Brianna Dalesandro

Wellcome Open Res. 2023 Oct 30;8:41. doi: 10.12688/wellcomeopenres.18716.2. eCollection 2023.

ABSTRACT

BACKGROUND: Streptococcus pyogenes (StrepA) causes a significant burden of disease globally from superficial infections to invasive disease. It is responsible for over 500,000 deaths each year, predominantly in low- and middle-income countries (LMIC). Superficial StrepA infections of the skin and pharynx can lead to rheumatic heart disease, the largest cause of StrepA-related deaths in LMIC. StrepA can also asymptomatically colonise normal skin and the pharynx (carriage), potentially increasing infection risk. Streptococcus dysgalactiae subsp. equisimilis (SDSE) carriage is also common in LMIC and may interact with StrepA. This study aims to investigate StrepA and SDSE carriage and infection epidemiology, transmission dynamics and naturally acquired immunity within households in The Gambia.

METHODS: A longitudinal household observational cohort study will be conducted over one year. 45 households will be recruited from the urban area of Sukuta, The Gambia, resulting in approximately 450 participants. Households will be visited monthly, and available participants will undergo oropharyngeal and normal skin swabbing. Incident cases of pharyngitis and pyoderma will be captured via active case reporting, with swabs taken from disease sites. Swabs will be cultured for the presence of group A, C and G beta-haemolytic streptococci. Isolates will undergo whole genome sequencing. At each visit, clinical, socio-demographic and social mixing data will be collected. Blood serum will be collected at baseline and final visit. Oral fluid and dried blood spot samples will be collected at each visit. Mucosal and serum anti-StrepA antibody responses will be measured.

OUTCOME: This study will report StrepA and SDSE clinical epidemiology, risk factors, transmission dynamics, and serological responses to carriage and infection. Detailed social mixing behaviour will be combined with phylogenetic relatedness to model the extent of transmission occurring withing and between households. The study will provide data to help meet global strategic StrepA research goals.

PMID:37954923 | PMC:PMC10638483 | DOI:10.12688/wellcomeopenres.18716.2

J Am Chem Soc. 2023 Nov 22;145(46):25056-25060. doi: 10.1021/jacs.2c05691. Epub 2023 Nov 8.

ABSTRACT

Probes that covalently label protein targets facilitate the identification of ligand-binding sites. Lysine residues are prevalent in the proteome, making them attractive substrates for covalent probes. However, identifying electrophiles that undergo amine-specific, regioselective reactions with binding site lysine residues is challenging. Squarates can engage in two sequential conjugate addition-elimination reactions with amines. Nitrogen donation reduces the second reaction rate, making the mono squaramide a mild electrophile. We postulated that this mild electrophilicity would demand a longer residence time near the amine, affording higher selectivity for binding site lysines. Therefore, we compared the kinetics of squarate and monosquaramide amine substitution to alternative amine bioconjugation handles. The data revealed that N-hydroxy succinimidyl esters react 4 orders of magnitude faster, consistent with their labeling promiscuity. Squarate reactivity can be tuned by a substitution pattern. Electron-withdrawing groups on the vinylogous ester or amide increase reaction rates. Dithionosquarates react more rapidly than squarates, while vinylogous thioester analogs, dithiosquarates, react more slowly. We assessed squarate selectively using the UDP-sugar processing enzyme GlfT2 from Mycobacterium tuberculosis, which possesses 21 surface-exposed lysines. The reaction predominately modified one lysine proximal to a binding site to afford covalent inhibition. These findings demonstrate the selectivity of squaric esters and squaramides, which is a critical feature for affinity-based chemoproteomic probes.

PMID:37938802 | PMC:PMC10935565 | DOI:10.1021/jacs.2c05691

Protein Pept Lett. 2023;30(11):959-965. doi: 10.2174/0109298665238177231020044054.

ABSTRACT

BACKGROUND: LinB, as a Haloalkane dehalogenase, has good catalytic activity for many highly toxic and recalcitrant compounds, and can realize the elimination of chemical weapons HD in a green non-toxic mode.

