft with at the lateral side of the nerve. Side-to-end (SE): the nerve was cut and sutured to the graft with end-to-end neurorrhaphy. Denervated: nerve was cut and both endings were buried into the muscle. The evaluation was done by walking track analysis, electrophysiology, body mass, cranial tibial muscle mass, nerve and muscle fibers morphometry., Results: The EE, ES and SE have the same potential of reinnervation., Conclusion: There is no functional or histological difference between these different types of neurorrhaphy.

Introduction: Chiari malformation type I (CM) is the main congenital malformation disease of the craniovertebral junction. The ideal surgical treatment is still controversial. Invasive procedures inside the cerebrospinal fluid (CSF) space and associated with dural repair are considered the gold standard; however, less invasive surgery with isolated bone decompression without dural opening may be possible in selected patients. Our study evaluates the efficacy of intraoperative CSF flow measurement with ultrasonography (USG) as a determining parameter in the selection of these patients., Methods: We analyzed prospectively 49 patients with CM operated on at the Hospital das Clínicas, College of Medicine, University of São Paulo. Patients underwent decompressive surgery with or without opening of the dura mater after intraoperative USG measuring flow rate. A value of 3 cm/second was considered a cutoff. Quality of life before and after surgery and the improvement of neck pain and headache were evaluated., Results: Among 49 patients enrolled, 36 patients (73%) had CSF flow >3 cm/second and did not undergo duraplasty. In 13 patients (27%) with initial flow <3 cm/second, dural opening was performed together with duraplasty. All patients improved when preoperative and postoperative scores were compared, and all clinical parameters evaluated did not differ between both surgical groups. Patients submitted to bone decompression alone had a lower complication rate., Conclusions: Intraoperative USG with measurement of CSF allows the proper selection of patients with CM for less invasive surgery with bone decompression without duraplasty., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Ofatumumab is a humanized anti-CD20 monoclonal antibody that has recently garnered interest as a potential therapeutic agent for nephrotic syndrome. We report our center's experience in administering ofatumumab to five pediatric patients with idiopathic nephrotic syndrome., Methods: Between March 2015 and November 2016, five patients were treated with ofatumumab. One patient had post-transplant recurrent focal segmental glomerulosclerosis (FSGS) which had been resistant to plasmapheresis and numerous immunosuppressive agents. Four patients had nephrotic syndrome in their native kidneys, one with initial steroid-resistant disease and the others with subsequent development of steroid resistance. Two of the patients were treated with a desensitization protocol after experiencing hypersensitivity reactions to ofatumumab., Results: One patient did not complete ofatumumab treatment due to infusion reactions. Of the four remaining patients, three achieved complete remission after treatment, and one achieved partial remission. One of the patients achieving complete remission represents the first reported case of successful treatment of post-transplant recurrent FSGS using ofatumumab. Two patients who received ofatumumab with our desensitization protocol were able to complete their treatments after initially experiencing hypersensitivity reactions., Conclusions: Ofatumumab may be an effective treatment for refractory childhood nephrotic syndrome and post-transplant recurrent FSGS. A desensitization protocol may be helpful to address hypersensitivity reactions.

Methods: Thirty cases, previously treated according to the new algorithm, were presented to four spine surgeons who were questioned about their personal suggestion for treatment, and the treatment suggested according to the application of the algorithm. After four weeks, the same questions were asked again to evaluate reliability (intra- and inter-observer) using the Kappa index., Results: The reliability of the treatment suggested by applying the algorithm was superior to the reliability of the surgeons' personal suggestion for treatment. When applying the upper cervical spine injury treatment algorithm, an agreement with the treatment actually performed was obtained in more than 89% of the cases., Conclusion: The system is safe and reliable for treating traumatic upper cervical spine injuries. The algorithm can be used to help surgeons in the decision between conservative versus surgical treatment of these injuries.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

Introduction: Crows' feet wrinkles are caused by the action of the orbicularis oculi muscle on overlying skin. Treatment options range from botulinum toxin to a multitude of surgical methods. We first described our technique in 2003 and refined it in 2006. We highlight the evolution of our technique and look at our results to assess the effectiveness of our technique., Objective: The aim of this article is to show our experience of 13 years in 134 patients with our technique of orbicularis oculi myectomy., Methods: From September 2000 to July 2013, we operated on 134 patients with an age range of 28-77 years. Of these patients, 104 had myectomies via lifting and 22 via a blepharoplasty approach. Five patients had myectomies for treatment of blepharospasm and a further three patients to restore symmetry in facial palsy. An evaluation of the results was performed by two medical students., Results: Our results showed reduction of the wrinkles in all cases. For the statistical analysis the Wilcoxon test was performed. The p value was less than 0.001 showing a significant reduction of crows' feet wrinkles in both sides when the orbicularis myectomy was performed, via blepharoplasty or lifting. For myectomy performed for blepharospasm or facial palsy, the statistical analysis was not done due to the low numbers., Conclusion: In the appropriate patient, orbicularis oculi myectomy is an effective and long-term treatment for crows' feet wrinkles with a low risk of complications and high patient satisfaction., Level of Evidence Iv: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Unlabelled: Paraclinoid aneurysms are lesions located adjacent to the clinoid and ophthalmic segments of the internal carotid artery. In recent years, flow diverter stents have been introduced as a better endovascular technique for treatment of these aneurysms., Method: From 2009 to 2014, a total of 43 paraclinoid aneurysms in 43 patients were surgically clipped. We retrospectively reviewed the records of these patients to analyze clinical outcomes., Results: Twenty-six aneurysms (60.5%) were ophthalmic artery aneurysms, while 17 were superior hypophyseal artery aneurysms (39.5%). The extradural approach to the clinoid process was used to clip these aneurysms. One hundred percent of aneurysms were clipped (complete exclusion in 100% on follow-up angiography). The length of follow-up ranged from 1 to 60 months (mean, 29.82 months)., Conclusion: Surgical clipping continues to be a good option for the treatment of paraclinoid aneurysms.