Residue concentrations of cloxacillin in milk after intramammary dry cow treatment considering dry period length

Dry cow treatment with an intramammary antibiotic is recommended to reduce the risk of mastitis at the beginning of the next lactation. The dry period may be shortened unintentionally, affecting antibiotic residue depletion and the time when residues reach concentrations below the maximum residue limit (MRL). The objective of this study was to evaluate residue depletion in milk after dry cow treatment with cloxacillin, considering dry periods of 14 (G14d), 21 (G21d), and 28 d (G28d). Overall, fifteen cows with 60 udder quarters were included in the study. For each cow, three of the udder quarters were treated with 1000 mg cloxacillin benzathine (2:1) on d 252, d 259, and d 266 of gestation; one quarter was left untreated. Milk samples were drawn until 20 DIM and milk composition, somatic cell count and cloxacillin residues were analyzed. The HPLC-MS/MS revealed different excretion kinetics for the compounds cloxacillin and cloxacillin benzathine (1:1). All cows showed a cloxacillin and cloxacillin benzathine (1:1) concentration below the MRL of 30 µg/kg after 5 d. In the udder quarters of G21d and G28d, the cloxacillin concentration was already below the MRL at first milking after calving. The cloxacillin benzathine (1:1) concentration in the milk of G28d, G21d, and G14d fell below 30 µg/kg on the 5th, 3rd, and 5th DIM, respectively. Shortening the dry period affects residue depletion after dry cow treatment with cloxacillin. The risk of exceeding the MRL, however, seems low, even with dry periods shorter than 14 d

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.</p

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology

GestaltMatcher Database - A global reference for facial phenotypic variability in rare human diseases

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.</p

Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair

Schwann cell development and peripheral nerve myelination require the serial expression of transcriptional activators, such as Sox10, Oct6 (also called Scip or Pou3f1) and Krox20 (also called Egr2). Here we show that transcriptional repression, mediated by the zinc-finger protein Zeb2 (also known as Sip1), is essential for differentiation and myelination. Mice lacking Zeb2 in Schwann cells develop a severe peripheral neuropathy, caused by failure of axonal sorting and virtual absence of myelin membranes. Zeb2-deficient Schwann cells continuously express repressors of lineage progression. Moreover, genes for negative regulators of maturation such as Sox2 and Ednrb emerge as Zeb2 target genes, supporting its function as an inhibitor of inhibitors in myelination control. When Zeb2 is deleted in adult mice, Schwann cells readily dedifferentiate following peripheral nerve injury and become repair cells. However, nerve regeneration and remyelination are both perturbed, demonstrating that Zeb2, although undetectable in adult Schwann cells, has a latent function throughout life

Das neu identifizierte Gen MACC1 ist ein Regulator des HGF/Met-Signalweges und ist prognostisch für die Metastasierung des Kolonkarzinoms

