Lactate regulates cell cycle by remodelling the anaphase promoting complex.

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.

Minimum information guidelines for experiments structurally characterizing intrinsically disordered protein regions.

An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.

An open-source drug discovery platform enables ultra-large virtual screens.

On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.

pUL36 Deubiquitinase Activity Augments Both the Initiation and the Progression of Lytic Herpes Simplex Virus Infection in IFN-Primed Cells.

The evolutionarily conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal deubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo, as it acts as a potent viral immune evasin. Interferon (IFN) induces the expression of hundreds of antiviral factors, including many ubiquitin modulators, which HSV-1 needs to neutralize to efficiently initiate a productive infection. Herein, we discover two functions of the conserved pUL36 DUB during lytic replication in cell culture in an understudied but equally important scenario of HSV-1 infection in IFN-treated cells. Our data indicate that the pUL36 DUB contributes to overcoming the IFN-mediated suppression of productive infection in both the early and late phases of HSV-1 infection. We show that incoming tegument-derived pUL36 DUB activity contributes to the IFN resistance of HSV-1 in IFN-primed cells to efficiently initiate lytic virus replication. Subsequently, the de novo expressed DUB augmented the efficiency of virus replication and increased the output of infectious virus. Notably, the DUB defect was only apparent when IFN was applied prior to infection. Our data indicate that IFN-induced defense mechanisms exist and that they work to both neutralize infectivity early on and slow the progression of HSV-1 replication in the late stages of infection. Also, our data indicate that pUL36 DUB activity contributes to the disarming of these host responses. IMPORTANCE HSV-1 is a ubiquitous human pathogen that is responsible for common cold sores and may also cause life-threatening disease. pUL36 is an essential, conserved herpesvirus protein with N-terminal deubiquitinating (DUB) activity. The DUB is dispensable for HSV-1 replication in cell culture but represents an important viral immune evasin in vivo. IFN plays a pivotal role in HSV-1 infection and suppresses viral replication both in vitro and in vivo. Here, we show that DUB activity contributes to overcoming IFN-induced cellular resistance in order to more efficiently initiate lytic replication and produce infectious virions. As such, DUB activity in the incoming virions increases their infectivity, while the de novo synthesized DUB augments productive infection. Thus, the HSV-1 DUB antagonizes the activity of IFN-inducible effector proteins to facilitate productive infection at multiple levels. Our findings underscore the importance of using more challenging cell culture systems to fully understand virus protein functions.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

VirtualFlow Ants-Ultra-Large Virtual Screenings with Artificial Intelligence Driven Docking Algorithm Based on Ant Colony Optimization.

The docking program PLANTS, which is based on ant colony optimization (ACO) algorithm, has many advanced features for molecular docking. Among them are multiple scoring functions, the possibility to model explicit displaceable water molecules, and the inclusion of experimental constraints. Here, we add support of PLANTS to VirtualFlow (VirtualFlow Ants), which adds a valuable method for primary virtual screenings and rescoring procedures. Furthermore, we have added support of ligand libraries in the MOL2 format, as well as on the fly conversion of ligand libraries which are in the PDBQT format to the MOL2 format to endow VirtualFlow Ants with an increased flexibility regarding the ligand libraries. The on the fly conversion is carried out with Open Babel and the program SPORES. We applied VirtualFlow Ants to a test system involving KEAP1 on the Google Cloud up to 128,000 CPUs, and the observed scaling behavior is approximately linear. Furthermore, we have adjusted several central docking parameters of PLANTS (such as the speed parameter or the number of ants) and screened 10 million compounds for each of the 10 resulting docking scenarios. We analyzed their docking scores and average docking times, which are key factors in virtual screenings. The possibility of carrying out ultra-large virtual screening with PLANTS via VirtualFlow Ants opens new avenues in computational drug discovery.

Evaluating the Spectrum of Cognitive-Motor Relationships During Dual-Task Jump Landing.

Cognitive function plays a role in understanding noncontact anterior cruciate ligament injuries, but the research into how cognitive function influences sport-specific movements is underdeveloped. The purpose of this study was to determine how various cognitive tasks influenced dual-task jump-landing performance along with how individuals' baseline cognitive ability mediated these relationships. Forty female recreational soccer and basketball players completed baseline cognitive function assessments and dual-task jump landings. The baseline cognitive assessments quantified individual processing speed, multitasking, attentional control, and primary memory ability. Dual-task conditions for the jump landing included unanticipated and anticipated jump performance, with and without concurrent working memory and captured visual attention tasks. Knee kinematics and kinetics were acquired through motion capture and ground reaction force data. Jumping conditions that directed visual attention away from the landing, whether anticipated or unanticipated, were associated with decreased peak knee flexion angle (P < .001). No interactions between cognitive function measures and jump-landing conditions were observed for any of the biomechanical variables, suggesting that injury-relevant cognitive-motor relationships may be specific to secondary task demands and movement requirements. This work provides insight into group- and subject-specific effects of established anticipatory and novel working memory dual-task paradigms on the neuromuscular control of a sport-specific movement.

FoxH1 represses miR-430 during early embryonic development of zebrafish via non-canonical regulation.

BACKGROUND: FoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression. RESULTS: We identify the conserved microRNA-430 family (miR-430) as a novel target of FoxH1. miR-430 levels are increased in foxH1 mutants, resulting in a reduced expression of transcripts that are targeted by miR-430 for degradation. To determine the underlying mechanism of miR-430 repression, we performed chromatin immunoprecipitation studies and overexpression experiments with mutant as well as constitutive active and repressive forms of FoxH1. Our studies reveal a molecular interaction of FoxH1 with miR-430 loci independent of the FHD. Furthermore, we show that previously described mutant forms of FoxH1 that disrupt DNA binding or that lack the C-terminal Smad Interaction Domain (SID) dominantly interfere with miR-430 repression, but not with the regulation of previously described FoxH1 targets. CONCLUSIONS: We were able to identify the distinct roles of protein domains of FoxH1 in the regulation process of miR-430. We provide evidence that the indirect repression of miR-430 loci depends on the connection to a distal repressive chromosome environment via a non-canonical mode. The widespread distribution of such non-canonical binding sites of FoxH1, found not only in our study, argues against a function restricted to regulating miR-430 and for a more global role of FoxH1 in chromatin folding.

Time course of nocturnal cough and wheezing in children with acute bronchitis monitored by lung sound analysis.

Cough and wheezing are the predominant symptoms of acute bronchitis. Hitherto, the evaluation of respiratory symptoms was limited to subjective methods such as questionnaires. The main objective of this study was to objectively determine the time course of cough and wheezing in children with acute bronchitis. The impact of nocturnal cough on parent's quality of life was assessed as secondary outcome. In 36 children (2-8 years), the frequency of nocturnal cough and wheezing was recorded during three nights by automated lung sound monitoring. Additionally, parents completed symptom logs, i.e., the Bronchitis Severity Score (BSS), as well as the Parent-proxy Children's Acute Cough-specific Quality of Life Questionnaire (PAC-QoL). During the first night, patients had 34.4 ± 52.3 (mean ± SD) cough epochs, which were significantly reduced in night 5 (13.5 ± 26.5; p < 0.001) and night 9 (12.8 ± 28.1; p < 0.001). Twenty-two patients had concomitant wheezing, which declined within the observation period as well. All subjective parameters (BSS, Cough log and PAC-QoL) were found to be significantly correlated with the objectively assessed cough parameters.Conclusion: Long-term recording of cough and wheezing offers a useful opportunity to objectively evaluate the time course of respiratory symptoms in children with acute bronchitis. To assess putative effects of pharmacotherapy on nocturnal bronchitis symptoms, future studies in more homogeneous patient groups are needed. What is Known: • Cough and wheezing are the predominant symptoms of acute bronchitis. • There is a diagnostic gap in long-term assessment of these respiratory symptoms, which needs to be closed to optimize individual therapies. What is New: • Long-term recording of nocturnal cough and wheezing allows for objective evaluation of respiratory symptoms in children with acute bronchitis and provides a tool to validate the efficacy of symptomatic bronchitis therapies.

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila.

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.

Long-term Recording of Night-Time Respiratory Symptoms in Patients with Stable COPD II-IV.

INTRODUCTION: Night-time respiratory symptoms have a considerable impact on sleep and life quality in patients with chronic obstructive pulmonary disease (COPD). Lack of awareness of night-time symptoms can lead to worsened COPD control. Automated long-term monitoring of respiratory symptoms with LEOSound enables assessment of nocturnal wheezing and cough. METHODS: In this observational study we investigated the prevalence and severity of cough and wheezing in patients with stable COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) II-IV] disease for two consecutive nights with the LEOSound system. 48 patients (30 males, 63%) were eligible for inclusion, median age was 67 years, and body mass index (BMI) was 25.3 kg/m2. RESULTS: In 15 out of 48 patients (31%), we found wheezing periods for at least 10-minute duration. Wheezing periods >30 minutes were monitored in seven patients and wheezing periods >60 minutes were monitored in three patients. The maximum duration of wheezing was 470 minutes in one patient with COPD II. The median wheezing rate differed between the COPD stages and between active and non-active smokers. Cough was found in 42 patients (87.5%) with a range of 1-326 events. The cough-period-index in night one was 0.83 n/hour (P25:0.33||P75: 2.04) and night two 0.97 n/hour (P25:0.25||P75: 1.9). Most of the cough events were non-productive with a median of 0.86. CONCLUSIONS: Night-time symptoms are common in COPD patients. LEOSound offers an opportunity to evaluate objectively night-time symptoms like wheezing and cough in patients with COPD which remain otherwise unnoticed. We found a high incidence of night-time wheezing in these patients, which was related to persistant smoking.

Cyclin G is involved in meiotic recombination repair in Drosophila melanogaster.

Cyclin G (CycG) belongs to the atypical cyclins, which have diverse cellular functions. The two mammalian CycG genes, CycG1 and CycG2, regulate the cell cycle in response to cell stress. Detailed analyses of the role of the single Drosophila cycG gene have been hampered by the lack of a mutant. We generated a null mutant in the Drosophila cycG gene that is female sterile and produces ventralised eggs. This phenotype is typical of the downregulation of epidermal growth factor receptor (EGFR) signalling during oogenesis. Ventralised eggs are also observed in mutants (for example, mutants of the spindle class) that are defective in meiotic DNA double-strand break repair. Double-strand breaks (DSBs) induce a meiotic checkpoint by activating Mei-41 kinase (the Drosophila ATR homologue), thereby indirectly causing dorsoventral patterning defects. We provide evidence for the role of CycG in meiotic checkpoint control. The increased incidence of DSBs in cycG mutant germaria may reflect inefficient DSB repair. Therefore, the downregulation of Mei-W68 (an endonuclease that induces meiotic DSBs), Mei-41, or Drosophila melanogaster Chk2 (a downstream kinase that initiates the meiotic checkpoint) rescues the cycG mutant eggshell phenotype. In vivo, CycG associates with Rad9 and BRCA2. These two proteins are components of the 9-1-1 complex, which is involved in sensing DSBs and in activating meiotic checkpoint control. Therefore, we propose that CycG has a role in an early step of meiotic recombination repair, thereby affecting EGFR-mediated patterning processes during oogenesis.

The interplay between autophagy and cGAS-STING signaling and its implications for cancer.

