Adhesin domains responsible for binding bacteria to surfaces they colonize project outwards from companion split domains.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly ß-sandwich extender modules, but other domains like a ß-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

Granulocytic myeloid-derived suppressor cell activity during biofilm infection is regulated by a glycolysis/HIF1a axis.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs), which are critical for orchestrating the antiinflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through HIF1a were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted Hif1a conditional KO mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, single-cell RNA-Seq (scRNA-Seq) analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia-response genes. These findings support the importance of a glycolysis/HIF1a axis in promoting G-MDSC antiinflammatory activity and biofilm persistence during PJI.

Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity.

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus. We demonstrate that acetate anions bind to and inhibit d-alanyl-d-alanine ligase (Ddl) activity in S. aureus. Ddl inhibition reduces intracellular d-alanyl-d-alanine (d-Ala-d-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a high intracellular d-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of d-Ala to d-glutamate by controlling d-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular d-Ala concentrations, partly to counter organic anion intoxication.

Metabolic diversity of human macrophages: potential influence on Staphylococcus aureus intracellular survival.

Staphylococcus aureus is a leading cause of medical device-associated biofilm infections. This is influenced by the ability of S. aureus biofilm to evade the host immune response, which is partially driven by the anti-inflammatory cytokine interleukin-10 (IL-10). Here, we show that treatment of human monocyte-derived macrophages (HMDMs) with IL-10 enhanced biofilm formation, suggesting that macrophage anti-inflammatory programming likely plays an important role during the transition from planktonic to biofilm growth. To identify S. aureus genes that were important for intracellular survival in HMDMs and how this was affected by IL-10, transposon sequencing was performed. The size of the S. aureus essential genome was similar between unstimulated HMDMs and the outgrowth control (18.5% vs 18.4%, respectively, with 54.4% overlap) but increased to 22.5% in IL-10-treated macrophages, suggesting that macrophage polarization status exerts differential pressure on S. aureus. Essential genes for S. aureus survival within IL-10-polarized HMDMs were dominated by negative regulatory pathways, including nitrogen and RNA metabolism, whereas S. aureus essential genes within untreated HMDMs were enriched in biosynthetic pathways such as purine and pyrimidine biosynthesis. To explore how IL-10 altered the macrophage intracellular metabolome, targeted metabolomics was performed on HMDMs from six individual donors. IL-10 treatment led to conserved alterations in distinct metabolites that were increased (dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, and acetyl-CoA) or reduced (fructose-6-phosphate, aspartic acid, and ornithine) across donors, whereas other metabolites were variable. Collectively, these findings highlight an important aspect of population-level heterogeneity in human macrophage responsiveness that should be considered when translating results to a patient population.IMPORTANCEOne mechanism that Staphylococcus aureus biofilm elicits in the host to facilitate infection persistence is the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Here, we show that exposure of human monocyte-derived macrophages (HMDMs) to IL-10 promotes S. aureus biofilm formation and programs intracellular bacteria to favor catabolic pathways. Examination of intracellular metabolites in HMDMs revealed heterogeneity between donors that may explain the observed variability in essential genes for S. aureus survival based on nutrient availability for bacteria within the intracellular compartment. Collectively, these studies provide novel insights into how IL-10 polarization affects S. aureus intracellular survival in HMDMs and the importance of considering macrophage heterogeneity between human donors as a variable when examining effector mechanisms.

Elucidating granulocytic myeloid-derived suppressor cell heterogeneity during Staphylococcus aureus biofilm infection.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated immature myeloid cells with immunosuppressive activity that expand during chronic inflammation, such as cancer and prosthetic joint infection (PJI). Myeloid-derived suppressor cells can be broadly separated into 2 populations based on surface marker expression and function: monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic myeloid-derived suppressor cells (G-MDSCs). Granulocytic myeloid-derived suppressor cells are the most abundant leukocyte infiltrate during PJI; however, how this population is maintained in vivo and cellular heterogeneity is currently unknown. In this study, we identified a previously unknown population of Ly6G+Ly6C+F4/80+MHCII+ MDSCs during PJI that displayed immunosuppressive properties ex vivo. We leveraged F4/80 and MHCII expression by these cells for further characterization using cellular indexing of transcriptomes and epitopes by sequencing, which revealed a distinct transcriptomic signature of this population. F4/80+MHCII+ MDSCs displayed gene signatures resembling G-MDSCs, neutrophils, and monocytes but had significantly increased expression of pathways involved in cytokine response/production, inflammatory cell death, and mononuclear cell differentiation. To determine whether F4/80+MHCII+ MDSCs represented an alternate phenotypic state of G-MDSCs, Ly6G+Ly6C+F4/80-MHCII- G-MDSCs from CD45.1 mice were adoptively transferred into CD45.2 recipients using a mouse model of PJI. A small percentage of transferred G-MDSCs acquired F4/80 and MHCII expression in vivo, suggesting some degree of plasticity in this population. Collectively, these results demonstrate a previously unappreciated phenotype of F4/80+MHCII+ MDSCs during PJI, revealing that a granulocytic-to-monocytic transition can occur during biofilm infection.

