Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii

Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.     

Inhibition of bacterial isoprenoid synthesis by fosmidomycin, a phosphonic acid-containing antibiotic

The mechanism of action of fosmidomycin (FR-31564), a phosphonic acid containing antibiotic, was examined against Escherichia coli, Micrococcus luteus and other bacteria. It converted growing Esch. coli cells into spheroplasts or swollen cells, but did not inhibit the enzymatic reactions of cell wall synthesis in ether-treated cells. Unlike bicyclomycin, phenethyl alcohol and mitomycin C, it did not reduce the amount of envelope lipoprotein, phospholipids, or DNA, but did reduce the amount of menaquinones and ubiquinones in growing Esch. coli cells. It also inhibited the biosynthesis of both carotenoids and menaquinones in M. luteus cells, suggesting that inhibition of the biosynthesis of a common precursor of these isoprenoids (possibly farnesyl pyrophosphate) might be the main site of its antibacterial activity.     

The molecular basis of resistance to anti-influenza drugs

Influenza A viruses resistant to adamantanes(amantadine and rimantadine) have drastically increased in the last few years. Moreover, these adamantane-resistant viruses have acquired resistance without compromising viral pathogenicity and transmissibility. Viruses resistant to the other anti-influenza drugs, neuraminidase (NA) inhibitors, have also been shown to emerge at a rate higher than previously thought. In addition, several recent observations suggest that human-to-human transmission of variants resistant to NA inhibitors may have occurred, contrary to earlier predictions that such variants were much less likely to be transmitted. Although the prevalence of NA inhibitor resistants remains low, surveillance of drug-resistant influenza viruses in communities is essential. Here, we review antiviral resistance in influenza viruses and the molecular mechanisms of the acquisition of resistance to these drugs.     

N-Formamide as a carbonyl precursor in the catalytic synthesis of Passerini adducts under aqua and mechanochemical conditions

A new simple, efficient, and environmentally friendly protocol is presented for the catalytic synthesis of α-acyloxycarboxamides using N-formamides as a carbonyl precursor under aqua and mechanochemical conditions. Immobilized sulfuric acid on silica gel was employed for the synthesis of desired products, via the reaction of benzoic acid, 1-napthylisocyanide and various heterocyclic N-formamides. After a careful optimization of the reaction conditions, the desired Passerini products were obtained in high to excellent yields in short reaction times (10–30 min) at room temperature. The highly efficient and environmentally friendly method provides a facile access to a library of α-acyloxycarboxamides derivatives for future research on bioactivity screening.

N-Formamides as carbonyl precursors in the synthesis of Passerini adducts under mechanochemical conditions.     

Tagetes erecta as an organic precursor: synthesis of highly fluorescent CQDs for the micromolar tracing of ferric ions in human blood serum

The present article illustrates the green synthesis of novel carbon quantum dots (CQDs) from biomass viz. Tagetes erecta (TE), and subsequently fabrication of a metal ion probe for the sensing of Fe³⁺ in real samples. TE-derived CQDs (TE-CQDs) have been synthesized by a facile, eco-friendly, bottom-up hydrothermal approach using TE as a carbon source. The successful synthesis and proper phase formation of the envisaged material has been confirmed by various characterization techniques (Raman, XRD, XPS, TEM, and EDS). Notably, the green synthesized TE-CQDs show biocompatibility, good solubility in aqueous media, and non-toxicity. The as-synthesized TE-CQDs show an intense photoluminescence peak at 425 nm and exhibit excitation dependent photoluminescence behavior. The proposed TE-CQD-based probe offers a remarkable fluorescence (FL) quenching for Fe³⁺ with high selectivity (K_q ∼ 10.022 × 10¹³ M⁻¹ s⁻¹) and a sensitive/rapid response in a linear concentration range 0–90 μM (regression coefficient R² ∼ 0.99) for the detection of Fe³⁺. The limit of detection (LOD) of the probe for Fe³⁺ has been found as 0.37 μM in the standard solution. It has further been applied for the detection of Fe³⁺ in real samples (human blood serum) and displays good performance with LOD ∼ 0.36 μM. The proposed TE-CQD-based ion sensing probe has potential prospects to be used effectively in biological studies and clinical diagnosis.

