Brianna Dalesandro

ACS Infect Dis. 2024 Mar 8;10(3):890-906. doi: 10.1021/acsinfecdis.3c00517. Epub 2024 Feb 24.

ABSTRACT

Increased resistance to current antimycobacterial agents and a potential bias toward relatively hydrophobic chemical entities highlight an urgent need to understand how current anti-TB drugs enter the tubercle bacilli. While inner membrane proteins are well-studied, how small molecules cross the impenetrable outer membrane remains unknown. Here, we employed mass spectrometry-based proteomics to show that octyl-β-d-glucopyranoside selectively extracts the outer membrane proteins of Mycobacterium tuberculosis. Differentially expressed proteins between nutrient-replete and nutrient-depleted conditions were enriched to identify proteins involved in nutrient uptake. We demonstrate cell surface localization of seven new proteins using immunofluorescence and show that overexpression of the proteins LpqY and ProX leads to hypersensitivity toward streptomycin, while overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane permeability, assessed through an EtBr accumulation assay. Further, proton NMR metabolomics suggests the role of six outer membrane proteins in glycerol uptake. This study identifies several outer membrane proteins that are involved in the permeation of small hydrophilic molecules and are potential targets for enhancing the uptake and efficacy of anti-TB drugs.

PMID:38400924 | DOI:10.1021/acsinfecdis.3c00517

Sci Rep. 2024 Mar 9;14(1):5797. doi: 10.1038/s41598-024-56520-5.

ABSTRACT

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

PMID:38461178 | PMC:PMC10925042 | DOI:10.1038/s41598-024-56520-5

Nat Commun. 2024 Mar 7;15(1):2007. doi: 10.1038/s41467-024-46321-9.

ABSTRACT

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.

PMID:38453922 | PMC:PMC10920689 | DOI:10.1038/s41467-024-46321-9

PLoS One. 2024 Mar 8;19(3):e0300069. doi: 10.1371/journal.pone.0300069. eCollection 2024.

ABSTRACT

INTRODUCTION: Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model.

METHODS: A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 μg and 200 μg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects.

RESULTS: In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 μg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 μg 4497-IgG-IRDye700DX compared to control groups (p = 0.037).

CONCLUSION: This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.

PMID:38457402 | PMC:PMC10923484 | DOI:10.1371/journal.pone.0300069

J Physiol. 2024 Mar 1. doi: 10.1113/JP286430. Online ahead of print.

NO ABSTRACT

PMID:38426245 | DOI:10.1113/JP286430

Skin Res Technol. 2024 Mar;30(3):e13638. doi: 10.1111/srt.13638.

ABSTRACT

BACKGROUND: Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate-based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux.

MATERIALS AND METHODS: Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild-type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile.

RESULTS: Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non-mitochondrial oxygen consumption than wild-type or type I diabetic (T1D) skin.

CONCLUSION: Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

PMID:38454567 | PMC:PMC10920985 | DOI:10.1111/srt.13638

Sci Adv. 2024 Mar;10(9):eadj3864. doi: 10.1126/sciadv.adj3864. Epub 2024 Feb 28.

ABSTRACT

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

PMID:38416829 | PMC:PMC10901376 | DOI:10.1126/sciadv.adj3864

Eur J Pharmacol. 2024 Feb 23:176440. doi: 10.1016/j.ejphar.2024.176440. Online ahead of print.

ABSTRACT

This study investigated the effects of Semaglutide (sema) on the gut microbiota of obese mice induced with high-fat diet (HFD). Male C57BL/6 J mice aged 6 weeks were enrolled and randomly distributed to four groups, which were provided with a normal control diet (NCD,NCD + Sema) and a 60% proportion of a high-fat diet (HFD,HFD + Sema), respectively. HFD was given for 10 weeks to develop an obesity model and the intervention was lasted for 18 days. The results showed Semaglutide significantly reduced body weight gain, areas under the curve (AUC) of glucose tolerance test and insulin resistance test, as well as adipose tissue weight in mice. Semaglutide effectively reduces lipid deposition and lipid droplet formation in the liver of obese mice, and regulates the expression of genes related to abnormal blood glucose regulation. Additionally, semaglutide influences the composition of gut microbiota, mitigating the microbial dysbiosis induced by a high-fat diet by impacting the diversity of the gut microbiota. After the high-fat diet intervention, certain strains such as Akkermansia, Faecalibaculum, and Allobaculum were significantly decreased, while Lachnospiraceae and Bacteroides were significantly increased. However, the application of semaglutide helped restore the lost flora and suppress excessive bacterial abundance. Moreover, semaglutide increased the content of tight junction proteins and repaired the damage to intestinal barrier function caused by the high-fat diet intervention. Furthermore, correlation analysis revealed an inverse relationship between Akkermansia levels and weight gain, blood glucose levels, and various obesity indicators. Correlation analysis also showed that Akkermansia level was negatively correlated with weight gain, blood glucose levels and a range of obesity indicators. This phenomenon may explain the anti-obesity effect of Semaglutide, which is linked to alterations in gut microbiota, specifically an increase in the abundance of Akkermansia. In summary, our findings indicate that Semaglutide has the potential to alleviate gut microbiota dysbiosis, and the gut microbiota may contribute to the obesity-related effects of the drug.

