KPC-39-mediated resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 clinical isolate

Resistance to ceftazidime–avibactam (CAZ-AVI) combination is being increasingly reported. Here, we report a CAZ-AVI resistant Klebsiella pneumoniae belonging to the high-risk ST307 clone and producing KPC-39, a single amino-acid variant of KPC-3 (A172T). Cloning experiments, steady state kinetic parameters and molecular dynamics simulations revealed a loss of carbapenemase activity and an increased affinity for ceftazidime. KPC-39 was identified in a patient without prior exposure to CAZ-AVI, suggesting silent dissemination in European healthcare settings.

Plant death caused by inefficient induction of antiviral R-gene-mediated resistance may function as a suicidal population resistance mechanism

Land plant genomes carry tens to hundreds of Resistance (R) genes to combat pathogens. The induction of antiviral R-gene-mediated resistance often results in a hypersensitive response (HR), which is characterized by virus containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. Alternatively, systemic HR (SHR) is sometimes observed in certain R gene–virus combinations, such that the virus systemically infects the plant and PCD induction follows the spread of infection, resulting in systemic plant death. SHR has been suggested to be the result of inefficient resistance induction; however, no quantitative comparison has been performed to support this hypothesis. In this study, we report that the average number of viral genomes that establish cell infection decreased by 28.7% and 12.7% upon HR induction by wild-type cucumber mosaic virus and SHR induction by a single-amino acid variant, respectively. These results suggest that a small decrease in the level of resistance induction can change an HR to an SHR. Although SHR appears to be a failure of resistance at the individual level, our simulations imply that suicidal individual death in SHR may function as an antiviral mechanism at the population level, by protecting neighboring uninfected kin plants.

Effect of L- to D-Amino Acid Substitution on Stability and Activity of Antitumor Peptide RDP215 against Human Melanoma and Glioblastoma

The study investigates the antitumor effect of two cationic peptides, R-DIM-P-LF11-215 (RDP215) and the D-amino acid variant 9D-R-DIM-P-LF11-215 (9D-RDP215), targeting the negatively charged lipid phosphatidylserine (PS) exposed by cancer cells, such as of melanoma and glioblastoma. Model studies mimicking cancer and non-cancer membranes revealed the specificity for the cancer-mimic PS by both peptides with a slightly stronger impact by the D-peptide. Accordingly, membrane effects studied by DSC, leakage and quenching experiments were solely induced by the peptides when the cancer mimic PS was present. Circular dichroism revealed a sole increase in β-sheet conformation in the presence of the cancer mimic for both peptides; only 9D-RDP215 showed increased structure already in the buffer. Ex vitro stability studies by SDS-PAGE as well as in vitro with melanoma A375 revealed a stabilizing effect of D-amino acids in the presence of serum, which was also confirmed in 2D and 3D in vitro experiments on glioblastoma LN-229. 9D-RDP215 was additionally able to pass a BBB model, whereupon it induced significant levels of cell death in LN-229 spheroids. Summarized, the study encourages the introduction of D-amino acids in the design of antitumor peptides for the improvement of their stable antitumor activity.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

IS THAT THE R171Q AMINO ACID VARIANT IN EXON 3 OF THE MEN1 GENE IS RELATED TO AGE, TO PLAY A MUTATING ROLE?

Background/aim: Several studies demonstrated that the R171Q amino acid variant in exon 3 of MEN1 gene is a polymorphism, and in some new studies it is probably a mutation. We found in our study of twelve cases, two young cases have this variant and developed multiple endocrine neoplasia type1. Materials and methods: twelve MEN1 young patients (7 female, 5 male) aged between 20 and 40 years old, were included in our study. After investigating each patient, biochemical and molecular researches is done. We sequenced exon 3 of the MEN1 gene of patients and some members of their families. Results: Ten patients have MEN1 syndrome and they have no mutation in MEN1 gene. Two patients from separated families have a c.512G>A heterozygote variant. Phenotypically the two cases have hyperparathyroidism in young age. One of them developed others tumors later. Conclusion: The R171Q variant is a mutation in some cases; causes hyperparathyroidism and will develop further MEN1 lesions later, and it is just a polymorphism in other cases. We believe that when this polymorphism combines with young age in severe depression, it will lead to MEN1 syndrome, which will make this polymorphism considered a genetic mutation