Autophagy is an intracellular process that targets various cargos for degradation, including members of the cGAS-STING signaling cascade. cGAS-STING senses cytosolic double-stranded DNA and triggers an innate immune response through type I interferons. Emerging evidence suggests that autophagy plays a crucial role in regulating and fine-tuning cGAS-STING signaling. Reciprocally, cGAS-STING pathway members can actively induce canonical as well as various non-canonical forms of autophagy, establishing a regulatory network of feedback mechanisms that alter both the cGAS-STING and the autophagic pathway. The crosstalk between autophagy and the cGAS-STING pathway impacts a wide variety of cellular processes such as protection against pathogenic infections as well as signaling in neurodegenerative disease, autoinflammatory disease and cancer. Here we provide a comprehensive overview of the mechanisms involved in autophagy and cGAS-STING signaling, with a specific focus on the interactions between the two pathways and their importance for cancer.

DistSNE: Distributed computing and online visualization of DNA methylation-based central nervous system tumor classification.

The current state-of-the-art analysis of central nervous system (CNS) tumors through DNA methylation profiling relies on the tumor classifier developed by Capper and colleagues, which centrally harnesses DNA methylation data provided by users. Here, we present a distributed-computing-based approach for CNS tumor classification that achieves a comparable performance to centralized systems while safeguarding privacy. We utilize the t-distributed neighborhood embedding (t-SNE) model for dimensionality reduction and visualization of tumor classification results in two-dimensional graphs in a distributed approach across multiple sites (DistSNE). DistSNE provides an intuitive web interface (https://gin-tsne.med.uni-giessen.de) for user-friendly local data management and federated methylome-based tumor classification calculations for multiple collaborators in a DataSHIELD environment. The freely accessible web interface supports convenient data upload, result review, and summary report generation. Importantly, increasing sample size as achieved through distributed access to additional datasets allows DistSNE to improve cluster analysis and enhance predictive power. Collectively, DistSNE enables a simple and fast classification of CNS tumors using large-scale methylation data from distributed sources, while maintaining the privacy and allowing easy and flexible network expansion to other institutes. This approach holds great potential for advancing human brain tumor classification and fostering collaborative precision medicine in neuro-oncology.

Influence of Fatigue on Cognitive-Motor Function During Unanticipated Landings.

BACKGROUND: Physical fatigue and cognitive performance have been suggested as risk factors for an anterior cruciate ligament (ACL) injury, and fatigue has also been demonstrated to reduce cognitive processing. The combined effects of fatigue and lower cognitive function during cognitive-challenging movements may increase knee mechanics associated with the ACL injury risk. HYPOTHESES: We hypothesized that (1) knee mechanics would be detrimentally affected by fatigue and associated with baseline cognitive function and (2) fatigue-induced deleterious changes in cognitive performance and knee mechanics would be correlated. STUDY DESIGN: Descriptive laboratory study. METHODS: A total of 22 athletes completed baseline cognitive testing. After performing maximal vertical jumps, they performed a jump-land-jump task based on unanticipated visual cues. Then, they completed a fatigue protocol including countermovement jumps, among other tasks, until the jump height decreased below 90% of their assessed maximum. Immediately after reaching the first fatigue point, they performed another set of jump-landing tasks, followed by repeating the fatigue protocol until the jump height decreased below 85% of their assessed maximum. After reaching the second fatigue point, they performed a final set of jump-landing tasks and repeated the initial cognitive assessment battery. RESULTS: Mixed-effects models revealed that knee flexion decreased through the fatigue protocol (baseline: 61.8°; midpoint: 61.1°; final: 60.1°; P = .003). Stepwise regression showed that fatigue-worsened attentional control corresponded to smaller knee abduction angles (R2adjusted = 51.68%; ßstandardized = 1.16; P = .001), and worse reaction time after fatigue correlated with increased knee abduction angles (ßstandardized = 0.85; P = .006) after accounting for the role of attentional control. CONCLUSION: Fatigue induced incremental modifications in sagittal-plane knee mechanics during an unanticipated sports movement. In addition, fatigue induced changes in cognitive function related to ACL injury-relevant knee mechanics. CLINICAL RELEVANCE: The novel findings regarding fatigue-dependent changes in injury-relevant biomechanics during cognitively challenging movements represent an extension of recent developments in understanding the role of cognition in the ACL injury risk.

