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ObjectiveThe anti-α4β7 integrin antibody vedolizumab is administered at a fixed dose for the treatment of IBDs. This leads to a wide range of serum concentrations in patients and previous studies had suggested that highest exposure levels are associated with suboptimal clinical response. We aimed to determine the mechanisms underlying these non-linear exposure-efficacy characteristics of vedolizumab.DesignWe characterised over 500 samples from more than 300 subjects. We studied the binding of vedolizumab to T cells and investigated the functional consequences for dynamic adhesion, transmigration, gut homing and free binding sites in vivo. Employing single-cell RNA sequencing, we characterised α4β7 integrin-expressing T cell populations ‘resistant’ to vedolizumab and validated our findings in vitro and in samples from vedolizumab-treated patients with IBD. We also correlated our findings with a post-hoc analysis of the Gemini II and III studies.ResultsRegulatory T (TReg) cells exhibited a right-shifted vedolizumab binding profile compared with effector T (TEff) cells. Consistently, in a certain concentration range, the residual adhesion, transmigration, homing of and availability of functional α4β7 on TReg cells in vivo was higher than that of/on TEff cells. We identified a vedolizumab-‘resistant’ α4β7-expressing β1+PI16+ TReg cell subset with pronounced regulatory properties as the substrate for this effect. Our observations correlated with exposure-efficacy data from Gemini II and III trials.ConclusionCompletely blocking TEff cell trafficking with vedolizumab, while simultaneously permitting residual homing of powerful TReg cells in an optimal ‘therapeutic window’ based on target exposure levels might be a strategy to optimise treatment outcomes in patients with IBD.

Lophodermium piceae is the most common endophyte of Norway spruce (Picea abies) needles, and it probably occurs in the total distribution area of Norway spruce. Its significance to the host tree is still largely unknown mainly because no methodology is known for generating endophyte-free saplings nor for inoculation of intact needles with this fungus. We report here how a daily heat treatment at 40 °C for 8 h eradicates endophytes of Norway spruce needles within a few days without causing visible harm to the plants. We compared various methods for inoculation of endophyte-free saplings. In order to obtain effective inocula, we investigated what factors activate dormant L. piceae infections in the needle tissue resulting in ascomata formation. Best inoculation success was achieved by suspending naturally shed, highly infected needles above the saplings. Infection frequencies of up to 70% of needles were achieved with repeated inoculations in the course of 3 years. Once established in needles, individual infections by L. piceae can persist for at least 5 years. Individual infections remain small in young needles, but at a needle age of 6 years, they start to grow and spread resulting in presence all over the needle from base to tip at a needle age of 7 years, but still without causing visible symptoms on the needles. The presented methodology for generating both endophyte-free and endophyte-infected saplings of Norway spruce provides a new approach for testing the significance of infections by L. piceae to insect herbivory and diseases of Norway spruce needles.

