Search

Methylene-selective C-H functionalization is a significant hurdle that remains to be addressed in the field of Pd(II) catalysis. We report a Pd(II)-catalyzed synthesis of benzocyclobutenes by methylene-selective C(sp3)-H arylation of ketones. The reaction utilizes glycine as a transient directing group and a 2-pyridone ligand, which may govern the methylene selectivity by making intimate molecular associations with the substrate during concerted metalation-deprotonation. This reaction is shown to be highly selective for intramolecular methylene C(sp3)-H arylation, thus enabling sequential C(sp3)-H functionalization.

Silver in the linings The bacterium Shewanella oneidensis is well known to use extracellular electron sinks, metal oxides and ions in nature or electrodes when cultured in a fuel cell, to power the catabolism of organic material. However, the power density of microbial fuel cells has been limited by various factors that are mostly related to connecting the microbes to the anode. Cao et al . found that a reduced graphene oxide–silver nanoparticle anode circumvents some of these issues, providing a substantial increase in current and power density (see the Perspective by Gaffney and Minteer). Electron microscopy revealed silver nanoparticles embedded or attached to the outer cell membrane, possibly facilitating electron transfer from internal electron carriers to the anode. —MAF

Rationale: Myofilament length-dependent activation (LDA) is the key underlying mechanism of cardiac heterometric autoregulation, commonly referred as the Frank-Starling Law of the heart. Although alterations in LDA are common in cardiomyopathic states, the precise structural and biochemical mechanisms underlying LDA remain unknown. Objective: Here, we examine the role of structural changes in the thick filament during diastole, in particular changes in the availability of myosin heads, in determining both calcium sensitivity and maximum contractile force during systole in permeabilized porcine cardiac fibers. Methods and Results: Permeabilized porcine fibers from ventricular myocardium were studied under relaxing conditions at short and long sarcomere length using muscle mechanics, biochemical measurements, and X-ray diffraction. Upon stretch, the porcine myocardium showed the increased calcium sensitivity and maximum calcium-activated force characteristic of LDA. Stretch increased diastolic ATP turnover, recruiting reserve myosin heads from the super-relaxed state at longer sarcomere length. Structurally, X-ray diffraction studies in the relaxed-muscle confirmed a departure from the helical ordering of the thick filament upon stretch which occurred concomitantly with a displacement of myosin heads towards actin, facilitating cross-bridge formation upon systolic activation. Mavacamten, a selective myosin-motor inhibitor known to weaken the transition to actin-bound power-generating states and to enrich the ordered super-relaxed state myosin population, reversed the structural effects of stretch on the thick filament, blunting the mechanical consequences of stretch; mavacamten did not, however, prevent other structural changes associated with LDA in the sarcomere, such as decreased lattice spacing or troponin-displacement. Conclusions: Our findings strongly indicate that in ventricular muscle, LDA and its systolic consequences are dependent on the population of myosin heads competent to form cross bridges and involves the recruitment of myosin heads from the reserve super-relaxed state pool during diastole.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1., Once DNA breaks occur, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other DNA repair factors to initiate the repair process. Here, the authors resolve the crystal structures of mouse and human HPF1, and human HPF1/PARP1 complex proving insights into PARP1 regulation.

In this study, the non-oxidative torrefaction of oil palm empty fruit bunch (OPEFB) pellets was investigated from 250 to 300 °C for 30 min in a horizontal fixed bed tubular reactor. The effects of the selected conditions on the yields, distributions and fuel characteristics of the torrefaction products were examined. The mass or solid yield (MY) decreased from 68.1 to 36.2%, whereas the liquid yield (LY) and gas yield (GY) increased from 19.4–40.1% and 12.5–23.7%, respectively, due to drying, devolatilization and depolymerisation during torrefaction. Physicochemical and calorific analyses showed that the torrefied OPEFB pellets have high carbon but low oxygen contents, which accounts for the high heating values (HHV = 22.83–25.81 MJ/kg). The torrefied OPEFB pellets also exhibit lower moisture (2–4%) and volatile matter (34.38–65.31 wt.%) but high ash (4–20 wt.%) and fixed carbon (28.69–41.62 wt.%) compared to the raw pellets. The OPEFB pellet fuel properties, namely pH that ranged from 6.65 to 7.74, hydrophobicity from 100 to 23.04% and grindability from 53.66 to 108, were markedly enhanced after torrefaction at 300 °C. The LY consisted of organics (67.64–62.62%) and water (32.36–37.38%) fractions characterised by high acidity (pH = 2.89–3.22) and dark hues formed by holocellulose and lignin thermal degradation at higher torrefaction temperatures. Based on the findings, the torrefied OPEFB pellets is a highly grindable, hydrophobic, thermally stable and promising solid biofuel for firing, co-firing or substituting coal in power plants provided the existing challenges that affect global biomass supply chains are addressed in detail.

