Phenotypic Screening Research Articles

First-Generation Radiolabeled Cyclic Peptides for Molecular Imaging of Platelet-Derived Growth Factor Receptor α.

Occult nodal spread and metastatic disease require longstanding imaging and biochemical assessments for thyroid cancer, a disease that has a propensity for diffuse, small-volume disease. We have developed a 64Cu-labeled platelet-derived growth factor receptor α (PDGFRA) antibody for immuno-PET of PDGFRA in metastatic papillary thyroid cancer (PTC). The present work describes the discovery of small cyclic PDGFRA-targeting peptides, their binding features, and radiolabeling with positron emitter gallium-68 (68Ga) for in vitro and in vivo characterization in thyroid cancer models. Phage-display technology with two separate libraries and seven different cell lines was used through three rounds of biopanning as well as flow cytometry and comparative analysis with recombinant protein to select specific peptide sequences. Phenotypic binding analysis was completed by using phosphorylation and cell migration assays. In vitro protein binding was analyzed with thermophoresis and flow cytometry using the fluorescent-labeled PDGFRA peptide. Peptide candidates were modified with the NOTA chelator for radiolabeling with 68Ga. In vitro cell uptake was studied in various thyroid cancer cell lines. In vivo studies of 68Ga-labeled peptides included metabolic stability and PET imaging. From the original library (1013 compounds), five different peptide groups were identified based on biopanning experiments with and without the α subunit of PDGFR, leading to ∼50 peptides. Subsequent phenotypic screening revealed two core peptide sequences (CP16 and CP18) that demonstrated significant changes in the level of PDGFRA phosphorylation and cell migration. Alanine scan sublibraries were created from these two lead peptide sequences, and peptides were radiolabeled using 68Ga-GaCl3 at pH 4.5, resulting in RCP > 95% within 34-40 min, including SPE purification. Cyclic peptide CP18.5 showed the strongest effects on cell migration, flow cytometry, and binding by visual interference color assay. 68Ga-labeled PDGFRA-targeting peptides showed elevated cell and tumor uptake in models of thyroid cancer, with 68Ga-NOTA-CP18.5 being the lead candidate. However, metabolic stability in vivo was compromised for 68Ga-NOTA-CP18.5 vs 68Ga-NOTA-CP18 but without impacting tumor uptake or clearance profiles. First-generation radiolabeled cyclic peptides have been developed as novel radiotracers, particularly 68Ga-NOTA-CP18.5, for the molecular imaging of PDGFRA in thyroid cancer.

Discovery of 5-phenyl-3-ureidothiophene-2-carboxamides as protective agents for ALS patient iPSC-derived motor neurons

We discovered novel neuroprotective compounds by phenotypic screening using SOD1-mutant amyotrophic lateral sclerosis (ALS) patient induced pluripotent stem cell (iPSC)-derived motor neurons. Mechanistic analysis showed that the protective effect of initial hit compound 1 was likely due to the inhibition of MAP4Ks, including MAP4K4, a member of the MAP4K kinase family. Structural transformation led to compound 15f, which showed improved MAP4K4 inhibitory activity and superior neuroprotective effects compared to 1 in motor neurons. The results suggest that structural optimization based on MAP4K4 inhibitory activity might improve the neuroprotective effect of this series of compounds.

Multi-omic lineage tracing predicts the transcriptional, epigenetic and genetic determinants of cancer evolution

Cancer is a highly heterogeneous disease, where phenotypically distinct subpopulations coexist and can be primed to different fates. Both genetic and epigenetic factors may drive cancer evolution, however little is known about whether and how such a process is pre-encoded in cancer clones. Using single-cell multi-omic lineage tracing and phenotypic assays, we investigate the predictive features of either tumour initiation or drug tolerance within the same cancer population. Clones primed to tumour initiation in vivo display two distinct transcriptional states at baseline. Remarkably, these states share a distinctive DNA accessibility profile, highlighting an epigenetic basis for tumour initiation. The drug tolerant niche is also largely pre-encoded, but only partially overlaps the tumour-initiating one and evolves following two genetically and transcriptionally distinct trajectories. Our study highlights coexisting genetic, epigenetic and transcriptional determinants of cancer evolution, unravelling the molecular complexity of pre-encoded tumour phenotypes.

SynBot is an open-source image analysis software for automated quantification of synapses

The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of neuroscience. Immunohistochemistry is a common tool for visualizing synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low-throughput analysis methods that are challenging to learn, and the results are variable between experimenters, especially when analyzing noisy images of brain tissue. We developed an open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems.