The reduction of antibiotic use in food producing animals becomes increasingly important. Therefore, suitable alternatives for mastitis treatment in dairy cows have to be considered. Oxytocin (OT) induces milk ejection and hence supports milk removal from infected mammary quarters. Beyond udder emptying, the injection of very high dosages of OT causes increased somatic cell counts (SCC) in milk and enables the transfer of immunoglobulins (Ig) from blood into milk through a reduced blood-milk barrier integrity. The aim of the present study was to investigate pathogen-specific changes of SCC, the blood derived milk components lactate dehydrogenase (LDH), serum albumin (SA), and IgG in milk of cows suffering from mastitis caused by different pathogens treated with two intravenous injections of high dosages of OT (100 IU). Milk samples from 184 dairy cows from different farms were collected on day 1 (day of clinical examination and mastitis diagnosis) and on days 2, 3, 14, and 28. Bacteriological examination (day 1) identified involved pathogens. Cows were randomly assigned to treatment (OT injections on days 1 and 2) or control group (no OT). Independently of the assigned experimental group, cows received the common therapy protocol of the veterinary practice after sample collection if the general condition was affected. Milk SCC, LDH, SA, and IgG changed specifically depending on involved pathogens. Highest values of all three parameters were measured in mastitis caused by Streptococcus uberis. Changes were less pronounced with other Streptococci spp., Staphylococci spp. or Corynebacterium bovis. Oxytocin treatment did not affect any of the studied parameters independent of the involved pathogen. Only in quarters infected with Staphylococci other than Staphylococcus aureus a decreased SCC and increased IgG concentrations in quarters, where no pathogens were detected, were observed. Thus, high dosage OT administration is obviously not suitable as a stand-alone mastitis treatment in dairy cows.Die Reduzierung des Antibiotikakonsums bei Tieren in der Lebensmittelproduktion wird immer wichtiger. Daher müssen geeignete Alternativen zur Mastitisbehandlung bei Milchkühen geprüft werden. Oxytocin (OT) induziert das Einschiessen der Milch in das Milchgangsystem und unterstützt somit das Ausmelken der Milch aus infizierten Vierteln der Milchdrüse. Über die Euterentleerung hinaus führt die Injektion sehr hoher OT-Dosierungen durch eine verringerte Integrität der Blut-Milch-Schranke zu einer erhöhten Anzahl somatischer Zellen (SCC) in der Milch und ermöglicht den Transfer von Immunglobulinen (Ig) aus der Blutbahn in die Milch. Das Ziel der vorliegenden Studie war es, bei an Mastitis erkrankten und mit zwei hohen Dosen Oxytocin (100 IE iv.) behandelten Kühen, in Abhängigkeit des Pathogens die Veränderungen von SCC, den aus Blut stammenden Milchkomponenten Lactatdehydrogenase (LDH), Serumalbumin (SA) und IgG in Milch zu untersuchen. Milchproben von 184 Milchkühen aus verschiedenen Betrieben wurden am Tag 1 (Tag der klinischen Untersuchung und Mastitisdiagnose) und an den Tagen 2, 3, 14 und 28 entnommen. Der Nachweis der Krankheitserreger erfolgte mittels einer bakteriologischen Untersuchung (Tag 1). Die Kühe wurden zufällig der Behandlungs- (OT-Injektionen an den Tagen 1 und 2) oder der Kontrollgruppe (kein OT) zugeordnet. Unabhängig von der zugewiesenen Versuchsgruppe wurden die Kühe mit reduziertem Allgemeinzustand nach der Probenentnahme gemäss dem Therapieprotokoll der Tierarztpraxis behandelt. Milch-SCC, LDH, SA und IgG änderten sich spezifisch in Abhängigkeit der beteiligten Pathogene. Die höchsten Werte aller drei Parameter wurden bei durch Streptococcus uberis verursachten Mastitiden gemessen. Die Veränderungen waren bei Streptococci spp., Staphylococci spp. oder Corynebacterium bovis weniger ausgeprägt. Unabhängig vom verursachenden Pathogen hatte die Oxytocin-Behandlung keinen Einfluss auf einen der untersuchten Parameter. Einzig bei Mastitiden verursacht durch andere Staphylokokken als Staphylococcus aureus wurden eine verminderte SCC gemessen und bei Mastitiden ohne Nachweis eines Pathogens konnte eine erhöhte IgG-Konzentration beobachtet werden. Daher ist eine hochdosierte OT-Verabreichung nicht als eigenständige Mastitisbehandlung bei Milchkühen geeignet.La réduction de l’utilisation d’antibiotiques chez les animaux destinés à l’alimentation devient de plus en plus importante. Par conséquent, des alternatives appropriées au traitement des mammites chez les vaches laitières doivent être envisagées. L’ocytocine (OT) induit l’éjection du lait et favorise donc l’élimination du lait des quartiers infectés. Au-delà de la vidange de la mamelle, l’injection de doses très élevées d’OT entraîne une augmentation du nombre de cellules somatiques (CSC) dans le lait et permet le transfert d’immunoglobulines (Ig) du sang vers le lait grâce à une réduction de l’intégrité de la barrière sang-lait. Le but de la présente étude était d’étudier les changements spécifiques aux agents pathogènes du CSC, les composants du lait dérivés du sang que sont la lactate déshydrogénase (LDH) et l’albumine sérique (SA) ainsi que les IgG dans le lait de vaches souffrant de mammites causées par différents agents pathogènes traités par deux injections intraveineuses de doses élevées d’OT (100 UI). Des échantillons de lait de 184 vaches laitières de différentes exploitations ont été prélevés au jour 1 (jour de l’examen clinique et diagnostic de mammite) et aux jours 2, 3, 14 et 28. L’examen bactériologique (jour 1) a identifié les agents pathogènes impliqués. Les vaches ont été assignées au hasard au traitement (injections d’OT les jours 1 et 2) ou au groupe témoin (pas d’OT). Indépendamment du groupe auquel elles étaient attribuées, les vaches ont reçu le protocole thérapeutique usuel du cabinet vétérinaire après le prélèvement de l’échantillon si leur état général était affecté. Le CSC, la LDH, la SA et les IgG du lait ont varié spécifiquement en fonction des agents pathogènes impliqués. Les valeurs les plus élevées des trois paramètres ont été mesurées dans les mammites causées par Streptococcus uberis. Les changements étaient moins prononcés avec d’autres Streptococci spp., Staphylococci spp. ou Corynebacterium bovis. Le traitement à l’ocytocine n’a affecté aucun des paramètres étudiés indépendamment de l’agent pathogène impliqué. On a uniquement observé, dans les mammites causées par des staphylocoques autres que Staphylococcus aureus, une diminution du CSC et, dans les mammites où aucun agent pathogène n’a été détecté, une augmentation des concentrations d’IgG dans les quartiers. Ainsi, l’administration d’OT à forte dose n’est pas appropriée comme traitement unique des mammites chez les vaches laitières.La riduzione dell’uso di antibiotici negli animali per la produzione alimentare sta assumendo un’importanza sempre maggiore. Di conseguenza, è opportuno considerare delle alternative adeguate per il trattamento della mastite nelle vacche da latte. L’ossitocina (OT) induce l’eiezione del latte e quindi aiuta la rimozione di questo dai quarti mammari infetti. L’iniezione di dosi molto elevate di OT provoca non solo lo svuotamento della mammella, ma pure un aumento del numero di cellule somatiche (SCC) nel latte permettendo quindi il trasferimento di immunoglobuline (Ig) dal sangue al latte attraverso una ridotta integrità della barriera tra sangue e latte. Lo scopo del presente studio era di esaminare i cambiamenti patogeno-dipendenti dal SCC, i componenti del latte derivati dal sangue del lattato deidrogenasi (LDH), l’albumina del siero (SA) e le IgG nel latte, delle vacche affette da mastite causata da diversi patogeni e trattate con due iniezioni intravenose ad alti dosaggi di OT (100 UI). I campioni di latte provenienti da 184 vacche da latte di diverse aziende sono stati raccolti il giorno 1 (giorno dell’esame clinico e della diagnosi di mastite) e nei giorni 2, 3, 14 e 28. L’esame batteriologico (giorno 1) ha identificato gli agenti patogeni coinvolti. Le vacche sono state assegnate in modo casuale al gruppo con il trattamento (iniezioni di OT il giorno 1 e 2) o al gruppo di controllo (nessuna OT). Indipendentemente dal gruppo sperimentale assegnato, se dopo la raccolta del campione le condizioni generali dell’animale risultavano compromesse, le vacche ricevevano un comune protocollo terapeutico dello studio veterinario. Si è rilevato che a seconda degli agenti patogeni coinvolti, l’SCC del latte, la LDH, la SA e le IgG cambiavano in modo specifico. I valori più alti fra tutti e tre i parametri sono stati misurati in caso di mastite causata da Streptococcus uberis. I cambiamenti erano meno pronunciati con gli Streptococchi spp., Staphylococci spp. o Corynebacterium bovis. Si è notato che il trattamento con ossitocina non influenzava nessuno dei parametri studiati, indipendentemente dal patogeno coinvolto. Si è osservato nei quarti infettati da stafilococchi diversi dallo Staphylococcus aureus una diminuzione della SCC mentre nei quarti dove non venivano rilevati agenti patogeni si evidenziava un aumento delle concentrazioni di IgG. Pertanto, la somministrazione di OT ad alto dosaggio non è ovviamente adatta al trattamento stand-alone della mastite nelle vacche da latte.