TE-CQDs synthesized via the hydrothermal method for the detection of Fe³⁺ in HBS.     

Carbapenem antibiotic stress increases bla KPC ‐2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae

BackgroundThe clinical isolation rates of carbapenem‐resistant Klebsiella pneumoniae (CR‐KP) continue to increase. In China, clinical CR‐KP isolates are mainly attributed to the bla _KPC‐2 gene carried on plasmids, and the bla _KPC‐2 copy number correlates with the expression of KPC enzymes, which can cause elevated carbapenem MICs.MethodsThirty‐seven CR‐KP isolates were collected at the Second People’s Hospital of Lianyungang City between January 2020 and March 2021, with no duplicate isolates, and were screened for the bla _KPC‐2 gene with PCR. Analysis of current CRKP resistance to clinically relevant antimicrobials using the bioMérieux VITEK^® 2 bacterial identification card. The multilocus sequence types of the strains were confirmed with PCR and DNA sequencing. Recombinant plasmids pET20b‐bla _KPC‐2 and pET20b‐CpsG were constructed, and the copy numbers of the recombinant plasmids per unit volume was calculated based on the molecular weight of the plasmids. After the genomes DNA of clinical isolates of K. pneumoniae carrying the bla _KPC‐2 gene were purified, the bla _KPC‐2 gene relative copy number in individual K. pneumoniae strains was indicated by the double standard curve method. Detection of MIC values changes of K. pneumoniae under imipenem selection pressure by broth microdilution method.ResultsAmong the 37 CR‐KP strains isolated, only the bla _KPC‐2 gene was detected in 30 strains, three strains were positive for the bla _NDM‐1 gene, two strains carried both the bla _KPC‐2 and bla _NDM‐1 genes, and two strains without detectable carbapenem resistance genes. The ST11 clone was predominant among the 37 carbapenem‐resistant K. pneumoniae isolates. Drug sensitivity testing showed that except for polymyxins (100% susceptible) and tigecycline (75.7% intermediate), the 37 CR‐KP strains were resistant to almost all antimicrobial drugs. The bla _KPC‐2 relative copy number in nine ST11 clinical isolates of K. pneumoniae was 7.64 ± 2.51 when grown on LB plates but 27.67 ± 13.04 when grown on LB plates containing imipenem. Among these nine isolates, five CRKP strains exhibited elevated MICs to imipenem, while the remaining four strains showed unchanged MIC values to imipenem.ConclusionCarbapenem‐resistant Klebsiella pneumoniae isolates may have multiple pathways to achieve high levels of carbapenem resistance, and moderate carbapenem pressure can increase the copy number of KPC enzyme genes in CRKP strains and enhance the degree of carbapenem resistance in the strains.     

Cytogenetic and molecular basis of multiple myeloma

Multiple myeloma(MM) originating from plasma cells is characterized by complex chromosomal aberrations. The most prominent chromosomal abnormalities of MM are aneuploidy and translocations affecting the immunoglobulin heavy chain locus on chromosome 14q32. Additionally, a variety of genetic aberrations such as ras mutations have been found in MM. Because these chromosomal and genetic abnormalities are closely associated with clinical behavior including prognosis, cytogenetic findings have a great impact on planning treatment strategy. Furthermore, studies of signal transductions and mechanisms of oncogenesis in association with these abnormalities will provide targets to develop novel therapeutic agents. Here we summarize the chromosomal and genetic abnormalities of MM and their clinical implications.     

Cellular and molecular basis of wound healing in diabetes

Diabetic foot ulcers (DFUs), a leading cause of amputations, affect 15% of people with diabetes. A series of multiple mechanisms, including decreased cell and growth factor response, lead to diminished peripheral blood flow and decreased local angiogenesis, all of which can contribute to lack of healing in persons with DFUs. In this issue of the JCI, Gallagher and colleagues demonstrate that in diabetic mice, hyperoxia enhances the mobilization of circulating endothelial progenitor cells (EPCs) from the bone marrow to the peripheral circulation (see the related article beginning on page 1249). Local injection of the chemokine stromal cell-derived factor-1alpha then recruits these EPCs to the cutaneous wound site, resulting in accelerated wound healing. Thus, Gallagher et al. have identified novel potential targets for therapeutic intervention in diabetic wound healing.     