OBJECTIVES: In order to display Haloalkane dehalogenase LinB on the surface of Bacillus subtilis spore.

METHODS: We have constituted the B. subtilis spore surface display system of halogenated alkanes dehalogenase LinB by gene recombination.

RESULTS: Data revealed that LinB can display on spore surface successfully. The hydrolyzing HD analogue 2-chloroethyl ethylsulfide (2-CEES) activity of displayed LinB spores was 4.30±0.09 U/mL, and its specific activity was 0.78±0.03U/mg. Meanwhile, LinB spores showed a stronger stress resistance activity on 2-CEES than free LinB. This study obtained B. subtilis spores of LinB (phingobium japonicum UT26) with enzyme activity that was not reported before.

CONCLUSION: Spore surface display technology uses resistance spore as the carrier to guarantee LinB activity, enhances its stability, and reduces the production cost, thus expanding the range of its application.

PMID:37946356 | DOI:10.2174/0109298665238177231020044054

N Engl J Med. 2023 Nov 11. doi: 10.1056/NEJMoa2307563. Online ahead of print.

ABSTRACT

BACKGROUND: Semaglutide, a glucagon-like peptide-1 receptor agonist, has been shown to reduce the risk of adverse cardiovascular events in patients with diabetes. Whether semaglutide can reduce cardiovascular risk associated with overweight and obesity in the absence of diabetes is unknown.

METHODS: In a multicenter, double-blind, randomized, placebo-controlled, event-driven superiority trial, we enrolled patients 45 years of age or older who had preexisting cardiovascular disease and a body-mass index (the weight in kilograms divided by the square of the height in meters) of 27 or greater but no history of diabetes. Patients were randomly assigned in a 1:1 ratio to receive once-weekly subcutaneous semaglutide at a dose of 2.4 mg or placebo. The primary cardiovascular end point was a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke in a time-to-first-event analysis. Safety was also assessed.

RESULTS: A total of 17,604 patients were enrolled; 8803 were assigned to receive semaglutide and 8801 to receive placebo. The mean (±SD) duration of exposure to semaglutide or placebo was 34.2±13.7 months, and the mean duration of follow-up was 39.8±9.4 months. A primary cardiovascular end-point event occurred in 569 of the 8803 patients (6.5%) in the semaglutide group and in 701 of the 8801 patients (8.0%) in the placebo group (hazard ratio, 0.80; 95% confidence interval, 0.72 to 0.90; P<0.001). Adverse events leading to permanent discontinuation of the trial product occurred in 1461 patients (16.6%) in the semaglutide group and 718 patients (8.2%) in the placebo group (P<0.001).

CONCLUSIONS: In patients with preexisting cardiovascular disease and overweight or obesity but without diabetes, weekly subcutaneous semaglutide at a dose of 2.4 mg was superior to placebo in reducing the incidence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke at a mean follow-up of 39.8 months. (Funded by Novo Nordisk; SELECT ClinicalTrials.gov number, NCT03574597.).

PMID:37952131 | DOI:10.1056/NEJMoa2307563

Circulation. 2023 Nov 12. doi: 10.1161/CIRCULATIONAHA.123.067505. Online ahead of print.