An array of l- and d-halotryptophans with different substituents at the indole moiety was synthesized employing either enzymatic halogenation by halogenases or incorporation of haloindoles using tryptophan synthase. Introduction of these Trp derivatives into RGD peptides as a benchmark system was performed to investigate their influence on bioactivity. Halotryptophan-containing RGD peptides display increased affinity toward integrin αvβ3 and enhanced selectivity over integrin α5β1. In addition, bromotryptophan was exploited as a platform for late-stage diversification by Suzuki–Miyaura cross-coupling (SMC), resulting in new-to-nature biaryl motifs. These peptides show enhanced affinity toward αvβ3, good affinity to αvβ8, and remarkable selectivity over α5β1 and αIIbβ3 while featuring fluorogenic properties. Their feasibility as a probe was demonstrated in vitro. Extensive molecular dynamics simulations were undertaken to elucidate NMR and high-performance liquid chromatography (HPLC) data for these late-stage diversified cyclic RGD peptides and to further characterize their conformational preferences.

Lophodermium piceae represents the most common Norway spruce needle endophyte. The aim of this study was to find out whether subpopulations of L. piceae in climatically different environments (in which Norway spruce occurs natively) are adapted to local thermal conditions. L. piceae’s ability for thermal adaptation was investigated by determining growth rates of 163 isolates in vitro at four different temperatures: 2, 6, 20 and 25 °C. Isolates were obtained between 1995 and 2010 from apparently healthy needles sampled in Finland, Poland, Switzerland, Italy and southeastern Siberia. The sampling sites represent seven climatically distinct locations. Results were evaluated in relation to the age and geographic origin of the isolate, in addition to the highest and lowest average monthly temperature of the sampling location. We found a significant correlation between the growth rate and the age of the isolate at 25 °C. Variation in growth rates between subpopulations was low compared to within subpopulations. Only at 2 °C did statistically significant differences between the average growth rates of subpopulations emerge. These results suggest that L. piceae covers the whole distribution area of Norway spruce but that generally the thermal reaction norm of its subpopulations has not changed according to local temperature ranges, despite high contrast in thermal conditions across this vast area. Therefore, it would appear that the thermal environment is not a crucial factor in assessing the fitness of this fungal species within the native range of Norway spruce.

Organotypic slice cultures (OTCs) have been employed in the laboratory since the early 1980s and have proved to be useful for the study of a number of neural systems. Our recent work focuses on the development of behavioral stress resilience induced by repeated daily injections of neuropeptide Y into the basolateral amygdala (BLA). Resilience develops over weeks, persisting to 8 weeks. To unravel the cellular mechanisms underlying neuropeptide Y-induced stress resilience we developed in vitro OTCs of the BLA. Here, we provide an optimized protocol that consistently yields viable and healthy OTCs containing the BLA and surrounding tissue using the interface method, prepared with slices taken from postnatal (P) day 14 rats. We explain key points to optimizing tissue viability and discuss mitigation or avoidance of pitfalls that can arise to aid in successful implementation of this technique. We show that principal neurons in BLA OTCs (8 weeks in vitro = equivalent postnatal day 70) develop into networks that are electrophysiologically very similar to those from acute slices obtained from older rats (P70) and respond to pharmacological treatments in a comparable way. Furthermore, we highlight how these cultures be used to further understand the molecular, cellular, and circuit-level neuropathophysiological changes underlying stress disorders. BLA OTCs provide long-term physiological and pharmacological results whose predictions were borne out in vivo, supporting the validity of the BLA OTC as a model to unravel BLA neurocircuitry. Recent preliminary results also support the successful application of this approach to preparing long-lived OTCs of BLA and neocortex from mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Organotypic slice culture Support Protocol 1: Changing medium Support Protocol 2: Drug incubations Basic Protocol 2: Excision of OTC slices from inserts Support Protocol 3: Fixation of slices.

Invasive candidiasis is a healthcare-associated fungal infection with a high mortality rate. Neutrophils, the first line of defense during fungal infections, express the immunoregulatory Candida albicans receptors CEACAM1, CEACAM3, and CEACAM6. We analyzed the effects of specific antibodies on C. albicans-induced neutrophil responses. CEACAM6 ligation by 1H7-4B and to some extent CEACAM1 ligation by B3-17, but not CEACAM3 ligation by 308/3-3, resulted in the immediate release of stored CXCL8 and altered transcriptional responses of the C. albicans-stimulated neutrophils. Integrated network analyses and dynamic simulations of signaling cascades predicted alterations in apoptosis and cytokine secretion. We verified that CEACAM6 ligation enhanced Candida-induced neutrophil apoptosis and increased long-term IL-1β/IL-6 release in responses to C. albicans. CEACAM3 ligation, but not CEACAM1 ligation, increased the long-term release of pro-inflammatory IL-1β/IL-6. Taken together, we demonstrated for the first time that ligation of CEACAM receptors differentially affects the regulation of C. albicans-induced immune functions in human neutrophils.

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

Non-classical thyroid hormone signalling via cell surface receptor integrin αvβ3, expressed on most cancer cells and proliferating endothelial cells, has been shown to drive tumour cell proliferation and survival, as well as angiogenesis. Tumours develop within a complex microenvironment that is composed of many different cell types, including mesenchymal stem cells. These multipotent progenitor cells actively home to growing tumours where they differentiate into cancer-associated fibroblast-like cells and blood vessel-stabilising pericytes and thus support the tumour’s fibrovascular network. Integrin αvβ3 expression on mesenchymal stem cells makes them susceptible to thyroid hormone stimulation. Indeed, our studies demonstrated – for the first time – that thyroid hormones stimulate the differentiation of mesenchymal stem cells towards a carcinoma-associated fibroblast-/pericyte-like and hypoxia-responsive, pro-angiogenic phenotype, characterised by the secretion of numerous paracrine pro-angiogenic factors, in addition to driving their migration, invasion, and recruitment to the tumour microenvironment in an experimental hepatocellular carcinoma model. The deaminated thyroid hormone metabolite tetrac, a specific inhibitor of thyroid hormone action at the integrin site, reverses these effects. The modulation of mesenchymal stem cell signalling and recruitment by thyroid hormones via integrin αvβ3 adds a further layer to the multifaceted effects of thyroid hormones on tumour progression, with important implications for the management of cancer patients and suggests a novel mechanism for the anti-tumour activity of tetrac.

