Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Elemental Fluorine Detection by Dielectric Barrier Discharge Coupled to Nanoelectrospray Ionization Mass Spectrometry for Nontargeted Analysis of Fluorinated Compounds.

The growing use of fluorochemicals has elevated the need for nontargeted detection of unknown fluorinated compounds and transformation products. Elemental mass spectrometry (MS) coupled to chromatography offers a facile approach for such analyses by using fluorine as an elemental tag. However, efficient ionization of fluorine has been an ongoing challenge. Here, we demonstrate a novel atmospheric-pressure elemental ionization method where fluorinated compounds separated by gas chromatography (GC) are converted to Na2F+ for nontargeted detection. The compounds are first introduced into a helium dielectric barrier discharge (DBD) for breakdown. The plasma products are subsequently ionized by interaction with a nanoelectrospray ionization (nano-ESI) plume of sodium-containing aqueous electrolytes. Our studies point to HF as the main plasma product contributing to Na2F+ formation. Moreover, the results reveal that Na2F+ is largely formed by the ion-neutral reaction between HF and Na2A(NaA)n+, gas-phase reagent ions produced by nano-ESI where A represents the anion of the electrolyte. Near-uniform fluorine response factors are obtained for a wide range of compounds, highlighting good efficiency of HF formation by DBD regardless of the chemical structure of the compounds. Detection limits of 3.5-19.4 pg of fluorine on-column are obtained using the reported GC-DBD-nano-ESI-MS. As an example of nontargeted screening, extractions from oil-and-water-repellent fabrics are analyzed via monitoring Na2F+, resulting in detection of a fluorinated compound on a clothing item. Notably, facile switching of the ion source to atmospheric-pressure chemical ionization with the exact same chromatographic method allows identification of the detected compound at the flagged retention time.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions.

Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity.IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.

Classifying single fibers based on fluorinated surface treatments.

Fibers are an important form of forensic evidence, but their evidential value can be severely limited when the identified characteristics of the fibers are common, such as blue cotton. Detecting chemical fiber treatments offers an avenue to further classify fibers and to improve their evidential value. In this report, we investigate the potential of fluoropolymer fiber coatings, used to impart oil and water-repellent properties in fabrics, for differentiating between fibers. The thin nature of these fiber surface modifications creates an analytical challenge for their detection on a single fiber, a typical sample size for forensic evidence. Specifically, pyrolysis-gas chromatography-mass spectrometry (py-GC-MS) has shown promising selectivity but the sensitivity of the method is not adequate for single-fiber analysis of fluorinated coatings. To overcome this challenge, we utilize a newly developed elemental ionization source, plasma-assisted reaction chemical ionization (PARCI). The high sensitivity of py-GC-PARCI-MS for elemental fluorine analysis offers selective and sensitive detection of fluorinated pyrolysates among the non-fluorinated pyrolysates of the fiber core. As a result, fluoropolymer coatings are detected from 10-mm single-fiber samples. The technique is applied for classification of 22 fiber types, resulting in 4 distinct groups via hierarchical cluster analysis based on similarity of fluorine pyrograms. These results present the first study to classify fibers based on fluorinated coatings, and highlight the potential of py-GC-PARCI-MS for forensic analyses. Graphical Abstract ᅟ.

Peripapillary and macular morpho-vascular changes in patients with genetic or clinical diagnosis of autosomal dominant optic atrophy: a case-control study.

PURPOSE: To evaluate the macular and peripapillary morpho-vascular changes in ADOA, using optical coherence tomography (OCT) and OCT angiography (OCTA). METHODS: Prospectively defined, cross-sectional case-control study. Consecutive patients with a genetic or clinical diagnosis of ADOA along with age- and sex-matched controls were included. The radial peripapillary capillary (RPC) density and vessel density (VD) in the parafoveal superficial and deep capillary plexuses (SCP and DCP, respectively) were evaluated with OCTA. The ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thickness were determined using structural OCT. We applied a previously validated customized macro (Fiji, SciJava Consortium) to compute RPC density. The remaining parameters were calculated by the built-in software. Non-parametric methods were used for data analysis. The target α level was 0.05, which was adjusted through Bonferroni's correction when multiple outcomes were tested. RESULTS: Fifty-eight eyes (n = 29 control; n = 29 ADOA) from 30 subjects (mean age 42.43 ± 15.30 years; 37.93% male) were included. Parafoveal SCP VD, GCC thickness, RPC VD in the temporal quadrant, as well as RNFL thickness in the nasal and temporal quadrants were decreased in ADOA eyes (all p < 0.001). When only patients with genetically confirmed diagnosis were included, capillary dropout in the circumpapillary superior and inferior quadrants also became evident (both p < 0.001). The GCC/parafoveal SCP ratio was increased in ADOA, relatively to matched controls. In contrast, none of the circumpapillary morpho-vascular ratios was significantly different in ADOA eyes. CONCLUSIONS: The microvascular and structural changes found in ADOA suggest that both the macular and peripapillary regions are involved, although the threshold for damage of the structural and vascular components may be different for each region. Larger series with longitudinal follow-up may validate OCTA biomarkers helpful for disease monitoring.

PERIPAPILLARY NEUROVASCULAR COUPLING IN THE EARLY STAGES OF DIABETIC RETINOPATHY.

PURPOSE: To study radial peripapillary capillary (RPC) density in the early stages of diabetic retinopathy (DR), using optical coherence tomography angiography. METHODS: A cross-sectional evaluation of RPCs was performed using optical coherence tomography angiography (Avanti RTVue-XR 100, Optovue Inc, Fremont, CA). Annular RPC density was the primary outcome. Global density and retinal nerve fiber layer thickness were secondary outcomes. Diabetic eyes were divided into three groups: no DR, mild nonproliferative DR (mild NPDR), and moderate NPDR. Multilevel mixed-effects univariate and multivariate linear regression models were used. RESULTS: We included 155 eyes (n = 42 control; n = 27 no DR; n = 28 mild NPDR; and n = 58 moderate NPDR) from 86 subjects (mean [SD] age 63.39 [10.70] years; 46.45% male). When compared with controls, a significant decrease in annular RPC density was found in all groups of diabetic eyes on multivariate analysis (no DR: ß = -2.95, P < 0.001; mild NPDR: ß = -1.76, P = 0.017; and moderate NPDR: ß = -2.82, P < 0.001). We also detected a significant decrease in retinal nerve fiber layer thickness in diabetic eyes (even in the no DR group). Furthermore, in diabetic eyes, annular RPC density and retinal nerve fiber layer thickness correlated significantly (R = 0.4874, P < 0.001). CONCLUSION: Peripapillary neurovascular changes occur early in the course of DR. Their significance in the progression of DR warrants further research.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Atmospheric-Pressure Dielectric Barrier Discharge as an Elemental Ion Source for Gas Chromatographic Analysis of Organochlorines.

Atmospheric-pressure dielectric barrier discharge (AP-DBD) plasma has emerged in recent years as a versatile plasma for molecular ionization and elemental spectroscopy. However, its capabilities as an elemental ion source have been less explored, partly because of difficulties in the detection of positive elemental ions from this low-gas-temperature plasma. In this work, we investigate the detection of negative elemental ions to enable elemental mass spectrometry (MS) using AP-DBD. A gas chromatograph is coupled to a helium AP-DBD apparatus and positioned in front of an atmospheric-pressure-sampling mass spectrometer with no modifications to the ion sampling interface. We demonstrate that Cl- ions are detected with a compound-independent efficiency, enabling elemental quantification of organochlorines. Further, addition of oxygen at low concentration (11 ppm, v/v) to the helium plasma improves the analytical performance by reducing postcolumn peak broadening, whereas high oxygen concentrations (>110 ppm, v/v) lead to loss of the compound-independent response. The optimized GC-AP-DBD-MS setup shows close to 2 orders of magnitude of linearity for its compound-independent Cl response and offers detection limits of 0.5-1 pg of Cl on column (0.6 pg/s), suitable for analysis of organochlorines in food samples. We demonstrate this capability by analyzing orange juice spiked with pesticides at 9 µg/L and a single internal standard. Importantly, we demonstrate that a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction followed by GC-AP-DBD-MS quantification using the single standard provides acceptable recoveries (80-120%). These results highlight uniform QuEChERS extraction of a range of compounds and the compound-independent response of AP-DBD for Cl, making the combination of the two methods desirable for the rapid quantification of organochlorines. Furthermore, we discuss ionization matrix effects in AP-DBD for chlorine detection and offer strategies to flag matrix-impacted analytes. These results suggest that AP-DBD has the potential to become a unified ion source for both elemental quantification and molecular identification of GC eluents on a single MS platform.

Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Diabetes Mellitus-Not Just Type 1 or Type 2 Anymore.

INTRODUCTION: Diabetes mellitus traditionally has been categorized as type 1 (insulin deficiency due to autoimmune destruction of islet cells) or type 2 (insulin resistance with the development of relative insulin deficiency). However, other pathophysiologic etiologies for diabetes must be considered in the evaluation of patients with new-onset diabetes. CASE PRESENTATION: We report the case of a 50-year-old man with a diagnosis of type 2 diabetes mellitus who-despite appropriate pharmacotherapy-developed worsening hyperglycemia. Further investigation revealed the presence of metastatic pancreatic cancer. DISCUSSION: Although an association between pancreatic cancer and diabetes has been noted widely in the gastroenterology, oncology, and endocrine literature, a paucity of primary care literature on the topic exists. Features of predominant insulin deficiency and new onset of diabetes in a patient without family history of type 2 diabetes should raise suspicion for undetected/early-stage pancreatic cancer. CONCLUSIONS: This case highlights the importance of considering all possible pathophysiologic etiologies when a patient has a new diagnosis of diabetes. Clinicians should consider the possibility of pancreatic cancer in patients with new-onset diabetes mellitus, especially when features not characteristic of type 2 diabetes are present. Understanding the relationship between diabetes and pancreatic cancer has the potential to improve early detection of pancreatic cancer and can provide an opportunity for early treatment and improved survival.

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2-V3 interaction and modulates neutralization sensitivity.

Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4(+) T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.

Ultrafast tissue staining with chemical tags.

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.

Ulnar neuropathy and medial elbow pain in women's fastpitch softball pitchers: a report of 6 cases.

BACKGROUND: Elite-level women's fastpitch softball players place substantial biomechanical strains on the elbow that can result in medial elbow pain and ulnar neuropathic symptoms. There is scant literature reporting the expected outcomes of the treatment of these injuries. This study examined the results of treatment in a series of these patients. METHODS: We identified 6 female softball pitchers (4 high school and 2 collegiate) with medial elbow pain and ulnar neuropathic symptoms. Trials of conservative care failed in all 6, and they underwent surgical treatment with subcutaneous ulnar nerve transposition. These patients were subsequently monitored postoperatively to determine outcome. RESULTS: All 6 female pitchers had early resolution of elbow pain and neuropathic symptoms after surgical treatment. Long-term follow-up demonstrated that 1 patient quit playing softball because of other injuries but no longer reported elbow pain or paresthesias. One player was able to return to pitching at the high school level but had recurrent forearm pain and neuritis 1 year later while playing a different sport and subsequently stopped playing competitive sports. Four patients continued to play at the collegiate level without further symptoms. CONCLUSIONS: Medial elbow pain in women's softball pitchers caused by ulnar neuropathy can be treated effectively with subcutaneous ulnar nerve transposition if nonsurgical options fail. Further study is necessary to examine the role of overuse, proper training techniques, and whether pitching limits may be necessary to avoid these injuries.

Atrioesophageal Fistula: A Rare Complication of Radiofrequency Ablation.

75-year-old woman was admitted with fever, chills, altered mentation, and right-sided weakness. A month earlier, she had undergone catheter radiofrequency ablation for treatment of chronic atrial fibrillation. A magnetic resonance imaging scan of her brain revealed septic emboli with multiple bilateral cerebral and cerebellar infarcts, as well as extensive bilateral leptomeningeal enhancement. Blood cultures were positive for Streptococcus mitis, Rothia mucilaginosa, Streptococcus pneumonia, and Candida albicans, which suggested a connection between gastrointestinal and cardiovascular systems. A chest computed tomography scan with contrast showed a curvilinear low attenuation structure communicating between the esophagus and the left pulmonary vein-an atrioesophageal fistula. Ten days after admission, the patient died from multiple cerebral septic emboli secondary to atrioesophageal fistula following radiofrequency ablation.

Charge neutralization of the central lysine cluster in prion protein (PrP) promotes PrP(Sc)-like folding of recombinant PrP amyloids.

The structure of the infectious form of prion protein, PrP(Sc), remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrP(Sc), especially within residues ∼90-160. Polyanionic cofactors can enhance infectivity and PrP(Sc)-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrP(Sc) multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101-110. For example, in our parallel in-register intermolecular ß-sheet model of PrP(Sc), not only would these lysines be clustered within the 101-110 region of the primary sequence, but they would have intermolecular spacings of only ∼4.8 Å between stacked ß-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant ß-sheet cores and infrared spectra that are more reminiscent of bona fide PrP(Sc). These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrP(Sc) formation.

Evaluation of Intensive "4R" Strategies for Decreasing Nitrous Oxide Emissions and Nitrogen Surplus in Rainfed Corn.

The "4R" approach of using the right rate, right source, right timing, and right placement is an accepted framework for increasing crop N use efficiency. However, modifying only one 4R component does not consistently reduce nitrous oxide (NO) emissions. Our objective was to determine if N fertilizer applied in three split applications (Sp), by itself or combined with changes in N source and rate, could improve N recovery efficiency (NRE) and N surplus (NS) and decrease NO emissions. Over two corn ( L.) growing seasons in Minnesota, NO emissions ranged from 0.6 to 0.9 kg N ha. None of the treatment combinations affected grain yield. Compared with urea applied in a single application at the recommended N rate, Sp by itself did not improve NRE or NS and did not decrease NO. Combining Sp with urease and nitrification inhibitors and/or a 15% reduction in N rate increased NRE from 57 to >73% and decreased NS by >20 kg N ha. The only treatment that decreased NO (by 20-53%) was Sp combined with inhibitors and reduced N rate. Emissions of NO were more strongly correlated with NS calculated from grain N uptake ( = 0.61) compared with whole-plant N uptake ( = 0.39), possibly because most N losses occurred before grain filling. Optimizing both application timing and N source can allow for a moderate reduction in N rate that does not affect grain yield but decreases NO. Grain-based NS may be a more useful indicator of NO emissions than whole-plant-based NS.

Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal.

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 µM) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 µM for imatinib, 3.72 µM for dasatinib, and 81.35 µM for nilotinib; for L3 larvae, 11.27 µM, 13.64 µM, and 70.98 µM, respectively; for adult males, 41.6 µM, 3.87 µM, and 68.22 µM, respectively; and for adult females, 42.89 µM, 9.8 µM, and >100 µM, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Parallel in-register intermolecular ß-sheet architectures for prion-seeded prion protein (PrP) amyloids.

Structures of the infectious form of prion protein (e.g. PrP(Sc) or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP(Sc) retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP(C)), but evidence is accumulating that these helices are absent in PrP(Sc) amyloid. Moreover, recombinant PrP(C) can form amyloid fibrils in vitro that have parallel in-register intermolecular ß-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily ß-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90-231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP(Sc) fibrils, can have stable parallel in-register ß-sheet cores. These simulations revealed that the C-terminal residues ∼124-227 more readily adopt stable tightly packed structures than the N-terminal residues ∼90-123 in the absence of cofactors. Variations in the placement of turns and loops that link the ß-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures.

The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.