Associations Between Cognitive Function and ACL Injury-Related Biomechanics: A Systematic Review.

CONTEXT: Does lower baseline cognitive function predispose athletes to ACL injury risk, especially when performing unplanned or dual-task movements? OBJECTIVE: To evaluate the association between cognitive function and biomechanics related to ACL injuries during cognitively challenging sports movements. DATA SOURCES: PubMed (MEDLINE), Web of Science, Scopus, and SciELO databases were searched; additional hand searching was also conducted. STUDY SELECTION: The following inclusion criteria had to be met: participants completed (1) a neurocognitive test, (2) a cognitively challenging sport-related task involving lower limbs, and (3) a biomechanical analysis. The following criteria determined exclusion from the review: studies involving participants with (1) recent or current musculoskeletal injuries; (2) recent or current concussion; (3) ACL surgical reconstruction, reviews of the literature, commentary or opinion articles, and case studies. STUDY DESIGN: Systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P) statement and registered at the International Prospective Register of Systematic Reviews (PROSPERO). LEVEL OF EVIDENCE: Level 3. DATA EXTRACTION: Two of authors independently extracted data and assessed the methodological quality of the articles with the Downs and Black and ROBINS-I checklists, to assess methodological quality and risk of bias, respectively. RESULTS: Six studies with different methodologies and confounding factors were included in this review. Of these 6 studies, 3 were ranked as high-quality, 3 demonstrated a low risk of bias, 2 a moderate risk, and 1 a severe risk. Five studies found a cognitive-motor relationship, with worse cognitive performance associated with increased injury risk, with 1 study reporting the opposite directionality for 1 variable. One study did not identify any interaction between cognitive function and biomechanical outcomes. CONCLUSION: Worse cognitive performance is associated with an increased injury risk profile during cognitively challenging movements.

Carrier protein mediated cargo sensing in quorum signal synthases.

Acyl-homoserine lactone synthases make specific AHL quorum sensing signals to aid virulence in Gram-negative bacteria. Here, we use solution NMR spectroscopy to demonstrate that the carrier protein-enzyme interface accurately reveals substrate recognition mechanisms in two quorum signal synthases.

Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity.

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5-cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8-12.2 µM. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin-resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli ΔtolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 µM respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 µg/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 µg/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

1H, 13C and 15N backbone and sidechain assignment of the Burkholderia mallei acyl carrier protein.

Acyl carrier proteins (ACPs) are universally conserved proteins amongst different species and are involved in fatty acid synthesis. Bacteria utilize ACPs as acyl carriers and donors for the synthesis of products such as endotoxins or acyl homoserine lactones (AHLs), which are used in quorum sensing mechanisms. In this study, wehave expressed isotopically labeled holo-ACP from Burkholderia mallei in Escherichia coli to assign 100% of non-proline backbone amide (HN) resonances, 95.5% of aliphatic carbon resonances and 98.6% of aliphatic hydrogen sidechain resonances.

SALL4B, not targeted by IMiD, is important for SALL4-mediated tumorigenesis.

Oncofetal transcription factor SALL4 is essential for cancer cell survival. 1-5 Recently, several groups reported that immunomodulatory imide drugs (IMiDs) could degrade SALL4 in a proteasome-dependent manner. 6,7 Intriguingly, we observed that IMiDs had no effect on SALL4-positive cancer cells. Further studies demonstrated that IMiDs could only degrade SALL4A, one of the SALL4 isoforms. This finding raises the possibility that SALL4B, the isoform not affected by IMiDs, may be essential for SALL4-mediated cancer cell survival. SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. SALL4B gain-of-function alone led to liver tumor formation in mice. Our observation that protein degraders can possess isoform-specific effects exemplifies the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.