Introduction Chronic histiocytic intervillositis of unknown etiology (CIUE) is a non-infectious, most probably immunologic placenta lesion. CIUE is associated with recurrent miscarriage, intrauterine growth restriction and stillbirth. Among the pathologic-anatomic defined placental lesions this entity displays the highest risk of recurrence in following pregnancies (about 67–100%). The histiocytic cells accumulate in the placental blood space but do not infiltrate into the villi or decidua. Sparsely known is the expression profile of these intervillous cells regarding histiocytic markers. Methods We analysed 5-22 markers by immunohistochemistry in a total of 41 placenta samples and evaluated decidual, villous and intervillous histiocytic cells. Results In CIUE, intervillous CD163+ histiocytes over-express CD11c/CD18 and down-regulate CD206/CD209, while CD163+ decidual and Hofbauer cells show low CD11c/CD18 and higher CD206/CD209 protein expressions. Discussion CD163 expression indicates a M2-like polarisation. CD11c and CD18 form the complement receptor 4 which could be related to a complement mediated trigger for aberrant cell accumulation in CIUE.

The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes, and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with a high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure. Biolayer interferometry data of the protein-protein interaction of EGF-mCherry with the soluble EGFR are comparable to that of unmodified EGF. Cell culture experiments demonstrated that this fusion replicates all important features of the natural ligand. Finally, fluorescent assays based on EGF-mCherry provided a simple and convenient method to compare EGFR levels on cells and to determine competition of EGFR-binding molecules. These assays will help to rank competitive properties of EGFR inhibitors.

Diversity of fungi was investigated in Scots pine and Norway spruce stumps showing symptoms of butt rot in managed coniferous forests located in the northernmost distribution area of Heterobasidion butt rot in Finland. During a survey of 101 recently clear-cut stands, a total of 479 stumps showing signs of decay were sampled. Fungi were isolated from wood chips taken at the transition zone between healthy and decayed wood. A subsample of 209 isolates from Norway spruce and nine from Scots pine was selected for sequencing ITS-DNA. In Norway spruce 59 species and in Scots pine six fungal species were detected based on ≥98% similarity and 62 genera based on ≥95% similarity with sequences in GenBank. Three sequences did not match to any identified sequence in GenBank. The majority of the isolates (74%) were Ascomycota while 18% were Basidiomycota and 6% Zygomycota. The dominance of Ascomycota over Basidiomycota was pronounced in spruce stumps of stands with a butt rot incidence above 20% and the decayed area was larger in sample discs from which only Ascomycota were isolated than in discs inhabited by Basidiomycota. The result suggests that there is a high site- and/or tree -specific variation of the mycoflora in the Norway spruce decaying butt. Based on these results no single fungal species can be suspected to act as causal agent of butt rot occurring in Norway spruce nor in Scots pine in Northern Ostrobothnia and Kainuu counties.

Animal tests are conducted in all fields of trauma research, but transferability of these data to humans is limited. For example, it is still unclear which animal species is most similar to humans in terms of physiology of blood coagulation. To improve transferability and raise awareness of the existing differences, we compared human coagulation to coagulation of different animals. Rotational thromboelastometry was used to analyse the blood of pigs, sheep, rabbits and dogs. Animal data were compared with human coagulation based on the number of significant differences of the test parameters and on a descriptive comparison of the extent of relative deviation of the single values. All animal species showed significant differences in coagulation properties when compared with humans. Coagulation parameters of dogs and sheep were on average most similar to humans. However, there is no animal which is most similar to humans concerning all aspects of coagulation. Differences in coagulation between humans and animals are significant. This must be taken into account when transferring animal test data to humans.

Objectives Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis , Entamoeba histolytica , Cryptosporidium spp., Blastocystis hominis , and Dientamoeba fragilis with routine laboratory procedures. Methods Stool specimens ( n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica / dispar , and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. Results D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar . Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar . One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs ( n = 3) and apathogenic amoebae ( n = 27). Conclusions The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB., publishedVersion

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes-parasites that cause lethal diseases in humans and livestock-employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes2. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space3-5. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation6,7. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.

Science advances 7(12), eabf1380 - (2021). doi:10.1126/sciadv.abf1380, For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution., Published by Assoc., Washington, DC [u.a.]

For the two proteins myoglobin (MB) and fluoroacetate dehalogenase (FAcD), we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of crystals, the same sample delivery device, as well as the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins reasonable data statistics can be obtained with approximately 5000 room temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that diffraction quality is rather linked to the properties of the crystals than to the radiation source. Time-resolved experiments can therefore be conducted at the source that best matches the desired time-resolution.

Invasive pulmonary aspergillosis (IPA) is a severe infection that is difficult to diagnose due to the ubiquitous presence of fungal spores, the underlying diseases of risk patients, and limitations of currently available markers. In this study, we performed a comprehensive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based identification of host and fungal proteins expressed during IPA in mice and humans. The proteomic analysis of bronchoalveolar lavage samples of individual IPA and control cases allowed the description of common host factors that had significantly increased abundance in both infected animals and IPA patients compared to their controls. Although increased levels of these individual host proteins might not be sufficient to distinguish bacterial from fungal infection, a combination of these markers might be beneficial to improve diagnosis. We also identified 16 fungal proteins that were specifically detected during infection and may be valuable candidates for biomarker evaluation., Graphical abstract

Background: The COVID 19 pandemic increased publication productivity enormously with numerous new COVID-19-related articles appearing daily, despite the fact that many health care workers in the partially overburdened national health care systems were faced with major challenges. Methods: In a cross-sectional, observational, retrospective study we compared and correlated 17 epidemiologic, health care system-related and health-economic factors from medical databases and intergovernmental organisations potentially influencing the COVID-19 and non-COVID-19 publication productivity between 1 January and 30 April 2020 amongst the 30 countries most severely affected by the pandemic. These factors were additionally correlated with the national pre-COVID-19 publication rate for the same pre-year period to identify potential changes in the general publication behaviour. Findings: COVID-19 and non-COVID-19 publication rates correlated strongest with access to and quality of health care (&rho, = 0.80 and 0.87, p &lt, 0.0001), COVID-19 cases per capita (&rho, = 0.78 and 0.72, p &lt, 0.0001), GDP per capita (&rho, = 0.69 and 0.76, p &lt, 0.0001), health spending per capita (&rho, = 0.61 and 0.73, p &lt, 0.0001) and the pre-COVID-19 Hirsch-Index (&rho, = 0.61 and 0.62, p = 0.002 and &lt, 0.0001). Ratios of publication rates for &ldquo, Cancer&rdquo, &ldquo, Diabetes&rdquo, and &ldquo, Stroke&rdquo, in 2020 versus the pre-year period were 0.88 &plusmn, 0.06, 1.02 &plusmn, 0.18 and 0.9 &plusmn, 0.20, resulting in a pooled ratio of 0.93 &plusmn, 0.06 for non-COVID-19 publications. Interpretation: There are marked geographic and national differences in publication productivity during the COVID-19 pandemic. Both COVID-19- and non-COVID-19 publication productivity correlates with epidemiologic, health care system-related and healtheconomic factors, and pre-COVID publication expertise. Countries with a stable scientific infrastructure appear to maintain non-COVID-19 publication productivity nearly at the pre-year level and at the same time use their resilience to produce COVID-19 publications at high rates.

Heterobasidion butt rot causes considerable losses to forestry in coniferous forests in many parts of Europe including southern Fennoscandia. In northern Fennoscandia, including central and northern Finland, both Heterobasidion annosum and H. parviporum are rare. This study aimed to determine the incidence of butt rot in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) north of the area commonly infested by Heterobasidion spp. in Finland. The incidence of butt rot in these conifers was investigated in Northern Ostrobothnia and Kainuu using two data sets: stump inspections in 101 regeneration cutting sites and the Finnish National Forest Inventory (NFI) data. According to stump inspections butt rot occurred in 1% of Scots pine trees and 10% of Norway spruce trees. In the most recent NFI data from 2009 to 2011 the incidence of trees with rot in mature managed stands of these two counties was 1% and 5% for Scots pine and Norway spruce, respectively. According to NFI, these values are nearly equal to the average incidence of butt rot in mature pines and spruces of the whole of Finland. Consequently, the low incidence of Heterobasidion butt rot in these parts of Finland does not mean lower occurrence of butt rot. Butt rot incidence varied greatly between sites: 0–24% of Norway spruce stumps in spruce dominated sites and 0–13% of Scots pine stumps in pine dominated sites were infected. High butt rot incidence in Norway spruce was associated with high site fertility, stand age, low organic matter content of soil and low temperature sum of the site. In Scots pine butt rot appeared after stand age exceeded 120 years while decay occurred already in 40–80 years old Norway spruces. High butt rot incidence in Scots pine was associated also with high flow accumulation of the site. Precipitation, plot slope, plot aspect, ditches, stand density and elevation were insignificant variables in models explaining decay incidence. During 1991–2013 butt rot incidence in Scots pine or Norway spruce has not changed in the study area. Similarly, to the more southern areas with high Heterobasidion risk, butt rot control in the North should focus especially on fertile mineral soil sites.

Heterobasidion parviporum and H. annosum are the most damaging root and butt rot pathogens of conifers in southern Fennoscandia where their distribution and frequency are reasonably well described in literature. However, in the most northern boreal forests of Europe the occurrence and significance of these pathogens is poorly known. In the future global warming may increase root and butt rot damages in the North. In this research the distribution of Heterobasidion spp. in northern Finland was compiled from literature and various available databases and from a field study in two counties of northern Finland, Northern Ostrobothnia and Kainuu. Conifer stumps from 101 regeneration cutting sites were inspected and sample discs from stumps with signs of decay were investigated for the presence of Heterobasidion spp. In all, 11,846 stumps were investigated. Decay was found in 429 Norway spruce stumps and 50 Scots pine stumps. H. parviporum was isolated from five spruce discs, each from a different site. H. annosum was not detected. According to previous records H. annosum has been identified only twice in northern Finland and these locations are over 600 km south of the northernmost Scots pine stands in Fennoscandia. Instead, H. parviporum occurs sporadically in the whole distribution area of Norway spruce in northern Finland. Most probably H. parviporum can be found in overmature dense stands at fertile sites but even there only at low frequencies. In northern Finland butt rot of conifers is caused mainly by other decay fungi than Heterobasidion. Low pH of the organic soil layer is probably a significant factor hampering H. parviporum from spreading in the North.

Over the last decades, the medical device industry has grown significantly. Complex and highly functionalized medical devices and implants are being developed to improve patient treatment and to enhance their health-related quality of life. However, medical devices from this new generation often cannot be sterilized by standard methods such as autoclaving or sterilizing gases, as they are temperature sensitive, containing electronic components like sensors and microchips, or consist of polymers. Gamma irradiation for sterilization of such products is also problematic due to long processing times under highly reactive conditions resulting in material degradation or loss of functionality. Low-energy electron-beam treatment could enable irradiation sterilization of medical surfaces within seconds. This method is very fast in comparison to gamma irradiation because of its high dose rate and therefore degradation processes of polymers can be reduced or even prevented. Additionally, electron penetration depth can be precisely controlled to prevent damage of sensitive components like electronics and semiconductors. The presented study focuses on two key aspects: 1.) Can new and highly functionalized medical products in future be sterilized using low-energy electron-beam irradiation; and 2.) Is the low-energy electron-beam technology suitable to be set up on-site to speed up sterilization processing or make it available “just-in-time”. To address these questions, different test specimens were chosen with complex geometry or electronic functional parts to gather information about the limitations and chances for this new approach. The test specimens were inoculated with clinical relevant test organisms (Pseudomonas aeruginosa) as well as with approved radiation resistant organisms (Deinococcus radiodurans and Bacillus pumilus) to prove the suitability of low-energy electron-beam treatment for the above-mentioned medical products. The calculation of the D10 value for B. pumilus revealed equal efficacy when compared to standard high-energy irradiation sterilization. All of the above-mentioned germs were successfully inactivated by low-energy electron-beam treatment when test specimens were inoculated with a germ load > 10^6 CFU and treated with doses ≥ 10 kGy (for B. pumilus and P. aeruginosa) and > 300 kGy (for D. radiodurans) respectively. As an example, for specialized electronic components to be sterilized, an impedance sensor for cell culture applications was sterilized and unimpaired functionality was demonstrated even after five repeated sterilization cycles to a total dose of 50 kGy. To address the second aspect of on-site suitability of this technology, the product handling for low-energy electron-beam treatment had to be adapted to minimize the size of the electron-beam facility. Therefore, a mini electron-beam source was used and a specialized sample holder and 3D-handling regime were developed to allow reproducible surface treatment for complex product geometries. Inactivation of B. pumilus inoculated medical screws (> 10^6 CFU) was successful using the developed handling procedure. In addition, a packaging material (PET12/PE50) for medical products was investigated for its suitability for low-energy irradiation sterilization. Biocompatibility assessment revealed the material to be eligible for this application as even overdoses did not impair the biocompatibility of the material. With these results, the principal suitability of low-energy electron-beam treatment for sterilization of medical products containing electronics like sensors is demonstrated. The low-energy technology and the specialized 3D-handling regime allow the on-site setup of the technology in hospitals, medical practices or any other point of care.

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene-therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, while additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with qPCR results demonstrated the technique’s future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

The spotted salamander (Ambystoma maculatum) shares a unique endosymbiotic relationship with the unicellular green alga, Oophila amblystomatis. Despite studies isolating and identifying O. amblystomatis in salamander eggs, the taxonomic identity of the alga remains a point of ongoing debate. In this study, the nuclear SSU rRNA gene was used to characterize two well-supported Oophila clades that include lineages identified from past studies in addition to new isolates from the current study. These two clades do not form a monophyletic group and, furthermore, O. amblystomatis appears to be paraphyletic with numerous other chlamydomonad algae. To gain further insight into the biogeographic variation of the host A. maculatum, the mitochondrial ND4 and control gene regions were examined and the phylogeography was observed to be similar to that noted in the literature. Additionally, the response of O. amblystomatis to atrazine and 2,4-dichlorophenoxyacetic acid following 96 h exposure and a 96 h recovery phase was characterized, as this is a plausible mechanism by which the development of the host salamander could be impaired by herbicides. At the 96 h growth rate, no-observed-effect concentrations were 64 μg/L and 30 mg/L for atrazine and 2,4-D, respectively. We observed full recovery ofO. amblystomatis at these concentrations within 96 h. These data suggest that atrazine and 2,4-D do not pose a significant risk to the symbiotic algae, or, indirectly, to the host salamander. In conclusion, we recommend a revision of the current taxonomy of O. amblystomatis, and demonstrate the need for species identification and thorough phylogenetic reconstruction in toxicity testing to accurately inform risk assessment.

Significance Skin forms a critical protective barrier between a mammal and its external environment. Baseline data on the mammalian skin microbiome elucidates which microorganisms are found on healthy skin and provides insight into mammalian evolutionary history. To our knowledge, this study represents the largest existing mammalian skin microbiome survey. Our findings demonstrate that human skin is distinct, not only from other Primates, but from all 10 mammalian orders sampled. Identifying significant similarities between branching of mammalian phylogenetic trees and relatedness trees for their corresponding microbial communities raises the possibility that mammals have experienced coevolution between skin microbiota and their corresponding host species., Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment, yet the factors that govern skin microbial community composition among mammals are poorly understood. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. Skin from the back, torso, and inner thighs of 177 nonhuman mammals was sampled, representing individuals from 38 species and 10 mammalian orders. Animals were sampled from farms, zoos, households, and the wild. The DNA extracts from all skin swabs were amplified by PCR and sequenced, targeting the V3-V4 regions of bacterial and archaeal 16S rRNA genes. Previously published skin microbiome data from 20 human participants, sampled and sequenced using an identical protocol to the nonhuman mammals, were included to make this a comprehensive analysis. Human skin microbial communities were distinct and significantly less diverse than all other sampled mammalian orders. The factor most strongly associated with microbial community data for all samples was whether the host was a human. Within nonhuman samples, host taxonomic order was the most significant factor influencing skin microbiota, followed by the geographic location of the habitat. By comparing the congruence between host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing evidence of phylosymbiosis between skin microbial communities and their hosts.

Two Great Lake embayments, Bay of Quinte (Lake Ontario) and Maumee Bay (Lake Erie) prone to toxic cyanobacteria blooms were sampled in early and late summer in 2006 to determine which cyanobacteria genotypes were present and how many of those were capable of producing microcystins. Microcystins, chlorophyll a, soluble reactive phosphorus and stoichiometric seston ratios of carbon, nitrogen and phosphorus were measured on the same samples to provide an ecological characterization of the phytoplankton. The V3 hypervariable region of the 16S rRNA and aminotransferase (AMT) genes, microcystin (mcyE) and nodularin (nda) were analyzed with PCR-DGGE and quantitative real-time PCR to describe cyanobacterial communities. DGGE results indicated species capable of producing microcystins were present at some point in all study sites and were most likely from species within the genus Microcystis. Concentrations of microcystins were greatest when hepatotoxic cell numbers were highest and when seston stoichiometry indicated cells were not nutrient stressed. This study demonstrated the effective use of a combination of molecular and ecological monitoring of cyanobacterial communities in regions that are susceptible to toxic cyanobacteria blooms.

Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

Background This study aimed to evaluate the diagnostic performance of a novel nonquantitative methylation-specific reverse hybridization (MSRH) assay to detect secreted frizzled-related protein 2 ( SFRP2) promotor methylation in fecal DNA. Methods SFRP2 promoter methylation was investigated in stool DNA isolated from 18 colorectal cancer (CRC) patients and 22 healthy controls using the MSRH assay based on methylation-specific DNA amplification followed by reverse hybridization of biotinylated amplicons to sequence-specific methylation detection probes, with MethyLight serving as a reference method. Results SFRP2 promotor methylation as determined by MSRH vs. MethyLight showed a sensitivity and specificity of 61.1% and 86.3% vs. 77.7% and 77.3%, respectively. Moderate agreement (κ = 0.54, 95% confidence interval [95% CI], 0.29-0.80, pConclusions Our findings, although limited by the small sample size, do not support the use of the MSRH assay for CRC screening in stool.

