Biology subjects

Mittal, N.

Publications and source records attributed to Mittal, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Phenolic Chemical Defense Contributes to Resistance against Multiple Soybean Cyst Nematode Populations in Wild Soybean

Soybean cyst nematode (Heterodera glycines, SCN) is one of the most damaging pathogens of soybean worldwide. Virulence diversity among SCN populations and reliance on a limited number of resistance sources present major challenges for durable SCN management. Here, we investigated a wild soybean (Glycine soja) genotype (WsR), that is resistant to two SCN populations, race 2 (HG type 1.2.5.7) and race 5 (HG type 2.5.7), and compared its transcriptional and metabolic responses with those of the susceptible genotype (WsS). WsR exhibited a substantially stronger defense-associated transcriptional response to both SCN populations, including preferential induction of genes associated with Ca2+/calmodulin and salicylic acid signaling. Integration of transcriptomic and time-resolved metabolomic analyses revealed convergence on phenolic metabolism, with enhanced accumulation of phenolic acids, flavonoids, and isoflavonoids in resistant WsR. Genes involved in phenolic biosynthesis and modification were also preferentially induced in WsR, linking transcriptional reprogramming with the observed metabolic response. Importantly, two resistance-associated phenolic compounds, 4-hydroxybenzaldehyde and 2,3-dihydroxybenzoic acid, directly increased mortality of SCN second-stage juveniles from both populations in a concentration-dependent manner. By connecting resistance-associated transcriptional responses and metabolic reprogramming with direct activity of specific phenolic compounds against SCN, our study provides functional evidence linking phenolic metabolism to chemical defense against SCN. Together, these findings identify enhanced phenolic chemical defense as a major component of resistance to multiple SCN populations in wild soybean and highlight wild soybean as a valuable source of molecular and biochemical diversity for SCN resistance.

plant biology↗

bioRxiv2026

Duplication of superoxide dismutase and a mutation in aquaglyceroporin mediates the sensitivity of Plasmodium falciparum to cryptosporin, a natural product derived from Acaromyces ingoldii

Cryptosporin, a fungal metabolite, exhibited potent antimalarial activity against both asexual blood stage Plasmodium falciparum and liver-stage Plasmodium berghei with minimal human HepG2 toxicity. Unlike atovaquone, cryptosporins mechanism is independent of mitochondrial electron transport. Minimum inoculum of resistance showed a low risk of resistance development. RNA-Seq analysis revealed the upregulation of genes associated with sexual development including many canonical markers such as Pfs25, and PfCCp3, suggesting a stress response that is also seen when parasites are treated with artemisinin. In vitro evolution and whole genome sequencing analysis identified a mutation (F138Y) in PfAQP (PF3D7_1132800) and duplications of the two superoxide dismutase genes, PfSOD-1 (PF3D7_0814900) and PfSOD-2 (PF3D7_0623500). CRISPR/Cas9 editing confirmed that the F138Y mutation in PfAQP was sufficient to confer resistance to cryptosporin. Alignment of the P. falciparum structure with that of HsAQP3 suggests the mutation may impact transport of hydrogen peroxide and the transition between open and closed conformations. Indeed, studies with BY4742 {Delta}fps1 yeast expressing PfAQP showed that the permeability of PfAQP was not affected by cryptosporin and that it is likely not a direct target. Taken together, this study highlights the role of PfAQP in the resistance development of cryptosporin. In addition, cryptosporin likely induces high levels of oxidative stress which results in the duplications of oxidative dismutase genes as part of the parasites defense response. These findings highlight the role of PfAQP in mediating drug resistance, the mechanism of which warrants further research.

genetics↗

bioRxiv2026

SYBR Gold staining of single stranded nucleic acids is strongly influenced by the presence of guanosines

