Brianna Dalesandro

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Establishing the Structure-Activity Relationship of Daptomycin.

ACS Med Chem Lett. 2020 Jul 09;11(7):1442-1449

Authors: Chow HY, Po KHL, Jin K, Qiao G, Sun Z, Ma W, Ye X, Zhou N, Chen S, Li X

Abstract
Daptomycin is effective in treating infections caused by antibiotic-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and vancomycin-resistant S. aureus (VRSA). Due to its distinct mechanism of action toward multidrug-resistant bacteria, daptomycin provides an attractive structural motif to generate new daptomycin-based antibiotics to combat the problem of bacterial resistance. In this study, we used the total synthesis method to produce daptomycin analogues with a variety in terms of types and sites of modifications. Five classes of daptomycin analogues were synthesized, and the antimicrobial activities of the analogues were analyzed by several biological assays. From this study, we established a comprehensive structure-activity relationship of daptomycin which will lay the foundation for the further development of daptomycin-based antibiotics.

PMID: 32676152 [PubMed]

Rationally designed redirection of natural killer cells anchoring a cytotoxic ligand for pancreatic cancer treatment.

J Control Release. 2020 Jul 16;:

Authors: Lee YE, Ju A, Choi HW, Kim JC, Kim EE, Kim TS, Kang HJ, Kim SY, Jang JY, Ku JL, Kim SC, Jun E, Jang M

Abstract
The emergence of T-cell engineering with chimeric antigen receptors (CARs) has led to attractive therapeutics; however, autologous CAR-T cells are associated with poor clinical outcomes in solid tumors because of low safety and efficacy. Therefore, the aim of our study was to develop a CAR therapy with enhanced cytotoxicity against solid cancer using allogeneic NK cells. In this study, we engineered "off-the-shelf" NK cells to redirect them towards pancreatic ductal adenocarcinoma (PDAC) by improving their target-specific cytotoxic potential. By integrated bioinformatic and clinicopathological analyses, folate receptor alpha (FRα) and death receptor 4 (DR4) were significantly highly expressed in patient-derived tumor cells. The combined expression of FRα and DR4/5 was associated with inferior clinical outcomes, therefore indicating their use as potential targets for biomolecular treatment. Thus, FRα and DR4 expression pattern can be a strong prognostic factor as promising therapeutic targets for the treatment of PDAC. For effective PDAC treatment, allogeneic CAR-NK cells were reprogrammed to carry an apoptosis-inducing ligand and to redirect them towards FRα and initiate DR4/5-mediated cancer-selective cell death in FRα- and DR4/5-positive tumors. As a result, the redirected cytotoxic ligand-loaded NK cells led to a significantly enhanced tumor-selective apoptosis. Accordingly, use of allogeneic CAR-NK cells that respond to FRα and DR4/5 double-positive cancers might improve clinical outcomes based on personal genome profiles. Thus, therapeutic modalities based on allogeneic NK cells can potentially be used to treat large numbers of patients with optimally selective cytotoxicity.

PMID: 32682905 [PubMed - as supplied by publisher]

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Developing PEGylated Reversed D-peptide as a Novel HER2-targeted SPECT Imaging Probe for Breast Cancer Detection.

Bioconjug Chem. 2020 Jul 13;:

Authors: Du S, Luo C, Yang G, Gao H, Wang Y, Li X, Zhao H, Luo Q, Ma X, Shi J, Wang F

Abstract
Human epidermal growth factor receptor-2 (HER2)-enriched breast cancer is characterized by strong invasiveness, high recurrence rate, and poor prognosis. HER2-specific imaging can help screening right patients for appropriate HER2-targeted therapies. Previously, we have developed a 99mTc-labeled HER2-targeted H6 peptide for SPECT imaging of breast cancer. However, the poor metabolic stability and high gallbladder uptake will hamper its clinical application. In this study, a retro-inverso D-peptide of H6 (RDH6) was designed to increase the metabolic stability. PEGylation was used to improve its water solubility and in vivo pharmacokinetics. The results showed that the D-amino acids in 99mTc-PEG4-RDH6 brought better metabolic stability than 99mTc-PEG4-H6, thus achieving higher tumor uptake. As the PEG chain length increased, the probe hydrophilicity gradually increased, and its liver uptake decreased. Compared with radiotracers modified by PEG4 and PEG12, 99mTc-PEG24-RDH6 had the comparable tumor uptake and the lowest liver radioactivity. The SPECT imaging demonstrated that 99mTc-PEG24-RDH6 could specifically distinguish HER2-positive tumors from HER2-negative tumors with better imaging contrast, which thus had the potential for clinical screening of HER2-positive breast patients.