Chenopodium quinoa is a halophytic pseudocereal crop that is being cultivated in an ever-growing number of countries. Because quinoa is highly resistant to multiple abiotic stresses and its seed has a better nutritional value than any other major cereals, it is regarded as a future crop to ensure global food security. We generated a high-quality genome draft using an inbred line of the quinoa cultivar Real. The quinoa genome experienced one recent genome duplication about 4.3 million years ago, likely reflecting the genome fusion of two Chenopodium parents, in addition to the γ paleohexaploidization reported for most eudicots. The genome is highly repetitive (64.5% repeat content) and contains 54 438 protein-coding genes and 192 microRNA genes, with more than 99.3% having orthologous genes from glycophylic species. Stress tolerance in quinoa is associated with the expansion of genes involved in ion and nutrient transport, ABA homeostasis and signaling, and enhanced basal-level ABA responses. Epidermal salt bladder cells exhibit similar characteristics as trichomes, with a significantly higher expression of genes related to energy import and ABA biosynthesis compared with the leaf lamina. The quinoa genome sequence provides insights into its exceptional nutritional value and the evolution of halophytes, enabling the identification of genes involved in salinity tolerance, and providing the basis for molecular breeding in quinoa.

Objectives Limited treatment options available for Mycobacterium abscessus infections include the parenteral β-lactam antibiotics cefoxitin and imipenem, which show moderate in vitro activity. Other β-lactam antibiotics (except meropenem) have no considerable in vitro activity, due to their rapid hydrolysis by a broad-spectrum β-lactamase (Bla_Mab). We here addressed the impact of β-lactamase production and β-lactam in vitro stability on M. abscessus MIC results and determined the epidemiological cut-off (ECOFF) values of cefoxitin, imipenem and meropenem. Methods By LC high-resolution MS (LC-HRMS), we assessed the in vitro stability of cefoxitin, imipenem and meropenem. M. abscessus ATCC 19977 strain and its isogenic blaMab deletion mutant were used for MIC testing. Based on MIC distributions for M. abscessus clinical strains, we determined ECOFFs of cefoxitin, imipenem and meropenem. Results A functional Bla_Mab increased MICs of penicillins, ceftriaxone and meropenem. LC-HRMS data showed significant degradation of cefoxitin, imipenem and meropenem during standard antibiotic susceptibility testing procedures. MIC, MIC50 and ECOFF values of cefoxitin, imipenem and meropenem are influenced by incubation time. Conclusions The results of our study support administration of imipenem, meropenem and cefoxitin, for treatment of patients infected with M. abscessus. Our findings on in vitro instability of imipenem, meropenem and cefoxitin explain the problematic correlation between in vitro susceptibility and in vivo activity of these antibiotics and question the clinical utility of susceptibility testing of these chemotherapeutic agents.

Chronic alcoholism is associated with widespread regional differences from controls in brain activity and connectivity dynamics measured by blood-oxygen-level-dependent (BOLD) signals. Identification of alcoholism-related neurofunctional power dynamics using functional magnetic resonance imaging (fMRI) that relate to cognition and behavior may serve as biomarkers of alcoholism. Previously, resting-state fMRI studies examined BOLD signals at a single low-frequency (LF) bandwidth. BOLD signals, however, oscillate systematically at different frequencies and are organized in a resting brain where LF oscillation facilitates long-distance communication between regions across cortical regions, whereas high-frequency (HF) oscillation occurs in closely localized, subcortical areas. Using a frequency power quantification approach, we investigated whether the organization of BOLD signal oscillations across all measured frequency bandwidths is altered in alcoholism and relates to cognitive performance. Frequency-dependent oscillation power differences between 56 sober alcoholics and 56 healthy controls occurred for all frequency bands. Alcoholics exhibited greater frequency oscillation power in the orbitofrontal cortex and less power in the posterior insula within the HF bandwidth than controls. Aberrant orbitofrontal HF power was associated with poorer memory performance and slower psychomotor speed in alcoholics. Middle-frequency and LF power proved sensitive in detecting altered frequency oscillation dynamics in parietal and postcentral cortical regions of alcoholics. This study is novel in identifying alcohol-related differences in BOLD oscillation power of the full fMRI frequency bandwidth. Specifically, HF power aberrations were associated with poorer cognitive functioning in alcoholism and may serve as a biomarker for identifying neural targets for repair.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca(2+) channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1β are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM–Munc13–RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis. SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca(2+) channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss? Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P.dactylifera. To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1-type anion channel is regulated by ABA kinase PdOST1. Energy-dispersive X-ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl(-) medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 perse. Only when nitrate was present at the extracellular face of the anion channel did the OST1-gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure. Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

Besides anthropogenic causes, lightning is the major reason for forest fire ignition worldwide. Information on lightning characteristics and impact points is missing or controversial, due to the difficulty of lightning stroke localization and the relation to single forest fire events. Austria as an Alpine country experiences a high number of thunderstorms and lightnings, which ignite more than one third of forest fires from June to August. With data from the “Austrian Lightning Detection and Information System” (ALDIS) it was possible to link single lightnings and their characteristics to the location and attributes of individual forest fires. Three subsets with lightning data were compiled and analyzed regarding their strength (kA), polarity (positive or negative) and multiplicity (number of re-strokes). Additional data on the time of ignition, burned area, sea level, exposition and burned vegetation were investigated. Two fire weather sub-indices (FFMC − Fine Fuel Moisture Code and BUI − Build Up Index) of the Canadian FWI (Fire Weather Index) were calculated for the location of the impact point of each lightning and over a period of twenty days, including the day of ignition. Positive lightnings were significantly more likely to induce a fire. Both the FFMC and BUI showed a significant mean decrease after the day of ignition. Precipitation was significant lower at those impact points where forest fires were ignited. Burned area was larger when lightnings ignited during day hours. Most lightning fires occurred at higher altitudes with southerly or western exposition, mainly in stands of Norway spruce (Picea abies L.). Pine species were four times more often affected than the natural tree species distribution would assume. The study results contribute to the international discussion on lightning fires and will help to provide recommendations for further modelling studies on the forecast of lightning caused forest fires.

Natural killer (NK) cell subpopulations from 8 HLA-matched but killer cell immunoglobulin-like receptor (KIR)/HLA-ligand-mismatched patient-donor pairs were analyzed in the course of allogeneic hematopoietic stem cell transplantation (HCT). The patients' post-transplantation NKG2A-/LIR-1- NK cells, which expressed only inhibitory KIRs for which the patient had no HLA class I ligands, showed higher cytotoxic capacity than the NKG2A-/LIR-1- NK cells lacking any inhibitory KIRs that remained tolerant throughout the course of HCT. The NKG2A+ NK cell subpopulations displayed the highest levels of cytotoxic activation, which appeared to be significantly enhanced in comparison with that in allogeneic graft's donors. LIR-1- NK cells were much more frequent after HCT than LIR-1+ NK cells and LIR-1 expression on NKG2A+ or NKG2A- NK cells was associated with significantly lower cytotoxic activities. Thus NKG2A-/LIR-1- NK cells expressing only HLA-mismatched KIRs show a partial break in tolerance in the first year following HCT. The failure to exclude LIR-1+ cells within the NKG2A- NK cell subset in previous studies could explain the earlier conflicting results. Thus systemic immune activation in patients following HCT augments the GvL effect through both increasing overall NK cell activities and partially breaking tolerance of unlicensed NK cells.

There are a variety of factors to be considered when choosing a particular species of microalgae for aquaculture feed, including growth rate, cell size, nutritional composition, digestibility and pigmentation. A good example of a microalga that fits these criteria is the chrysophyte Boekelovia hooglandii . This study investigates the growth rate and nutritional profile [protein, lipid (34 fatty acids), carbohydrate, and pigment (7 pigments)] of B. hooglandii to assess suitability as a feed. This study also identifies the peak concentrations and correlations among nutritional constituents. Under the tested conditions B. hooglandii exhibited a relatively high biomass productivity of 0.52 g L − 1 day − 1 dry weight (dw), in comparison with reported growth rates for three commonly used aquaculture feed algae, Isochrysis galbana (0.16 g L − 1 day − 1 dw), Tetraselmis suecica (0.27 g L − 1 day − 1 dw), and Pavlova sp. (0.28 g L − 1 day − 1 dw). In addition, the proportion of lipid, protein, and carbohydrate in the algal biomass on days 35 and 63 is suitable for bivalve larvae and juvenile oysters, respectively. A notable correlation identified in this study was between percent protein and growth rate (r = 0.934, p = 0.020), which highlights the nutritional changes occurring during culturing. As a consequence of the relatively high biomass and nutritional profile, B. hooglandii appears to be a suitable candidate as aquaculture feed for bivalve larvae and juvenile oysters. Statement of relevance Identified an algal species with high growth & ideal nutrients for oyster feed that is significantly greater in growth when compared to other commonly used species.

Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For this purpose, progressing soil water deficit is communicated from roots to shoots. Abscisic acid (ABA) is the key signal in stress-induced stomatal closure, but ABA as an early xylem-delivered signal is still a matter of debate. In this study, poplar plants (Populus × canescens) were exposed to water stress to investigate xylem sap sulfate and ABA, stomatal conductance, and sulfate transporter (SULTR) expression. In addition, stomatal behavior and expression of ABA receptors, drought-responsive genes, transcription factors, and NCED3 were studied after feeding sulfate and ABA to detached poplar leaves and epidermal peels of Arabidopsis (Arabidopsis thaliana). The results show that increased xylem sap sulfate is achieved upon drought by reduced xylem unloading by PtaSULTR3;3a and PtaSULTR1;1, and by enhanced loading from parenchyma cells into the xylem via PtaALMT3b. Sulfate application caused stomatal closure in excised leaves and peeled epidermis. In the loss of sulfate-channel function mutant, Atalmt12, sulfate-triggered stomatal closure was impaired. The QUAC1/ALMT12 anion channel heterologous expressed in oocytes was gated open by extracellular sulfate. Sulfate up-regulated the expression of NCED3, a key step of ABA synthesis, in guard cells. In conclusion, xylem-derived sulfate seems to be a chemical signal of drought that induces stomatal closure via QUAC1/ALMT12 and/or guard cell ABA synthesis.

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is a receptor for Candida albicans. It is crucial for the immune response of intestinal epithelial cells to this opportunistic pathogen. Moreover, CEACAM1 is of importance for the mucosal colonization by different bacterial pathogens. We therefore studied the influence of the human CEACAM1 receptor in human CEACAM1-transgenic mice on the C. albicans colonization and infection utilizing a colonization/dissemination and a systemic infection mouse model. Our results showed no alterations in the host response between the transgenic mice and the wild-type littermates to the C. albicans infections. Both mouse strains showed comparable C. albicans colonization and mycobiota, similar fungal burdens in various organs, and a similar survival in the systemic infection model. Interestingly, some of the mice treated with anti-bacterial antibiotics (to prepare them for C. albicans colonization via oral infection) also showed a strong reduction in endogenous fungi instead of the normally observed increase in fungal numbers. This was independent of the expression of human CEACAM1. In the systemic infection model, the human CEACAM1 expression was differentially regulated in the kidneys and livers of Candida-infected transgenic mice. Notably, in the kidneys, a total loss of the largest human CEACAM1 isoform was observed. However, the overwhelming immune response induced in the systemic infection model likely covered any CEACAM1-specific effects in the transgenic animals. In vitro studies using bone marrow-derived neutrophils from both mouse strains also revealed no differences in their reaction to C. albicans. In conclusion, in contrast to bacterial pathogens interacting with CEACAM1 on different mucosal surfaces, the human CEACAM1-transgenic mice did not reveal a role of human CEACAM1 in the in vivo candidiasis models used here. Further studies and different approaches will be needed to reveal a putative role of CEACAM1 in the host response to C. albicans. CA extern