PMID:38402930 | DOI:10.1016/j.ejphar.2024.176440

Int J Mol Sci. 2024 Feb 15;25(4):2297. doi: 10.3390/ijms25042297.

ABSTRACT

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

PMID:38396974 | PMC:PMC10889169 | DOI:10.3390/ijms25042297

MAbs. 2024 Jan-Dec;16(1):2316872. doi: 10.1080/19420862.2024.2316872. Epub 2024 Feb 21.

ABSTRACT

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

PMID:38381460 | PMC:PMC10883104 | DOI:10.1080/19420862.2024.2316872

Cell Chem Biol. 2024 Jun 20;31(6):1219-1230.e5. doi: 10.1016/j.chembiol.2024.01.002. Epub 2024 Feb 2.

ABSTRACT

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

PMID:38309277 | DOI:10.1016/j.chembiol.2024.01.002

IUCrJ. 2024 Mar 1;11(Pt 2):133-139. doi: 10.1107/S2052252524000289.

ABSTRACT

Vancomycin is a glycopeptide antibiotic that for decades has been a mainstay of treatment for persistent bacterial infections. However, the spread of antibiotic resistance threatens its continued utility. In particular, vancomycin-resistant enterococci (VRE) have become a pressing clinical challenge. Vancomycin acts by binding and sequestering the intermediate Lipid II in cell-wall biosynthesis, specifically recognizing a D-alanine-D-alanine dipeptide motif within the Lipid II molecule. VRE achieve resistance by remodeling this motif to either D-alanine-D-lactate or D-alanine-D-serine; the former substitution essentially abolishes recognition by vancomycin of Lipid II, whereas the latter reduces the affinity of the antibiotic by roughly one order of magnitude. The complex of vancomycin bound to D-alanine-D-serine has been crystallized, and its 1.20 Å X-ray crystal structure is presented here. This structure reveals that the D-alanine-D-serine ligand is bound in essentially the same position and same pose as the native D-alanine-D-alanine ligand. The serine-containing ligand appears to be slightly too large to be comfortably accommodated in this way, suggesting one possible contribution to the reduced binding affinity. In addition, two flexible hydroxyl groups - one from the serine side chain of the ligand, and the other from a glucose sugar on the antibiotic - are locked into single conformations in the complex, which is likely to contribute an unfavorable entropic component to the recognition of the serine-containing ligand.

PMID:38277167 | PMC:PMC10916290 | DOI:10.1107/S2052252524000289

J Immunol. 1981 Sep;127(3):959-63.

ABSTRACT

An in vitro model for the propagation and expansion of the memory B lymphocyte population is described. DNP-BGG immune cells were mixed with OVA immune cells and challenged immediately with DNP-OVA. After the 1st response had begun to wane, the cells were rechallenged with DNP-OVA (day 11 of culture). An average of 13-fold more PFC were observed after delayed challenge (day 11). This expansion in the PFC response was an antigen-dependent process and did not involve recruitment of new memory cells from the virgin lymphocyte pool. The level of expansion of the memory cell pool was also calculated using limiting dilution analysis and was found to fall in a range of 16- to 67-fold increase in precursor frequency. In addition to the expansion of the memory B cell population, we also observed the development of 2 immunoregulatory cycles previously observed only in vivo. First, in the presence of persistent antigen, a cyclical PFC response was seen. Second, after day 10 of culture, optimal PFC numbers were observed only when DNP-lysine was added to the plaque assay. Such hapten-augmentable PFC responses have been reported by other investigators as indicative of anti-idiotypic regulation. This possibility is examined more extensively in the following communication.

PMID:7021677

bioRxiv. 2024 Jan 25:2024.01.22.576741. doi: 10.1101/2024.01.22.576741. Preprint.

ABSTRACT

Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

PMID:38328117 | PMC:PMC10849550 | DOI:10.1101/2024.01.22.576741

Diabetes. 2024 Mar 1;73(3):357-358. doi: 10.2337/dbi23-0033.

NO ABSTRACT

PMID:38377448 | DOI:10.2337/dbi23-0033

Int J Mol Sci. 2024 Jan 18;25(2):1193. doi: 10.3390/ijms25021193.

ABSTRACT

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the F_OF₁-ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial F_OF₁-ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of F_OF₁-ATPase supports mitochondrial polarization.

PMID:38256264 | PMC:PMC10816083 | DOI:10.3390/ijms25021193