Disulfiram: Mechanisms, Applications, and Challenges.

Background: Since disulfiram's discovery in the 1940s and its FDA approval for alcohol use disorder, other indications have been investigated. This review describes potential clinical applications, associated risks, and challenges. Methods: For this narrative review, a PubMed search was conducted for articles addressing in vivo studies of disulfiram with an emphasis on drug repurposing for the treatment of human diseases. The key search terms were "disulfiram" and "Antabuse". Animal studies and in vitro studies highlighting important mechanisms and safety issues were also included. Results: In total, 196 sources addressing our research focus spanning 1948-2022 were selected for inclusion. In addition to alcohol use disorder, emerging data support a potential role for disulfiram in the treatment of other addictions (e.g., cocaine), infections (e.g., bacteria such as Staphylococcus aureus and Borrelia burgdorferi, viruses, parasites), inflammatory conditions, neurological diseases, and cancers. The side effects range from minor to life-threatening, with lower doses conveying less risk. Caution in human use is needed due to the considerable inter-subject variability in disulfiram pharmacokinetics. Conclusions: While disulfiram has promise as a "repurposed" agent in human disease, its risk profile is of concern. Animal studies and well-controlled clinical trials are needed to assess its safety and efficacy for non-alcohol-related indications.

A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20-30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell's phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

Are Men at High Risk for Osteoporosis Underscreened? A Quality Improvement Project.

CONTEXT: Osteoporosis is a major cause of morbidity and mortality in both men and women. The mortality rate in men within 1 year of hip fracture is 37.5%, which is 51% higher than in women. Although clear guidelines exist for osteoporosis screening in women, these are less clear for men. The available guidelines recommend screening high-risk men; however, screening does not appear to be a standard practice. OBJECTIVE: To increase screening rates of osteoporosis in high-risk men in our primary care clinic by 50%. DESIGN: The screening rate of osteoporosis was determined in high-risk male veterans more than 50 years of age enrolled in the resident physician- and nurse practitioner-staffed primary care clinics at a Veterans Affairs Medical Center in Cleveland, OH. High-risk factors included prolonged use of steroids; hypogonadism; and autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus, which are known to be associated with osteoporosis. We surveyed health care professional trainees and nurses to explore their barriers to screening for osteoporosis in high-risk men. MAIN OUTCOME MEASURES: After creating awareness about the importance of this condition among the health care professionals, we analyzed whether this education had any impact on the screening rate. RESULTS: The baseline screening rate in high-risk men was 11%. After phased surveys and awareness building, the screening rate increased to 20%. CONCLUSION: Osteoporosis in high-risk men is under-screened. Creating more awareness about the impact of this condition among health professional trainees and nurses can lead to improved screening rates.

Formation of the ether lipids archaetidylglycerol and archaetidylethanolamine in Escherichia coli.

In archaea, the membrane phospholipids consist of isoprenoid hydrocarbon chains that are ether-linked to a sn-glycerol1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these micro-organisms to extreme environments, but it also reflects an evolutionary marker that distinguishes archaea from bacteria and eukaryotes. CDP-archaeol is the central precursor for polar head group attachment. We examined various bacterial enzymes involved in the attachment of L-serine and glycerol as polar head groups for their promiscuity in recognizing CDP-archaeol as a substrate. Using a combination of mutated bacterial and archaeal enzymes, archaetidylethanolamine (AE) and archaetidylglycerol (AG) could be produced in vitro using nine purified enzymes while starting from simple building blocks. The ether lipid pathway constituted by a set of archaeal and bacterial enzymes was introduced into Escherichia coli, which resulted in the biosynthesis of AE and AG. This is a further step in the reprogramming of E. coli for ether lipid biosynthesis.

Biosynthesis of archaeal membrane ether lipids.

A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria.

Identification of CDP-archaeol synthase, a missing link of ether lipid biosynthesis in Archaea.

Archaeal membrane lipid composition is distinct from Bacteria and Eukarya, consisting of isoprenoid chains etherified to the glycerol carbons. Biosynthesis of these lipids is poorly understood. Here we identify and characterize the archaeal membrane protein CDP-archaeol synthase (CarS) that catalyzes the transfer of the nucleotide to its specific archaeal lipid substrate, leading to the formation of a CDP-activated precursor (CDP-archaeol) to which polar head groups are attached. The discovery of CarS enabled reconstitution of the entire archaeal lipid biosynthesis pathway in vitro, starting from simple isoprenoid building blocks and using a set of five purified enzymes. The cell free synthetic strategy for archaeal lipids we describe opens opportunity for studies of archaeal lipid biochemistry. Additionally, insights into archaeal lipid biosynthesis reported here allow addressing the evolutionary hypothesis of the lipid divide between Archaea and Bacteria.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili.

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the ß-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

Characterization of the single stranded DNA binding protein SsbB encoded in the Gonoccocal Genetic Island.

BACKGROUND: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg(2+) or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity. CONCLUSIONS/SIGNIFICANCE: We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island.

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.

Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria.

Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.

A novel relaxase homologue is involved in chromosomal DNA processing for type IV secretion in Neisseria gonorrhoeae.

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single-stranded and blocked at the 5' end. Mutation of traI identified Tyr93 as required for DNA secretion, while substitution of Tyr201 resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N-terminal hydrophobic region. The HD domain residue Asp120 was required for wild-type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N-terminal region promotes membrane interaction. We propose that Tyr93 initiates DNA processing and Tyr201 is required for termination or acts in DNA binding. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.

"Plasmo2D": an ancillary proteomic tool to aid identification of proteins from Plasmodium falciparum.

Bioinformatics tools to aid gene and protein sequence analysis have become an integral part of biology in the post-genomic era. Release of the Plasmodium falciparum genome sequence has allowed biologists to define the gene and the predicted protein content as well as their sequences in the parasite. Using pI and molecular weight as characteristics unique to each protein, we have developed a bioinformatics tool to aid identification of proteins from Plasmodium falciparum. The tool makes use of a Virtual 2-DE generated by plotting all of the proteins from the Plasmodium database on a pI versus molecular weight scale. Proteins are identified by comparing the position of migration of desired protein spots from an experimental 2-DE and that on a virtual 2-DE. The procedure has been automated in the form of user-friendly software called "Plasmo2D". The tool can be downloaded from http://144.16.89.25/Plasmo2D.zip.

Proteomic analysis of urinary protein markers for accurate prediction of diabetic kidney disorder.

AIM OF THE STUDY: Microalbuminuria is currently the only diagnostic tool available for early diagnosis of diabetic nephropathy. The test is based on immunological detection of small quantities of albumin in the urinary samples of diabetes patients. There are several limitations of the use of microalbuminuria as an index of renal function. It is therefore desirable to identify additional protein markers that would augment prediction of diabetic nephropathy. The aim of this study is to identify urinary protein markers for specific and more accurate prediction of nephropathy in diabetes patients. DESIGN: 100 registered Type II diabetic patients were studied. Abundant proteins of microalbuminuria positive urinary samples of these patients were analyzed by proteomics approaches of 2-Dimentional Gel Electrophoresis (2DGE) and mass spectrometry. RESULTS: 2-DGE analysis of the urine sample revealed four main proteins along with albumin in these samples. These were zinc alpha-2 glycoprotein, alpha-1 acid glycoprotein, alpha-1 microglobulin and IgG as identified by Matrix Assisted Laser Desorption Ionization-Tune of Flight (MALDI-ToF) and by western blot. Twenty control samples and three cases with microalbuminuria negative to positive transition does suggest the early and co-appearance of the markers with albumin. We have also analyzed full length spectrum of these samples by MALDI-ToF. CONCLUSION: Our study shows the presence of additional proteins in urine samples of microalbuminuria positive diabetes patients. These proteins can be used as markers for specific and accurate clinical analysis of Diabetic nephropathy. We propose a mass spectrometry based high throughput diagnostic approach to detect these markers in the urine sample.