PMID: 32660241 [PubMed - as supplied by publisher]

Bacteria and bacterial derivatives as drug carriers for cancer therapy.

J Control Release. 2020 Jul 15;:

Authors: Cao Z, Liu J

Abstract
The application of bacteria and bacteria-derived membrane vesicles (MVs) has promising potential to make a great impact on the development of controllable targeted drug delivery for combatting cancer. Comparing to most other traditional drug delivery systems, bacteria and their MVs have unique capabilities as drug carriers for cancer treatment. They can overcome physical barriers to target and accumulate in tumor tissues and initiate antitumor immune responses. Furtherly, they are able to be modified both genetically and chemically, to produce and transport anticancer agents into tumor tissues with improved safety and efficacy of cancer treatment but decreased cytotoxic effects to normal cells. In this review, we present some examples of tumor-targeting bacteria and bacteria-derived MVs for the delivery of anticancer drugs, including chemo-therapeutic, radio-therapeutic, photothermal-therapeutic, and immuno-therapeutic agents. We also discuss the advantages as well as the limitations of these tumor-targeting bacteria and their MVs used as platforms for controlled delivery of anticancer therapeutic agents, and further highlight their great potential on clinical translation.

PMID: 32681947 [PubMed - as supplied by publisher]

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Investigating the Role of Mucin as Frontline Defense of Mucosal Surfaces against Mycobacterium avium Subsp. hominissuis.

J Pathog. 2020;2020:9451591

Authors: Bechler J, Bermudez LE

Abstract
Mycobacterium avium is a human and animal pathogen that infects the host through the mucosal surfaces. Past work has demonstrated that the bacterium can interact with both the respiratory and gastrointestinal tracts. Those surfaces in the body are covered by a bilayer of a glycoprotein, mucin, which works as a physical barrier and a gel which contains antibacterial and antivirus properties. This current work shows that different strains of M. avium, in contrast to Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes, are not able to bind to mucins, MUC2 and MUC5b, the main mucins in the gastrointestinal and respiratory tracts, respectively. The lack of binding is due to the characteristics of the cell wall and is impaired by altering lipids, proteins, or glycolipids. M. avium, in contrast to E. coli, interacts with epithelial cells equally in the presence or absence of the mucin, suggesting that the cell wall of the pathogen can facilitate the bacterial movement through the mucin layer, towards the mucosal wall. In conclusion, the study has shown that M. avium can avoid the mucin barrier, which explains its ability to interact with the mucosal epithelium, even in absence of motion-related structures.

PMID: 32665865 [PubMed]

A 4-strain Probiotic Supplement Influences Gut Microbiota Composition and Gut Wall Function in Patients with Ulcerative Colitis.

Int J Pharm. 2020 Jul 14;:119648

Authors: Ghyselinck J, Verstrepen L, Moens F, Van den Abbeele P, Said J, Smith B, Bjarnason I, Basit AW, Gaisford S

Abstract
Symprove, a multi-strain probiotic, has been shown to exert a mild anti-inflammatory effect in patients with ulcerative colitis (UC). We examined stool samples from 3 patients with UC in order to create microbiotas in an in-vitro gut model. The effects of Symprove on bacterial diversity and metabolic activity in the microbiotas was evaluated over 48h. In addition, the influence of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. The relative proportions of the main bacterial phyla in UC patients differed from those of healthy subjects studied previously; levels of Firmicutes were lowered and levels of Bacteroidetes were raised. Addition of Symprove changed the bacterial composition in the microbiotas over a 48h period. Several other factors generally implicated in good gut health changed after dosing with probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased, levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased, epithelial tight junction integrity improved and wound healing occurred faster than a control. The results imply it is not the simple addition of probiotic bacteria that improves gut health. Rather, the probiotic bacteria generate lactate, which then stimulates growth of commensal gut bacteria, raising SCFA levels (particularly butyrate). The increased butyrate concentration positively influences inflammation response and time of wound healing.

PMID: 32679260 [PubMed - as supplied by publisher]

Bacillus Calmette–Guérin (BCG) immunotherapy for bladder cancer is the only bacterial cancer therapy approved for clinical use. Although presumed to induce T cell-mediated immunity, whether tumor elimination depends on bacteria-specific or tumor-specific immunity is unknown. Herein we show that BCG-induced bladder tumor elimination requires CD4 and CD8 T cells, although...

