Insights into the development of Ixodes scapularis: a resource for research on a medically important tick species

Ticks (Acari: Ixodida) are arthropod ectoparasites dependent on a bloodmeal from a vertebrate host at each developmental stage for completion of their life cycle. This tick feeding cycle impacts animal health by causing damage to hides, secondary infections, immune reactions and diseases caused by transmission of pathogens. The genus Ixodes includes several medically important species that vector diseases, including granulocytic anaplasmosis and Lyme disease. I. scapularis, commonly called the black-legged or deer tick, is a medically-important tick species in North America and therefore was the first tick genome to be sequenced, thus serving as an important resource for tick research. This Primer focuses on the normal developmental cycle and laboratory rearing of I. scapularis. Definition of normal morphology, along with a consistent source of laboratory-reared I. scapularis, are fundamental for all aspects of future research, especially the effects of genetic manipulation and the evaluation of tick vaccine efficacy. Recent research important for the advancement of tick research, namely the development of tick cell culture systems for study of ticks and tick-borne pathogens, RNA interference for genetic manipulation of ticks and discovery of candidate antigens for development of tick vaccines, are briefly presented along with areas to target for future research.The preparation of this chapter was partially supported by the EU FP7 ANTIGONE project number 278976, and the Walter R. Sitlington Endowed Chair for Food Animal Research to K.M. Kocan.Peer Reviewe

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/2/ange202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/1/ange202004991.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/3/ange202004991_am.pd

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/3/anie202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/2/anie202004991_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/1/anie202004991.pd

CKD-MBD after kidney transplantation

Successful kidney transplantation corrects many of the metabolic abnormalities associated with chronic kidney disease (CKD); however, skeletal and cardiovascular morbidity remain prevalent in pediatric kidney transplant recipients and current recommendations from the Kidney Disease Improving Global Outcomes (KDIGO) working group suggest that bone disease—including turnover, mineralization, volume, linear growth, and strength—as well as cardiovascular disease be evaluated in all patients with CKD. Although few studies have examined bone histology after renal transplantation, current data suggest that bone turnover and mineralization are altered in the majority of patients and that biochemical parameters are poor predictors of bone histology in this population. Dual energy X-ray absorptiometry (DXA) scanning, although widely performed, has significant limitations in the pediatric transplant population and values have not been shown to correlate with fracture risk; thus, DXA is not recommended as a tool for the assessment of bone density. Newer imaging techniques, including computed tomography (quantitative CT (QCT), peripheral QCT (pQCT), high resolution pQCT (HR-pQCT) and magnetic resonance imaging (MRI)), which provide volumetric assessments of bone density and are able to discriminate bone microarchitecture, show promise in the assessment of bone strength; however, future studies are needed to define the value of these techniques in the diagnosis and treatment of renal osteodystrophy in pediatric renal transplant recipients

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Insights into the development of Ixodes scapularis: a resource for research on a medically important tick species

Abstract Ticks (Acari: Ixodida) are arthropod ectoparasites dependent on a bloodmeal from a vertebrate host at each developmental stage for completion of their life cycle. This tick feeding cycle impacts animal health by causing damage to hides, secondary infections, immune reactions and diseases caused by transmission of pathogens. The genus Ixodes includes several medically important species that vector diseases, including granulocytic anaplasmosis and Lyme disease. I. scapularis, commonly called the black-legged or deer tick, is a medically-important tick species in North America and therefore was the first tick genome to be sequenced, thus serving as an important resource for tick research. This Primer focuses on the normal developmental cycle and laboratory rearing of I. scapularis. Definition of normal morphology, along with a consistent source of laboratory-reared I. scapularis, are fundamental for all aspects of future research, especially the effects of genetic manipulation and the evaluation of tick vaccine efficacy. Recent research important for the advancement of tick research, namely the development of tick cell culture systems for study of ticks and tick-borne pathogens, RNA interference for genetic manipulation of ticks and discovery of candidate antigens for development of tick vaccines, are briefly presented along with areas to target for future research

Sheep experimentally infected with a human isolate of Anaplasma phagocytophilum serve as a host for infection of Ixodes scapularis ticks

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. . A. phagocytophilum is transmitted by . Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of . A. phagocytophilum which then served as a host for infection of . I. scapularis nymphs and adults. . A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an . Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While . A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female . I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with . A. phagocytophilum as determined by PCR of guts (80-87%) and salivary glands (67-100%). Female . I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted . A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of . I. scapularis infected with this human isolate of . A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface.This research was supported by the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, a Center for Veterinary Health Sciences, Research Advisory Committee grant, and the Spanish Ministerio de Ciencia e Innovación (MICINN) project BFU2008-01244/BMC to JF.Peer Reviewe

Small molecules inhibitors of the heterogeneous ribonuclear protein A18 (hnRNP A18): a regulator of protein translation and an immune checkpoint.

We have identified chemical probes that simultaneously inhibit cancer cell progression and an immune checkpoint. Using the computational Site Identification by Ligand Competitive Saturation (SILCS) technology, structural biology and cell-based assays, we identify small molecules that directly and selectively bind to the RNA Recognition Motif (RRM) of hnRNP A18, a regulator of protein translation in cancer cells. hnRNP A18 recognizes a specific RNA signature motif in the 3\u27UTR of transcripts associated with cancer cell progression (Trx, VEGF, RPA) and, as shown here, a tumor immune checkpoint (CTLA-4). Post-transcriptional regulation of immune checkpoints is a potential therapeutic strategy that remains to be exploited. The probes target hnRNP A18 RRM in vitro and in cells as evaluated by cellular target engagement. As single agents, the probes specifically disrupt hnRNP A18-RNA interactions, downregulate Trx and CTLA-4 protein levels and inhibit proliferation of several cancer cell lines without affecting the viability of normal epithelial cells. These first-in-class chemical probes will greatly facilitate the elucidation of the underexplored biological function of RNA Binding Proteins (RBPs) in cancer cells, including their effects on proliferation and immune checkpoint activation