The molecular basis of the Rhesus antigen Ew

BACKGROUND: The Rhesus antigen Ew (ISBT designation 004 011) was first described in 1955. It is defined by a specific antibody, but its molecular genetic basis has not yet been resolved. STUDY DESIGN AND METHODS: Two individuals serologically characterized to express the rare Rhesus antigen Ew were analyzed by sequencing of all 10 exons of the RHCE gene. RESULTS: A nucleotide exchange at position 500 (T500A) resulting in a Met167Lys amino acid substitution was found in both individuals. Moreover, we show that an individual carrying the Ew antigen is capable to produce an alloantibody against the wild-type E antigen. CONCLUSION: The single-point mutation T500A in exon 4 of the RHCE gene is a molecular basis of the rare Rhesus antigen Ew.     

The molecular and genetic basis of colon cancer

Remarkable progress has been accomplished in understanding the molecular basis of genetic colon cancer syndromes including FAP and HNPCC, and their variants; of sporadic colon cancer; and of the rare hamartomatous polyp syndromes. This molecular progress now has to be translated into clinical progress in molecular diagnosis, and in pharmacologic therapy for colonic polyps and cancers. It is hoped that such progress will impact on the frequency and mortality of this very common and frequently fatal cancer.     

The regulation of gene expression and the molecular basis of cancer

Over the last few years, the advent of new genetic techniques allowed a detailed analysis of the structural and functional organization of the genome and the genes in a living cell. After the discovery of cellular oncogenes, genes which are implicated in cell growth control and differentiation, recent investigations provided evidence to a crucial role played by certain gene regulatory DNA elements and oncoproteins in the molecular mechanisms of carcinogenesis.     

Molecular basis of rifampicin resistance in Haemophilus influenzae

OBJECTIVES: To determine the molecular basis of rifampicin resistance in Haemophilus influenzae. METHODS: Mutations in the rifampicin-resistance determining region of the rpoB gene of H. influenzae were analysed by gene amplification and sequencing in 12 rifampicin resistant, one intermediate and four susceptible isolates. RESULTS: All clinical resistant isolates except one had at least one amino acid substitution in the beta-subunit of RNA polymerase. Eleven resistant isolates had amino acid changes at codons 513, 516, 518, 526 and 533 of cluster I, with the most common amino acid substitution being Asp-516-->Val. Only one resistant isolate also had a second mutation Asn-518-->Asp in cluster I; transformants obtained with DNA of this isolate also had both mutations. All the amino acid changes in cluster I were detected in isolates with a high level of rifampicin resistance (MIC > or =32 mg/L), except the Asp-516-->Ala mutation in a low-level resistant isolate (MIC 4 mg/L). Only one serotype f isolate with an MIC of 2 mg/L had a mutation in cluster II. Cluster III presented no amino acid changes. In in vitro-generated high-level rifampicin-resistant mutants, only amino acid changes at codons 516 and 526 were seen, with new amino acid changes appearing at codon 526 of cluster I, while His-526-->Asn was associated with low-level resistance. CONCLUSIONS: Rifampicin resistance in H. influenzae is due to point mutations in the rpoB gene, and the resistance levels are dependent on both the location and the nature of amino acid substitution.     

Genetic basis of erythromycin resistance in oral bacteria

We determined the prevalence of erythromycin-resistant bacteria in the oral cavity and identified mef and erm(B) as the most common resistance determinants. In addition, we demonstrate the genetic linkage, on various Tn1545-like conjugative transposons, between erythromycin and tetracycline resistance in a number of isolates.     

Seroprevalence and molecular detection of Toxoplasma gondii in healthy blood donors in southwest Iran

Background

Toxoplasmosis is a cosmopolitan parasitic disease caused by Toxoplasma gondii (T. gondii). Blood transfusion is a probable route of T. gondii transmission. Due to lack of information about seroprevalence of T. gondii in healthy blood donors, this study was aimed to determine the chronic and acute infection using serological and molecular methods.