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Exploring the antibacterial mechanism of essential oils by membrane permeability, apoptosis and biofilm formation combination with proteomics analysis against methicillin-resistant staphylococcus aureus.

Int J Med Microbiol. 2020 Jul;310(5):151435

Authors: Tang C, Chen J, Zhang L, Zhang R, Zhang S, Ye S, Zhao Z, Yang D

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important causes of food poisoning and infectious diseases worldwide, it can produce a large number of virulence factors, enhance the colonization ability of the host so that it can quickly colonize and spread on the surface of the objects. Essential oil (EO) is one of the natural products with antimicrobial properties, can be used as an important source of antibacterial agent discovery, and has a broad development prospect. However, the unclear mechanisms of antibacterial action have become an obstacle to its further development and use. Hence, the objective of the present study was to reveal the antibacterial mechanism of EO from Amomum villosum Lour (A villosum Lour) against MRSA using label-free quantitative proteomics, investigate the effect of EO on the bacterial proteome, enzymatic activities and leakage of bacterial intracellular biomacromolecule. Proteomic analysis of MRSA in the presence of EO found that a total of 144 differential expressed proteins (DEPs) between the control and treatment group, in which 42 proteins were distinctly up-regulated and 102 proteins were down-regulated. Besides, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, determination of cell membrane permeability and apoptosis, scanning electron microscopy (SEM) observations, bacterial surface hydrophobicity, and biofilm formation measurement were performed. Collectively, the above results indicated that the cell membrane damage by EO leads to the loss of membrane integrity and causes leakage of intracellular macromolecular substances, inhibition of protein, and biofilm synthesis. These findings manifested that EO exerts antibacterial effect by multiple avenues and expands our understanding of the antibacterial mechanism, it has potential application value in food preservative and pharmaceutical industries.

PMID: 32654773 [PubMed - in process]

In combating viral infections, the Fab portion of an antibody could mediate virus neutralization, whereas Fc engagement of Fc-γ receptors (FcγRs) could mediate an array of effector functions. Evidence abounds that effector functions are important in controlling infections by influenza, Ebola, or HIV-1 in animal models. However, the relative contribution...

Nature Reviews Microbiology, Published online: 10 July 2020; doi:10.1038/s41579-020-0417-9

A recent study reports that gut-colonizing commensal bacteria produce a neurotransmitter that modulates the sensory behaviour of their host.

Nature Biotechnology, Published online: 13 July 2020; doi:10.1038/s41587-020-0601-5

Off-the-shelf CAR T cells avoid immune rejection in mouse models of allogeneic cancer cell therapy.

ABSTRACT

We investigated the requirement of 15 human butyrate-producing gut bacterial strains for eight B vitamins and the proteinogenic amino acids by a combination of genome sequence analysis and in vitro growth experiments. The Ruminococcaceae species Faecalibacterium prausnitzii and Subdoligranulum variabile were auxotrophic for most of the vitamins and the amino acid tryptophan. Within the Lachnospiraceae, most species were prototrophic for all amino acids and several vitamins, but biotin auxotrophy was widespread. In addition, most of the strains belonging to Eubacterium rectale and Roseburia spp., but few of the other Lachnospiraceae strains, were auxotrophic for thiamine and folate. Synthetic coculture experiments of five thiamine or folate auxotrophic strains with different prototrophic bacteria in the absence and presence of different vitamin concentrations were carried out. This demonstrated that cross-feeding between bacteria does take place and revealed differences in cross-feeding efficiency between prototrophic strains. Vitamin-independent growth stimulation in coculture compared to monococulture was also observed, in particular for F. prausnitzii A2-165, suggesting that it benefits from the provision of other growth factors from community members. The presence of multiple vitamin auxotrophies in the most abundant butyrate-producing Firmicutes species found in the healthy human colon indicates that these bacteria depend upon vitamins supplied from the diet or via cross-feeding from other members of the microbial community.

IMPORTANCE Microbes in the intestinal tract have a strong influence on human health. Their fermentation of dietary nondigestible carbohydrates leads to the formation of health-promoting short-chain fatty acids, including butyrate, which is the main fuel for the colonic wall and has anticarcinogenic and anti-inflammatory properties. A good understanding of the growth requirements of butyrate-producing bacteria is important for the development of efficient strategies to promote these microbes in the gut, especially in cases where their abundance is altered. The demonstration of the inability of several dominant butyrate producers to grow in the absence of certain vitamins confirms the results of previous in silico analyses. Furthermore, establishing that strains prototrophic for thiamine or folate (butyrate producers and non-butyrate producers) were able to stimulate growth and affect the composition of auxotrophic synthetic communities suggests that the provision of prototrophic bacteria that are efficient cross feeders may stimulate butyrate-producing bacteria under certain in vivo conditions.

