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Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Activity-based protein profiling (ABPP) is a commonly utilized technique to globally characterize the endogenous activity of multiple enzymes within a related family. While it has been used extensively to identify enzymes that are differentially active across various mammalian tissues, recent efforts have expanded this technique to studying bacteria. As ABPP is applied to diverse sets of bacterial strains found in microbial communities, there is also an increasing need for robust tools for assessing the conservation of enzymes across closely related bacterial species and strains. In this chapter, we detail the integration of gel-based ABPP with basic bioinformatic tools to enable the analysis of enzyme activity, distribution, and homology. We use as an example the family of serine hydrolases identified in the skin commensal bacterium Staphylococcus epidermidis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis ( Mtb ). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The bacterial genus Staphylococcus comprises diverse species that colonize the skin as commensals but can also cause infection. Previous work identified a family of serine hydrolases termed fluorophoshonate-binding hydrolases (Fphs) in the pathogenic bacteria Staphylococcus aureus , one of which, FphB, functions as a virulence factor. Using a combination of bioinformatics and activity-based protein profiling (ABPP), we identify homologues of these enzymes in the related commensal bacteria Staphylococcus epidermidis . Two of the S. aureus Fph enzymes were not identified in S. epidermidis . Using ABPP, we identified several candidate hydrolases that were not previously identified in S. aureus that may be functionally related to the Fphs. Interestingly, the activity of the Fphs vary across clinical isolates of S. epidermidis . Biochemical characterization of the FphB homologue in S. epidermidis (SeFphB) suggests it is a functional homologue of FphB in S. aureus , but our preliminary studies suggest it may not have a role in colonization in vivo . This potential difference in biological function between the Fphs of closely related staphylococcal species may provide mechanisms for specific inhibition of S. aureus infection without perturbing commensal communities of related bacteria.

Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilized extensively to characterize human disease states and identify druggable targets in diverse disease conditions. ABPP has also recently found applications in microbiology. This includes using activity-based probes (ABPs) for functional studies of pathogenic bacteria as well as complex communities within a microbiome. This review will focus on recent advances in the use of ABPs to profile enzyme activity in disease models, screen for selective inhibitors of key enzymes, and develop imaging tools to better understand the host-bacterial interface., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Presenilin 1 (PS1), the catalytic component of gamma secretase, associates with synaptotagmin 1 (Syt-1). This interaction is decreased in the brains of patients with sporadic Alzheimer's disease. However, it remains unclear how this interaction changes during normal aging. Because aging is a risk factor for Alzheimer's disease, we sought to identify changes in PS1 and Syt-1 association during aging in primary neurons in vitro and mouse brain sections ex vivo. We also tested the effect of aging on the calcium dependence of the interaction by treating neurons aged in vitro with KCl. We found that PS1 and Syt-1 increase their association with age, an effect that is more robust in neuronal processes than cell bodies. Treatment with KCl triggered the interaction in both young and old neurons. Baseline calcium levels and calcium influx in response to KCl treatment were significantly higher in older neurons, which can partially explain the increase in PS1/Syt-1 binding with age. These results suggest a compensatory mechanism during normal aging to offset detrimental age-associated effects., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Context: A spinal epidural abscess (SEA) is a serious condition that may be managed with antibiotics alone or with decompressive surgery combined with antibiotics., Purpose: The objectives of this study were to assess the clinical outcomes of SEA after surgical management and to identify the patient-level factors that are associated with outcomes following surgical decompression and removal of SEA., Study Design/setting: Retrospective chart review analysis., Patient Sample: An analysis of 154 consecutive patients who initially presented to a tertiary-care, academic medical center with SEA, and were subsequently treated with surgery between 2010 and 2015 was performed., Outcome Measures: Postoperative predischarge American Spinal Injury Association Impairment Scale (AIS) scores, 6-month follow-up encounter AIS scores, need for revision surgery, and mortality during SEA surgery were the primary outcomes.Physiological Measures: AIS scores., Method: Fisher's exact and Wilcoxon rank-sum tests were used to assess the associations between patient-level factors and surgical outcomes. Moreover, an interactive, predictive model for postoperative predischarge AIS score was developed using a proportional odds regression model. There was no funding secured for this study and there is no conflict of interest-associated biases., Results: One hundred fifty-four patients (mean age of 58 years) were treated using surgical decompression in addition to antibiotics. The majority of patients were Caucasian (81%) and male (61%). No intraoperative mortality was reported. A second SEA surgery was performed in 8% of patients. A comparison of the preoperative and postoperative predischarge AIS scores showed that 49% of patients maintained a score of E or improved, while 45% remained at their preoperative status and 6% worsened. Among a subset of patients (n=36; 23%) for whom a 6-month follow-up encounter occurred, 75% maintained an AIS score of E or improved, 19% remained at their preoperative status, and 6% worsened. Both the presence and longer duration of preoperative paresis was associated with an increased risk of remaining at the same AIS score or worsening at the predischarge encounter (both p< .001). A predictive model for predischarge AIS scores was developed based on several patient characteristics., Conclusions: Surgical decompression can contribute to improving or maintaining AIS scores in a high percentage of SEA patients. The presence and duration of preoperative paresis are prognostic for poorer outcomes and suggest that rapid surgical intervention before paresis develops may lead to improved postoperative outcomes. Our modeling tool enables an estimation of probabilities of patients' predischarge condition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single-cell level., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Alzheimer's disease (AD)-linked protein, presenilin 1 (PS1), is present at the synapse, and the knock-out of presenilin in mice leads to synaptic dysfunction. On the other hand, synaptic activity was shown to influence PS1-dependent generation of distinct amyloid β (Aβ) species. However, the precise nature of these regulations remains unclear. The current study reveals novel role of PS1 at the synapse, and deciphers how PS1 and synaptic vesicle-associated protein, synaptotagmin 1 (Syt1) modulate each other functions in neurons via direct activity-triggered interaction. Additionally, the therapeutic potential of fostering PS1-Syt1 binding is investigated as a synapse-specific strategy for AD prevention., Methods: PS1-based cell-permeable peptide targeting PS1-Syt1 binding site was designed to inhibit PS1-Syt1 interaction in neurons. PS1 conformation, synaptic vesicle exocytosis and trafficking were assayed by fluorescence lifetime imaging microscopy (FLIM), glutamate release/synaptopHluorin assay, and fluorescence recovery after photobleaching, respectively. Syt1 level and interaction with PS1 in control and sporadic AD brains were determined by immunohistochemistry and FLIM. AAV-mediated delivery of Syt1 into mouse hippocampi was used to investigate the therapeutic potential of strengthening PS1-Syt1 binding in vivo. Statistical significance was determined using two-tailed unpaired Student's t-test, Mann-Whitney's U-test or two-way ANOVA followed by a Bonferroni's post-test., Results: We demonstrate that targeted inhibition of the PS1-Syt1 binding in neurons, without changing the proteins' expression level, triggers "pathogenic" conformational shift of PS1, and consequent increase in the Aβ42/40 ratio. Moreover, our data indicate that PS1, by binding directly to Syt1, regulates synaptic vesicle trafficking and facilitates exocytosis and neurotransmitter release. Analysis of human brain tissue revealed that not only Syt1 levels but also interactions between remaining Syt1 and PS1 are diminished in sporadic AD. On the other hand, overexpression of Syt1 in mouse hippocampi was found to potentiate PS1-Syt1 binding and promote "protective" PS1 conformation., Conclusions: The study reports novel functions of PS1 and Syt1 at the synapse, and demonstrates the importance of PS1-Syt1 binding for exocytosis and safeguarding PS1 conformation. It suggests that reduction in the Syt1 level and PS1-Syt1 interactions in AD brain may present molecular underpinning of the pathogenic PS1 conformation, increased Aβ42/40 ratio, and impaired exocytosis.

