Notes

Spectral framing involving fiber Bragg gratings based on non-rigid origami.
It leads to radical treatment of the disease like therapeutic penetrating keratoplasty but in a less invasive manner and without a donor graft.
Posterior lamellar keratectomy is a simple, effective and inexpensive technique for management of small, peripheral, deep-seated recalcitrant keratitis. It leads to radical treatment of the disease like therapeutic penetrating keratoplasty but in a less invasive manner and without a donor graft.Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino acid composition of the peptide is unique, with l-Ala added at the first position in most bacteria but with l-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of l-Ser instead of l-Ala into peptide stems, but its mechanistic function is unknown. Here, we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor by-product UDP-MurNAc-l-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis. IMPORTANCE YfiH is a peptidoglycan (PG)-editing factor required for the maintenance of specific amino acid compositions of the stem peptides. However, the activity of YfiH has not been deciphered, and the editing mechanism involving YfiH has remained a mystery. Through X-ray crystallographic and biochemical analyses, we demonstrate that YfiH is a hydrolase with a previously unknown activity specific for the UDP-MurNAc-monopeptide, one of the nucleotide precursors from the cytoplasmic steps of the PG biosynthesis pathway. YfiH selectively hydrolyzes UDP-MurNAc-Ser, an incorrect by-product of the biosynthesis reaction, to ensure that only the correct PG precursor, UDP-MurNAc-Ala, is incorporated. Therefore, this work reveals coupled synthetic and editing reactions in the cytoplasmic steps of PG biosynthesis.Escherichia coli, a ubiquitous commensal/pathogenic member from the Enterobacteriaceae family, accounts for high infection burden, morbidity, and mortality throughout the world. With emerging multidrug resistance (MDR) on a massive scale, E. coli has been listed as one of the Global Antimicrobial Resistance and Use Surveillance System (GLASS) priority pathogens. Understanding the resistance mechanisms and underlying genomic features appears to be of utmost importance to tackle further spread of these multidrug-resistant superbugs. While a few of the globally prevalent sequence types (STs) of E. coli, such as ST131, ST69, ST405, and ST648, have been previously reported to be highly virulent and harboring MDR, there is no clarity if certain ST lineages have a greater propensity to acquire MDR. In this study, large-scale comparative genomics of a total of 5,653 E. coli genomes from 19 ST lineages revealed ST-wide prevalence patterns of genomic features, such as antimicrobial resistance (AMR)-encoding genes/mutatata but also to identify the relevant features that could be linked to a particular class/target. This is perhaps one of the pioneering studies that has attempted to classify a large repertoire of E. coli genome data sets (5,653 genomes) belonging to 19 different STs (including well-studied as well as understudied STs) using machine learning approaches. Important features identified by these approaches have revealed ST-specific signature proteins, which could be further studied to predict possible associations with the phenotypic profiles, thereby providing a better understanding of virulence and the resistance mechanisms among different clonal lineages of E. coli.There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.Human parainfluenza virus type 3 (HPIV-3) is a significant cause of lower respiratory tract infections, with the most severe disease in young infants, immunocompromised individuals, and the elderly. HPIV-3 infections are currently untreatable with licensed therapeutics, and prophylactic and therapeutic options are needed for patients at risk. To complement existing human airway models of HPIV-3 infection and develop an animal model to assess novel intervention strategies, we evaluated infection and transmission of HPIV-3 in ferrets. A well-characterized human clinical isolate (CI) of HPIV-3 engineered to express enhanced green fluorescent protein (rHPIV-3 CI-1-EGFP) was passaged on primary human airway epithelial cells (HAE) or airway organoids (AO) to avoid tissue culture adaptations. rHPIV3 CI-1-EGFP infection was assessed in vitro in ferret AO and in ferrets in vivo. Undifferentiated and differentiated ferret AO cultures supported rHPIV-3 CI-1-EGFP replication, but the ferret primary airway cells from AO wections in vitro and in vivo to evaluate novel prophylaxis and treatment options. Currently existing animal models lack the potential for studying animal-to-animal transmission or the effect of immunosuppressive therapy. Here, we describe the use of the ferret model in combination with authentic clinical viruses to further complement human ex vivo models, providing a platform to study approaches to prevent and treat HPIV-3 infection. Although we did not detect ferret-to-ferret transmission in our studies, these studies lay the groundwork for further refinement of the ferret model to immunocompromised ferrets, allowing for studies of severe HPIV-3-associated disease. Such models for preclinical evaluation of prophylaxis and antivirals can contribute to reducing the global health burden of HPIV-3.Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotidional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory farR gene (farR*) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in farR* alters the activity of the regulator, FarR*, in such a way that not only its own gene, farR*, but also the divergently transcribed farE gene and genes controlled by the global regulator, agr, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as farE deletion in either the parent or the RomR strain (RomR ΔfarE) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the ΔfarE mutants. Since the PG content in the supernatant (2,244 ng/optical density [OD]) was more than 100-fold higher than that of fatty acids (FA), we anraveled the underlying resistance mechanism, which is based on a point mutation in the farR regulator gene, causing overexpression of FarE, which most likely acts as a phospholipid (PG) efflux pump, as large amounts of PG were found in the supernatant and the pellet fraction. We show that PG can bind to Rom, thereby abrogating its antimicrobial activity. The direct interaction of Rom with PG suggests that Rom's actual target is the cytoplasmic membrane. Antibiotics that interact with PG are rare. Since Rom can be chemically synthesized, it serves as a lead compound for synthesis of improved variants.Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi apparatus is required for the budding-to-filamentous-growth transition in the human-pathogenic fungus Candida albicans; however, the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e., Efr3, Ypp1, and Stt4. The amounts of plasma membrane PI(4)P in the efr3Δ/Δ and ypp1Δ/Δ mutants were ∼60% and ∼40%, respectively, of that in the wild-type strain, whereas it was nearly undetectable in the stt4Δ/Δ mutant. All three mutants had reduced plas7ma membrane phosphatidylserine (PS). Although these mutants had normal yeast-phase growth, they were defective in filamentous growth, exhibited defect albicans, we generated deletion mutants of the three putative plasma membrane PI-4-kinase complex components and quantified the levels of plasma membrane PI(4)P in each of these strains. Our work reveals that this phosphatidylinositol phosphate is specifically critical for the yeast-to-hyphal transition, cell wall integrity, and virulence in a mouse systemic infection model. Biocytin solubility dmso The significance of this work is in identifying a plasma membrane phospholipid that has an infection-specific role, which is attributed to the loss of plasma membrane PI(4)P resulting in β(1,3)-glucan unmasking.
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