ABSTRACT

Background: Patients with heart failure (HF) with preserved ejection fraction (HFpEF) and obesity experience high burden of symptoms and functional impairment, and a poor quality of life. In the STEP-HFpEF trial, once-weekly semaglutide 2.4 mg improved symptoms, physical limitations and exercise function, and reduced inflammation and body weight. This prespecified analysis investigated the effects of semaglutide on the primary and confirmatory secondary endpoints across the range of the Kansas City Cardiomyopathy Questionnaire (KCCQ) scores at baseline; and on all key summary and individual KCCQ domains. Methods: STEP-HFpEF randomized 529 participants with symptomatic HF, EF ≥45% and BMI of ≥30 kg/m² to once-weekly semaglutide 2.4 mg or placebo for 52 weeks. Dual primary endpoints were change in KCCQ-Clinical Summary Score (CSS) and body weight. Confirmatory secondary endpoints included change in 6-minute-walk-distance (6MWD), a hierarchical composite endpoint (death, HF events and change in KCCQ-CSS and 6MWD) and change in C-reactive protein. Patients were stratified by KCCQ-CSS tertiles at baseline. Semaglutide effects on the primary, confirmatory secondary and select exploratory endpoints (NTproBNP) were examined across these subgroups. Semaglutide effects on additional KCCQ domains (Total Symptom Score [including symptom burden and frequency], Physical Limitations Score, Social Limitations Score, Quality of Life Score, and Overall Summary Score) were also evaluated. Results: Baseline median KCCQ-CSS across tertiles was 37, 59 and 77 points, respectively. Semaglutide consistently improved primary endpoints across KCCQ tertiles 1-3 (estimated treatment differences (ETD;95% CI): for KCCQ-CSS, 10.7 (5.4,16.1), 8.1 (2.7,13.4), 4.6 (-0.6,9.9) points; for body weight, -11 (-13.2,-8.8), -9.4 (-11.5,-7.2), -11.8 (-14.0,-9.6)%, respectively; Pinteraction=0.28 and 0.29, respectively); same was observed for, confirmatory secondary and exploratory endpoints (Pinteraction>0.1 for all). Semaglutide-treated patients experienced improvements in all key KCCQ domains (ETD: 6.7-9.6 points across domains; P≤0.001 for all). Greater proportion of semaglutide- versus placebo-treated patients experienced at least 5-, 10-, 15- and 20-point improvements in all KCCQ domains (Odds Ratios: 1.6-2.9 across domains; P<0.05 for all). Conclusions: In patients with HFpEF and obesity, semaglutide produced large improvements in HF-related symptoms, physical limitations, exercise function, inflammation, body weight and NTproBNP regardless of baseline health status. Benefits of semaglutide extended to all key KCCQ domains.

PMID:37952180 | DOI:10.1161/CIRCULATIONAHA.123.067505

Sci Rep. 2023 Nov 11;13(1):19664. doi: 10.1038/s41598-023-46776-8.

ABSTRACT

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, β-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca²⁺ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca²⁺-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

PMID:37952048 | PMC:PMC10640643 | DOI:10.1038/s41598-023-46776-8

Front Immunol. 2023 Oct 26;14:1258136. doi: 10.3389/fimmu.2023.1258136. eCollection 2023.

ABSTRACT

INTRODUCTION: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines.

METHODS: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level.

RESULTS: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination.

DISCUSSION: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

PMID:37954588 | PMC:PMC10637626 | DOI:10.3389/fimmu.2023.1258136

Cancer Discov. 2023 Nov 9:OF1. doi: 10.1158/2159-8290.CD-NB2023-0083. Online ahead of print.

ABSTRACT

Preclinical data presented at the Society for Immunotherapy of Cancer Annual Meeting show the potential of cancer-targeted myeloid cells, reprogrammed in the body through the use of chimeric antigen receptor-encoding mRNA encapsulated in lipid nanoparticles, to shrink metastases and suppress tumor growth.

PMID:37943022 | DOI:10.1158/2159-8290.CD-NB2023-0083

J Natl Cancer Inst. 2023 Nov 7:djad226. doi: 10.1093/jnci/djad226. Online ahead of print.

ABSTRACT

BACKGROUND: Poliovirus receptor (PVR) interacts with three receptors: T-cell immunoglobulin immunoreceptor tyrosine-based inhibitory motif (TIGIT), CD96, DNAX accessory molecule-1 (DNAM), that are predominantly expressed on T cells and natural killer cells. Many solid tumors, including IDH-wildtype glioblastoma, have been reported to overexpress PVR, and this overexpression is associated with poor prognosis. However, there is no preclinical or clinical trials investigating the use of cell-based immunotherapies targeting PVR in IDH-wildtype glioblastoma.

METHODS: We analyzed PVR expression in transcriptome sequencing databases and specimens from IDH-wildtype glioblastoma patients. We developed PVR targeting chimeric antigen receptor (CAR)-T cells using lentivirus. The antitumor activity of CAR-T cells was demonstrated in patient-derived glioma stem cells (GSCs), intracranial and subcutaneous mouse xenograft models.

RESULTS: We verified PVR expression in primary GSCs, surgical specimens from IDH-wildtype glioblastoma patients and organoids. Accordingly, we developed PVR receptor-based second-generation CAR-T cells. The antitumor activity of CAR-T cells was demonstrated in GSCs and xenograft models. Tumor recurrence occurred in intracranial xenograft model because of antigen loss. The combinational therapy of TIGIT extracellular domain-based CAR-T cells and NK-92 cells markedly suppressed tumor recurrence and prolonged survival.

CONCLUSIONS: PVR receptor-based CAR-T cells were capable of killing GSCs and suppressing tumor recurrence when combined with NK-92 cells.

PMID:37944044 | DOI:10.1093/jnci/djad226

EMBO Rep. 2023 Nov 9:e49561. doi: 10.15252/embr.201949561. Online ahead of print.

ABSTRACT

Multidrug-resistant bacteria present a major threat to public health that urgently requires new drugs or treatment approaches. Here, we conduct integrated proteomic and metabolomics analyses to screen for molecular candidates improving survival of mice infected with Vibrio parahaemolyticus, which indicate that L-Alanine metabolism and phagocytosis are strongly correlated with mouse survival. We also assess the role of L-Alanine in improving mouse survival by in vivo bacterial challenge experiments using various bacteria species, including V. parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Functional studies demonstrate that exogenous L-Alanine promotes phagocytosis of these multidrug-resistant pathogen species. We reveal that the underlying mechanism involves two events boosted by L-Alanine: TLR4 expression and L-Alanine-enhanced TLR4 signaling via increased biosynthesis and secretion of fatty acids, including palmitate. Palmitate enhances binding of lipopolysaccharide to TLR4, thereby promoting TLR4 dimer formation and endocytosis for subsequent activation of the PI3K/Akt and NF-κB pathways and bacteria phagocytosis. Our data suggest that modulation of the metabolic environment is a plausible approach for combating multidrug-resistant bacteria infection.

PMID:37943703 | DOI:10.15252/embr.201949561

J Am Pharm Assoc (2003). 2023 Nov 6:S1544-3191(23)00370-9. doi: 10.1016/j.japh.2023.10.037. Online ahead of print.

ABSTRACT

BACKGROUND: Tirzepatide is a dual GLP-1 and gastric inhibitory polypeptide (GIP) receptor agonist whose mechanism of action leads to a greater effect of gastric emptying (GE) than typical Glucagon-like peptide-1 receptor agonists (GLP-1 RAs). After the first dose of tirzepatide, gastric emptying is most substantially delayed. The drug then undergoes tachyphylaxis after subsequent doses. While data on GLP1-RAs has historically demonstrated a lack of impact on bioavailability of oral hormonal contraceptives, manufacturer recommendations for tirzepatide indicate an interaction exists.

OBJECTIVES: The objectives of this literature review were to review trial data on differences in impact of tirzepatide and GLP-1 RAs on oral hormonal contraceptives as well as provide an analysis of safety data between oral contraceptives and incretin agents.

METHODS: PubMed and Google Scholar were searched using the generic name for the GLP-1/GIP agent, the generic names for GLP-1 RAs and hormonal contraceptives, followed by the generic names plus the interacting medication. A total of six clinical trials were selected for inclusion in the literature review.

RESULTS: Of the six articles included in the review, one investigated the use of tirzepatide and showed a statistically significant reduction in AUC, Cmax, and Tmax when tirzepatide was concomitantly administered with an oral hormonal contraceptive. The remaining five studies involving GLP-1 RAs did not show a significant difference of impact of the agents on oral hormonal contraceptives.

CONCLUSION: In conclusion, it could be suggested that tirzepatide had a greater impact on absorption of oral hormonal contraceptives compared to other GLP-1 RAs. The rapid dose escalation and greater delay on GE enhanced the impact on oral medications such as contraceptives. This differed from other GLP-1 RAs and creates a unique need for enhanced provider and patient education regarding the management of this interaction as well as future studies to evaluate this interaction further.

PMID:37940101 | DOI:10.1016/j.japh.2023.10.037

Front Physiol. 2023 Oct 23;14:1257739. doi: 10.3389/fphys.2023.1257739. eCollection 2023.

ABSTRACT

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DY_m). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DY_m indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DY_m depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DY_m, mitochondrial ROS, and cytosolic Ca²⁺ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DY_m and other indicators in live cell imaging applications.

PMID:37936577 | PMC:PMC10627182 | DOI:10.3389/fphys.2023.1257739

Front Physiol. 2023 Oct 23;14:1257739. doi: 10.3389/fphys.2023.1257739. eCollection 2023.

ABSTRACT

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DY_m). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DY_m indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DY_m depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DY_m, mitochondrial ROS, and cytosolic Ca²⁺ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DY_m and other indicators in live cell imaging applications.

PMID:37936577 | PMC:PMC10627182 | DOI:10.3389/fphys.2023.1257739

Methods Mol Biol. 2024;2715:197-205. doi: 10.1007/978-1-0716-3445-5_12.

ABSTRACT

Most bacterial secretion systems are large machines that cross the cell envelope to deliver effectors outside the cell or directly into target cells. The peptidoglycan layer can therefore represent a physical barrier for the assembly of these large machines. Secretion systems and their counterparts such as type IV pili, flagella, and conjugation machines have therefore evolved or hijacked enzymes with peptidoglycan degradation activity. These enzymes are usually glycoside hydrolases that cleave the glycan chains of the peptidoglycan. Their activities are spatially controlled to avoid cell lysis and to create local rearrangement of the cell wall. In addition, peptidoglycan hydrolases may not be only required for the proper assembly of the secretion systems but may directly participate to the release of the effectors. Finally, several antibacterial effectors possess peptidoglycan degradation activity that damage the cell wall once delivered in the target cell. Here, we describe protocols to test the peptidoglycan degradation activity of these proteins in vitro and in solution.

PMID:37930529 | DOI:10.1007/978-1-0716-3445-5_12

Mol Med Rep. 2023 Dec;28(6):234. doi: 10.3892/mmr.2023.13121. Epub 2023 Nov 3.

ABSTRACT

Insulin growth factor‑1 (IGF‑1) is an endocrine regulator that plays an important role in normal growth and development. IGF‑1 mediated effects may result in protecting macrophages from immunometabolic response. However, it is unclear whether IGF‑1 has a protective effect on fatty acid‑induced macrophages damage. In the present study, THP‑1 cells were differentiated into macrophages and stimulated with palmitic acid (PA) in the absence or presence of IGF‑1. Macrophages apoptosis was measured by Cell Counting Kit‑8 assay, flow cytometry, Hoechst 33342 staining and western blotting. The mitochondrial damage was evaluated using JC‑1 staining and mitochondrial reactive oxygen species detection. The activation of mitophagy was assessed using immunofluorescence and western blotting. As a result, IGF‑1 significantly restored the survival rate in macrophages, while the apoptosis was inhibited through mitochondrial pathway. In addition, IGF‑1 protected the mitochondrial damage induced by PA. Furthermore, PA induced mitophagy via phosphatase and tensin homolog‑induced putative kinase protein 1/Parkin, which was reversed by IGF‑1. Taken together, the present study demonstrated the protective effect of IGF‑1 on PA‑induced mitochondrial apoptosis in macrophages, which might provide a potential therapeutic strategy for treatment of lipotoxicity.

PMID:37921069 | DOI:10.3892/mmr.2023.13121

Nat Commun. 2023 Nov 4;14(1):7093. doi: 10.1038/s41467-023-42546-2.

ABSTRACT

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

PMID:37925433 | PMC:PMC10625600 | DOI:10.1038/s41467-023-42546-2

J Phys Chem B. 2023 Nov 3. doi: 10.1021/acs.jpcb.3c03473. Online ahead of print.

ABSTRACT

The uncoupling protein 1 (UCP1) dissipates the transmembrane (TM) proton gradient in the inner mitochondrial membrane (IMM) by leaking protons across the membrane and producing heat in the process. Such a nonshivering production of heat in the brown adipose tissue can combat obesity-related diseases. UCP1-associated proton leak is activated by free fatty acids and inhibited by purine nucleotides. The mechanism of proton leak and the binding sites of the activators (fatty acids) remain unknown, while the binding site of the inhibitors (nucleotides) was described recently. Using molecular dynamics simulations, we generated a conformational ensemble of UCP1. Using metadynamics-based free energy calculations, we obtained the most likely ATP-bound conformation of UCP1. Our conformational ensemble provides a molecular basis for a breadth of prior biochemical data available for UCP1. Based on the simulations, we make the following testable predictions about the mechanisms of activation of proton leak and proton leak inhibition by ATP: (1) R277 plays the dual role of stabilizing ATP at the binding site for inhibition and acting as a proton surrogate for D28 in the absence of a proton during proton transport, (2) the binding of ATP to UCP1 is mediated by residues R84, R92, R183, and S88, (3) R92 shuttles ATP from the E191-R92 gate in the intermembrane space to the nucleotide binding site and serves to increase ATP affinity, (4) ATP can inhibit proton leak by controlling the ionization states of matrix facing lysine residues such as K269 and K56, and (5) fatty acids can bind to UCP1 from the IMM either via the cavity between TM1 and TM2 or between TM5 and TM6. Our simulations set the platform for future investigations into the proton transport and inhibition mechanisms of UCP1.

PMID:37921649 | DOI:10.1021/acs.jpcb.3c03473

Tanaffos. 2023 Jan;22(1):53-60.

ABSTRACT

BACKGROUND: In severe COVID-19 cases, a hypercoagulable state may occur. Antiphospholipid syndrome-related auto-antibodies (APSRAs) contribute to coagulopathy, but their role in COVID- 19 remains unclear. We aimed to investigate the prevalence of positive APSRAs and their effect on clinical outcomes in confirmed COVID-19 patients.

MATERIALS AND METHODS: In this cross-sectional study, severe hospitalized COVID-19 cases were enrolled. Demographic and clinical data were obtained from the day of admission. APSRAs including IgG and/or IgM anticardiolipin (aCL) and anti-β2-glycoprotein1 (anti-β2GP1) as well as lupus anticoagulant (LAC) were measured.

RESULTS: In this study, 54 severe COVID-19 cases with positive RT-PCR and chest CT scans were recruited. Positive APSRAs were found in 7 (12.9%) patients. Positive LAC was a more prevalent marker as compared to other tests (11.1%). The prevalence of positive aCL (IgM or IgG) and anti-ß2 GPI (IgM or IgG) was 1.8% (in an elderly woman). Lower oxygen saturation was found in the positive APSRAs group as opposed to the negative APSRAs group (70.3±9 vs. 84.8±9.7%). The mortality rate in the positive APSRAs group was significantly higher relative to the negative APSRAs group (83.3% vs. 27.1%; P-value: 0.01). Likewise, the mechanical ventilation requirement in the positive group was also higher (50% vs. 27.1%, P-value: 0.28).

CONCLUSION: This study indicated that LAC might be associated with critical cases and high mortality of COVID-19. Nonetheless, the mortality was not related to macrothrombotic incidence.

PMID:37920325 | PMC:PMC10618584

Biomolecules. 2023 Oct 11;13(10):1508. doi: 10.3390/biom13101508.

ABSTRACT

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar K_d) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

PMID:37892190 | PMC:PMC10605039 | DOI:10.3390/biom13101508

Front Endocrinol (Lausanne). 2023 Oct 11;14:1252141. doi: 10.3389/fendo.2023.1252141. eCollection 2023.