Nicotinamide for Skin-Cancer Chemoprevention in Transplant Recipients.

BACKGROUND: Immunosuppressed organ-transplant recipients have an increased incidence of, and mortality from, skin cancer. Nicotinamide (vitamin B3) enhances the repair of ultraviolet (UV) radiation-induced DNA damage, reduces the cutaneous immunosuppressive effects of UV radiation, and reduces the incidence of keratinocyte cancers (including squamous-cell and basal-cell carcinomas) and actinic keratoses among high-risk immunocompetent patients. Whether oral nicotinamide is useful for skin-cancer chemoprevention in organ-transplant recipients is unclear. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, organ-transplant recipients who had had at least two keratinocyte cancers in the past 5 years to receive 500 mg of nicotinamide or placebo twice daily for 12 months. Participants were examined for skin lesions by dermatologists at 3-month intervals for 12 months. The primary end point was the number of new keratinocyte cancers during the 12-month intervention period. Secondary end points included the numbers of squamous-cell and basal-cell carcinomas during the 12-month intervention period, the number of actinic keratoses until 6 months after randomization, safety, and quality of life. RESULTS: A total of 158 participants were enrolled, with 79 assigned to the nicotinamide group and 79 to the placebo group. The trial was stopped early owing to poor recruitment. At 12 months, there were 207 new keratinocyte cancers in the nicotinamide group and 210 in the placebo group (rate ratio, 1.0; 95% confidence interval, 0.8 to 1.3; P = 0.96). No significant between-group differences in squamous-cell and basal-cell carcinoma counts, actinic keratosis counts, or quality-of-life scores were observed. Adverse events and changes in blood or urine laboratory variables were similar in the two groups. CONCLUSIONS: In this 12-month, placebo-controlled trial, oral nicotinamide therapy did not lead to lower numbers of keratinocyte cancers or actinic keratoses in immunosuppressed solid-organ transplant recipients. (Funded by the National Health and Medical Research Council; ONTRANS Australian New Zealand Clinical Trials Registry number, ACTRN12617000599370.).

Suppressing phase disproportionation in quasi-2D perovskite light-emitting diodes.

Electroluminescence efficiencies and stabilities of quasi-two-dimensional halide perovskites are restricted by the formation of multiple-quantum-well structures with broad and uncontrollable phase distributions. Here, we report a ligand design strategy to substantially suppress diffusion-limited phase disproportionation, thereby enabling better phase control. We demonstrate that extending the π-conjugation length and increasing the cross-sectional area of the ligand enables perovskite thin films with dramatically suppressed ion transport, narrowed phase distributions, reduced defect densities, and enhanced radiative recombination efficiencies. Consequently, we achieved efficient and stable deep-red light-emitting diodes with a peak external quantum efficiency of 26.3% (average 22.9% among 70 devices and cross-checked) and a half-life of ~220 and 2.8 h under a constant current density of 0.1 and 12 mA/cm2, respectively. Our devices also exhibit wide wavelength tunability and improved spectral and phase stability compared with existing perovskite light-emitting diodes. These discoveries provide critical insights into the molecular design and crystallization kinetics of low-dimensional perovskite semiconductors for light-emitting devices.

Role of Staphylococcus aureus Formate Metabolism during Prosthetic Joint Infection.

Biofilms are bacterial communities characterized by antibiotic tolerance. Staphylococcus aureus is a leading cause of biofilm infections on medical devices, including prosthetic joints, which represent a significant health care burden. The major leukocyte infiltrate associated with S. aureus prosthetic joint infection (PJI) is granulocytic myeloid-derived suppressor cells (G-MDSCs), which produce IL-10 to promote biofilm persistence by inhibiting monocyte and macrophage proinflammatory activity. To determine how S. aureus biofilm responds to G-MDSCs and macrophages, biofilms were cocultured with either leukocyte population followed by RNA sequencing. Several genes involved in fermentative pathways were significantly upregulated in S. aureus biofilm following G-MDSC coculture, including formate acetyltransferase (pflB), which catalyzes the conversion of pyruvate and coenzyme-A into formate and acetyl-CoA. A S. aureus pflB mutant (ΔpflB) did not exhibit growth defects in vitro. However, ΔpflB formed taller and more diffuse biofilm compared to the wild-type strain as revealed by confocal microscopy. In a mouse model of PJI, the bacterial burden was significantly reduced with ΔpflB during later stages of infection, which coincided with decreased G-MDSC influx and increased neutrophil recruitment, and ΔpflB was more susceptible to macrophage killing. Although formate was significantly reduced in the soft tissue surrounding the joint of ΔpflB-infected mice levels were increased in the femur, suggesting that host-derived formate may also influence bacterial survival. This was supported by the finding that a ΔpflBΔfdh strain defective in formate production and catabolism displayed a similar phenotype to ΔpflB. These results revealed that S. aureus formate metabolism is important for promoting biofilm persistence.

Degradation and Self-Healing in Perovskite Solar Cells.

Organic-inorganic halide perovskites are well-known for their unique self-healing ability. In the presence of strong external stimuli, such as light, temperature, and moisture, high-energy defects are created which can be healed by removing the perovskite from the degradation source. This self-healing ability has been showcased in devices with recoverable performance and day-and-night cycling operation to dramatically extend the device lifetime and even mechanical durability. However, to date, the mechanistic details and theory around this captivating trait are sparse and convoluted by the complex nature of perovskites. With a clear understanding of the intrinsic self-healing property, perovskite solar cells with extended lifetimes and durability can be designed to realize the large-scale commercialization of perovskite solar cells. Here, we spotlight the relevant degradation and self-healing literature and then propose design strategies to help conceptualize future research.

Computer-Generated R.E.N.A.L. Nephrometry Scores Yield Comparable Predictive Results to Those of Human-Expert Scores in Predicting Oncologic and Perioperative Outcomes.

PURPOSE: We sought to automate R.E.N.A.L. (for radius, exophytic/endophytic, nearness of tumor to collecting system, anterior/posterior, location relative to polar line) nephrometry scoring of preoperative computerized tomography scans and create an artificial intelligence-generated score (AI-score). Subsequently, we aimed to evaluate its ability to predict meaningful oncologic and perioperative outcomes as compared to expert human-generated nephrometry scores (H-scores). MATERIALS AND METHODS: A total of 300 patients with preoperative computerized tomography were identified from a cohort of 544 consecutive patients undergoing surgical extirpation for suspected renal cancer at a single institution. A deep neural network approach was used to automatically segment kidneys and tumors, and geometric algorithms were developed to estimate components of R.E.N.A.L. nephrometry score. Tumors were independently scored by medical personnel blinded to AI-scores. AI- and H-score agreement was assessed using Lin's concordance correlation and their predictive abilities for both oncologic and perioperative outcomes were assessed using areas under the curve. RESULTS: Median age was 60 years (IQE 51-68), and 40% were female. Median tumor size was 4.2 cm and 91.3% had malignant tumors, including 27%, 37% and 24% with high stage, grade and necrosis, respectively. There was significant agreement between H-scores and AI-scores (Lin's ⍴=0.59). Both AI- and H-scores similarly predicted meaningful oncologic outcomes (p <0.001) including presence of malignancy, necrosis, and high-grade and -stage disease (p <0.003). They also predicted surgical approach (p <0.004) and specific perioperative outcomes (p <0.05). CONCLUSIONS: Fully automated AI-generated R.E.N.A.L. scores are comparable to human-generated R.E.N.A.L. scores and predict a wide variety of meaningful patient-centered outcomes. This unambiguous artificial intelligence-based scoring is intended to facilitate wider adoption of the R.E.N.A.L. score.

Ligand-Driven Grain Engineering of High Mobility Two-Dimensional Perovskite Thin-Film Transistors.

Controlling grain growth is of great importance in maximizing the charge carrier transport for polycrystalline thin-film electronic devices. The thin-film growth of halide perovskite materials has been manipulated via a number of approaches including solvent engineering, composition engineering, and post-treatment processes. However, none of these methods lead to large-scale atomically flat thin films with extremely large grain size and high charge carrier mobility. Here, we demonstrate a novel π-conjugated ligand design approach for controlling the thin-film nucleation and growth kinetics in two-dimensional (2D) halide perovskites. By extending the π-conjugation and increasing the planarity of the semiconducting ligand, nucleation density can be decreased by more than 5 orders of magnitude. As a result, wafer-scale 2D perovskite thin films with highly ordered crystalline structures and extremely large grain size are readily obtained. We demonstrate high-performance field-effect transistors with hole mobility approaching 10 cm2 V-1 s-1 with ON/OFF current ratios of ∼106 and excellent stability and reproducibility. Our modeling analysis further confirms the origin of enhanced charge transport and field and temperature dependence of the observed mobility, which allows for clear deciphering of the structure-property relationships in these nascent 2D semiconductor systems.

Multifunctional Conjugated Ligand Engineering for Stable and Efficient Perovskite Solar Cells.