Silver nanoparticles (AgNPs) are widely sought after for a variety of biomedical and environmental applications due to their antimicrobial and catalytic properties. We present here a green and simple synthesis of AgNPs utilizing traditional Chinese medicinal herbs. The screening of 20 aqueous herb extracts shows that Sheng Di Huang (Rehmannia glutinosa) had the most promising potential in producing AgNPs of 30±6 nm, with narrow size distribution and high crystallinity. The antimicrobial activities of these AgNPs conducted on E. coli cells were found to be superior in comparison to poly(vinylpyrrolidone)-capped AgNPs synthesized using common chemical method. Additionally, the AgNPs obtained possess excellent catalytic performance in the reduction of 4-nitrophenol to 4-aminophenol. We compared the phytochemical and FTIR spectral analyses of the herb extract before and after synthesis, in order to elucidate the phytochemicals responsible for the reduction of Ag+ ions and the capping of the AgNPs produced.

Paclitaxel-coated devices have been shown to decrease restenosis when used in the femoropopliteal artery. Recent reports have suggested a possible risk of increased late mortality in patients treated with paclitaxel. It has been suggested that younger patients and those with limited comorbidities may be at higher risk. Our objective was to analyze long-term mortality based on patient age comparing treatment with paclitaxel to uncoated devices.We performed a retrospective review of 1,170 consecutive patients who underwent femoropopliteal percutaneous intervention by angioplasty, atherectomy, stent placement, or combination between 2011 and 2018. Patients were grouped by age at the time of procedure:60 years old (n = 244, 20.9%), 60-80 years old (n = 635, 54.3%), and80 years old (n = 291, 24.9%). Within each group, patients were further divided by use of paclitaxel. The primary outcome measure was survival assessed by Kaplan-Meier analysis. Differences between the groups were analyzed with analysis of variance. Multivariable analysis was performed using Cox proportional hazard models.Of the 1,170 patients who underwent femoropopliteal percutaneous intervention, 654 (55.9%) received a paclitaxel-coated device during treatment and 516 (44.1%) did not. Mean age of the overall patient cohort was 70.4 ± 12.6 years and 663 (56.7%) were male. When comparing the groups by age we found an increase in age but a decrease in the proportion of patients who smoke. The use of paclitaxel-coated devices was similar across the groups (60 years old, 56.2%; 60-80 years old, 57.0%;80 years old, 52.6%; P = 0.45). Demographics and comorbidities were similar between the patients treated with and without paclitaxel within each age group except more males in the60-year-old group treated without paclitaxel and more patients with chronic limb threatening ischemia in the80-year-old group treated with paclitaxel. In patients60 and 60-80 years old paclitaxel use was associated with increased survival at 4 years:60 (80.7% vs. 64.4%; P = 0.04); 60-80 (63.2% vs. 55.1%; P = 0.04). Survival was similar in the80-year-old group (46.6% vs. 32.8%; P = 0.65).Our data suggest that the use of paclitaxel-coated arterial devices is not associated with increased mortality. On the contrary, our data show that younger patients treated with paclitaxel show improved survival compared with those treated without paclitaxel. Paclitaxel-coated devices may be used with continued caution especially in patients at high risk for restenosis.

Objectives To characterise the ion release, pH changes and apatite formation of a phosphate free bioactive glass. Methods A SiO2-CaO-CaF2-Na2O glass was synthesized by a melt route with a composition close to the reactive glass in the commercial Cention N® composite. The glass was characterized after immersion in three media: Artificial Saliva pH4 (AS4) Artificial Saliva pH7 (AS7) and in a high phosphate artificial saliva at pH6.5 (AS6.5). The pH and fluoride release were measured using a pH meter and an ion selective electrode. The concentration of Ca, P, Na and Si were measured by ICP-OES. The glass powders after immersion were characterized by FTIR, X-ray powder diffraction and 19F MAS-NMR. Results The glass increased the pH in all three media. Fluoride was detected in all three media but was much higher in AS 6.5. Calcium fluoride formed in AS4 with a small amount of fluorapatite at long immersion times. Fluorapatite and calcium fluoride formed in AS7, whilst in AS6.5 fluorapatite formed. The ion concentrations in solution after immersion reflected the glass composition and the immersion media with fluorapatite being favoured by higher pHs and phosphate contents in the media. Significance The results demonstrated the ability of the glass to increase the pH and to form fluorapatite in phosphate containing media. This may explain the low incidence of secondary caries found in the commercial composite. Unlike the commercial composite evidence was found for the precipitation of fluorite, which will act to reduce the release of fluoride for preventing secondary caries.