ABSTRACT

Subcellular organelles dysfunction is implicated in various diseases, including metabolic diseases, neurodegenerative diseases, cancer, and cardiovascular diseases. BAM15, a selective mitochondrial uncoupler, has emerged as a promising therapeutic agent due to its ability to enhance mitochondrial respiration and metabolic flexibility. By disrupting the coupling between electron transport and ATP synthesis, BAM15 dissipates the proton gradient, leading to increased mitochondrial respiration and energy expenditure. This review provides a comprehensive overview of BAM15, including its mechanism of action and potential therapeutic applications in diverse disease contexts. BAM15 has shown promise in obesity by increasing energy expenditure and reducing fat accumulation. In diabetes, it improves glycemic control and reverses insulin resistance. Additionally, BAM15 has potential in non-alcoholic fatty liver disease, sepsis, and cardiovascular diseases by mitigating oxidative stress, modulating inflammatory responses, and promoting cardioprotection. The safety profile of BAM15 is encouraging, with minimal adverse effects and remarkable tolerability. However, challenges such as its high lipophilicity and the need for alternative delivery methods need to be addressed. Further research is necessary to fully understand the therapeutic potential of BAM15 and optimize its application in clinical settings.

PMID:37900126 | PMC:PMC10600450 | DOI:10.3389/fendo.2023.1252141

Front Endocrinol (Lausanne). 2023 Oct 11;14:1252141. doi: 10.3389/fendo.2023.1252141. eCollection 2023.

ABSTRACT

Subcellular organelles dysfunction is implicated in various diseases, including metabolic diseases, neurodegenerative diseases, cancer, and cardiovascular diseases. BAM15, a selective mitochondrial uncoupler, has emerged as a promising therapeutic agent due to its ability to enhance mitochondrial respiration and metabolic flexibility. By disrupting the coupling between electron transport and ATP synthesis, BAM15 dissipates the proton gradient, leading to increased mitochondrial respiration and energy expenditure. This review provides a comprehensive overview of BAM15, including its mechanism of action and potential therapeutic applications in diverse disease contexts. BAM15 has shown promise in obesity by increasing energy expenditure and reducing fat accumulation. In diabetes, it improves glycemic control and reverses insulin resistance. Additionally, BAM15 has potential in non-alcoholic fatty liver disease, sepsis, and cardiovascular diseases by mitigating oxidative stress, modulating inflammatory responses, and promoting cardioprotection. The safety profile of BAM15 is encouraging, with minimal adverse effects and remarkable tolerability. However, challenges such as its high lipophilicity and the need for alternative delivery methods need to be addressed. Further research is necessary to fully understand the therapeutic potential of BAM15 and optimize its application in clinical settings.

PMID:37900126 | PMC:PMC10600450 | DOI:10.3389/fendo.2023.1252141

Commun Biol. 2023 Oct 28;6(1):1095. doi: 10.1038/s42003-023-05444-3.

ABSTRACT

Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM⁺) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to Dutch donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy.

PMID:37898698 | PMC:PMC10613281 | DOI:10.1038/s42003-023-05444-3

ACS Omega. 2023 Oct 9;8(42):39035-39040. doi: 10.1021/acsomega.3c03837. eCollection 2023 Oct 24.

ABSTRACT

Vancomycin is a potent and broad-spectrum antibiotic that binds to the d-Ala-d-Ala moiety of the growing bacterial cell wall and kills bacteria. This fascinating binding model prompted us to design and synthesize d-Ala-d-Ala silica gels for the establishment of a new physicochemical (PC) screening method. In this report, we confirmed that vancomycin binds to d-Ala-d-Ala silica gel and can be eluted with MeOH containing 50 mM TFA. Finally, d-Ala-d-Ala silica gel enables to purify vancomycin from the culture broth of a vancomycin-producing strain, Amycolatopsis orientalis.

PMID:37901494 | PMC:PMC10601077 | DOI:10.1021/acsomega.3c03837