The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its 'phosphorylated' and 'dephosphorylated' states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer's disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic 'closed' conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment., Competing Interests: The authors declare that no competing interests exist.

Objectives: Two-dimensional speckle tracking (2D-ST) is a new method to measure tissue velocity (TV), strain and strain rate (SR), but it is unclear if results are comparable to color tissue Doppler imaging (TDI). The objective was therefore to compare the two modalities 2D-ST and TDI., Animals: 100 healthy dogs, Methods: TDI images were acquired from the interventricular septal wall (IVS) and the left ventricular free wall (LVFW) to measure longitudinal TV, strain, and SR, and grayscale images were collected for 2D-ST analysis. A software program was developed, that allowed extraction of single points (SP) from the 2D-ST data set to compare SP with averages of segments (AOS) results, which are usually displayed by the 2D-ST software., Results: A good agreement was found between AOS and SP measurements using 2D-ST. Although most data were within limits of agreement, significant differences were found between TDI and 2D-ST measurements for selected parameters. The differences were small in the IVS, but higher and of clinical relevance in the LVFW. 2D-ST was feasible and reproducible in the IVS, but less reliable in the LVFW., Conclusions: 2D-ST and color TDI can be used interchangeably in the IVS, but the methods reveal different results in the LVFW., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Dilated cardiomyopathy (DCM) in Doberman Pinschers is an autosomal dominant inherited disease. The prevalence of DCM in Doberman Pinschers of various age groups in Europe is currently unknown, but this information would be important to develop recommendations for screening programs., Objectives: To evaluate the prevalence of cardiomyopathy in various age groups of Dobermans., Animals: Seven hundred and seventy-five examinations in 412 Doberman Pinschers., Methods: Dogs were included in a prospective longitudinal cohort study. Each examination included echocardiography and 24-hour ECG (Holter) examination. A cut-off value of >100 ventricular premature contractions (VPCs) per 24 hours on Holter examination or abnormal echocardiography was considered diagnostic for cardiomyopathy. The cumulative prevalence included all dogs with DCM and healthy dogs >7 years of age., Results: DCM prevalence in various age groups was as follows: age group 1 (1 to <2 years) 3.3%, age group 2 (2 to <4 years) 9.9%, age group 3 (4 to <6 years) 12.5%, age group 4 (6 to <8 years) 43.6%, and age group 5 (>8 years) 44.1%. The cumulative prevalence of Doberman Pinscher cardiomyopathy was 58.2%. There was an equal sex distribution, but male dogs showed earlier echocardiographic changes than did female dogs, which had significantly more VPCs., Conclusions and Clinical Importance: The prevalence of Doberman cardiomyopathy is very high in Europe. Disease manifestation and progression are different between male and female dogs. Yearly screening for DCM by Holter examination and echocardiography is recommended, starting at 2 years of age.