Miracle-dz/UniFAR-FTUs huggingface dataset:
The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies

P. aeruginosa is classified as a priority one pathogen by the World Health Organisation, and new drugs are urgently needed, due to the emergence of multidrug-resistant (MDR) strains. Antimicrobial-resistant nosocomial pathogens such as P. aeruginosa pose unwavering and increasing threats. Antimicrobial stewardship has been a challenge during the COVID-19 pandemic, with a majority of those hospitalized with SARS-CoV2 infection given antibiotics as a safeguard against secondary bacterial infection. This increased usage, along with increased handling of sanitizers and disinfectants globally, may further accelerate the development and spread of cross-resistance to antibiotics. In addition, P. aeruginosa is the primary causative agent of morbidity and mortality in people with the life-shortening genetic disease cystic fibrosis (CF). Prolonged periods of selective pressure, associated with extended antibiotic treatment and the actions of host immune effectors, results in widespread adaptive and acquired resistance in P. aeruginosa found colonizing the lungs of people with CF. This review discusses the arsenal of resistance mechanisms utilized by P. aeruginosa, how these operate under high-stress environments such as the CF lung and how their interconnectedness can result in resistance to multiple antibiotic classes. Intrinsic, adaptive and acquired resistance mechanisms will be described, with a focus on how each layer of resistance can serve as a building block, contributing to multi-tiered resistance to antimicrobial activity. Recent progress in the development of anti-resistance adjuvant therapies, targeting one or more of these building blocks, should lead to novel strategies for combatting multidrug resistant P. aeruginosa. Anti-resistance adjuvant therapy holds great promise, not least because resistance against such therapeutics is predicted to be rare. The non-bactericidal nature of anti-resistance adjuvants reduce the selective pressures that drive resistance. Anti-resistance adjuvant therapy may also be advantageous in facilitating efficacious use of traditional antimicrobials, through enhanced penetration of the antibiotic into the bacterial cell. Promising anti-resistance adjuvant therapeutics and targets will be described, and key remaining challenges highlighted. As antimicrobial stewardship becomes more challenging in an era of emerging and re-emerging infectious diseases and global conflict, innovation in antibiotic adjuvant therapy can play an important role in extending the shelf-life of our existing antimicrobial therapeutic agents.
Deletion of the ATP2 Gene in Candida albicans Blocks Its Escape From Macrophage Clearance

Macrophages provide the first-line defense against invasive fungal infections and, therefore, escape from macrophage becomes the basis for the establishment of Candida albicans invasive infection. Here, we found that deletion of ATP2 (atp2Δ/Δ) in C. albicans resulted in a dramatic decrease from 69.2% (WT) to 1.2% in the escape rate in vitro. The effect of ATP2 on macrophage clearance stands out among the genes currently known to affect clearance. In the normal mice, the atp2Δ/Δ cells were undetectable in major organs 72 h after systemic infection, while WT cells persisted in vivo. However, in the macrophage-depleted mice, atp2Δ/Δ could persist for 72 h at an amount comparable to that at 24 h. Regarding the mechanism, WT cells sustained growth and switched to hyphal form, which was more conducive to escape from macrophages, in media that mimic the glucose-deficient environment in macrophages. In contrast, atp2Δ/Δ cells can remained viable but were unable to complete morphogenesis in these media, resulting in them being trapped within macrophages in the yeast form. Meanwhile, atp2Δ/Δ cells were killed by oxidative stress in alternative carbon sources by 2- to 3-fold more than WT cells. Taken together, ATP2 deletion prevents C. albicans from escaping macrophage clearance, and therefore ATP2 has a functional basis as a drug target that interferes with macrophage clearance.
Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.
Class II LBD genes ZmLBD5 and ZmLBD33 regulate gibberellin and abscisic acid biosynthesis

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors. Class I LBD members are widely reported to be pivotal for organ development, however, the role of class II members is unknown in cereal crops. Class II LBD proteins are distinguished from class I by the lack of a Gly-Ala-Ser (GAS) peptide and leucine-zipper-like coiled-coil domain, which is thought to be essential for protein dimerization. In this study, ZmLBD5 and ZmLBD33 form homo- and hetero-dimers, like class I members. At seedling stage, ZmLBD5 promoted biomass accumulation (shoot dry weight and root dry weight), root development (root length, root number, and root volume), and organ expansion (leaf area), while ZmLBD33 repressed these processes and display a dwarf phenotype. Both ZmLBD5 and ZmLBD33 displayed negative roles in drought tolerance mainly by increasing stomatal density and stomatal aperture. RNA sequencing, gene ontology enrichment analysis, and transient luciferase expression assays indicated that ZmLBD5 and ZmLBD33 are mainly involved in the regulation of the TPS-KS-GA2ox gene module, which comprises key enzymatic genes upstream of GA and ABA biosynthesis. GA1 content increased in ZmLBD5-overexpressing seedlings, while GA3 and abscisic acid content decreased in both transgenic seedlings. Consequently, exogenous GA1 or GA3 undoubtedly rescued the dwarf phenotype of ZmLBD33-overexpressing plants, with GA1 performing better. The study of ZmLBD5 and ZmLBD33 sheds light on the function of the class II LBD gene family in maize.
Biotext: Exploiting Biological-Text Format for Text Mining

The large amount of existing textual data justifies the development of new text mining tools. Bioinformatics tools can be brought to Text Mining, increasing the arsenal of resources. Here, we present BIOTEXT, a package of strategies for converting natural language text into biological-like information data, providing a general protocol with standardized functions, allowing to share, encode and decode textual data for amino acid and DNA. The package was used to encode the arbitrary information present in the headings of the biological sequences found in a BLAST survey. The protocol implemented in this study consists of 12 steps, which can be easily executed and/ or changed by the user, depending on the study area. BIOTEXT empowers users to perform text mining using bioinformatics tools. BIOTEXT is freely available at https://pypi.org/project/BIOTEXT/ (Python package) and https://sourceforge.net/projects/BIOTEXTtools/files/AMINOcode_GUI/ (Standalone tool).
Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as a protein that resides in α-cell secretory granules, and showed that it regulates glucagon secretion by directing glucagon towards the endolysosomal system in αTC1-6 cells. Here, we hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ) to induce diabetes, Arg-stimulated secretion of glucagon and Stmn2 was augmented. However, cell glucagon content was significantly increased (p<0.001), but Stmn2 levels were reduced (p<0.01) in STZ-treated mice, as measured by both ELISA and immunofluorescence intensity. Expression of Gcg mRNA increased ~4.5 times, while Stmn2 mRNA levels did not change. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p<0.001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p<0.01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for two weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome towards the secretory Lamp1+lysosome.
Integrative systems biology approach identified crucial genes and transcription factors associated with gallbladder cancer pathogenesis

Gallbladder cancer (GBC) has a lower incidence rate among the population relative to other cancer types but majorly contributes to the total cancer cases of the biliary tract system. GBC is distinguished from other malignancies due to its high mortality, marked geographical variation and poor prognosis. To date no systemic targeted therapy is available for GBC. The main objective of this study is to determine the molecular signatures correlated with GBC development using integrative system level approaches. We performed analysis of publicly available transcriptomic data to identify differentially regulated genes and pathways. Co-expression network analysis and differential regulatory network analysis identified hub genes and hub transcription factors (TFs) associated with GBC pathogenesis and progression. We then assessed the epithelial-mesenchymal transition (EMT) status of the hub genes using a combination of three scoring methods. The hub genes such as; CDC6, MAPK15, CCNB2, BIRC7, L3MBTL1 identified are regulators of cell cycle components which suggests that cell cycle regulatory genes are significantly linked to GBC pathogenesis and progression.
Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is mediated at least in part by the junctional component DLG-1 discs large, which appears to restrict protrusive activity to the apical domain. Finally, we demonstrate that the Rho-family GTPases CED-10 Rac and CDC-42 can regulate seam cell outgrowth and may also function downstream of LET-413. Our data uncover multiple essential functions for LET-413 in larval development and shed new light on the regulation of polarized outgrowth of the seam cells.
Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.
Resolving leaf level metabolism of C4Setaria viridis acclimated to low light through a biochemical model

When C4 plants are exposed to low light, CO2 concentration in the bundle sheath (BS) decreases, causing an increase in photorespiration, leakiness (the ratio of CO2 leak rate out of the BS over the rate of supply via C4 acid decarboxylation), and a consequent reduction in biochemical efficiency. This can to some extent be mitigated by complex acclimation syndromes, which are of primary importance for crop productivity, but not well studied. We unveil a strategy of leaf-level low light acclimation involving regulation of electron transport processes. Firstly, we characterise anatomy, gas-exchange and electron transport of Setaria viridis grown under low light. Through a newly developed biochemical model we resolve the photon fluxes, and reaction rates to explain how these concerted acclimation strategies sustain photosynthetic efficiency. Smaller BS in low light-grown plants limited leakiness but sacrificed light harvesting and ATP production. To counter ATP shortage and maintain high assimilation rates, plants facilitated light penetration through mesophyll and upregulated cyclic electron flow in the BS. This novel shade tolerance mechanism based on optimisation of light reactions is more efficient than the known mechanisms involving the rearrangement of dark reactions and can potentially lead to innovative strategies for crop improvement.We mechanistically link the optical cross section with the rate of electron transport, the engagement of cyclic electron flow, the relative rate of ATP and NADPH generation, and ultimately the apportioning of dark reactions between mesophyll and bundle sheath cells. The striking capacity of S. viridis to acclimate light reactions presents a novel and perhaps the most efficient shade tolerance strategy, potentially leading to novel possibilities for crop improvement.
Visualizing the organization and differentiation of the male-specific nervous system of C. elegans

Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode C. elegans. While 294 neurons are shared between the two sexes, the nervous system of the male contains an additional 93 malespecific neurons, most of which have received very little attention so far. To make these neurons amenable for future study, we describe here how a multicolor, multipromoter reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male specific neurons. We used this tool to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes. We demonstrate the usage of NeuroPAL for cellular fate analysis by analyzing the effect of removal of developmental patterning genes, including a HOX cluster gene (egl-5), a miRNA (lin-4) and a proneural gene (lin-32/Ato), on neuronal identity acquisition within the male-specific nervous system. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage that is concomitant with male tale retraction. This wave of differentiation couples neuronal maturation programs with the appearance of sexual organs. We call this wave “just-in-time” differentiation by its analogy to the mechanism of “just-in-time” transcription of metabolic pathway genes.
Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis. Author summary SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.
Prefrontal coding of learned and inferred knowledge during REM & NREM sleep

Idling brain activity has been proposed to facilitate inference, insight, and innovative problem-solving1-4. However, it remains unclear how and when the subconscious brain can create novel ideas. Here, we show coordinated roles of non-rapid eye movement (NREM) and rapid-eye movement (REM) sleep in weaving inferential information from learned information. In a transitive inference paradigm, mice gained the inference one day, but not shortly, after complete training. Inhibiting the neuronal computations in the anterior cingulate cortex (ACC) during post-learning either NREM or REM sleep, but not wakefulness, disrupted the inference without affecting the learned knowledge. In vivo Ca2+ imaging showed gradual development of inference-related representation, peaking at post-training REM sleep. Neurons representing learned information co-reactivated significantly during post-training NREM sleep, while their co-reactivation with neurons representing inferential information was significantly higher during REM sleep. Furthermore, after insufficient learning, artificial activation of medial entorhinal cortex-ACC dialogue during only REM sleep created inferential knowledge. These findings suggest that NREM sleep organizes the scattered learned knowledge in a complete hierarchy, while REM sleep computes the inferential information from the organized hierarchy. Collectively, our study provides a mechanistic insight on NREM and REM coordination in weaving inferential knowledge, thus highlighting the power of idling brain in cognitive flexibility.