The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Anisotropic diffusion creep in postperovskite provides a new model for deformation at the core-mantle boundary.

The lowermost portion of Earth's mantle (D″) above the core-mantle boundary shows anomalous seismic features, such as strong seismic anisotropy, related to the properties of the main mineral MgSiO3 postperovskite. But, after over a decade of investigations, the seismic observations still cannot be explained simply by flow models which assume dislocation creep in postperovskite. We have investigated the chemical diffusivity of perovskite and postperovskite phases by experiment and ab initio simulation, and derive equations for the observed anisotropic diffusion creep. There is excellent agreement between experiments and simulations for both phases in all of the chemical systems studied. Single-crystal diffusivity in postperovskite displays at least 3 orders of magnitude of anisotropy by experiment and simulation (Da = 1,000 Db; Db ≈ Dc) in zinc fluoride, and an even more extreme anisotropy is predicted (Da = 10,000 Dc; Dc = 10,000 Db) in the natural MgSiO3 system. Anisotropic chemical diffusivity results in anisotropic diffusion creep, texture generation, and a strain-weakening rheology. The results for MgSiO3 postperovskite strongly imply that regions within the D″ region of Earth dominated by postperovskite will 1) be substantially weaker than regions dominated by perovskite and 2) develop a strain-induced crystallographic-preferred orientation with strain-weakening rheology. This leads to strain localization and the possibility to bring regions with significantly varying textures into close proximity by strain on narrow shear zones. Anisotropic diffusion creep therefore provides an attractive alternative explanation for the complexity in observed seismic anisotropy and the rapid lateral changes in seismic velocities in D″.

Application of Adaptive Image Receive Coil Technology for Whole-Brain Imaging.

OBJECTIVE. The Adaptive Image Receive (AIR) radiofrequency coil is an emergent technology that is lightweight and flexible and exhibits electrical characteristics that overcome many of the limitations of traditional rigid coil designs. The purpose of this study was to apply the AIR coil for whole-brain imaging and compare the performance of a prototype AIR coil array with the performance of conventional head coils. SUBJECTS AND METHODS. A phantom and 15 healthy adult participants were imaged. A prototype 16-channel head AIR coil was compared with conventional 8-and 32-channel head coils using clinically available MRI sequences. During consensus review, two board-certified neuroradiologists graded the AIR coil compared with an 8-channel coil and a 32-channel coil on a 5-point ordinal scale in multiple categories. One- and two-sided Wilcoxon signed rank tests were performed. Noise covariance matrices and geometry factor (g-factor) maps were calculated. RESULTS. The signal-to-noise ratio, structural sharpness, and overall image quality scores of the prototype 16-channel AIR coil were better than those of the 8-channel coil but were not as good as those of the 32-channel coil. Noise covariance matrices showed stable performance of the AIR coil across participants. The median g-factors for the 16-channel AIR coil were, overall, less than those of the 8-channel coil but were greater than those of the 32-channel coil. CONCLUSION. On average, the prototype 16-channel head AIR coil outperformed a conventional 8-channel head coil but did not perform as well as a conventional 32-channel head coil. This study shows the feasibility of the novel AIR coil technology for imaging the brain and provides insight for future coil design improvements.

Secondary Products from Intramolecular Cycloadditions of Azidoalkyl Enol Ethers and Azidoalkyl Vinyl Bromides: 1-Azadienes, Their Reactions with Diphenylketene, and Radical Cyclizations To Form Bi- and Tricyclic Lactams.

Azidoalkyl enol ethers undergo intramolecular 1,3-dipolar cycloaddition to generate stable triazolines; in contrast, the cycloadducts formed by heating analogous azidoalkyl vinyl bromides are unstable with respect to elimination of N2 and HBr, affording 1-azadienes (2-alkenyl cyclic imines). These primary products may be isolated or treated directly with diphenylketene to produce bi- and tricyclic 3,4-dihydropyridin-2(1H)-ones; similar ring systems may also be produced from the azadienes by N-acylation and radical cyclization.

Transition-Metal-Free Reductive Hydroxymethylation of Isoquinolines.

A transition-metal-free reductive hydroxymethylation reaction has been developed, enabling the preparation of tetrahydroisoquinolines bearing C4-quaternary centers from the corresponding isoquinolines. Deuterium labelling studies and control experiments enable a potential mechanism to be elucidated which features a key Cannizzaro-type reduction followed by an Evans-Tishchenko reaction. When isoquinolines featuring a proton at the 4-position are used, a tandem methylation-hydroxymethylation occurs, leading to the formation of 2 new C-C bonds in one pot.