The carbon dioxide (CO2) torrefaction of oil palm empty fruit bunch (OPEFB) pellets was investigated at various temperatures from 250 to 300 °C and residence times from 15 to 60 minutes. The objective was to investigate the effects of CO2 torrefaction on the yield and characteristics of the torrefied products. The optimal conditions for maximum mass yield (MY) of CO2 torrefaction were also examined by response surface methodology (RSM) using full factorial design. Results revealed that temperature and time significantly influenced the mass (MY), liquid (LY) and gas (GY) yields. The MY and energy yield (EY) decreased with increasing severity of torrefaction, whereas the LY, GY, energy density (DE) and higher heating value (HHV) increased during the process. Characterisation revealed substantial improvements in the microstructure, pH, hydrophobicity and grindability of the torrefied pellets compared with the raw pellets. The thermal ignition and degradation characteristics of the OPEFB pellets were also significantly transformed after torrefaction. The liquid torrefaction products contained an acidic, turbid and pungent mixture of water and organic compounds. RSM optimisation revealed the optimal conditions: temperature of 275 °C and residence time of 35 minutes with the predicted MY of 50.54%, mass loss of ML = 49.46% and HHV = 24.47 MJ/kg. The findings revealed that CO2 torrefaction is a practical approach to clean energy recovery.

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment., SCOPUS: ar.j, info:eu-repo/semantics/published

Using glycopolymer surfaces, we have stimulated Shewanella oneidensis bacterial colonization and induced where the bacteria attach on a molecular pattern. When adherent bacteria were rinsed with methyl α-d-mannopyranoside, the glycopolymer-functionalized surfaces retained more cells than self-assembled monolayers terminated by a single mannose unit. These results suggest that the three-dimensional multivalency of the glycopolymers both promotes and retains bacterial attachment. When the methyl α-d-mannopyranoside competitor was codeposited with the cell culture, however, the mannose-based polymer was not significantly different from bare gold surfaces. The necessity for equilibration between methyl α-d-mannopyranoside and the cell culture to remove the enhancement suggests that the retention of cells on glycopolymer surfaces is kinetically controlled and is not a thermodynamic result of the cluster glycoside effect. The MshA lectin appears to facilitate the improved adhesion observed. Our findings that the surfaces studied here can induce stable initial attachment and influence the ratio of bacterial strains on the surface may be applied to harness useful microbial communities.

Emerging infectious diseases (EIDs) are a significant burden on global economies and public health to any country in the world. With the extensive application of traditional Chinese medicines (TCMs) for EID treatment, the underlying molecular mechanisms have caught more attention than before. The ShuFengJieDu capsule (abbreviated as SFJD) is a TCM prescription used for treating upper respiratory infection (URI) with symptoms of fever, sore throat, headaches, nasal congestion, and cough for more than 30 years in China. SFJD is also widely used for the prevention and treatment of viral infectious diseases, especially for the EIDs. In this study, a bioactivity-integrated method of ultraperformance liquid chromatography quadrapole/time-of-flight mass spectrometry combined with methyl thiazolyl tetrazolium assay was applied to screen potential antivirus compounds in SFJD on the H1N1-infected RAW264.7 cell models. Three compounds (forsythoside E, verbenalin, and emodin) exert the advantages of protective effects in cell vitality during H1N1 infection. The isobaric tags for relative and absolute quantification (iTRAQ)-coupled liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) analysis and the subsequent quantitative proteome analysis were performed to investigate the potential molecular mechanisms triggered by these three bioactive compound-triggered molecular mechanisms in H1N1-infected RAW264.7 cells. Dysregulated proteins were involved in regulating the levels of proinflammatory cytokines, the IFN (interferon)-stimulated gene signal in the Type I IFN, TBK/IRF3, and MAPK/NF-κB signaling pathways. In conclusion, we identified the main bioactive compounds in SFJD exerting antiviral effects and illuminated that Type I IFN and MAPK/NF-κB signaling pathways are involved in the anti-H1N1 infection effects of SFJD. Our study not only provides solid theoretical support for the clinic application of SFJD but also sheds light on the novel research methods for TCM study.

As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer’s disease and cancer2–7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8. The structure of human ACAT1, which catalyses the transfer of an acyl group from acyl-coenzyme A to cholesterol to form cholesteryl ester, is resolved by cryo-electron microscopy.

The management of diabetic wounds remains a major therapeutic challenge in clinics. Herein, we report a personalized treatment using 3D scaffolds consisting of radially or vertically aligned nanofibers in combination with bone marrow mesenchymal stem cells (BMSCs). The 3D scaffolds have customizable sizes, depths, and shapes, enabling them to fit a variety of type 2 diabetic wounds. In addition, the 3D scaffolds are shape-recoverable in atmosphere and water following compression. The BMSCs-laden 3D scaffolds are capable of enhancing the formation of granulation tissue, promoting angiogenesis, and facilitating collagen deposition. Further, such scaffolds inhibit the formation of M1-type macrophages and the expression of pro-inflammatory cytokines IL-6 and TNF-α and promote the formation of M2-type macrophages and the expression of anti-inflammatory cytokines IL-4 and IL-10. Taken together, BMSCs-laden, 3D nanofiber scaffolds with controlled structure and alignment hold great promise for the treatment of diabetic wounds. Statement of Significance In this study, we developed 3D radially and vertically aligned nanofiber scaffolds to transplant bone marrow mesenchymal stem cells (BMSCs). We personalized 3D scaffolds that could completely match the size, depth, and shape of diabetic wounds. Moreover, both the radially and vertically aligned nanofiber scaffolds could completely recover their shape and maintain structural integrity after repeated loads with compressive stresses. Furthermore, the BMSCs-laden 3D scaffolds are able to promote granulation tissue formation, angiogenesis, and collagen deposition, and switch the immune responses to the pro-regenerative direction. These 3D scaffolds consisting of radially or vertically aligned nanofibers in combination with BMSCs offer a robust, customizable platform potentially for a significant improvement of managing diabetic wounds.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α–β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4’s α-helices are membrane disruptive, while XCL1’s helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α–β chemokine structure). Here, we investigate XCL1’s antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular “Swiss army knife” that can refold into different structures for distinct context-dependent functions: whereas the α–β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1’s β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.

Metallothioneins (MTs) are ubiquitous proteins vital for essential metal homeostasis and heavy metal detoxification. The twenty-cysteinyl mammalian metallothioneins protect the proteome by sequestering heavy metals into thermodynamically stable metal thiolate structures when metalated with seven Cd2+. At physiological pH, the first metal (M) thiolate (SCys) structures formed involve M(SCys)4 terminal thiolates. With higher metal loading, M4(SCys)11 and M3(SCys)9 clusters form. As a regulator of the metallome, it is necessary to understand metal sequestration properties of MT in solution with other metalloproteins. We report that the association between apo-MT and apo-carbonic anhydrase (CA) enhances the formation of the protective mode of MT, in which Cd4(SCys)11-clusters form at much lower concentration levels than for the free apo-MT at physiological pH. Using stopped-flow kinetics and electrospray ionization mass spectrometry, we quantified this protective effect, determining that it is significant at pH 7.4, but the effect diminishes at pH 5.0. We report for the first time, the absolute stepwise binding constants of Cd2+ binding to human MT1a both in the presence and absence of CA through calibration by the known binding constant of Cd2+ to bovine CA. We report that this protein association affects the Cd2+ metalation rates of MT. The data support the physiological role of MTs as protectors of the metalloproteome from the toxic effects of Cd2+.

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1.ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

A strategy to control the diastereoselectivity of bond formation at a prochiral attached-ring bridgehead is reported. An unusual stereodivergent Michael reaction relies on basic vs. Lewis acidic conditions and non-covalent interactions to control re- vs. si- facial selectivity en route to fully substituted attached-rings. This divergency reflects differential engagement of one rotational isomer of the attached-ring system. The successful synthesis of an erythro subtarget diastereomer ultimately leads to a short formal synthesis of merrilactone A.

Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to be an ideal drug target for treating COVID-19. Leupeptin, a broad-spectrum covalent inhibitor of serine, cysteine, and threonine proteases, showed inhibitory activity against Mpro, with a 50% inhibitory concentration (IC50) value of 127.2 µM in vitro in our study here. In addition, leupeptin can also inhibit SARS-CoV-2 in Vero cells, with 50% effective concentration (EC50) values of 42.34 µM. More importantly, various strains of streptomyces that have a broad symbiotic relationship with medicinal plants can produce leupeptin and leupeptin analogs to regulate autogenous proteases. Fingerprinting and structure elucidation using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), respectively, further proved that the Qing-Fei-Pai-Du (QFPD) decoction, a traditional Chinese medicine (TCM) formula for the effective treatment of COVID-19 during the period of the Wuhan outbreak, contains leupeptin. All these results indicate that leupeptin at least contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This also reminds us to pay attention to the microbiomes in TCM herbs as streptomyces in the soil might produce leupeptin that will later infiltrate the medicinal plant. We propose that plants, microbiome, and microbial metabolites form an ecosystem for the effective components of TCM herbs. IMPORTANCE A TCM formula has played an important role in the treatment of COVID-19 in China. However, the mechanism of TCM action is still unclear. In this study, we identified leupeptin, a metabolite produced by plant-symbiotic actinomyces (PSA), which showed antiviral activity in both cell culture and enzyme assays. Moreover, leupeptin found in the QFPD decoction was confirmed by both HPLC fingerprinting and HRMS. These results suggest that leupeptin likely contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This result gives us important insight into further studies of the PSA metabolite and medicinal plant ecosystem for future TCM modernization research.

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, swarming motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-swarming cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. In contrast, flagellated cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species.ImportanceOur study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of the larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and non-motile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP(1). X-ray structural studies and theoretical calculations show that apoMP(1) induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and “turn off” diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP(1) dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP(1) inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Methylene-selective C‒H functionalization is a significant hurdle that remains to be addressed in the field of Pd(II) cataly-sis. In this paper we report a Pd(II) catalyzed synthesis of benzocyclobutenes by methylene-selective C(sp3)‒H arylation of ketones using a transient directing group. The reaction utilizes glycine as a transient directing group and an electron poor pyridone ligand which is expected to promote concerted metallation deprotonation, but also controls methylene selectivity by intimate interactions with the substrate. These reaction conditions are shown to be highly selective for in-tramolecular methylene C(sp3)‒H arylation, and thus enable sequential C(sp3)‒H functionalization.

Highlights • GILT is upregulated in chemoresistant LSC-enriched CD34+ progenitor cells. • Inhibition of GILT in AML cells sensitized them to Ara-C treatment through ROS-mediated mitochondrial damage and apoptosis. • PI3K/Akt/NRF2 pathway inhibition is critical for the intracellular oxidative state in GILT-suppression AML cells after Ara-C treatment. • GILT expression is related to a poor prognosis in AML patients., The persistence of leukemia stem cells (LSCs) is one of the leading causes of chemoresistance in acute myeloid leukemia (AML). To explore the factors important in LSC-mediated resistance, we use mass spectrometry to screen the factors related to LSC chemoresistance and defined IFN-γ-inducible lysosomal thiol reductase (GILT) as a candidate. We found that the GILT expression was upregulated in chemoresistant CD34+ AML cells. Loss of function studies demonstrated that silencing of GILT in AML cells sensitized them to Ara-C treatment both in vitro and in vivo. Further mechanistic findings revealed that the ROS-mediated mitochondrial damage plays a pivotal role in inducing apoptosis of GILT-inhibited AML cells after Ara-C treatment. The inactivation of PI3K/Akt/ nuclear factor erythroid 2-related factor 2 (NRF2) pathway, causing reduced generation of antioxidants such as SOD2 and leading to a shifted ratio of GSH/GSSG to the oxidized form, contributed to the over-physiological oxidative status in the absence of GILT. The prognostic value of GILT was also validated in AML patients. Taken together, our work demonstrated that the inhibition of GILT increases AML chemo-sensitivity through elevating ROS level and induce oxidative mitochondrial damage-mediated apoptosis, and inhibition of the PI3K/Akt/NRF2 pathway enhances the intracellular oxidative state by disrupting redox homeostasis, providing a potentially effective way to overcome chemoresistance of AML., Graphical abstract GILT inhibition overcomes chemoresistance in AML Left panel. Despite that Ara-C stimulates ROS generation and causes oxidative stress, chemoresistance can still develop as a result of compensatory elevation of antioxidants that detoxify the harmful oxidants. Activation of P13K/Akt/NRF2 pathwaypromotes expressionofantioxidants. Right panel. GILT knockdown facilitates disturbance of redox homeostasis of AML cell by (i) enhancing Ara-C-stimulated ROS over-production, (ii) reducing antioxidant level which is associated with inactivation of the P13K/Akt/NRF2 axis. The resulting severe oxidative stress leads to mitochondrial damage and apoptosis. Image, graphical abstract

Background: Cixutumumab, a monoclonal antibody targeting insulin-like growth factor I receptor, did not improve undetectable PSA rate at 28 weeks when combined with androgen deprivation in the randomized phase II SWOG S0925 trial for patients with new metastatic hormone-sensitive prostate cancer. We now present mature survival analyses, along with pre-specified secondary and exploratory endpoints. Methods: We randomized 210 patients to androgen deprivation with or without cixutumumab, 105 per treatment arm. We used Kaplan-Meier curves to analyze overall survival, radiographic progression-free survival, and castration resistance-free survival by treatment arm, disease volume, and risk group. We explored differences in survival by treatment arm via covariate-adjusted Cox proportional hazards models adjusted for disease volume and risk. Results: No difference was seen between treatment arms in overall survival (HR 1.01 [0.70-1.45]; p=0.97), radiographic progression-free survival (HR 1.17 [0.85-1.60]; p=0.35), or castration resistance-free survival (HR 1.02 [0.75-1.41]; p=0.88). At baseline, 105/198 (53.0%) patients had high risk features and 119/210 (56.7%) had high volume disease; 16.7% of patients had discordant classifications of high or low category for risk and volume. Adjusting for risk or volume yielded no differences in overall survival between arms. Inferior survival was observed in high risk (HR 1.89 [1.29-2.80]; p=0.001) and high volume (HR 2.75 [1.84-4.10]; p0.2 to ≤4.0 ng/mL (HR 3.72 [1.99-6.95]; p4.0 ng/mL (HR 7.13 [4.24-11.9]; p

In pursuit of 18F-labeled nucleosides for positron emission tomography (PET) imaging, we report on the chemical and radiochemical synthesis of two thymidine (dT) analogs, dT-C5-AMBF3 and dT-N3-AMBF3, that are radiofluorinated by isotope exchange (IEX) and studied as PET imaging agents in mice with tumor xenografts. dT-C5-AMBF3 shows preferential, and tumor-specific, uptake over dT-N3-AMBF3. This work provides a new synthetic method in order to access new nucleoside tracers for PET imaging.

Sierra Cheng,1 Vanessa Arciero,1 Hanan Goldberg,2 Camilla Tajzler,3 Aileen Manganaro,4 Natascha Kozlowski,4 Leigha Rowbottom,1 Rachel McDonald,1 Ronald Chow,1 Gaurav Vasisht,3 Sharon Shaji,3 Emily Chu Lee Wong,3 Michele Petrovic,2 Liying Zhang,1 Cameron Phillips,1 Pawel Zalewski,4 Anil Kapoor,3 Neil E Fleshner,2 Edward Chow,1 Urban Emmenegger1 1Sunnybrook Odette Cancer Centre, Toronto, Ontario, Canada; 2Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 3Juravinski Cancer Centre, Hamilton, Ontario, Canada; 4Durham Regional Cancer Centre, Oshawa, Ontario, CanadaCorrespondence: Urban EmmeneggerSunnybrook Odette Cancer Centre, 2075 Bayview Avenue, Toronto, Ontario M4N3M5, CanadaTel +1-416-480 4928Fax +1-416-480 6002Email urban.emmenegger@sunnybrook.caIntroduction: Radium-223 (Ra223) prolongs the survival and improves the quality of life of men with metastatic, castration-resistant prostate cancer (mCRPC) to bones. However, compared to other mCRPC therapies, using Ra223 comes with its unique challenges. Hence, we aimed to identify Ra223 utilization patterns under real-world conditions, as well as factors predicting treatment completion and outcome.Methods: In this retrospective chart analysis, 198 mCRPC patients were identified that had received Ra223 outside of clinical trials or access programs from January 2015 to October 2016 at four cancer centres in Ontario. The main outcomes studied were Ra223 completion rate, reasons for early treatment discontinuation, overall survival, and survival differences in patients completing Ra223 therapy versus patients receiving

Metal complexes with a [n × n] gridlike structure are discussed as attractive building blocks for various materials chemistry applications in molecular nanotechnology and electronics, which often rely on the grids’ magnetic and redox properties. Most of the known metallogrids are homometallic, though heterometallic systems that comprise two or more different metals promise higher level functionalities. However, heterometallic [n × n] grids are relatively rare, mostly because of the more challenging synthetic strategies. To that end a new heterometallic [2 × 2] grid complex [L4Ru2Co2](BF4)4 (2) based on a known pyrazolate-bridged bis(tridentate) compartmental N-donor ligand [L]− is presented in this work, along with its doubly oxidized congener [L4Ru2Co2](BF4)6 (3). In order to prevent scrambling of the different metal ions, a stepwise synthetic approach was implemented in which an inert RuII “corner complex” [(HL)2Ru](BF4)2 (1) was isolated first, followed by addition of the more labile CoII. This exclusi...

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

A significant challenge to making targeted CFTR modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rareCFTRmutation R352Q – with potential role contributing to intrapore chloride ion permeation – in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach ofin vitropatient-derived cell models andin silicoMD simulations to characterize rareCFTRmutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

The Mars Science Laboratory (MSL) recently discovered nitrates in Gale Crater (e.g., Stern et al., 2015; Sutter et al., 2017). One possible mechanism for ancient nitrate deposition on Mars is through HNOx formation and rain out in the atmosphere, for which lightning-induced NO is likely the fundamental source. This study investigates nitrogen (N₂) fixation in early Mars' atmosphere, with implications for early Mars' habitability. We consider a 1 bar atmosphere of background CO₂, with abundance of N₂, hydrogen, and methane varied from 1% to 10% to explore a swath of potential early Mars climates. We derive lightning-induced thermochemical equilibrium fluxes of NO and HCN by coupling the lightning-rate parametrization from the study of Romps et al. (2014) with chemical equilibrium with applications, and we use a Geant4 simulation platform to estimate the effect of solar energetic particle events. These fluxes are used as input into KINETICS, the Caltech/JPL coupled photochemistry and transport code, which models the chemistry of 50 species linked by 495 reactions to derive rain-out fluxes of HNOx and HCN. We compute equilibrium concentrations of cyanide and nitrate in a putative northern ocean at early Mars, assuming hydrothermal vent circulation and photoreduction act as the dominant loss mechanisms. We find average oceanic concentrations of ∼0.1–2 nM nitrate and ∼0.01–2 mM cyanide. HCN is critical for protein synthesis at concentrations >0.01 M (e.g., Holm and Neubeck, 2009), and our result is astrobiologically significant if secondary local concentration mechanisms occurred. Nitrates may act as high-potential electron acceptors for early metabolisms, although the minimum concentration required is unknown. Our study derives concentrations that will be useful for future laboratory studies to investigate the habitability at early Mars. The aqueous nitrate concentrations correspond to surface nitrate precipitates of ∼1–8 × 10⁻⁴ wt % that may have formed after the evaporation of surface waters, and these values roughly agree with recent MSL measurements.