Das Kolonkarzinom ist eine der häufigsten Tumorerkrankungen weltweit. Etwa 50 % der Patienten entwickeln Fernmetastasen. Diese haben eine sehr schlechte Überlebensprognose. Deshalb fokussiert die Forschung auf die Identifizierung neuer, molekularer Marker für eine verbesserte Metastasierungsvorhersage. Identifizierte Hochrisiko-Patienten könnten somit rechtzeitig eine individualisierte, intensivere Therapie erhalten. MACC1 (Metastasis-associated in colon cancer 1) ist ein neu identifiziertes Gen, das in Kolonkarzinomen und deren Fernmetastasen überexprimiert wird. Die Domänenstruktur von MACC1 ist kennzeichnend für Proteine der Rezeptor-Tyrosinkinase-Signalwege. Ziel dieser Arbeit war die Aufklärung der zellulären Funktion von MACC1 und seiner Rolle in der Tumorprogression sowie die Evaluierung von MACC1 als molekularer Metastasierungsmarker. MACC1-überexprimierende Tumorzellen zeigten in Abhängigkeit von der Domänenstruktur in in vitro Assays ein erhöhtes migratorisches, invasives und proliferatives Potential. Der Einfluss von MACC1 auf die Metastasierungskapazität von Tumorzellen konnte auch im Tiermodell belegt werden. Der Hepatocyte-growth-factor (HGF) induziert die epitheliale-mesenchymale Transition MACC1-exprimierender Zellen und die nukleäre Translokation von MACC1. Die Expression des HGF-Rezeptors Met war in diesen Zellen stark erhöht. Reportergen-Studien bestätigten die transkriptionelle Regulation von Met durch MACC1. Die Analyse humaner Kolonkarzinome ergab eine signifikant höhere MACC1 Expression in Primärtumoren mit metachroner Fernmetastasierung. MACC1 ist ein neu identifizierter Regulator des HGF/Met-Signalweges und trägt somit entscheidend zur Determinierung des metastatischen Potentials von Tumorzellen bei. MACC1 hat großes Potential als neuer, prognostischer Marker für die Metastasierung des Kolonkarzinoms und ist ein Kandidatengen als Ziel effektiver, molekularer Interventionsstrategien zur Metastasierungs-Prävention.Colon cancer is one of the most frequent malignant diseases worldwide. About 50% of the patients develop distant metastasis. These patients have only few therapy options and very poor survival rates. Therefore cancer research focuses on the identification of novel molecular markers to provide a better prognosis of the metastatic risk. Identified high-risk patients would get access to an early, individualized therapy. MACC1 (metastasis associated in colon cancer 1) is a newly identified gene that is overexpressed in colon carcinomas and their distant metastases. The MACC1 domain structure is characteristic for proteins of the receptor tyrosine kinase signalling pathways. Aim of this study was the analysis of the cellular function of MACC1, its role in tumor progression and its evaluation as a molecular, prognostic marker for metastasis. MACC1 overexpressing tumor cells revealed higher migratory, invasive, and proliferative potential in in vitro assays. The impact of MACC1 on the metastatic potential of tumors was also shown in mouse models. The hepatocyte growth factor (HGF) induced epithelial-mesenchymal-transition in MACC1 positive cells and MACC1´s nuclear translocation. Expression of the HGF receptor Met was strongly elevated in these cells. Reporter gene experiments confirmed the transcriptional regulation of Met by MACC1. Analyses in human colon carcinomas showed a significantly higher MACC1 expression in tumors that developed distant metastases. MACC1 is a newly identified regulator of the HGF/Met signalling pathway. It contributes decisively to the metastatic capacity of tumor cells. MACC1 has great potential as new prognostic marker for colon cancer metastasis and is a promising candidate as target for effective, molecular intervention strategies for metastasis prevention

Intervening in β-Catenin Signaling by Sulindac Inhibits S100A4-Dependent Colon Cancer Metastasis12

Colon cancer metastasis is often associated with activation of the Wnt/β-catenin signaling pathway and high expression of the metastasis mediator S100A4. We previously demonstrated the transcriptional regulation of S100A4 by β-catenin and the importance of the interconnection of these cellular programs for metastasis. Here we probe the hypothesis that the nonsteroidal anti-inflammatory drug sulindac sulfide can inhibit colon cancer metastasis by intervening in β-catenin signaling and thereby interdicting S100A4. We treated colon cancer cell lines heterozygous for gain-of-function and wild-type β-catenin with sulindac. We analyzed sulindac's effects on β-catenin expression and subcellular localization, β-catenin binding to the T-cell factor (TCF)/S100A4 promoter complex, S100A4 promoter activity, S100A4 expression, cell motility, and proliferation. Mice intrasplenically transplanted with S100A4-overexpressing colon cancer cells were treated with sulindac. Tumor growth and metastasis, and their β-catenin and S100A4 expressions, were determined. We report the expression knockdown of β-catenin by sulindac, leading to its reduced nuclear accumulation. The binding of β-catenin to TCF was clearly lowered, resulting in reduced S100A4 promoter activity and expression. This correlated well with the inhibition of cell migration and invasion, which could be rescued by ectopic S100A4 expression. In mice, sulindac treatment resulted in reduced tumor growth in the spleen (P = .014) and decreased liver metastasis in a human colon cancer xenograft model (P = .025). Splenic tumors and liver metastases of sulindac-treated mice showed lowered β-catenin and S100A4 levels. These results suggest that modulators of β-catenin signaling such as sulindac offer potential as antimetastatic agents by interdicting S100A4 expression

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology