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Recent outbreaks of cardiac surgery-associated Mycobacterium chimaera infections have highlighted the importance of species differentiation within the Mycobacterium avium complex and pointed to a lack of antibiotic susceptibility data for M. chimaera Using the MGIT 960/EpiCenter TB eXiST platform, we have determined antibiotic susceptibility patterns of 48 clinical M. chimaera isolates and 139 other non-tuberculous mycobacteria including 119 members of the M. avium complex and 20 Mycobacterium kansasii towards clofazimine and other drugs used to treat infections with slowly growing nontuberculous mycobacteria (NTM). MIC 50 , MIC 90 and tentative epidemiological cutoff (ECOFF) values for clofazimine were 0.5 mg/L, 1 mg/L and 2 mg/L for M. chimaera. Comparable values were observed for other M. avium complex members, lower MIC 50 (≤0.25 mg/L), MIC 90 (0.5 mg/L) and ECOFF (1 mg/L) values were found for M. kansasii Susceptibility to clarithromycin, ethambutol, rifampin, rifabutin, amikacin, moxifloxacin and linezolid was in general similar for M. chimaera and other members of the M. avium complex but increased for M. kansasii The herein determined MIC distributions, MIC 90 and ECOFF values of clofazimine for M. chimaera and other NTM provide the basis for the definition of clinical breakpoints. Further studies are needed to establish correlation of in vitro susceptibility and clinical outcome., (Copyright © 2021 American Society for Microbiology.)

Peptide backbone N-methylation, as seen in cyclosporin A, has been considered to be exclusive to nonribosomal peptides. We have identified the first post-translationally modified peptide or protein harboring internal α-N-methylations through discovery of the genetic locus for the omphalotins, cyclic N-methylated peptides produced by the fungus Omphalotus olearius. We show that iterative autocatalytic activity of an N-methyltransferase fused to its peptide substrate is the signature of a new family of ribosomally encoded metabolites.

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.

Basal rate of metabolism (BMR) and temperature regulation are described for Goeldi's monkey (Callimico goeldii), a threatened New World primate species of the family Callitrichidae. Measurements were conducted on sleeping individuals during the night, using a special nestbox designed to serve as a respirometry chamber, such that test animals remained undisturbed in their customary surroundings. Oxygen consumption was measured at ambient temperatures between 17.5 and 32 degrees C for 10 individuals with an average body mass of 557 g. Average BMR was 278+/-41 ml O(2) h(-1), which is lower than the value predicted on the basis of body mass. Individual differences in BMR were significant even when body mass was accounted for. Body temperature was measured in five individuals below thermoneutrality and averaged 36+/-0.3 degrees C. The corresponding thermal conductance averaged 29.3+/-2.2 ml O(2) h(-1) degrees C(-1), which is similar to the expected value. The metabolic and thermoregulatory patterns observed in C. goeldii resemble those of the closely related marmosets and tamarins. Low BMR is presumably associated with limited access to energy resources and may be directly linked with phylogenetic dwarfing in the family Callitrichidae.

The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, DeltaF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three-M3A7, MATG 1104, and cc24-met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of DeltaF508 CFTR. The antibodies exhibited different sensitivities for detecting DeltaF508 CFTR.

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.

Sphingolipids are highly enriched in the outer leaflet of the plasma membrane lipid bilayer. However, the first glycolipid, glucosylceramide, is synthesized in the opposite, cytosolic leaflet of the Golgi membrane. This has led us to experiments which suggest that the level of glucosylceramide in the cytosolic surface is carefully regulated both by the balance between synthesis and hydrolysis and by transport away from this surface through translocators, multidrug transporters, the same molecules that make cancer cells resistant to chemotherapy. Our data suggest a role for newly synthesized glucosylceramide not only in the formation of domains in the luminal leaflet of the Golgi but also on the cytosolic surface of this organelle.

Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation DeltaF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from DeltaF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In DeltaF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas DeltaF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of DeltaF508 CFTR expression from null to apparently normal amounts indicates that DeltaF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in DeltaF508 CF respiratory and intestinal disease.

Background: In normal adult pseudostratified human nasal surface epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) is localized to the apical domain of the ciliated cells, whereas in cystic fibrosis (CF), the mutated delta F 508 CFTR exhibits an abnormal cytoplasmic localization. Frequent airway injuries either in CF or non-CF patients may induce a remodeling of the surface epithelium characterized by a change in the morphological structure from normal columnar pseudostratified epithelium to either basal cell hyperplasia, mucous cell hyperplasia, or squamous metaplasia., Experimental Design: The localization of CFTR parallel to markers of cell differentiation, such as cytokeratin 14 (CK14, a marker of basal cells), cytokeratin 18 (CK 18, a marker of ciliated and mucous cells), cytokeratin 13 (CK13, a marker of squamous metaplasia cells), and desmoplakins (DP) 1 and 2 (markers of desmosomes) was analyzed by indirect immunofluorescence., Results: In normal pseudostratified epithelium, CFTR was detected at the apical plasma membrane of the ciliated cells, CK14 was identified in basal cells of focal areas, CK18 was localized in both ciliated and mucous cells, CK 13 was detected in all basal cells, and DP 1 and 2 were preferentially detected at the interface between columnar and basal cells. In basal cell hyperplasia, CFTR was poorly expressed in the cytoplasm of the more superficial cells, CK14 and CK13 were localized in basal cell multilayers, CK18 labeling was present in the more superficial cell layers, and DP 1 and 2 were preferentially detected at the interface between the more basal cells. In squamous metaplasia, CFTR labeling was either very low or even undetectable, CK14 was found in focal areas of the more basal cell layers, CK18 labeling was either very low or undetectable, CK13 expression was restricted to the flattened cells toward the epithelial surface, and DP 1&2 were intensively present between all the epithelial cells., Conclusions: These results suggest that the localization of CFTR in human nasal surface epithelium is related to the differentiation state of this epithelium. Abnormally low expression of the CFTR protein may not only be caused by CFTR gene mutations but can also be associated with airway surface epithelium dedifferentiation and remodeling.

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.

The generalized exocrinopathy cystic fibrosis (CF) is the most common severe genetic disease in Caucasian populations. A panel of more than 700 chromosomes from German and Turkish CF patients was screened for disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch, single strand conformation polymorphism, restriction analysis and direct sequencing of genomic DNA amplified by polymerase chain reaction. Besides the major 3-bp deletion, delta F508 that was found on 73% of German CF chromosomes, more than 50 other missense, nonsense, frame-shift, and splice-site mutations have already been identified. In general, a CFTR mutation is linked with a single 10-marker haplotype which indicates that in most cases a particular mutation spread from a common ancestor. The comparison of mutation genotypes with the disease phenotype emphasized the causative role of the type and localization of the CFTR mutation for clinical course and prognosis. Pancreatic status and the risk of colonization of airways with opportunistic pathogens are genetically determined. Most patients who are harbouring mutations in the nucleotide binding folds were suffering from severe CF disease. Mild or even aberrant forms of CF were observed for many missense mutations located in the putative transmembrane domains or for mutations that are expected to result in a truncated protein of half of wild-type CFTR.

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of hetero-duplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-delta F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-delta F508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the "delta F508 haplotype" and that contribute to its overpresentation among German non-delta F508 alleles that are associated with severe forms of disease.

In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German non-CF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed delta F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.

German cystic fibrosis (CF) chromosomes were screened for molecular lesions in exon 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch. An 3884G-to-A transition was detected in two patients which leads to an exchange of a serine by an asparagine in the Walker motif A of the second nucleotide binding fold. The affected serine residue is evolutionarily strongly conserved among the pro- and eukaryotic members of the protein superfamily of traffic ATPases. The two S1251N alleles were linked to the benign missense mutation F508C which is located in another conserved region of CFTR, the center region of the first nucleotide binding fold. Both patients with the complex allele F508C-S1251N are carrying delta F508 on the other CF chromosome and are suffering from severe pulmonary and gastrointestinal CF disease. Although F508C has been classified as a neutral sequence variation because of its discovery in healthy delta F508 gene carriers, it may nevertheless influence CFTR dysfunction caused by the S1251N mutation.