SYBR Gold is one of the most strongly fluorescent members of the SYBR family of asymmetrical cyanine dyes commonly used for staining double and single-stranded DNA and RNA. The dye fluorescence increases dramatically upon nucleic acid binding. The primary mode of binding to double-stranded DNA is through intercalation between the base-paired deoxyribonucleotides, while the precise mode of binding to single-stranded DNA and to RNA is less well understood. Here we show that the fluorescence activity of SYBR Gold is strongly influenced by the presence of guanines in single-stranded nucleic acids. We expect this insight to aid in the evaluation of SYBR Gold-based measurements of nucleic acid abundance and spark further studies into the binding behaviour of the SYBR family dyes.

molecular biology↗

bioRxiv2026

Ribosome profiling reveals extensive translational alterations in steatohepatitis-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one the most lethal cancer types. While infections with hepatitis B or hepatitis C viruses remain the primary risk factors for HCC development worldwide, metabolic disease-associated steatohepatitis has become the fastest growing etiology of HCC, particularly in the West. Our study characterizes, for the first time, the remodeling of the translation landscape in steatohepatitis-associated HCC using ribosome profiling and mass spectrometry techniques. In striking contrast with the transcriptional alterations, which do not show strong dependence on associated co-morbidity, translational alterations are broader in the steatohepatitis compared to the viral background. These alterations affect hundreds of genes, whose products are often involved in subcellular protein localization. By quantifying the relationship between the ribosome occupancy of upstream or downstream open reading frames (uORFs or dORFs) and coding regions (CDSs), we provide evidence of uORFs attenuating the translation efficiency of CDSs in HCC. We also identify numerous novel translated regions, including from RNAs currently annotated as non-coding, some yielding peptides that can be reproducibly identified in mass spectrometry datasets. Our study thus provides novel molecular data on HCC with a focus on the steatohepatitis etiology, reveals unexpectedly extensive translational control in this co-morbidity, and gives multiple hints and candidate targets for follow-up experiments.

systems biology↗

bioRxiv2026

Regulated RIAM-Talin Engagement Controls Adhesion Stability and Mechanical Output

RIAM (Rap1-GTP-interacting adaptor molecule) links Rap1 to talin-1 to promote integrin activation and is normally enriched at nascent adhesions. How the duration of RIAM-talin engagement influences adhesion dynamics and force transmission remains unclear. Here, we used a RIAM chimera in which the native talin-binding site was replaced with the talin-binding motif of Kank2 to enforce sustained talin association and redistribute RIAM modules to mature adhesions. This talin-tethered RIAM chimera enhanced integrin activation, accelerated both adhesion assembly and disassembly, reduced adhesion lifetime, increased the fraction of nascent adhesions nucleated by RIAM, and elevated cellular traction forces. Notably, while integrin activation by the chimera was largely Rap1-independent, Rap1 binding remained necessary for optimal adhesion turnover kinetics and full traction force generation. These findings demonstrate that prolonged talin engagement of RIAM is sufficient to reprogram adhesion dynamics and reveal a separable role for Rap1 in coordinating force transmission downstream of integrin activation. Together, our results highlight the importance of regulating the residence and engagement mode of RIAM at talin for controlling adhesion plasticity and mechanotransduction.

cell biology↗

bioRxiv2025

Heat shock induces silent ribosomes and reorganizes mRNA turnover

mRNAs associate with single or multiple ribosomes; these ribosomal assemblies -- monosomes and polysomes -- translate the mRNAs before degradation. The impact of heat stress on this mRNA turnover remains unclear. In heat-shocked yeast cells, the proportion of monosomes within the ribosomal assemblies rises without a corresponding increase in the number of mRNAs associated with them. As a result, most monosomes are devoid of mRNAs and silent, lacking translational initiation factors and proteins facilitating posttranslational folding. The accumulation of silent ribosomes generally reduces the rate of association of transcripts with the ribosomes. However, elevated temperatures enhance the ribosomal association of specific mRNAs, primarily those encoding heat-shock proteins, allowing them to balance their increased degradation rates. Additionally, reduced binding of the Xrn1 exonuclease to mRNAs diminishes the influence of codon optimality on mRNA stability. These mechanisms reorganize mRNA turnover to prioritize heat-shock protein synthesis over ribosome biogenesis.

genomics↗

bioRxiv2025

Identification of new interactors of eIF3f by endogenous proximity-dependent biotin labelling in human muscle cells.