An effector Peptide family required for Drosophila toll-mediated immunity.

In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

A Flexible Strategy for the Regiocontrolled Synthesis of Pyrazolo[1,5-a]pyrazines.

A four-step protocol for the synthesis of pyrazolo[1,5-a]pyrazines has been developed. Commercially available pyrazoles were alkylated and formylated in a regiocontrolled manner to give pyrazole-5-aldehydes bearing 2,2-dialkoxyethyl substitution on N-1. Efficient conditions for the subsequent deprotection and cyclization of these intermediates allowed access to pyrazolo[1,5-a]pyrazines with multiple substitution patterns. The versatility of the pyrazole-5-aldehyde intermediates was further demonstrated through a deprotection and double-reductive amination sequence to give 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazines.

Cryptococcus neoformans var. grubii-Induced Arthritis with Encephalitic Dissemination in a Dog and Review of Published Literature.

This article describes the clinical, pathological, and immunohistochemical findings associated with Cryptococcus neoformans var. grubii in a 4-year-old female Boxer dog from Uberlândia, Minas Gerais, Southeastern Brazil. Clinically, there was a swelling at the right metatarsal region and the hock joint with enlargement of regional lymph nodes. Radiographical evaluation revealed lysis of the tarsal bone; cytology demonstrated cryptococcal intralesional organisms at the swollen joint. Despite empirical antifungals therapeutic, the animal developed neurological cryptococcosis and died spontaneously. Significant pathological alterations included arthritis, lymphadenitis, and encephalitic cryptococcomas associated with numerous intralesional narrow-necked budding encapsulated yeasts. Immunohistochemistry utilising monoclonal antibodies that label C. neoformans sp. complex capsule, characterised the yeasts as C. neoformans var. grubii. Collectively, the pathological and immunohistochemical findings of this dog indicate that the intralesional organisms observed within the articular surface of the hock joint, lymph nodes, and the brain were C. neoformans var. grubii, confirming the participation of this fungal pathogen in the development of cryptococcal arthritis. In this case, the most likely pathogenesis was percutaneous inoculation with resultant abscess-like lesion, which resulted in the draining sinus, swelling of the right hind limb with progression to the articular disease. Thereafter, the fungal pathogen probably compromised the adjacent lymph nodes with subsequent haematogenous distribution to the brain, terminating with cryptococcal arthritis, lymphadenitis, and encephalitis.

Acute lymphoblastic leukemia in a pygmy hippopotamus (Hexaprotodon liberiensis).

A captive, 31-yr-old, intact male pygmy hippopotamus presented with nonspecific signs of weight loss, inappetence, diarrhea, and lethargy. After 5 wk of diagnostic investigation and symptomatic treatment, an acute leukemic process with concurrent polycystic kidney disease was suspected. The animal's condition continued to deteriorate prompting euthanasia. Necropsy, histopathologic, and immunohistochemical examination confirmed acute T-cell lymphoblastic leukemia and polycystic kidneys. Acute T-cell lymphoblastic leukemia has not previously been documented in this species; however, polycystic kidney disease has been reported. This case report adds to the increasing number of pygmy hippopotamuses diagnosed with polycystic kidney disease and describes acute T-cell lymphoblastic leukemia, a previously unreported disease of this species.

The Future of Vestibular Schwannoma Management.

The future of the management of both sporadic and neurofibromatosis type 2-asscoiated vestibular schwannomas (VSs) will be shaped by cutting-edge technologic and biomedical advances to enable personalized, precision medicine. This scoping review envisions the future by highlighting the most promising developments published, ongoing, planned, or potential that are relevant for VS, including integrated omics approaches, artificial intelligence algorithms, biomarkers, liquid biopsy of the inner ear, digital medicine, inner ear endomicroscopy, targeted molecular imaging, patient-specific stem cell-derived models, ultra-high dose rate radiotherapy, optical imaging-guided microsurgery, high-throughput development of targeted therapeutics, novel immunotherapeutic strategies, tumor vaccines, and gene therapy.

Mitigating disease risk in an endangered pinniped: early hookworm elimination optimizes the growth and health of Australian sea lion pups.

The Australian sea lion (Neophoca cinerea) experiences high pup mortality of seasonally alternating severity, partly attributed to endemic hookworm (Uncinaria sanguinis) infection. To further explore health outcomes of early hookworm elimination, a treatment trial was conducted at Seal Bay Conservation Park, South Australia, over consecutive lower and higher mortality breeding seasons (2019, 19.2%; 2020-1; 28.9%). Pups (n = 322) were stratified into two age cohorts (median 14 d and 24 d recruitment ages) and randomly assigned to treated (topical ivermectin 500 µg/kg) or control (untreated) groups. A younger prepatent cohort <14 d old (median 10 d) was identified a posteriori. A seasonally independent growth benefit resulted from hookworm elimination across all age cohorts. The greatest relative improvements (bodyweight + 34.2%, standard length + 42.1%; p ≤ 0.001) occurred in the month post-treatment, in the youngest prepatent cohort. A significant benefit of lesser magnitude (bodyweight + 8.6-11.6%, standard length + 9.5-18.4%; p ≤ 0.033) persisted up to 3 months across all age cohorts - greatest in the youngest pups. Treatment resulted in immediate improvement in hematological measures of health - decreased anemia and inflammation severity (p ≤ 0.012). These results enhance our understanding of host-parasite-environment interactions within the context of hematological ontogenesis, confirm the seasonally independent benefits of hookworm disease intervention, and further inform conservation recommendations for this endangered species.

Identification of optimal conditions for human placental explant culture and extracellular vesicle release.

Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile in vitro model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs. We also determined the time frame in which trophoblast viability and functionality remain optimal. Multiplex vesicle flow cytometry with classical EV and placenta-specific markers revealed three separate populations of explant-derived EVs: small CD63+ EVs; large PLAP+ EVs; and CD63-/PLAP- EVs. These culture and analytical approaches will enable in vitro modeling of short-term effects of environmental perturbations associated with pregnancy complications on placental function and EV release.

Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection.

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

Extensive production of Neospora caninum tissue cysts in a carnivorous marsupial succumbing to experimental neosporosis.

Experimental infections of Sminthopsis crassicaudata, the fat-tailed dunnart, a carnivorous marsupial widely distributed throughout the arid and semi-arid zones of Australia, show that this species can act as an intermediate host for Neospora caninum. In contrast to existing models that develop relatively few N. caninum tissue cysts, dunnarts offer a new animal model in which active neosporosis is dominated by tissue cyst production. The results provide evidence for a sylvatic life cycle of N. caninum in Australia between marsupials and wild dogs. It establishes the foundation for an investigation of the impact and costs of neosporosis to wildlife.

A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea).

We detail a novel presentation of tuberculosis associated with intestinal perforation in an endangered Australian sea lion (Neophoca cinerea) from South Australian waters and confirm the presence of this disease in the region of highest pup production. In February 2017, a 3-yr-old juvenile male died shortly after hauling out at the Kingscote beach on Kangaroo Island. On postmortem examination, we found a mid-jejunal intestinal perforation and partial obstruction (from a strangulating fibrous and granulomatous mesenteric mass), a marked multicentric abdominal fibrosing granulomatous lymphadenitis, and a large volume serosanguinous peritoneal effusion. Acid-fast bacteria were detected postmortem in cytologic preparations of the mesenteric lymph node and in histologic sections of jejunum and the encircling mass. Mycobacterial infection was confirmed by positive culture after 3 wk. Molecular typing using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing with 12-locus analysis identified Mycobacterium pinnipedii. This case highlights the need for vigilance of zoonotic disease risk when handling pinnipeds, including in the absence of specific respiratory signs or grossly apparent pulmonary pathology. Increased serologic population surveillance is recommended to assess the species' risk from this and other endemic diseases, especially given its endangered status.

Topical ivermectin is a highly effective seal 'spot-on': A randomised trial of hookworm and lice treatment in the endangered Australian sea lion (Neophoca cinerea).