Phloretin Targets Polyphosphate Kinase 1 to Attenuate Acinetobacter baumannii Virulence and Persistence In Vitro and In Vivo

Acinetobacter baumannii (A. baumannii) is well known for its virulence and persistence, particularly in intensive care units. Therefore, new strategies and candidates to treat A. baumannii infection are urgently needed considering the emergence of drug-resistant bacteria. Polyphosphate kinase 1 (PPK1) is required for bacterial survival as it is involved in maintaining antibiotic resistance or tolerance, pathogenesis, and adversity resistance. Multiple phenotypic assays related to virulence and persistence were performed in this study, and phloretin was shown to attenuate A. baumannii virulence and persistence by inhibiting PPK1 activity. Phloretin hampered mobility, interfered with biofilm formation and decreased resistance to ampicillin, heat, and hydrogen peroxide stress in A. baumannii. The therapeutic effect was also examined in a mouse pneumonia infection model. Molecular simulation and site-directed mutagenesis revealed that ARG-22, MET-622, ASN57, and ARG-65 were the sites of phloretin action against PPK1. Phloretin treatment led to changes in metabolic pathways associated with A. baumannii virulence and persistence, including glycerophospholipid metabolism and fatty acid biosynthesis. Furthermore, phloretin alleviated pneumonic injury in a mouse pneumonia infection model in vivo, indicating that phloretin is a promising compound for preventing A. baumannii infection resistance by targeting PPK1.

P01-10 Cell painting PLUS: an enhanced multiplexed phenotypic assay for chemical hazard screening

P01-10 Cell painting PLUS: an enhanced multiplexed phenotypic assay for chemical hazard screening

Synthesis at the Interface of Chemistry and Biology.

ConspectusChemical synthesis as a tool to control the structure and properties of matter is at the heart of chemistry─from the synthesis of fine chemicals and polymers to drugs and solid-state materials. But as the field evolves to tackle larger and larger molecules and molecular complexes, the traditional tools of synthetic chemistry become limiting. In contrast, Mother Nature has developed very different strategies to create the macromolecules and molecular systems that make up the living cell. Our focus has been to ask whether we can use the synthetic strategies and machinery of Mother Nature, together with modern chemical tools, to create new macromolecules, and even whole organisms with properties not existing in nature. One such example involves reprogramming the complex, multicomponent machinery of ribosomal protein synthesis to add new building blocks to the genetic code, overcoming a billion-year constraint on the chemical nature of proteins. This methodology exploits the concept of bioorthogonality to add unique codons, tRNAs and aminoacyl-tRNA synthetases to cells to encode amino acids with physical, chemical and biological properties not found in nature. As a result, we can make precise changes to the structures of proteins, much like those made by chemists to small molecules and beyond those possible by biological approaches alone. This technology has made it possible to probe protein structure and function in vitro and in vivo in ways heretofore not possible, and to make therapeutic proteins with enhanced pharmacology. A second example involves exploiting the molecular diversity of the humoral immune system together with synthetic transition state analogues to make catalytic antibodies, and then expanding this diversity-based strategy (new to chemists at the time) to drug discovery and materials science. This work ushered in a new nature-inspired synthetic strategy in which large libraries of natural or synthetic molecules are designed and then rationally selected or screened for new function, increasing the efficiency by which we can explore chemical space for new physical, chemical and biological properties. A final example is the use of large chemical libraries, robotics and high throughput phenotypic cellular screens to identify small synthetic molecules that can be used to probe and manipulate the complex biology of the cell, exemplified by druglike molecules that control cell fate. This approach provides new insights into complex biology that complements genomic approaches and can lead to new drugs that act by novel mechanisms of action, for example to selectively regenerate tissues. These and other advances have been made possible by using our knowledge of molecular structure and reactivity hand in hand with our understanding of and ability to manipulate the complex machinery of living cells, opening a new frontier in synthesis. This Account overviews the work in my lab and with our collaborators, from our early days to the present, that revolves around this central theme.

Resensitization to colistin results in rapid and stable recovery of adherence, serum resistance and ompW in Acinetobacter baumannii.

Colistin resistance in Acinetobacter baumannii is an emerging problem that limits antimicrobial therapy options. We isolated two pairs of colistin susceptible and colistin-resistant A. baumannii (K1007/K1006 and K408/K409) from two patients diagnosed with carbapenem-resistant A. baumannii infection. Colistin susceptible isolates were exposed to in vitro colistin induction for 50 generations. The selected cell populations were subjected to DNA and RNA sequencing and phenotypic assays. In the in vitro induction assay, K408 gained colistin resistance on the corresponding day of clinical resistance (K408-G25) and got resensitized to colistin in the consecutive generation (K408-G26). A significant upregulation of ompW, ata, adeFGH genes on K408-G25 was followed by a downregulation upon resensitization to colistin (G26). Despite the upregulation of the ompW gene in transcriptomic analysis, the ompW protein disappeared on K408-G25 and recovered in the resensitized generation (G26). In parallel, disrupted cell membrane integrity recovered in K408-G26. In the K408-G25, downregulation of pbpG and upregulation of pbp1a/pbp3 genes decreased serum-resistance which was reversed in the resensitized generation (G26). The K1007 did not gain colistin resistance amongst 50-generations, however, the generation corresponding to clinical resistance day (K1007-G9) had a similar trend with K408-G25. The clinical colistin-resistant K409 and K1006 had SNPs on pmrA and pmrB genes. In this study, we observed that A. baumannii regulates adhesion, efflux pumps and serum-resistance associated genes as an early response to colistin stress. Besides, the ompW protein disappears in the cell membrane of colistin resistant cells which recovers after resensitization to colistin. The lack of ompW protein in colistin-resistant cells should be taken into consideration for escape mutants in development of antivirulence vaccination or treatment options.

Identification of Senkyunolide I as a novel modulator of hepatic steatosis and PPARα signaling in zebrafish and hamster models

Ethnopharmacological relevanceNon-alcoholic fatty liver disease (NAFLD) is the leading cause of liver-related morbidity and mortality, with hepatic steatosis being the hallmark symptom. Salvia miltiorrhiza Bunge (Smil, Dan-Shen) and Ligusticum striatum DC (Lstr, Chuan-Xiong) are commonly used to treat cardiovascular diseases and have the potential to regulate lipid metabolism. However, whether Smil/Lstr combo can be used to treat NAFLD and the mechanisms underlying its lipid-regulating properties remain unclear. PurposeTo assess the feasibility and reliability of a short-term high-fat diet (HFD) induced zebrafish model for evaluating hepatic steatosis phenotype and to investigate the liver lipid-lowering effects of Smil/Lstr, as well as its active components. MethodsThe phenotypic alterations of liver and multiple other organ systems were examined in the HFD zebrafish model using fluorescence imaging and histochemistry. The liver-specific lipid-lowering effects of Smil/Lstr combo were evaluated endogenously. The active molecules and functional mechanisms were further explored in zebrafish, human hepatocytes, and hamster models. ResultsIn 5-day HFD zebrafish, significant lipid accumulation was detected in the blood vessels and the liver, as evidenced by increased staining with Oil Red O and fluorescent lipid probes. Hepatic hypertrophy was observed in the model, along with macrovesicular steatosis. Smil/Lstr combo administration effectively restored the lipid profile and alleviated hepatic hypertrophy in the HFD zebrafish. In oleic-acid stimulated hepatocytes, Smil/Lstr combo markedly reduced lipid accumulation and cell damage. Subsequently, based on zebrafish phenotypic screening, the natural phthalide senkyunolide I (SEI) was identified as a major molecule mediating the lipid-lowering activities of Smil/Lstr combo in the liver. Moreover, SEI upregulated the expression of the lipid metabolism regulator PPARα and downregulated fatty acid translocase CD36, while a PPARα antagonist sufficiently blocked the regulatory effect of SEI on hepatic steatosis. Finally, the roles of SEI on hepatic lipid accumulation and PPARα signaling were further verified in the hamster model. ConclusionsWe proposed a zebrafish-based screening strategy for modulators of hepatic steatosis and discovered the regulatory roles of Smil/Lstr combo and its component SEI on liver lipid accumulation and PPARα signaling, suggesting their potential value as novel candidates for NAFLD treatment.

Nonlipogenic ABCA1 Inducers (NLAI) for Alzheimer's Disease Validated in a Mouse Model Expressing Human APOE3/APOE4.

Therapeutics enhancing apolipoprotein (APOE) positive function are a priority, because APOE4 is the major genetic risk factor for Alzheimer's disease (AD). The function of APOE, the key constituent of lipoprotein particles that transport cholesterol and lipids in the brain, is dependent on lipidation by ABCA1, a cell-membrane cholesterol transporter. ABCA1 transcription is regulated by liver X receptors (LXR): agonists have been shown to increase ABCA1, often accompanied by unwanted lipogenesis and elevated triglycerides (TG). Therefore, nonlipogenic ABCA1-inducers (NLAI) are needed. Two rounds of optimization of an HTS hit, derived from a phenotypic screen, gave lead compound 39 that was validated and tested in E3/4FAD mice that express human APOE3/4 and five mutant APP and PSEN1 human transgenes. Treatment with 39 increased ABCA1 expression, enhanced APOE lipidation, and reversed multiple AD phenotypes, without increasing TG. This NLAI/LXR-agonist study is the first in a human APOE-expressing model with hallmark amyloid-β pathology.

Prevalence and Molecular Characterization of Carbapenem and Colistin-Resistance Genes in Clinical Strains of Pseudomonas aeruginosa Isolated from Diabetic Patients

Abstract Introduction Pseudomonas aeruginosa is one of the important opportunistic nosocomial pathogens that are increasingly becoming resistant to many drugs including the drugs of last resort, carbapenems, and colistin. Methods The study involved the examination of clinical isolates of P. aeruginosa recovered from diabetic patients for the presence of acquired genes implied in carbapenem and colistin resistance. Results A total of 100 clinical isolates from diabetic patients' wound and sputum were investigated for their susceptibility to imipenem and colistin followed by phenotypic assay and resistance gene screening. The study revealed 27 sputum isolates being resistant to imipenem further confirmed by a modified Hodge test and 28 wound isolates were resistant to colistin. The resistant isolates of wound and sputum were found to possess blaOXA-48 , blaVIM , mcr-1, and mcr-5 genes. The strains had a coexistence of carbapenem- and colistin-resistant genes. Conclusion Considering the fact that infection by such resistant isolates in diabetics could be frightening, clinical laboratories must employ rapid diagnostic methods to detect resistance which can aid in the treatment decision-making.

Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries.

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

First identification and genomic features of multidrug-resistant Citrobacter freundii ST669 strain isolated from a domesticated duck in Bangladesh

ObjectivesCitrobacter freundii is a prevalent source of nosocomial infections and a well-known cause of diarrheal diseases. In recent years, it has also become increasingly resistant to various antimicrobials. In this study, we screened and characterized a multidrug-resistant (MDR) C. freundii isolate obtained from a domesticated diseased duck to better understand the genetic features, molecular epidemiology, and underlying factors linked to the antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) of the isolate. MethodsThe C. freundii BAU_TM8 strain was isolated using culturing, staining, biochemical, polymerase chain reaction, and Matrix-assisted laser desorption/ionization-time of flight methods. The MDR properties of the strain were determined by a disk diffusion test. The genomic sequence of C. freundii BAU_TM8 was performed using the Illumina NextSeq2000 platform. The ARGs, VFGs, and genomic functional characteristics of the C. freundii BAU_TM8 strain were identified using several open-source databases. ResultsThe sequence type of this strain was ST669, and the pathogenicity index of the strain was 0.919. Moreover, the strain had an estimated genome length of 5,797,806 bp, harboring 62 contigs, a G + C content of 54.32 %, and five contig L50s with an N50 value of 443,947 bp. Using phylogenetic analysis, this strain was closely related to two strains isolated from human and environmental samples in the USA and China despite huge geographical distances. The C. freundii BAU_TM8 strain consisted of 40 AGRs encoding resistance to 19 antimicrobial categories, e.g., fluoroquinolones, macrolides, folate pathway antagonists, aminoglycosides, tetracyclines, cephalosporins, and others. According to the phenotypic assay and genome sequence, the sensitivity and specificity of resistance profiles of the strain were 100 % and 20 %, respectively. Moreover, the virulence factor database detected 66 VFGs in this strain. This strain contained 1581 subsystems, having 33 % subsystem coverage and 2275 genes encoding amino acid derivatives, carbohydrate metabolism, protein metabolism, cofactors, vitamins, prosthetic groups, pigments, respiration, motility and chemotaxis, stress response, DNA metabolism, nucleosides and nucleotides, and others. ConclusionsTo the best of our knowledge, this is the first WGS report of C. freundii from a domesticated duck in Bangladesh. The ubiquitous occurrence of ARGs and VFGs in the C. freundii BAU_TM8 strain detected in this study highlights the growing concern about antimicrobial resistance in humans, animals, and environments.

Phenotypic Screening of Rice Cultures for Resistance against Brown Plant Hopper (Nilaparvata lugens Stal) at Warangal, India

Brown planthopper (BPH), Nilaparvatalugens Stal (Homoptera: Delphacidae), is one of the most destructive insect pests causing significant yield losses in rice production. During Kharif, 2021, a set of 91 rice cultures were screened against brown plant hopper (BPH) through standard seed box technique in screen house at Regional Agricultural Research Station, Warangal. Resistant checks RP 2068-18-3-5 recorded Damage Score (DS) of <3, Mo-1 recorded DS of 3.67, PTB 33 recorded DS of 3.29. Mean damage score was 8.94 in the susceptible check Taichung Native 1(TN-1) against BPH. The 2 entries viz., RPGP-1066-36-12-1-2 and WS-18-OYT-72/BPH were found to be highly resistant to BPH. RPGP-1386-3-1-1-1 was found to be resistant to BPH. 5 entries viz., BPT 3025, RPGP-1011-100-56-1-4-1, RPGP-2212-8-1-2-2, KNM 12505 and WS-18-AYT-10/BPH were found to be moderately resistant to BPH. Cultivation of resistant cultures will reduce the cost of plant protection against BPH in addition to protection of natural enemies and environment. The rice cultures exhibiting resistance against BPH may be helpful in crossing programmes to develop BPH resistant varieties which shall be used in IPM as a component.

Phenotypic and genomic analysis of the hypervirulent methicillin-resistant Staphylococcus aureus ST630 clone in China.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is a rarely reported lineage worldwide. This study aimed to trace the dissemination of the emerging MRSA ST630 clones in China and investigate their virulence potential. We collected 22 ST630-MRSA isolates from across China and performed whole-genome sequencing analysis and virulence characterization on these isolates. Epidemiological results showed that MRSA ST630 isolates were primarily isolated from pus/wound secretions, mainly originating from Jiangxi province, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type V (SCCmec V) predominated (11/22, 50.0%) among the MRSA ST630 isolates. Interestingly, nearly half (45.5%) of the 22 ST630-MRSA isolates tested lacked intact SCCmec elements. Phylogenetic analysis demonstrated that ST630-MRSA could be divided into two distinct clades, with widespread dissemination mainly in Chinese regions. Five representative isolates were selected for phenotypic assays, including hemolysin activity, real-time fluorescence quantitative PCR, western blot analysis, hydrogen peroxide killing assay, blood killing assay, cell adhesion and invasion assay, and mouse skin abscess model. The results showed that, compared to the USA300-LAC strain, ST630 isolates exhibited particularly strong invasiveness and virulence in the aforementioned phenotypic assays. This study described the emergence of a highly virulent ST630-MRSA lineage and improved our insight into the molecular epidemiology of ST630 clones in China.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is an emerging clone with an increasing isolation rate in China. This study raises awareness of the hypervirulent MRSA ST630 clones in China and alerts people to their widespread dissemination. ST630-staphylococcal cassette chromosome mec V is a noteworthy clone in China, and we present the first comprehensive genetic and phenotypic analysis of this lineage. Our findings provide valuable insights for the prevention and control of infections caused by this emerging MRSA clone.

Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2).

CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders.

Identification of DDX5 as a Potential Therapeutic Target of Osteosarcoma Using Thiazolone Probes.

Osteosarcoma (OS) is a rare malignant tumor that has predominantly affected children and adolescents in the past 50 years. The genomes of OS tumors exhibit a high degree of complexity, which leads to the great challenge of target identification for anti-OS. To date, no efficient therapeutic target for the treatment of OS has been validated in clinical practice. In our previous drug hunting for the treatment of OS by phenotypic screening, we found that thiazolone derivate (R)-8i was an effective and selective inhibitor against OS in MNNG/HOS cells and in vivo. However, the mechanism of action and specific molecular targets of (R)-8i remain unclear. In this study, we design and synthesize the photo-cross-linking probes based on the lead compound (R)-8i and identify DDX5 as a potential target protein using an activity-based protein profiling strategy. Further experiments including Western blot, shRNA knockdown experiments, cell colony formation, wound healing assays, and cellular thermal shift assays support that (R)-8i binds to DDX5 and induces its degradation, which affect cell proliferation and migration through the PI3K-AKT-mTOR signaling pathway. The research shows that DDX5 is a potential therapeutic target for the treatment of OS.

Identification of novel small molecule-based strategies of COL7A1 upregulation and readthrough activity for the treatment of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.

Pseudomonas fragariae sp. nov., a novel bacterial species causing leaf spots on strawberry (Fragaria×ananassa).

In Florida, angular leaf spot, caused by Xanthomonas fragariae, was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417T (17T=LMG 32456T=DSM 113340 T) as the type strain, in relation to Fragaria×ananassa, the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

The phosphatase inhibitor BVT-948 can be used to efficiently screen functional sexual development proteins in the malaria parasite Plasmodium berghei

BackgroundStudying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites. MethodsDifferences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte–ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development. ResultsThe phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte–ookinete conversion and transmission to mosquitoes. ConclusionsSeven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening.