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This paper describes the ‘4DN Data Portal’ that hosts data generated by the 4D Nucleome network, including Hi-C and other chromatin conformation capture assays, as well as various sequencing-based and imaging-based assays. Raw data have been uniformly processed to increase comparability and the portal is implemented with visualization tools to browse the data without download.

The Antarctic Weddell inhabits one of the most extreme environments on Earth. Comparative genomics with close relatives reveals the specific genetic adaptations for cardiovascular and fat metabolism that enable it to thrive.

Triple negative breast cancer (TNBC) is an aggressive disease with a 5-y relative survival rate of 11% after distant metastasis. To survive the metastatic cascade, tumor cells remodel their signaling pathways by regulating energy production and upregulating survival pathways. AMP-activated protein kinase (AMPK) and Akt regulate energy homeostasis and survival, however, the individual or synergistic role of AMPK and Akt isoforms during lung colonization by TNBC cells is unknown. The purpose of this study was to establish whether targeting Akt, AMPKα or both Akt and AMPKα isoforms in circulating cancer cells can suppress TNBC lung colonization. Transient silencing of Akt1 or Akt2 dramatically decreased metastatic colonization of lungs by inducing apoptosis or inhibiting invasion, respectively. Importantly, transient pharmacologic inhibition of Akt activity with MK-2206 or AZD5363 inhibitors did not prevent colonization of lung tissue by TNBC cells. Knockdown of AMPKα1, AMPKα2, or AMPKα1/2 also had no effect on metastatic colonization of lungs. Taken together, these findings demonstrate that transient decrease in AMPK isoforms expression alone or in combination with Akt1 in circulating tumor cells does not synergistically reduce TNBC metastatic lung colonization. Our results also provide evidence that Akt1 and Akt2 expression serve as a bottleneck that can challenge colonization of lungs by TNBC cells.

Abstract Background In contrast to typical measures employed to assess outcomes in healthcare such as mortality or recovery rates, it is difficult to define which specific outcomes of care are the most important in caring for dying individuals. Despite a variety of tools employed to assess different dimensions of palliative care, there is no consensus on a set of core outcomes to be measured in the last days of life. In order to optimise decision making in clinical practice and comparability of interventional studies, we aim to identify and propose a set of core outcomes for the care of the dying person. Methods Following the COMET initiative approach, the proposed study will proceed through four stages to develop a set of core outcomes: In stage 1, a systematic review of the literature will identify outcomes measured in existing peer reviewed literature, as well as outcomes derived through qualitative studies. Grey literature, will also be included. Stage 2 will allow for the identification and determination of patient and proxy defined outcomes of care at the end of life via quantitative and qualitative methods at an international level. In stage 3, from a list of salient outcomes identified through stages 1 and 2, international experts, family members, patients, and patient advocates will be asked to score the importance of the preselected outcomes through a Delphi process. Stage 4 consists of a face-to-face consensus meeting of international experts and patient/family representatives in order to define, endorse, and propose the final Core Outcomes Set. Discussion Core Outcome Sets aim at promoting uniform assessment of care outcomes in clinical practice as well as research. If consistently employed, a robust set of core outcomes for the end of life, and specifically for the dying phase, defined by relevant stakeholders, can ultimately be translated into best care for the dying person. Patient care will be improved by allowing clinicians to choose effective and meaningful treatments, and research impact will be improved by employing internationally agreed clinically relevant endpoints and enabling accurate comparison between studies in systematic reviews and/or in meta-analyses.

Abstract Background In patients seen in the emergency department (ED) with acute stone obstruction many risk factors that indicate need for urgent renal drainage are known. However, in patients discharged from ED without renal drainage factors that can minimize revisit to the emergency department are not fully identified. We evaluated SIRS (systemic inflammatory response syndrome) as a risk factor for urgent renal drainage and revisit to the ED in patients with acute stone colic during their ED visit. Methods Retrospective review was performed of patients presenting to a tertiary academic emergency department (ED) from an obstructing ureteral or UPJ stone with hydronephrosis confirmed on an abdominal and pelvic CT scan. Data evaluated over a 3-year period included stone size, presence of UTI, presence or absence of SIRS and other clinical variables as risk factors for urgent renal drainage and ED revisits. Results 1983 patients with urolithiasis were seen at the ED and 649 patients had obstructive urolithiasis on CT scan. SIRS was diagnosed in 15% (99/649) patients. 54/99 (55%) patients with SIRS underwent urgent renal drainage compared to 99/550 (17%) in non-SIRS patients. In a multivariate analysis SIRS was a predictor of urgent intervention compared to non-SIRS patients (odds ratio 4.6, p

The characterization of immortalized canine osteosarcoma (OS) cell lines used for research has historically been based on phenotypic features such as cellular morphology and expression of bone specific markers. With the increasing use of these cell lines to investigate novel therapeutic approaches prior to in vivo translation, a much more detailed understanding regarding the genomic landscape of these lines is required to ensure accurate interpretation of findings. Here we report the first whole genome characterization of eight canine OS cell lines, including single nucleotide variants, copy number variants and other structural variants. Many alterations previously characterized in primary canine OS tissue were observed in these cell lines, including TP53 mutations, MYC copy number gains, loss of CDKN2A, PTEN, DLG2, MAGI2, and RB1 and structural variants involving SETD2, DLG2 and DMD. These data provide a new framework for understanding how best to incorporate in vitro findings generated using these cell lines into the design of future clinical studies involving dogs with spontaneous OS.

Abstract Background Provider recognition of level of functioning may be suboptimal in the dialysis setting, and this lack of recognition may lead to less patient-centered care. We aimed to assess whether delivery of an app-based, individualized functioning report would improve patients’ perceptions of patient-centeredness of care. Methods In this pre-post pilot study at three outpatient dialysis facilities in metropolitan Atlanta, an individualized functioning report—including information on physical performance, perceived physical functioning, and community mobility—was delivered to patients receiving hemodialysis (n = 43) and their providers. Qualitative and quantitative approaches were used to gather patient and provider feedback to develop and assess the report and app. Paired t test was used to test for differences in patient perception of patient-centeredness of care (PPPC) scores (range, 1 = most patient-centered to 4 = least patient-centered) 1 month after report delivery. Results Delivery of the reports to both patients and providers was not associated with a subsequent change in patients’ perceptions of patient-centeredness of their care (follow-up vs. baseline PPPC scores of 2.35 vs. 2.36; P > 0.9). However, patients and providers generally saw the potential of the report to improve the patient-centeredness of care and reacted positively to the individualized reports delivered in the pilot. Patients also reported willingness to undergo future assessments. However, while two-thirds of surveyed providers reported always or sometimes discussing the reports they received, most (98%) participating patients reported that no one on the dialysis care team had discussed the report with them within 1 month. Conclusions Potential lack of fidelity to the intervention precludes definitive conclusions about effects of the report on patient-centeredness of care. The disconnect between patients’ and providers’ perceptions of discussions of the report warrants future study. However, this study introduces a novel, individualized, multi-domain functional report that is easily implemented in the setting of hemodialysis. Our pilot study provides guidance for improving its use both clinically and in future pragmatic research studies, both within and beyond the dialysis population.

Objective To determine whether and how cognitive assessment data should be included in a report for patients with SLE and their providers.Methods Leveraging experiences from prior studies, we created a cognitive report that included a hypothetical patient’s results on tests of multiple domains based on the NIH Toolbox Fluid Cognition Battery. In focus groups that comprised patients with SLE (two groups) and their providers (two groups), feedback was sought on the presentation of results as well as the potential value of the report in the clinical setting.Results Feedback regarding the presentation of the report was generally positive. Both patients with SLE and their providers liked its simple graphics and use of a colour-gradated scale to indicate performance. However, both groups stressed the importance of using non-stigmatising language in describing results. Several potential purposes of the report, including distinguishing cognitive versus other issues, explaining cognitive challenges, improving patient–provider interactions, guiding decision-making, improving functioning or preventing impairment and tracking cognitive function over time, were noted by the participants. Potential barriers, such as inadequate clinical staffing or time and lack of potential treatments for identified issues, were also discussed.Conclusion In this exploratory study, we found that both patients with SLE and their providers were receptive to the idea of a patient-friendly report of cognitive test results. This study provides important information to guide future pragmatic research to optimise the delivery of cognitive information to patients with SLE.