The Australian sea lion (Neophoca cinerea) is an endangered and declining otariid species, with a high rate of pup mortality associated with endemic hookworm (Uncinaria sanguinis) infection a suspected contributor to this decline. Injected ivermectin is an effective treatment for Uncinaria sp. in otariids, with optimal outcomes achieved by the early treatment of pups prior to disease development. This randomised controlled trial evaluated the effectiveness of the novel use of a topical ivermectin formulation against hookworm infection and lice (Antarctophthirus microchir) infestation, in comparison with injected ivermectin. During the 2017 breeding season at Dangerous Reef, South Australia, pups ≤ 70 cm in standard length (≤ 2 weeks of age; n = 85) were randomised to single dose topical (500 µg/kg spot-on; n = 27) or injected (200 µg/kg subcutaneous; n = 29) ivermectin treatment groups, or to an untreated control group (n = 29). Topical ivermectin was highly effective for U. sanguinis elimination, and not significantly different to the injected formulation (estimated effectiveness 96.4% and 96.8%, respectively; P > 0.05). Its application resulted in an 81.6% reduction and 62.7% additional clearance for A. microchir infestation by 15-24 days post-treatment, compared with untreated control pups (also not significantly different to injected ivermectin; 83.1% and 59.4%, respectively; P > 0.05). Treatment with either ivermectin formulation significantly ameliorated increases in inflammatory markers detected in the blood of untreated control pups - peripheral blood eosinophil counts (persisting to 36-41 days post-recruitment P < 0.05) and increased plasma protein concentrations (15-24 days post-recruitment; P < 0.05). Further, an initial short-term decrease in body condition in the control group was not observed in either of the treatment groups. This study demonstrates that topical ivermectin is an effective antiparasitic treatment in N. cinerea. It offers an alternative administration method for ivermectin delivery to a young pup cohort in this species, and an alternative, minimally invasive management tool for species conservation.

Cutaneous Chromatophoromas in Four Species of Australian Elapid Snake.

This report documents the clinicopathological features of cutaneous chromatophoromas in four wild-caught, captive Australian elapid snakes: a strap-snouted brown snake (Pseudonaja aspidoryncha), a tiger snake (Notechis scutatus), an Eastern brown snake (Pseudonaja textilis) and a Mengden's brown snake (Pseudonaja mengdeni). All tumours were subclassified as melanophoromas, with three assessed as malignant on the basis of invasive growth or presence of intracoelomic metastases. The chromatophoromas were single or multiple, black or orange pigmented, cutaneous, sometimes ulcerated, plaques or nodules. Microscopically, the neoplastic cells were often spindle shaped with low or variable pigmentation. Neoplastic cells in one tumour were notable for their pleomorphic round cell morphology and high mitotic rate. One snake with late-stage metastasis survived for over 5 years. There are few reports of chromatophoromas in elapid snakes and, to our knowledge, this is the first report of these tumours in Australian elapid snakes.

Renal Crest Proliferative Lesions in Cats with Chronic Kidney Disease.

In a histopathological study of the renal crest (RC) of kidneys of cats with chronic kidney disease (CKD), 58/90 (64%) had epithelial proliferation. Of these, 33 cats had hyperplasia of the collecting duct (CD) epithelium (CDH) alone, eight had hyperplasia of the urothelium covering the RC (RCUH), of which one had concurrent abaxial renal pelvic urothelial hyperplasia (UH), and eight had both CDH and RCUH. CDH or RCUH were present in five cats with marked dysplasia of the CD epithelium (CDD) and four cats with invasive carcinomas, which also had epithelial dysplasia. All nine cats with marked dysplasia or neoplasia of the RC also had substantially altered RC contours due to focal haemorrhage, papillary necrosis or fibrosis. Three of the carcinomas had a strong desmoplastic response. In control cats, both urothelial (RC and renal pelvis) and tubular (CD and distal tubular) cells were immunopositive for cytokeratin (CK; AE1/AE3), tubular epithelial cells were positive for vimentin (Vim) and aquaporin 2 (Aq2), while urothelial cells were positive for p63. PAX8 immunolabelling was difficult to validate. CD and UH labelling was similar to control tissue. While urothelial dysplasia had the same immunolabelling pattern as UH and control tissue, CDD was generally immunonegative for Aq2. As immunolabelling of the four carcinomas did not distinguish between tubular and urothelial origin, with three positive for both Vim and p63, all were broadly designated as RC carcinomas. Overall, proliferative epithelial lesions are common in cats with CKD and form a continuum from simple hyperplasia to neoplasia of the urothelium or CD of the RC.

The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi.

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Δdaisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.

Rhodium catalysed C-3/5 methylation of pyridines using temporary dearomatisation.

Pyridines are ubiquitous aromatic rings used in organic chemistry and are crucial elements of the drug discovery process. Herein we describe a new catalytic method that directly introduces a methyl group onto the aromatic ring; this new reaction is related to hydrogen borrowing, and is notable for its use of the feedstock chemicals methanol and formaldehyde as the key reagents. Conceptually, the C-3/5 methylation of pyridines was accomplished by exploiting the interface between aromatic and non-aromatic compounds, and this allows an oscillating reactivity pattern to emerge whereby normally electrophilic aromatic compounds become nucleophilic in the reaction after activation by reduction. Thus, a set of C-4 functionalised pyridines can be mono or doubly methylated at the C-3/5 positions.