Surface passivation is an effective way to boost the efficiency and stability of perovskite solar cells (PSCs). However, a key challenge faced by most of the passivation strategies is reducing the interface charge recombination without imposing energy barriers to charge extraction. Here, a novel multifunctional semiconducting organic ammonium cationic interface modifier inserted between the light-harvesting perovskite film and the hole-transporting layer is reported. It is shown that the conjugated cations can directly extract holes from perovskite efficiently, and simultaneously reduce interface non-radiative recombination. Together with improved energy level alignment and the stabilized interface in the device, a triple-cation mixed-halide medium-bandgap PSC with an excellent power conversion efficiency of 22.06% (improved from 19.94%) and suppressed ion migration and halide phase segregation, which lead to a long-term operational stability, is demonstrated. This strategy provides a new practical method of interface engineering in PSCs toward improved efficiency and stability.

Thermal decomposition of cyclohexane by flash pyrolysis vacuum ultraviolet photoionization time-of-flight mass spectrometry: a study on the initial unimolecular decomposition mechanism.

Thermal decomposition of cyclohexane at temperatures up to 1310 K was performed using flash pyrolysis coupled with vacuum ultraviolet (118.2 nm) photoionization time-of-flight mass spectrometry. The experimental results revealed that the major initiation reaction of cyclohexane decomposition was C-C bond fission leading to the formation of 1,6-hexyl diradical. The 1,6-hexyl diradical could isomerize to 1-hexene and decompose into ˙C3H7 + ˙C3H5 and ˙C4H7 + ˙C2H5. The 1,6-hexyl diradical could also undergo direct dissociation; the C4H8 fragment via the 1,4-butyl diradical intermediate was observed, serving as evidence of the 1,6-hexyl diradical mechanism. Quantum chemistry calculations at UCCSD(T)/cc-pVDZ level of theory on the initial reaction pathways of cyclohexane were performed and found to be consistent with the experimental conclusions. Cyclohexyl radical was not observed as an initial intermediate in the pyrolysis. Benzene was produced from sequential H2 eliminations of cyclohexane at high temperatures.

Gene Expression and Data Analysis Pipeline Using Cancer BioPortal in the Classroom.

At institutions with an emphasis on authentic research experiences as an integral part of the biology curriculum, COVID created a huge challenge for course instructors whose learning objectives were designed for such experiences. Moving such laboratory experiences online when remote learning became necessary has resulted in a new model for CUREs that utilizes free online databases to provide not only a novel research experience for students, but also the opportunity to engage in big data analysis. Cancer BioPortal (cBioPortal) is an open-access collective cancer research resource for storing and exploring clinical, genomic, proteomic, and transcriptomic data. cBioPortal eliminates the computational barrier of interpreting complex genomic data by providing easily understandable visualization that can be interpreted and translated into relevant biological insights. Because no prior computational knowledge is required, cBioPortal is an ideal educational tool for either in-person or distance learning environments. We developed a pedagogical approach, video tutorials, and data analysis workflows centered on using cBioPortal. Pedagogically, students develop an initial research outline that is continually updated and graded throughout the project. Progress during the project or course is assessed by a series of student presentations that are 5 to 15 minutes in length and are aimed at explaining the approach used in data acquisition, interpretation of the data, and relevance to the initial hypothesis. While cancer-specific, this analysis platform appeals to a wide range of classes and student interests. Further, the project has been successfully done both as an independent research experience and as part of a virtual class-based research project.

Lead-Free Organic-Perovskite Hybrid Quantum Wells for Highly Stable Light-Emitting Diodes.

Two-dimensional perovskites that could be regarded as natural organic-inorganic hybrid quantum wells (HQWs) are promising for light-emitting diode (LED) applications. High photoluminescence quantum efficiencies (approaching 80%) and extremely narrow emission bandwidth (less than 20 nm) have been demonstrated in their single crystals; however, a reliable electrically driven LED device has not been realized owing to inefficient charge injection and extremely poor stability. Furthermore, the use of toxic lead raises concerns. Here, we report Sn(II)-based organic-perovskite HQWs employing molecularly tailored organic semiconducting barrier layers for efficient and stable LEDs. Utilizing femtosecond transient absorption spectroscopy, we demonstrate the energy transfer from organic barrier to inorganic perovskite emitter occurs faster than the intramolecular charge transfer in the organic layer. Consequently, this process allows efficient conversion of lower-energy emission associated with the organic layer into higher-energy emission from the perovskite layer. This greatly broadened the candidate pool for the organic layer. Incorporating a bulky small bandgap organic barrier in the HQW, charge transport is enhanced and ion migration is greatly suppressed. We demonstrate a HQW-LED device with pure red emission, a maximum luminance of 3466 cd m-2, a peak external quantum efficiency up to 3.33%, and an operational stability of over 150 h, which are significantly better than previously reported lead-free perovskite LEDs.