Regulation of protein synthesis is central to maintaining skeletal muscle integrity and its understanding is important for the treatment of muscular and neuromuscular pathologies. The eIF3f subunit of the translation initiation factor eIF3 has a key role, as it stands at the crossroad between protein-synthesis-associated hypertrophy and MAFbx/atrogin-1-dependent. To decipher the molecular mechanisms underpinning the role of eIF3f in regulating muscle mass, we established a cellular model that enables interrogation of eIF3f functionality via identification of proximal interactors. Using CRISPR-Cas9 molecular scissors, we generated single cell clones of immortalised human muscle cells expressing eIF3f fused to the BirA biotin ligase (eIF3f-BioID1 chimera) from the endogenous EIF3F locus. Biotinylated proteins, representing interactors of eIF3f in nanometer range distance, were identified by streptavidin pull-downs and mass spectrometry. In both proliferating and differentiated muscle cells, the eIF3f-BioID1 chimera co-sedimented with ribosomal complexes in polysome profiles and interacted mainly with components of the eIF3 complex, and with the eIF4E, eIF4G, and eIF5 initiation factors. Surprisingly, we identified several nucleus-localised interactors of eIF3f, and the immunofluorescence analyses revealed a previously unknown nuclear localization of eIF3f in both myoblasts and myotubes. We also identified novel cytoplasmic partners of eIF3f, responsible for the maintenance of skeletal muscle ultrastructure (sarcomeric/Z-disc (SYNPO2) bound proteins) and proteins of the lysosomal compartment (LAMP1). The established tagging system should be useful to further advance studies of eIF3f function in hypertrophic and atrophic conditions in skeletal muscle.

molecular biology↗

bioRxiv2024

Differential cell-ECM interaction of rhabdomyosarcoma subtypes regulated by PAX3-FOXO1

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, with two subtypes: Fusion-positive RMS (FPRMS), which has the PAX3-FOXO1 fusion gene, and fusion-negative RMS (FNRMS). Despite their distinct characteristics, treatments mainly rely on conventional chemotherapies without considering these differences. This study highlights that FNRMS cells exhibit significantly heightened interaction with the extracellular matrix (ECM) compared to FPRMS cells. Using single-cell RNA sequencing of skeletal muscle tissues and RNA sequencing of RMS samples, we identified the upregulation of genes related to cell-ECM interaction and TGF{beta} signaling in FNRMS compared to FPRMS. We also confirmed enhanced cell-ECM interaction stimulated by TGF{beta} signaling in FNRMS cells, using confocal reflection microscopy to monitor dynamic cell-ECM interaction and a live-cell sensor to quantitatively assess TGF{beta} signaling activity. Additionally, we discovered that the PAX3-FOXO1 fusion gene, characteristic of FPRMS, stimulated nitric oxide synthesis, which suppresses TGF{beta} signaling and reduces cell-ECM interaction. These findings suggest that PAX3-FOXO1 determines the diminished cell-ECM interactions in FPRMS. Experimental data show higher sensitivity of FNRMS to cell-ECM interaction disruption and TGF{beta} inhibition. Furthermore, the diminished cell-ECM interaction in FPRMS, allowing cells to survive in the ectopic environment through circulation, may partly explain its higher metastatic potential compared to FNRMS.

cell biology↗

bioRxiv2024

Improved prediction of stabilizing mutations in proteins by incorporation of mutational effects on ligand binding

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, due to their relative rarity. We tested {Delta}{Delta}G0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using eighty-two mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1{degrees}C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3{degrees}C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.

biophysics↗

bioRxiv2024

Deep Learning Analysis on Images of iPSC-derived Motor Neurons Carrying fALS-genetics Reveals Disease-Relevant Phenotypes