The Personalized Proteome: Comparing Proteogenomics and Open Variant Search Approaches for Single Amino Acid Variant Detection

Discovery of variant peptides such as single amino acid variant (SAAV) in shotgun proteomics data is essential for personalized proteomics. Both the resolution of shotgun proteomics methods and the search engines have improved dramatically, allowing for confident identification of SAAV peptides. However, it is not yet known if these methods are truly successful in accurately identifying SAAV peptides without prior genomic information in the search database. We studied this in unprecedented detail by exploiting publicly available long-read RNA seq and shotgun proteomics data from the gold standard reference cell line NA12878. Searching spectra from this cell line with the state-of-the-art open modification search engine ionbot against carefully curated search databases resulted in 96.7% false positive SAAVs and an 85% lower true positive rate than searching with peptide search databases that incorporate prior genetic information. While adding genetic variants to the search database remains indispensable for correct peptide identification, inclusion of long-read RNA sequences in the search database contributes only 0.3% new peptide identifications. These findings reveal the differences in SAAV detection that result from various approaches, providing guidance to researchers studying SAAV peptides and developers of peptide spectrum identification tools.

Oxytocin amino acid variation within Neotropical primates: new genetic variants in hormone and receptor sequences and evidence for evolutionary forces driving this unexpected diversity

Oxytocin is a mammalian neuropeptide hormone that mediates behaviours important to reproduction. Despite almost universal amino acid sequence conservation across most groups of mammals, several unique forms have been reported across Neotropical primates. To explore sequence diversity, we investigated the genes encoding oxytocin and its receptor across the Atelidae, which was known to contain at least three unique oxytocin sequences. Additionally, we included the genus Cebus, within the Cebidae, to further explore the ubiquity of the Pro8 variant in this family. We found a novel amino acid variant (Val3) within the Atelidae radiation, bringing the total number of oxytocin sequences within Neotropical primates to seven. Analyses of physicochemical properties revealed conservative substitutions that are likely tolerated within the selective constraints imposed by receptor binding. Furthermore, we report radical substitutions at the eighth codon and evidence for co-evolution between Pro8 and a ligand-binding region of the oxytocin receptor in the Atelidae, supporting the notion that this variant may affect binding specificity. Overall, we suggest that selective constraint on binding specificity may maintain proper oxytocin function and that the diversification of amino acid sequence is likely due to a variety of processes such as relaxed constraint, neutral mutation, positive selection and coevolution.

Plant expression of NifD protein variants resistant to mitochondrial degradation

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)–linker–NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)–linker–NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Single Amino Acid Variant (SAV) Percentage and Monomer Modeling of Spike Protein of SARS-CoV-2 in Jordan

Spike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human endothelial cells of the respiratoty system for the virus to be engulfed causing COVID-19 disease after priming by type II transmembrane protease TMPRSS2 and then binding with the angiotensin-converting enzyme 2 (ACE2). Therefore, mutations and amino acid variants analysis are essential in understanding the mechanism of binding of spike protein with its receptor to have an insights on possibilities to design a peptide or nucleotide-based vaccine for COVID-19. Here, we employed Iterative Threading Assembly Refinement (I-TASSER) and Multiple Alignment using Fast Fourier Transform (MAFFT) to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 and to analyze the amino acid variants for protein sequences from GISAID database for samples collected from Jordan in a try to find an explanation for the low confirmed number of COVID-19 in Jordan. Our Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) findings showed four single amino acid variants (SAV) found in 20 samples of SARS-CoV-2. What is equal to 5% of samples showed tyrosine deletion at Y144 located in the SARS-CoV-like_Spike_S1_NTD (N terminal domain), 62% showed aspartate substitution to glycine at D614G located in the SARS-CoV-2_Spike_S1_RBD (spike recognition binding site), 5% showed aspartate substitution to tyrosine at D1139Y and 5% showed glycine substitution to serine at G1167S both located in the Corona_S2 domain. The findings have shown lower mutational sensitivity in all variants that might not affect the function of spike glycoprotein except for D614G, which has the highest mutational sensitivity score (5 out of 9) indicating a higher likelihood to affect the function of the spike protein. This might suggest, in general, a reduced transmitability of SARS-CoV-2 in Jordan.