ABSTRACT

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.

IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.

Nature, Published online: 08 July 2020; doi:10.1038/s41586-020-2474-7

A combination of gnotobiotic mouse models, transcriptomics, circuit tracing and chemogenetic manipulations identifies neuronal circuits that integrate microbial signals in the gut with regulation of the sympathetic nervous system.

DarT and DarG form a toxin‐antitoxin pair in M. tuberculosis. In the absence of DarG, the toxin ADP‐ribosylates DNA leading to cell death. In the presence of DarG, DarG binds to and neutralizes the toxin, and plausibly also recruits DNA repair machinery to remove the ADP‐ribosyl moiety and repair the DNA.

Abstract

Of the ~80 putative toxin‐antitoxin (TA) modules encoded by the bacterial pathogen Mycobacterium tuberculosis (Mtb), three contain antitoxins essential for bacterial viability. One of these, Rv0060 (DNA ADP‐ribosyl glycohydrolase, DarG _Mtb ), functions along with its cognate toxin Rv0059 (DNA ADP‐ribosyl transferase, DarT _Mtb ), to mediate reversible DNA ADP‐ribosylation (Jankevicius et al., 2016). We demonstrate that DarT _Mtb ‐DarG _Mtb form a functional TA pair and essentiality of darG_Mtb is dependent on the presence of darT_Mtb , but simultaneous deletion of both darT_Mtb‐darG_Mtb does not alter viability of Mtb in vitro or in mice. The antitoxin, DarG _Mtb , forms a cytosolic complex with DNA‐repair proteins that assembles independently of either DarT _Mtb or interaction with DNA. Depletion of DarG _Mtb alone is bactericidal, a phenotype that is rescued by expression of an orthologous antitoxin, DarG _Taq , from Thermus aquaticus. Partial depletion of DarG _Mtb triggers a DNA‐damage response and sensitizes Mtb to drugs targeting DNA metabolism and respiration. Induction of the DNA‐damage response is essential for Mtb to survive partial DarG _Mtb ‐depletion and leads to a hypermutable phenotype.

Scientists have discovered that one of the good bacteria found in the human gut has a benefit that has remained unrecognized until now: The potential to reduce the risk for heart disease.

As humanity fights against the coronavirus, the battle against antibiotic-resistant bacteria continues. Scientists at UCLouvain have succeeded in capturing unique images of protein soldiers that help bacteria resist drugs. This discovery is published in the prestigious scientific journal Nature Chemical Biology. The image recordings will make it possible to develop new attacks on bacteria and thus produce more effective antibiotics.

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Impact of ligand size and conjugation chemistry on the performance of universal chimeric antigen receptor T-cells for tumor killing.

Bioconjug Chem. 2020 Jun 09;:

Authors: Pellegrino C, Favalli N, Sandholzer M, Volta L, Bassi G, Millul J, Cazzamalli S, Matasci M, Villa A, Myburgh R, Manz M, Neri D

Abstract
All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CART-cells. We have used fluoresce-in-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad con-centration range. Antibodies could be conveniently used both in IgG format and as smaller Diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.

PMID: 32515934 [PubMed - as supplied by publisher]

The chemical structures of cell wall polysaccharides (CWPS) of seven distinct lactococcal strains were elucidated. Comparative genome analysis of the gene clusters that encode these structures of 107 lactococcal strains has led to the identification of four distinct genotypes that correlates to distinct chemical structures. The combined genome and structural data allow predictions of characteristics of lactococcal CWPS based on sequence information, while a model for the biosynthetic pathway of the CWPS of the prototypical B‐type strain IL1403 is proposed.

Abstract

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A–C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan‐embedded rhamnan backbone polymer to which a surface‐exposed, poly/oligosaccharidic side‐chain is covalently linked. Chemical structures of the side‐chain of seven lactococcal strains were elucidated, highlighting their diverse and strain‐specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase‐encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side‐chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side‐chain (for C‐type strains), (d) the presence of Galf and phosphodiester bonds in the side‐chain, and (e) the presence of glycerol phosphate substituents in the side‐chain.