Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 mu g/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 mu g/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased

Differential Interaction of Platelet-Derived Extracellular Vesicles With Circulating Immune Cells: Roles of TAM Receptors, CD11b, and Phosphatidylserine

Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca++. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding

LZTR1 loss-of-function variants associated with café au lait macules with or without freckling

Pathogenic variants in the leucine zipper-like transcriptional regulator 1 gene (LZTR1) have been identified in schwannomatosis and Noonan syndrome. Here, we expand the phenotype spectrum of LZTR1 variants. We identified four loss-of-function heterozygous LZTR1 variants in five children with multiple café au lait macules and one adult with multiple café au lait macules and axillar freckling, by applying gene panel analysis in four families. The three LZTR1 variants, namely, c.184del/p.Glu62Serfs*39, c.1927C  T/ p.Arg340* had been previously reported in a patient with schwannomatosis. Similar to what is known from other LZTR1-associated conditions, penetrance of the skin manifestations was reduced in two carriers of the familial variants. Our study expands the LZTR1 phenotype to the presence of isolated café au lait macules with or without freckling. Thus, variants in the LZTR1 gene should be considered in patients with multiple café au lait macules

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation

Schlussbericht

Im Rahmen des Forschungsprogramms des DZL wurde am Translational Lung Research Center Heidelberg (TLRC), Partnereinrichtung Thoraxklinik, in der Förderperiode 2016-2020, ein koordiniertes translationales Forschungsprogramm zur Verbesserung des Verständnisses der Krankheitsentstehung, Prävention, Diagnostik und Therapie von chronischen und malignen Lungenerkrankungen durchgeführt. Im Mittelpunkt der Forschungstätigkeiten standen die Krankheitsbereiche COPD, Lungenkrebs, interstitielle Lungenerkrankungen und, Pulmonale Hypertonie. Des Weiteren leisteten die Plattformen Biobank / Datenmanagement und Bildgebung einen wichtigen Beitrag zum Forschungs-programm. Im Bereich COPD wurden maßgebliche Analysen für die COSYCONET Kohorte durchgeführt und ein neuer prädikativer Marker entwickelt (oxygeniertes Hämoglobin). Ebenfalls wurden die interventionellen Verfahren für medikamentös austherapierte Patienten in die klinische Routine überführt. Langzeitbeobachtungen zeigen ein deutlich verbessertes Überleben bei entsprechender Patientenselektion. Im Bereich Lungenkrebs wurden neue Biomarker identifiziert. Therapeutisch bedeutend wurde eine Fusionsvariante als Hochrisiko-Faktor identifiziert und anhand der gewonnenen Ergebnisse die Therapiesteuerung verbessert. Bezüglich der pulmonalen Hypertonie wurde eine Regulation für das PAH-Gene erstbeschrieben und miRNA Biomarker für Therapiezwecke entdeckt. Im Bereich ILD wurden ebenfalls neue Biomarker identifiziert, die den Krankheitsverlauf und ggfs. Therapieansprechen prognostizieren können. Damit hat die Thoraxklinik entscheidend zur translationalen Forschung im TLRC/ DZL beigetragen und gute Voraussetzungen für die Fortsetzung des Forschungsprogramms in der Förderperiode 2021-23 geschaffen. Die Forschungsergebnisse wurden über eine Vielzahl von Publikationen in renommierten Fachjournalen, Pressemitteilungen und über die TLRC/DZL Homepage an das interessierte Fachpublikum und die Öffentlichkeit weitergegeben

Schlussbericht zum Verwendungsnachweis für das Vorhaben FKZ 82DZL004B2

Im Mittelpunkt der Forschung der Partnereinrichtung Thoraxklinik Heidelberg stehen translationale und klinische Projektvorhaben in den Krankheitsbereichen Asthma & Allergie (AA), chronisch obstruktive Lungenerkrankungen (COPD), Mukoviszidose (CF), Interstitielle (diffus parenchymatöse) Lungenerkrankung (DPLD), pulmonale Hypertonie und Lungenkrebs. Die klinische Datenbank und Lungenbiobank der Thoraxklinik ist ein Katalysator für standortübergreifende Zusammenarbeiten vor allem im Bereich LC, aber auch zunehmen für COPD und DPLD. Das Forschungsprogramm aller Krankheitsbereiche hat zum Ziel die neuesten im Labor gewonnen Erkenntnisse in die Entwicklung neuer diagnostischer Methoden und therapeutischer Strategien umzusetzen. Dieser Schlussbericht fasst die in der Förderperiode 2021 – 2023 generierten Forschungsergebnissen zusammen. Die Forschungsergebnisse wurden als Publikationen in renommierten Fachjournalen, Pressemitteilungen und über die TLRC/DZL Homepage an das interessierte Fachpublikum und die Öffentlichkeit weitergegeben

Prospective evaluation of NGS-based sequencing in epilepsy patients: results of seven NASGE-associated diagnostic laboratories