Summary: Long-lived rodents have become an attractive model for the studies on aging. To understand evolutionary paths to long life, we prepare chromosome-level genome assemblies of the two longest-lived rodents, Canadian beaver (Castor canadensis) and naked mole rat (NMR, Heterocephalus glaber), which were scaffolded with in vitro proximity ligation and chromosome conformation capture data and complemented with long-read sequencing. Our comparative genomic analyses reveal that amino acid substitutions at “disease-causing” sites are widespread in the rodent genomes and that identical substitutions in long-lived rodents are associated with common adaptive phenotypes, e.g., enhanced resistance to DNA damage and cellular stress. By employing a newly developed substitution model and likelihood ratio test, we find that energy and fatty acid metabolism pathways are enriched for signals of positive selection in both long-lived rodents. Thus, the high-quality genome resource of long-lived rodents can assist in the discovery of genetic factors that control longevity and adaptive evolution.

Hemophagocytic lymphohistiocytosis (HLH) is also known as hemophagocytic syndrome. It is a lethal hematologic condition due to a dysregulated immune response which results in inappropriately activated macrophages damaging host tissues. Based on the etiology, HLH can be primary (genetic) or secondary (acquired). The most common cause of a secondary HLH is an infection. Viral infections are the most common cause of secondary HLH. Among the viral causes of secondary HLH, Epstein–Barr virus is the most common etiologic agent. Cytomegalovirus (CMV) is a common causative pathogen in the immunocompromised host but is rare in an immunocompetent adult. In infection- associated secondary HLH, treatment includes antimicrobial therapy. HLH carries a high mortality and morbidity rate as it is an underdiagnosed clinical condition. Successful early diagnosis allows for adequate time for curative therapy. Treatment for HLH includes chemotherapy, immunomodulators, and a hematopoietic stem-cell transplant. The 2004 diagnostic criteria set by the Histiocyte Society serves as a guide to make an earlier clinical diagnosis. A review of PubMed literature revealed only five reported cases of CMV-induced HLH. We describe the sixth case of CMV pneumonitis-induced HLH and syndrome of inappropriate antidiuretic hormone secretion in a 72-year-old White male. He was treated successfully with oral valganciclovir and corticosteroids.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE—detected in the Yoruba population of Nigeria—conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the “emerging” nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz’s critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder with symptoms including intrusive thoughts and time-consuming repetitive behaviors. Here Noh and colleagues identify genes enriched for functional variants associated with increased risk of OCD.

PurposeTriple negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer. AMP-activated protein kinase (AMPK) is a major energy regulator that suppresses tumor growth, and 1-(3-chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) is a novel AMPK activator that inhibits growth and induces apoptosis in colon cancer. The purpose of this project was to test the effects of FND-4b on AMPK activation, proliferation, and apoptosis in breast cancer with a particular emphasis on TNBC.Materials and methods(i) Estrogen-receptor positive breast cancer (ER+BC; MCF-7, and T-47D), TNBC (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND-4b for 24h. Immunoblot analysis assessed AMPK, acetyl-CoA carboxylase (ACC), ribosomal protein S6, cyclin D1, and cleaved PARP. (ii) Sulforhodamine B growth assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. Proliferation was also assessed by counting cells after 72h of FND-4b treatment. (iii) Cell death ELISA assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h.Results(i) FND-4b increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in all subtypes. (ii) FND-4b decreased proliferation in all cells, while dose-dependent growth decreases were found in ER+BC and TNBC. (iii) Increases in apoptosis were observed in ER+BC and the MDA-MB-231 cell line with FND-4b treatment.ConclusionsOur findings indicate that FND-4b decreases proliferation for a variety of breast cancers by activating AMPK and has notable effects on TNBC. The growth reductions were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+BC cells were more susceptible to FND-4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose treatment. Further development of FND compounds could result in a novel therapeutic for TNBC.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US with the majority of deaths due to metastatic disease. Current chemotherapeutic regimens involve highly toxic agents, which limits their utility; therefore, more effective and less toxic agents are required to see a reduction in CRC mortality. Novel fluorinated N,N'-diarylureas (FND) were developed and characterized by our group as potent activators of adenosine monophosphate-activated kinase (AMPK) that inhibit cell cycle progression. The purpose of this study was to determine the effect of a lead FND compound, FND-4b, either alone or combined with PI-103 (a dual PI3K/mTOR inhibitor) or SN-38 (active metabolite of irinotecan) on cell cycle arrest and apoptosis of CRC cell lines (both commercially-available and novel lines established from our patient population). Treatment with FND-4b for 24h resulted in a marked induction of phosphorylated AMPK expression and a concomitant reduction in markers of cell proliferation, such as cyclin D1, in all CRC cell lines. Apoptosis was also notably increased in CRC cells treated with FND-4b. Regardless of the genetic profile of the CRC cells, FND-4b treatment alone resulted in decreased cell proliferation. Moreover, the combination of FND-4b with PI-103 resulted in increased cell death in all cell lines, while the combination of FND-4b with SN-38 resulted in increased cell death in select cell lines. Our findings identify FND-4b, which activates AMPK at micromolar concentrations, as a novel and effective inhibitor of CRC growth either alone or in combination with PI-103 and SN-38.

Patients with advanced-stage gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have a poor overall prognosis despite chemotherapy and radiotherapy (e.g., peptide receptor radionuclide therapy (PRRT)). Better treatment options are needed to improve disease regression and patient survival. The purpose of this study was to examine a new treatment strategy by combining PI3K/mTOR dual inhibition and radiotherapy. First, we assessed the efficacy of two PI3K/mTOR dual inhibitors, PF-04691502 and PKI-402, to inhibit pAkt and increase apoptosis in NET cell lines (BON and QGP-1) and patient-derived tumor spheroids as single agents or combined with radiotherapy (XRT). Treatment with PF-04691502 decreased pAkt (Ser473) expression for up to 72 h compared with the control; in contrast, decreased pAkt expression was noted for less than 24 h with PKI-402. Simultaneous treatment with PF-04691502 and XRT did not induce apoptosis in NET cells; however, the addition of PF-04691502 48 h after XRT significantly increased apoptosis compared to PF-04691502 or XRT treatment alone. Our results demonstrate that schedule-dependent administration of a PI3K/mTOR inhibitor, combined with XRT, can enhance cytotoxicity by promoting the radiosensitivity of NET cells. Moreover, our findings suggest that radiotherapy, in combination with timed PI3K/mTOR inhibition, may be a promising therapeutic regimen for patients with GEP-NET.

Chemical defoliation of seed corn production fields accelerates seed maturation and desiccation and expedites seed harvest. Early seed harvest is important to minimize the risk of frost damage while in the field. This newly adopted seed production practice also allows seed companies to plan harvest and manage dryer space more efficiently. However, premature defoliation may interfere with the migration of oil bodies within embryo cells during desiccation and affect seed germination and vigor. The objective of this study was to investigate the effect of chemical defoliation on the migration patterns of oil bodies within embryo cells during desiccation. Chemically defoliated and non-defoliated plants from five commercial hybrid seed corn fields were sampled in 2014 and 2015. Whole ears with husks were harvested before and after defoliant application at 600 g H2O kg−1 fresh weight (fw), and weekly thereafter until seed reached approximately 300–350 g H2O kg−1 fw. Ten embryos extracted from center-row seeds were fixed to stop metabolic processes, then sliced, processed, and photographed using scanning transmission electron microscopy. The oil bodies within embryo cells followed normal migration patterns according to seed moisture content, regardless of defoliation treatment. Seed germination and vigor were verified and were not significantly affected by defoliation. Chemical defoliation is a viable production practice to accelerate seed corn desiccation and to manage harvest and seed dryer availability more efficiently without negatively affecting seed germination and vigor.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by poor survival. Radiotherapy plays an important role in treating TNBC. The purpose of this study was to determine whether inhibiting the AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) pathways alone or in combination potentiates radiotherapy in TNBC. AMPKα1 and AMPKα2 knockdown diminished cyclin D1 expression and induced G1 cell cycle arrest but did not induce apoptosis alone or in combination with radiotherapy. Next, we analyzed the role of PI3K p85α, p85β, p110α, p110β, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction. Akt1 and p110α knockdown diminished cyclin D1 expression and induced apoptosis. Silencing Akt1 promoted synergistic apoptosis induction during radiotherapy and further reduced survival after radiation. Treatment with the Akt inhibitor, MK-2206 48 h after radiotherapy decreased Akt1 levels and potentiated radiation-induced apoptosis. Together, our results demonstrate that AMPKα, p110α, and Akt1 promote TNBC proliferation and that Akt1 is a key regulator of radiosensitivity in TNBC. Importantly, combining radiotherapy with the pharmacological inhibition of Akt1 expression is a potentially promising approach for the treatment of TNBC.

The domestic dog, Canis familiaris, is a valuable model for studying human diseases. The publication of the latest Canine genome build and annotation, CanFam3.1 provides an opportunity to enhance our understanding of gene regulation across tissues in the dog model system. In this study, we used the latest dog genome assembly and small RNA sequencing data from 9 different dog tissues to predict novel miRNAs in the dog genome, as well as to annotate conserved miRNAs from the miRBase database that were missing from the current dog annotation. We used both miRCat and miRDeep2 algorithms to computationally predict miRNA loci. The resulting, putative hairpin sequences were analysed in order to discard false positives, based on predicted secondary structures and patterns of small RNA read alignments. Results were further divided into high and low confidence miRNAs, using the same criteria. We generated tissue specific expression profiles for the resulting set of 811 loci: 720 conserved miRNAs, (207 of which had not been previously annotated in the dog genome) and 91 novel miRNA loci. Comparative analyses revealed 8 putative homologues of some novel miRNA in ferret, and one in microbat. All miRNAs were also classified into the genic and intergenic categories, based on the Ensembl RefSeq gene annotation for CanFam3.1. This additionally allowed us to identify four previously undescribed MiRtrons among our total set of miRNAs. We additionally annotated piRNAs, using proTRAC on the same input data. We thus identified 263 putative clusters, most of which (211 clusters) were found to be expressed in testis. Our results represent an important improvement of the dog genome annotation, paving the way to further research on the evolution of gene regulation, as well as on the contribution of post-transcriptional regulation to pathological conditions.

Esophageal diverticulum is a rare but important cause of dysphagia in children. We present a case of a two year old female with dysphagia from six months of age. Her symptoms were refractory to acid suppressive and prokinetic therapies. Upper GI revealed an anterior proximal esophageal diverticulum consistent with Killian-Jamieson Diverticulum. This was confirmed with EGD and corrected with right cervical exploration with resection of the diverticulum. There are three types of esophageal diverticula based on location in the esophagus. Killian-Jamieson diverticulum is very rare, with less than 30 cases reported, all in the elderly. This is the first reported pediatric case of Killian-Jamieson diverticulum.

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.

The domestic dog, Canis familiaris, is a well-established model system for mapping trait and disease loci. While the original draft sequence was of good quality, gaps were abundant particularly in promoter regions of the genome, negatively impacting the annotation and study of candidate genes. Here, we present an improved genome build, canFam3.1, which includes 85 MB of novel sequence and now covers 99.8% of the euchromatic portion of the genome. We also present multiple RNA-Sequencing data sets from 10 different canine tissues to catalog ∼175,000 expressed loci. While about 90% of the coding genes previously annotated by EnsEMBL have measurable expression in at least one sample, the number of transcript isoforms detected by our data expands the EnsEMBL annotations by a factor of four. Syntenic comparison with the human genome revealed an additional ∼3,000 loci that are characterized as protein coding in human and were also expressed in the dog, suggesting that those were previously not annotated in the EnsEMBL canine gene set. In addition to ∼20,700 high-confidence protein coding loci, we found ∼4,600 antisense transcripts overlapping exons of protein coding genes, ∼7,200 intergenic multi-exon transcripts without coding potential, likely candidates for long intergenic non-coding RNAs (lincRNAs) and ∼11,000 transcripts were reported by two different library construction methods but did not fit any of the above categories. Of the lincRNAs, about 6,000 have no annotated orthologs in human or mouse. Functional analysis of two novel transcripts with shRNA in a mouse kidney cell line altered cell morphology and motility. All in all, we provide a much-improved annotation of the canine genome and suggest regulatory functions for several of the novel non-coding transcripts.

On modern computers memory access patterns and cache utilization are as important, if not more important, than operation count in obtaining high-performance implementations of algorithms. In this work, the memory behavior of a large family of algorithms for computing the Walsh-Hadamard transform, an important signal processing transform related to the fast Fourier transform, is investigated. Empirical evidence shows that the family of algorithms exhibit a wide range of performance, despite the fact that all algorithms perform the same number of arithmetic operations. Different algorithms, while having the same number of memory operations, access memory in different patterns and consequently have different numbers of cache misses. A recurrence relation is derived for the number of cache misses and is used to determine the distribution of cache misses over the space of WHT algorithms.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.