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.

Background: Diabetes is a risk factor for postoperative complications. Previous meta-analyses have shown that elevated glycated hemoglobin (HbA1c) levels are associated with postoperative complications in various surgical populations. However, this is the first meta-analysis to investigate the association between preoperative HbA1c levels and postoperative complications in patients undergoing elective major abdominal surgery.Methods: PRISMA guidelines were adhered to for this study. Six databases were searched up to April 1, 2020. Primary studies investigating the effect of HbA1c levels on postoperative complications after elective major abdominal surgery were included. Risk of bias and quality of evidence assessments were performed. Data were pooled using a random effects model. Meta-regression was performed to evaluate different HbA1c cut-off values. Results: Twelve observational studies (25,036 patients) were included. Most studies received a ‘good’ and ‘moderate quality’ score using the NOS and GRADE, respectively. Patients with a high HbA1c had a greater risk of anastomotic leaks (odds ratio [OR]: 2.80, 95% CI [1.63, 4.83], P < 0.001), wound infections (OR: 1.21, 95% CI [1.08, 1.36], P = 0.001), major complications defined as Clavien-Dindo [CD] 3–5 (OR: 2.16, 95% CI [1.54, 3.01], P < 0.001), and overall complications defined as CD 1–5 (OR: 2.12, 95% CI [1.48, 3.04], P < 0.001).Conclusions: An HbA1c between 6% and 7% is associated with higher risks of anastomotic leaks, wound infections, major complications, and overall postoperative complications. Therefore, guidelines with an HbA1c threshold > 7% may be putting pre-optimized patients at risk. Future randomized controlled trials are needed to explore causation before policy changes are made.

Spexin (SPX), a highly conserved neuropeptide, is known to have diverse functions and has been implicated/associated with pathological conditions, including obesity, diabetes, anorexia nervosa, and anxiety/mood disorders. Although most of the studies on SPX involved the mouse model, the solution structure of mouse SPX, structural aspects for SPX binding with its receptors GalR2/3, and its cellular expression/distribution in mouse tissues are largely unknown. Using CD and NMR spectroscopies, the solution structure of mouse SPX was shown to be in the form of a helical peptide with a random coil from Asn1 to Pro4 in the N-terminal followed by an α-helix from Gln5 to Gln14 in the C-terminus. The molecular surface of mouse SPX is largely hydrophobic with Lys11 as the only charged residue in the α-helix. Based on the NMR structure obtained, docking models of SPX binding with mouse GalR2 and GalR3 were constructed by homology modeling and MD simulation. The models deduced reveal that the amino acids in SPX, especially Asn1, Leu8, and Leu10, could interact with specific residues in ECL1&2 and TMD2&7 of GalR2 and GalR3 by H-bonding/hydrophobic interactions, which provides the structural evidence to support the idea that the two receptors can act as the cognate receptors for SPX. For tissue distribution of SPX, RT-PCR based on 28 tissues/organs harvested from the mouse demonstrated that SPX was ubiquitously expressed at the tissue level with notable signals detected in the brain, GI tract, liver, gonad, and adrenal gland. Using immunohistochemical staining, protein signals of SPX could be located in the liver, pancreas, white adipose tissue, muscle, stomach, kidney, spleen, gonad, adrenal, and hypothalamo-pituitary axis in a cell type-specific manner. Our results, as a whole, not only can provide the structural information for ligand/receptor interaction for SPX but also establish the anatomical basis for our on-going studies to examine the physiological functions of SPX in the mouse model.

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

BackgroundRespiratory viruses are transmitted and acquired via the nasal mucosa, and thereby may influence the nasal metabolome composed of biochemical products produced by both host cells and microbes. Studies of the nasal metabolome demonstrate virus-specific changes that sometimes correlate with viral load and disease severity. Here, we evaluate the nasopharyngeal metabolome of COVID-19 infected individuals and report several small molecules that may be used as potential therapeutic targets. Specimens were tested by qRT-PCR with target primers for three viruses: Influenza A (INFA), respiratory syncytial virus (RSV), and SARS-CoV-2, along with asymptomatic controls. The nasopharyngeal metabolome was characterized using an LC-MS/MS-based small-molecule screening kit capable of quantifying 141 analytes. A machine learning model identified 28 discriminating analytes and correctly categorized patients with a viral infection with an accuracy of 96% (R2=0.771, Q2=0.72). A second model identified 5 analytes to differentiate COVID19-infected patients from those with INFA or RSV with an accuracy of 85% (R2=0.442, Q2=0.301). Specifically, LysoPCaC18:2 concentration was significantly increased in COVID19 patients (P< 0.0001), whereas beta-hydroxybutyric acid, Met SO, succinic acid, and carnosine concentrations were significantly decreased (P< 0.0001). This study demonstrates that COVID19 infection results in a unique NP metabolomic signature with carnosine and LysoPCaC18:2 as potential therapeutic targets.Significance StatementEfforts to elucidate how SARS-CoV-2 interacts with the host has become a global priority. To identify biomarkers for potential therapeutic interventions, we used a targeted metabolomics approach evaluating metabolite profiles in the nasal mucosa of COVID-19 patients and compared metabolite profiles to those of other respiratory viruses (influenza A, RSV). We identified a COVID-19-specific signature characterized by changes to LysoPCaC18:2, beta-hydroxybutyric acid, Met SO, succinic acid, and carnosine. Carnosine is a promising potential target against SARS-CoV-2 as it has been shown to interfere with binding of SARS-CoV-2 to the ACE2 receptor. This study provides compelling evidence for the use of metabolomics as an avenue for the identification of novel drug targets for viral respiratory infections in the nasopharynx.

PDCD10, also known as CCM3, is a gene found to be associated with the human disease cerebral cavernous malformations (CCMs). PDCD10 forms a complex with GCKIII kinases including STK24, STK25, and MST4. Studies in C. elegans and Drosophila have shown a pivotal role of the PDCD10-GCKIII complex in maintaining epithelial integrity. Here, we found that mice deficient of Pdcd10 or Stk24/25 in the kidney tubules developed polyuria and displayed increased water consumption. Although the expression levels of aquaporin genes were not decreased, the levels of total and phosphorylated aquaporin 2 (Aqp2) protein in the apical membrane of tubular epithelial cells were decreased in Pdcd10- and Stk24/25-deficient mice. This loss of Aqp2 was associated with increased expression and membrane targeting of Ezrin and phosphorylated Ezrin, Radixin, Moesin (p-ERM) proteins and impaired intracellular vesicle trafficking. Treatment with Erlotinib, a tyrosine kinase inhibitor promoting exocytosis and inhibiting endocytosis, normalized the expression level and membrane abundance of Aqp2 protein, and partially rescued the water reabsorption defect observed in the Pdcd10-deficient mice. Our current study identified the PDCD10-STK-ERM signaling pathway as a potentially novel pathway required for water balance control by regulating vesicle trafficking and protein abundance of AQP2 in the kidneys.

Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5′-hydroxy-6′-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide., Elucidating a highly diastereoselective sulfoxidation of S-deoxy-amanitin to afford α-amanitin and a more synthetically accessible analog of near-native toxicity.

The outer membrane of Salmonella enterica plays an important role in combating stress encountered in the environment and hosts. The transport and insertion of lipopolysaccharides (LPS) into the outer membrane involves lipopolysaccharide transport proteins (LptA-F) and mutations in the genes encoding for these proteins are often lethal or result in the transport of atypical LPS that can alter stress tolerance in bacteria. During studies of heterogeneity in bile salts tolerance, S. enterica serovar Typhimurium E40 was segregated into bile salts tolerant and sensitive cells by screening for growth in TSB with 10% bile salts. An isolate (E40V) with a bile salts MIC >20% was selected for further characterization. Whole-genome sequencing of E40 and E40V using Illumina and PacBio SMRT technologies revealed a non-synonymous single nucleotide polymorphism (SNP) in lptG. Leucine at residue 26 in E40 was substituted with proline in E40V. In addition to growth in the presence of 10% bile salts, E40V was susceptible to novobiocin while E40 was not. Transcriptional analysis of E40 and E40V, in the absence of bile salts, revealed significantly greater (p < 0.05) levels of transcript in three genes in E40V; yjbE (encoding for an extracellular polymeric substance production protein), yciE (encoding for a putative stress response protein), and an uncharacterized gene annotated as an acid shock protein precursor (ASPP). No transcripts of genes were present at a greater level in E40 compared to E40V. Corresponding with the greater level of these transcripts, E40V had greater survival at pH 3.35 and staining of Calcofluor-binding polysaccharide (CBPS). To confirm the SNP in lptG was associated with these phenotypes, strain E40E was engineered from E40 to encode for the variant form of LptG (L26P). E40E exhibited the same differences in gene transcripts and phenotypes as E40V, including susceptibility to novobiocin, confirming the SNP was responsible for these differences.

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated buddingin vitro, providing a biophysical explanation for thein-vivophenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an “off” switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.