BackgroundEpilepsy is one of the most common and disabling neurological disorders. It is highly prevalent in children with neurodevelopmental delay and syndromic diseases. However, epilepsy can also be the only disease-determining symptom. The exact molecular diagnosis is essential to determine prognosis, comorbidity, and probability of recurrence, and to inform therapeutic decisions.Methods and materialsHere, we describe a prospective cohort study of patients with epilepsy evaluated in seven diagnostic outpatient centers in Germany. Over a period of 2 months, 07/2022 through 08/2022, 304 patients (317 returned result) with seizure-related human phenotype ontology (HPO) were analyzed. Evaluated data included molecular results, phenotype (syndromic and non-syndromic), and sequencing methods.ResultsSingle exome sequencing (SE) was applied in half of all patients, followed by panel (P) testing (36%) and trio exome sequencing (TE) (14%). Overall, a pathogenic variant (PV) (ACMG cl. 4/5) was identified in 22%; furthermore, a significant number of patients (12%) carried a reported clinically meaningful variant of unknown significance (VUS). The average diagnostic yield in patients ≤ 12 y was higher compared to patients &gt;12 y cf. Figure 2B vs. Figure 3B. This effect was more pronounced in cases, where TE was applied in patients ≤ 12 vs. &gt;12 y [PV (PV + VUS): patients ≤ 12 y: 35% (47%), patients &gt; 12 y: 20% (40%)]. The highest diagnostic yield was achieved by TE in syndromic patients within the age group ≤ 12 y (ACMG classes 4/5 40%). In addition, TE vs. SE had a tendency to result in less VUS in patients ≤ 12 y [SE: 19% (22/117) VUS; TE: 17% (6/36) VUS] but not in patients &gt;12 y [SE: 19% (8/42) VUS; TE: 20% (2/10) VUS]. Finally, diagnostic findings in patients with syndromic vs. non-syndromic symptoms revealed a significant overlap of frequent causes of monogenic epilepsies, including SCN1A, CACNA1A, and SETD1B, confirming the heterogeneity of the associated conditions.ConclusionIn patients with seizures—regardless of the detailed phenotype—a monogenic cause can be frequently identified, often implying a possible change in therapeutic action (36.7% (37/109) of PV/VUS variants); this justifies early and broad application of genetic testing. Our data suggest that the diagnostic yield is highest in exome or trio-exome-based testing, resulting in a molecular diagnosis within 3 weeks, with profound implications for therapeutic strategies and for counseling families and patients regarding prognosis and recurrence risk

LZTR1 loss-of-function variants associated with café au lait macules with or without freckling

Pathogenic variants in the leucine zipper-like transcriptional regulator 1 gene (LZTR1) have been identified in schwannomatosis and Noonan syndrome. Here, we expand the phenotype spectrum of LZTR1 variants. We identified four loss-of-function heterozygous LZTR1 variants in five children with multiple café au lait macules and one adult with multiple café au lait macules and axillar freckling, by applying gene panel analysis in four families. The three LZTR1 variants, namely, c.184del/p.Glu62Serfs*39, c.1927C &lt; T/p.Gln643*, and c.857_858delinsT/p.Gly286Valfs*65, were novel, whereas the variant c.1018C &gt; T/ p.Arg340* had been previously reported in a patient with schwannomatosis. Similar to what is known from other LZTR1-associated conditions, penetrance of the skin manifestations was reduced in two carriers of the familial variants. Our study expands the LZTR1 phenotype to the presence of isolated café au lait macules with or without freckling. Thus, variants in the LZTR1 gene should be considered in patients with multiple café au lait macules

Effect of allopurinol in addition to hypothermia treatment in neonates for hypoxic-ischemic brain injury on neurocognitive outcome (ALBINO):Study protocol of a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III)

Background: Perinatal asphyxia and resulting hypoxic-ischemic encephalopathy is a major cause of death and long-term disability in term born neonates. Up to 20,000 infants each year are affected by HIE in Europe and even more in regions with lower level of perinatal care. The only established therapy to improve outcome in these infants is therapeutic hypothermia. Allopurinol is a xanthine oxidase inhibitor that reduces the production of oxygen radicals as superoxide, which contributes to secondary energy failure and apoptosis in neurons and glial cells after reperfusion of hypoxic brain tissue and may further improve outcome if administered in addition to therapeutic hypothermia. Methods: This study on the effects of ALlopurinol in addition to hypothermia treatment for hypoxic-ischemic Brain Injury on Neurocognitive Outcome (ALBINO), is a European double-blinded randomized placebo-controlled parallel group multicenter trial (Phase III) to evaluate the effect of postnatal allopurinol administered in addition to standard of care (including therapeutic hypothermia if indicated) on the incidence of death and severe neurodevelopmental impairment at 24 months of age in newborns with perinatal hypoxic-ischemic insult and signs of potentially evolving encephalopathy. Allopurinol or placebo will be given in addition to therapeutic hypothermia (where indicated) to infants with a gestational age ≥ 36 weeks and a birth weight ≥ 2500 g, with severe perinatal asphyxia and potentially evolving encephalopathy. The primary endpoint of this study will be death or severe neurodevelopmental impairment versus survival without severe neurodevelopmental impairment at the age of two years. Effects on brain injury by magnetic resonance imaging and cerebral ultrasound, electric brain activity, concentrations of peroxidation products and S100B, will also be studied along with effects on heart function and pharmacokinetics of allopurinol after iv-infusion. Discussion: This trial will provide data to assess the efficacy and safety of early postnatal allopurinol in term infants with evolving hypoxic-ischemic encephalopathy. If proven efficacious and safe, allopurinol could become part of a neuroprotective pharmacological treatment strategy in addition to therapeutic hypothermia in children with perinatal asphyxia. Trial registration: NCT03162653, www.ClinicalTrials.gov, May 22, 2017.</p