Amyotrophic lateral sclerosis (ALS) is a devastating condition with very limited treatment options. It is a heterogeneous disease with complex genetics and unclear etiology, making the discovery of disease-modifying interventions very challenging. To discover novel mechanisms underlying ALS, we leverage a unique platform that combines isogenic, induced pluripotent stem cell (iPSC)-derived models of disease-causing mutations with rich phenotyping via high-content imaging and deep learning models. We introduced eight mutations that cause familial ALS (fALS) into multiple donor iPSC lines, and differentiated them into motor neurons to create multiple isogenic pairs of healthy (wild-type) and sick (mutant) motor neurons. We collected extensive high-content imaging data and used machine learning (ML) to process the images, segment the cells, and learn phenotypes. Self-supervised ML was used to create a concise embedding that captured significant, ALS-relevant biological information in these images. We demonstrate that ML models trained on core cell morphology alone can accurately predict TDP-43 mislocalization, a known phenotypic feature related to ALS. In addition, we were able to impute RNA expression from these image embeddings, in a way that elucidates molecular differences between mutants and wild-type cells. Finally, predictors leveraging these embeddings are able to distinguish between mutant and wild-type both within and across donors, defining cellular, ML-derived disease models for diverse fALS mutations. These disease models are the foundation for a novel screening approach to discover disease-modifying targets for familial ALS.

neuroscience↗

bioRxiv2023

Impact of Low Shear Stress Preconditioning on Endothelial Cell Traction and Alignment in Response to High Shear Stress

Within the vascular system, endothelial cells (ECs) are exposed to fluid shear stress (FSS), a mechanical force exerted by blood flow that is critical for regulating cellular tension and maintaining vascular homeostasis. The way ECs react to FSS varies significantly; while high, laminar FSS supports vasodilation and suppresses inflammation, low or disturbed FSS can lead to endothelial dysfunction and increase the risk of cardiovascular diseases. Yet, the adaptation of ECs to dynamically varying FSS remains poorly understood. This study focuses on the dynamic responses of ECs to brief periods of low FSS, examining its impact on endothelial traction--a measure of cellular tension that plays a crucial role in how endothelial cells respond to mechanical stimuli. By integrating traction force microscopy (TFM) with a custom-built flow chamber, we analyzed how human umbilical vein endothelial cells (HUVECs) adjust their traction in response to shifts from low to high shear stress. We discovered that initial exposure to low FSS prompts a marked increase in traction force, which continues to rise over 10 hours before slowly decreasing. In contrast, immediate exposure to high FSS causes a quick spike in traction followed by a swift reduction, revealing distinct patterns of traction behavior under different shear conditions. Importantly, the direction of traction forces and the resulting cellular alignment under these conditions indicate that the initial shear experience dictates long-term endothelial behavior. Our findings shed light on the critical influence of short-lived low-shear stress experiences in shaping endothelial function, indicating that early exposure to low FSS results in enduring changes in endothelial contractility and alignment, with significant consequences for vascular health and the development of cardiovascular diseases.

cell biology↗

bioRxiv2023

Resident memory T cell precursors in tumor draining lymph nodes require type-1 IFN for optimal differentiation

Resident memory (Trm) cells play an essential role in anti-tumor immunity. However, little is known about the precursors that differentiate into protective Trm populations against cancer. Here we employed an established model of B16 melanoma neoadjuvant anti-CD4 therapy, to track tumor antigen-specific CD8+ T cells through tissues and across time; from their priming as effectors to their differentiation into Trm. We show that tumor-draining lymph nodes (TDLNs) contain Teff cells that begin to express canonical Trm markers CD103 and CD69. These tumor-specific Teff cells seeded skin and tumor during the effector phase of the response, although egress from these tissues was not required Trm development in LNs. Paired scRNAseq/scTCRseq was used to identify Teff clonotypes in TDLNs and trace their differentiation, in real-time, into Trm populations. We found that expanded clonotypes favored the Trm fate and were unlikely to co-differentiate into other lineages. Precursors of Trm (pre-Trm) clonotypes that subsequently seeded populations throughout tumors, LNs, and skin, were characterized by early expression of tissue residency, stemness, and type-1 IFN sensing genes. These multipotent pre-Trm cells sensed plasmacytoid dendritic cell-derived type-1 interferons in TDLNs, and their expression of interferon alpha receptor was required for their formation of Trm populations in LNs but not in skin. These findings reveal the defining features of pre-Trm cells in response to tumor antigens, and reveal a previously unappreciated role for type-1 IFNs in programming regional Trm immunity to cancer. One Sentence SummaryAnti-tumor effector CD8 T cells adopt early characteristics of tissue residency and stemness, and rely on the sensing of type-1 interferons for their local differentiation into resident memory T cells.

immunology↗

bioRxiv2023

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding ofproteins with alpha helical domains

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we have quantified the expression of RPL39L in human pluripotent cells, cancer cell lines and tissue samples, and in mouse germ cells. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread proteasomal activity. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

molecular biology↗

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗

bioRxiv2022

Plasmodium exoerythrocytic parasites redirect trafficking of human proteins to the parasitophorous vacuole

Changes in host cell morphology and transcription after apicomplexan parasite infection have long been noted, but there have been few studies of the functional consequences of host cell remodeling. Here we show, using time-dependent immunofluorescence microscopy of multiple human cell lines (HepG2, HC-04, Huh7.5.1 and primary human hepatocytes), infected with multiple Plasmodium species (Plasmodium berghei, P. falciparum and P. vivax (hypnozoites and schizonts)), and antibodies to multiple human proteins (HsNR4A3, HsMUC13, HsGOLGA8A, HsCGA, HsBiP, HsCXCL2), that human protein trafficking is extensively modified in Plasmodium infected cells. Using conventional as well as ultrastructure expansion microscopy we show that newly-synthesized human proteins are trafficked to the parasitophorous vacuole instead of the infected-cell plasma membrane, nucleus or extracellular space. Universal redirection of human signaling proteins cells the parasitophorous vacuole may provide a mechanistic explanation for how apicomplexan parasites can block host cells response to infection.

cell biology↗

bioRxiv2022

A fast-killing tyrosine amide ((S)-SW228703) with blood and liver-stage antimalarial activity associated with the Cyclic Amine Resistance Locus (PfCARL)

Current malaria treatments are threatened by drug resistance and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their target(s) are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for activity of (S)-SW703, suggesting differences in mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity on multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

microbiology↗

bioRxiv2022

A small-molecule myosin inhibitor as a targeted multi-stage antimalarial

Malaria is a devastating disease that resulted in an estimated 627,000 deaths in 2020. About 80% of those deaths were among children under the age of five. Our approach is to develop small molecule inhibitors against cytoskeletal targets that are vital components of parasite function, essential at multiple stages of parasite infection, can be targeted with high specificity, and are highly druggable. Here we describe KNX-115, which inhibits purified Plasmodium falciparum myosin A (PfMyoA) actin-activated ATPase with a potency in the 10s of nanomolar range and >50-fold selectivity against cardiac, skeletal, and smooth muscle myosins. KNX-115 inhibits the blood and liver stages of Plasmodium with an EC50 of about 100 nanomolar, with negligible liver cell toxicity. In addition, KNX-115 inhibits sporozoite cell traversal and blocks the gametocyte to oocyst conversion in the mosquito. KNX-115 displays a similar killing profile to pyrimethamine and parasites are totally killed after 96 hours of treatment. In line with its novel mechanism of action, KNX-115 is equally effective at inhibiting a panel of Plasmodium strains resistant to experimental and marketed antimalarials. In vitro evolution data likely suggests a refractory potential of KNX-115 in developing parasite resistance.

pharmacology and toxicology↗

bioRxiv2022

A novel oral GyrB/ParE dual binding inhibitor effective against multidrug resistant Neisseria gonorrhoeae and other high-threat pathogens

Drug resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance, as well as limit development of new resistance. We describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6- fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2- oxa-8-azaspiro[4.5]decan-3-yl)methanol], which simultaneously binds to ATP binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae including drug-resistant strains. A phosphate prodrug JSF-2659 was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram positive organisms, and JSF-2659 was shown in a deep tissue model of VRSA and a model of C. difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

microbiology↗