Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome-depleted regions.

Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single-molecule fluorescence studies show that the Cbf1 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones, whereas the Pho4 HLH region does not. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single-molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by PFs and how this translates to facilitating chromatin opening inside cells.

Cryo-EM structure of the ß3-adrenergic receptor reveals the molecular basis of subtype selectivity.

The ß3-adrenergic receptor (ß3AR) is predominantly expressed in adipose tissue and urinary bladder and has emerged as an attractive drug target for the treatment of type 2 diabetes, obesity, and overactive bladder (OAB). Here, we report the cryogenic electron microscopy structure of the ß3AR-Gs signaling complex with the selective agonist mirabegron, a first-in-class drug for OAB. Comparison of this structure with the previously reported ß1AR and ß2AR structures reveals a receptor activation mechanism upon mirabegron binding to the orthosteric site. Notably, the narrower exosite in ß3AR creates a perpendicular pocket for mirabegron. Mutational analyses suggest that a combination of both the exosite shape and the amino-acid-residue substitutions defines the drug selectivity of the ßAR agonists. Our findings provide a molecular basis for ßAR subtype selectivity, allowing the design of more-selective agents with fewer adverse effects.

Boosting exciton dissociation and charge transfer in CsPbBr3 QDs via ferrocene derivative ligation for CO2 photoreduction.

Photo-catalytic CO2 reduction with perovskite quantum dots (QDs) shows potential for solar energy storage, but it encounters challenges due to the intricate multi-electron photoreduction processes and thermodynamic and kinetic obstacles associated with them. This study aimed to improve photo-catalytic performance by addressing surface barriers and utilizing multiple-exciton generation in perovskite QDs. A facile surface engineering method was employed, involving the grafting of ferrocene carboxylic acid (FCA) onto CsPbBr3 (CPB) QDs, to overcome limitations arising from restricted multiple-exciton dissociation and inefficient charge transfer dynamics. Kelvin Probe Force Microscopy and XPS spectral confirmed successfully creating an FCA-modulated microelectric field through the Cs active site, thus facilitating electron transfer, disrupting surface barrier energy, and promoting multi-exciton dissociations. Transient absorption spectroscopy showed enhanced charge transfer and reduced energy barriers, resulting in an impressive CO2-to-CO conversion rate of 132.8 µmol g-1 h-1 with 96.5% selectivity. The CPB-FCA catalyst exhibited four-cycle reusability and 72 h of long-term stability, marking a significant nine-fold improvement compared to pristine CPB (14.4 µmol g-1 h-1). These results provide insights into the influential role of FCA in regulating intramolecular charge transfer, enhancing multi-exciton dissociation, and improving CO2 photoreduction on CPB QDs. Furthermore, these findings offer valuable knowledge for controlling quantum-confined exciton dissociation to enhance CO2 photocatalysis.

Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Unexpected catalytic activity of nanorippled graphene.

Graphite is one of the most chemically inert materials. Its elementary constituent, monolayer graphene, is generally expected to inherit most of the parent material's properties including chemical inertness. Here, we show that, unlike graphite, defect-free monolayer graphene exhibits a strong activity with respect to splitting molecular hydrogen, which is comparable to that of metallic and other known catalysts for this reaction. We attribute the unexpected catalytic activity to surface corrugations (nanoscale ripples), a conclusion supported by theory. Nanoripples are likely to play a role in other chemical reactions involving graphene and, because nanorippling is inherent to atomically thin crystals, can be important for two-dimensional (2D) materials in general.

Mmp-induced fat body cell dissociation promotes pupal development and moderately averts pupal diapause by activating lipid metabolism.

In Lepidoptera and Diptera, the fat body dissociates into single cells in nondiapause pupae, but it does not dissociate in diapause pupae until diapause termination. Using the cotton bollworm, Helicoverpa armigera, as a model of pupal diapause insects, we illustrated the catalytic mechanism and physiological importance of fat body cell dissociation in regulating pupal development and diapause. In nondiapause pupae, cathepsin L (CatL) activates matrix metalloproteinases (Mmps) that degrade extracellular matrix proteins and cause fat body cell dissociation. Mmp-induced fat body cell dissociation activates lipid metabolism through transcriptional regulation, and the resulting energetic supplies increase brain metabolic activity (i.e., mitochondria respiration and insulin signaling) and thus promote pupal development. In diapause pupae, low activities of CatL and Mmps prevent fat body cell dissociation and lipid metabolism from occurring, maintaining pupal diapause. Importantly, as demonstrated by chemical inhibitor treatments and CRISPR-mediated gene knockouts, Mmp inhibition delayed pupal development and moderately increased the incidence of pupal diapause, while Mmp stimulation promoted pupal development and moderately averted pupal diapause. This study advances our recent understanding of fat body biology and insect diapause regulation.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Characterization of the degradation products of lignocellulosic biomass by using tandem mass spectrometry experiments, model compounds, and quantum chemical calculations.

Biomass-derived degraded lignin and cellulose serve as possible alternatives to fossil fuels for energy and chemical resources. Fast pyrolysis of lignocellulosic biomass generates bio-oil that needs further refinement. However, as pyrolysis causes massive degradation to lignin and cellulose, this process produces very complex mixtures. The same applies to degradation methods other than fast pyrolysis. The ability to identify the degradation products of lignocellulosic biomass is of great importance to be able to optimize methodologies for the conversion of these mixtures to transportation fuels and valuable chemicals. Studies utilizing tandem mass spectrometry have provided invaluable, molecular-level information regarding the identities of compounds in degraded biomass. This review focuses on the molecular-level characterization of fast pyrolysis and other degradation products of lignin and cellulose via tandem mass spectrometry based on collision-activated dissociation (CAD). Many studies discussed here used model compounds to better understand both the ionization chemistry of the degradation products of lignin and cellulose and their ions' CAD reactions in mass spectrometers to develop methods for the structural characterization of the degradation products of lignocellulosic biomass. Further, model compound studies were also carried out to delineate the mechanisms of the fast pyrolysis reactions of lignocellulosic biomass. The above knowledge was used to assign likely structures to many degradation products of lignocellulosic biomass.

Limitation of life sustaining therapy in disorders of consciousness: ethics and practice.

Clinical conversations surrounding the continuation or limitation of life-sustaining therapies (LLST) are both challenging and tragically necessary for patients with disorders of consciousness (DoC) following severe brain injury. Divergent cultural, philosophical and religious perspectives contribute to vast heterogeneity in clinical approaches to LLST-as reflected in regional differences and inter-clinician variability. Here we provide an ethical analysis of factors that inform LLST decisions among patients with DoC. We begin by introducing the clinical and ethical challenge and clarifying the distinction between withdrawing and withholding life-sustaining therapy. We then describe relevant factors that influence LLST decision-making including diagnostic and prognostic uncertainty, perception of pain, defining a 'good' outcome, and the role of clinicians. In concluding sections, we explore global variation in LLST practices as they pertain to patients with DoC and examine the impact of cultural and religious perspectives on approaches to LLST. Understanding and respecting the cultural and religious perspectives of patients and surrogates is essential to protecting patient autonomy and advancing goal-concordant care during critical moments of medical decision-making involving patients with DoC.

Spontaneous exciton dissociation in organic photocatalyst under ambient conditions for highly efficient synthesis of hydrogen peroxide.

SignificanceHydrogen peroxide is a highly competitive ready-to-use product for solar energy transformation. Nevertheless, the contemporary photosynthetic systems are not efficient enough, due to severe charge recombination caused by high activation energy and binding energy of the exciton. Herein, we achieve spontaneous exciton dissociation at room temperature. Moreover, the photosynthesis of H2O2 reaches between 9,366 and 12,324 µmol·g-1 from 9 AM to 4 PM in ambient conditions, that is, sunlight irradiation, real water including fresh water and seawater, room temperature, and open air. The ultrahigh photocatalytic efficiency in ambient conditions allows the solar-to-chemical conversion in a real cost-effective and sustainable way, which represents an important step toward real applications.

Atomistic measurement and modeling of intrinsic fracture toughness of two-dimensional materials.

Quantifying the intrinsic mechanical properties of two-dimensional (2D) materials is essential to predict the long-term reliability of materials and systems in emerging applications ranging from energy to health to next-generation sensors and electronics. Currently, measurements of fracture toughness and identification of associated atomistic mechanisms remain challenging. Herein, we report an integrated experimental-computational framework in which in-situ high-resolution transmission electron microscopy (HRTEM) measurements of the intrinsic fracture energy of monolayer MoS2 and MoSe2 are in good agreement with atomistic model predictions based on an accurately parameterized interatomic potential. Changes in crystalline structures at the crack tip and crack edges, as observed in in-situ HRTEM crack extension tests, are properly predicted. Such a good agreement is the result of including large deformation pathways and phase transitions in the parameterization of the inter-atomic potential. The established framework emerges as a robust approach to determine the predictive capabilities of molecular dynamics models employed in the screening of 2D materials, in the spirit of the materials genome initiative. Moreover, it enables device-level predictions with superior accuracy (e.g., fatigue lifetime predictions of electro- and opto-electronic nanodevices).

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and Minimizing Cellular Stress.

The human lung is a complex organ comprised of diverse populations of epithelial, mesenchymal, vascular and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short), which provides a high-yield single cell suspension suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared to four other dissociation protocols. Resulting single cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and non-hematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue with no reduction in viability when compared to previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared to four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Optimization of mouse kidney digestion protocols for single-cell applications.

Single-cell technologies such as flow cytometry and single-cell RNA sequencing have allowed for comprehensive characterization of the kidney cellulome. However, there is a disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterizing kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: a scRNAseq protocol (P1), a multi-tissue digestion kit from Miltenyi Biotec (P2), and a protocol established in our laboratory (P3). Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal, and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B intercalating cells compared with the other techniques. P1 and P3 produced similar yields for most cell types; however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich specific kidney cell types may benefit from this comparative analysis.NEW & NOTEWORTHY This study is the first to evaluate published single-cell suspension preparation protocols and their ability to produce high-quality cellular yields from the mouse kidney. Three single-cell digestion protocols were compared and each produced significant differences in kidney cellular heterogeneity. These findings highlight the importance of the digestion protocol when using single-cell technologies. This study may help future single-cell science research by guiding researchers to choose protocols that enrich certain cell types of interest.

Mechanisms underlying category learning in the human ventral occipito-temporal cortex.

The human ventral occipito-temporal cortex (VOTC) has evolved into specialized regions that process specific categories, such as words, tools, and animals. The formation of these areas is driven by bottom-up visual and top-down nonvisual experiences. However, the specific mechanisms through which top-down nonvisual experiences modulate category-specific regions in the VOTC are still unknown. To address this question, we conducted a study in which participants were trained for approximately 13 h to associate three sets of novel meaningless figures with different top-down nonvisual features: the wordlike category with word features, the non-wordlike category with nonword features, and the visual familiarity condition with no nonvisual features. Pre- and post-training functional MRI (fMRI) experiments were used to measure brain activity during stimulus presentation. Our results revealed that training induced a categorical preference for the two training categories within the VOTC. Moreover, the locations of two training category-specific regions exhibited a notable overlap. Remarkably, within the overlapping category-specific region, training resulted in a dissociation in activation intensity and pattern between the two training categories. These findings provide important insights into how different nonvisual categorical information is encoded in the human VOTC.

Unconsciousness or unresponsiveness in akinetic mutism? Insights from a multimodal longitudinal exploration.

The clinical assessment of patients with disorders of consciousness (DoC) relies on the observation of behavioural responses to standardised sensory stimulation. However, several medical comorbidities may directly impair the production of reproducible and appropriate responses, thus reducing the sensitivity of behaviour-based diagnoses. One such comorbidity is akinetic mutism (AM), a rare neurological syndrome characterised by the inability to initiate volitional motor responses, sometimes associated with clinical presentations that overlap with those of DoC. In this paper, we describe the case of a patient with large bilateral mesial frontal lesions, showing prolonged behavioural unresponsiveness and severe disorganisation of electroencephalographic (EEG) background, compatible with a vegetative state/unresponsive wakefulness syndrome (VS/UWS). By applying an unprecedented multimodal battery of advanced imaging and electrophysiology-based techniques (AIE) encompassing spontaneous EEG, evoked potentials, event-related potentials, transcranial magnetic stimulation combined with EEG and structural and functional MRI, we provide the following: (i) a demonstration of the preservation of consciousness despite unresponsiveness in the context of AM, (ii) a plausible neurophysiological explanation for behavioural unresponsiveness and its subsequent recovery during rehabilitation stay and (iii) novel insights into the relationships between DoC, AM and parkinsonism. The present case offers proof-of-principle evidence supporting the clinical utility of a multimodal hierarchical workflow that combines AIEs to detect covert signs of consciousness in unresponsive patients.

Effect of ST36 electroacupuncture on the switch of skeletal muscle fibres in mice with sciatic nerve dissociation.

Skeletal muscle is striated muscle that moves autonomously and is innervated by peripheral nerves. Peripheral nerve injury is very common in clinical treatment. However, the commonly used treatment methods often focus on the regeneration of the injured nerve but overlook the pathological changes in the injured skeletal muscle. Acupuncture, as the main treatment for denervated skeletal muscle atrophy, is used extensively in clinical practice. In the present study, a mouse model of lower limb sciatic nerve detachment was constructed and treated with electroacupuncture Stomach 36 to observe the atrophy of lower limb skeletal muscle and changes in skeletal muscle fibre types before and after electroacupuncture Stomach 36 treatment. Mice with skeletal muscle denervation showed a decrease in the proportion of IIa muscle fibres and an increase in the proportion of IIb muscle fibres, after electroacupuncture Stomach 36. The changes were reversed by specific activators of p38 MAPK, which increased IIa myofibre ratio. The results suggest that electroacupuncture Stomach 36 can reverse the change of muscle fibre type from IIb to IIa after denervation of skeletal muscle by inhibiting p38 MAPK. The results provide an important theoretical basis for the treatment of clinical peripheral nerve injury diseases with electroacupuncture, in addition to novel insights that could facilitate the study of pathological changes of denervated skeletal muscle.

Biophysical library screening using a Thermo-FMN assay to identify and characterize Clostridioides difficile FabK inhibitors.

Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism's fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme's FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.