Proteome-wide probing of the dual NMT-dependent myristoylation tradeoff unveils potent, mechanism-based suicide inhibitors

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a major and ubiquitous lipid modification. Originally thought to modify only N-terminal glycine alpha-amino groups (G-myristoylation), NMTs are now known to modify lysine epsilon-amino groups (K-myristoylation), the significance of which is uncertain. Here we exploited systematic structural proteomics analyses and a novel pipeline involving the Shigella IpaJ protease to discriminate K- and G-myristoylation with unprecedented accuracy and identify the specific features driving each modification. NMT-dependent K-myristoylation occurs post-translationally and only on lysines 1, 2, or 3 following G-myristoylation or caspase cleavage. Direct interactions between the substrate′s reactive amino group and the NMT catalytic base slow K-myristoylation catalysis. IpaJ unmasked novel K-myristoylation sites in a dozen human proteins. The unique properties of NMT-driven K-myristoylation allowed us to design potent, mechanism-based suicide NMT inhibitors. These analyses unravel the respective paths towards K-myristoylation, G-myristoylation, or NMT inhibition, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

P530 Proactive therapeutic drug monitoring of anti-TNF agents is associated with higher mucosal healing rates in Crohn’s disease

Background Reactive therapeutic drug monitoring (TDM) is routinely used in managing secondary loss of response to anti-TNF agents. Proactive TDM use has been associated with better clinical outcomes but its use in routine practice is still controversial. We aimed to investigate whether using proactive TDM strategy will result in higher mucosal healing and clinical remission rates. Methods After review of electronic case files, all patients that received anti-TNF treatment for active disease from 01.01.2017-01.02.2021. and responded to induction were included. Patients starting treatment for postoperative prophylaxis were excluded from the study. In the proactive group TDM was performed in week 14 and patients with subtherapeutic trough levels were dose optimized and underwent subsequent TDM measurments until the target trough level was reached. In the reactive group TDM was performed in the case of loss of response. Mucosal healing was defined as SES CD 3 for patients with CD and Mayo endoscopic score of £ 1 for UC patients. Clinical remission after 52 weeks was defined by the attending physician. Results A total of 161 IBD patients were included, 109 patients with CD and 52 patients with UC. There were 69 patients in the proactive group and 92 patients in the reactive group. No significant difference regarding age at diagnosis, age at treatment initiation, disease duration , prior immunomodulator use and time do endoscopy was observed between groups. A higher proportion of patients in the proactive group achieved mucosal healing but the difference between groups was not significant in the total cohort (proactive 52.4% vs reactive 42.2%; p=0.250). A significantly higher proportion of CD patients (n=109) in the proactive group achieved mucosal healing compared to the reactive group (56.8% vs 34.8%; p=0.039). No significant difference in mucosal healing rates between the groups was observed in UC cases. No significant difference in clinical remission rates was observed between the proactive and reactive group in the total cohort, CD and UC patients respectively. Conclusion Proactive TDM strategy is a valuable tool in managing CD patients resluting in higher rates of mucosal healing. Further studies are needed to define the optimal timepoints for proactive TDM.

An ultra-highly sensitive and selective self-enhanced AIECL sensor for public security early warning in a nuclear emergency via a co-reactive group poisoning mechanism

A mechanism of co-reactive group poisoning (CGP) was discovered for designing efficient I2 vapor sensors for early warning of a nuclear emergency, which give an ultra-low detection limit of 0.13 ppt as well as high selectivity.

Clinical and Laboratory Profile of Dogs Seroreactive to Ehrlichiosis Treated at the Veterinary Medical Teaching Hospital in Niterói, State of Rio de Janeiro, Brazil

Background: Ehrlichiosis is a tick-borne disease highly prevalent in Brazil, and is relevant in canine clinical practice due to its high morbidity and mortality. Its clinical signs are nonspecific and its phases are acute, lasting 2 to 4 weeks; subclinical, i.e., asymptomatic; and chronic, resembling an autoimmune disease. The purpose of this study was to identify the occurrence of reactivity to Ehrlichia canis of bitches treated at the Veterinary Medical Teaching Hospital of the Universidade Federal Fluminense (UFF) - Niterói, RJ, Brazil, based on serological examination by iELISA, and to compare the hematological, biochemical, urinary protein-creatinine and urinary density profiles of reactive and non-reactive animals. Materials, Methods & Results: This study involved solely bitches, regardless of breed, starting at 1 year of age. One hundred and thirty bitches, 1 to 16 year-old (mean age 7.02 ± 4.00), weighing 1.5 to 50 kg (mean weight 12.12 ± 10.65) were subjected to clinical examination and abdominal ultrasound. Complete blood count, biochemical measurements, urinalysis and serology for E. canis were also performed. The serum was used in the iELISA to identify immunoglobulin G (IgG), using a canine Ehrlichia Imunotest® diagnostic kit (Imunodot®, Jaboticabal, SP, Brazil) according to the manufacturer’s instructions. Sixty animals (46.20%) were reactive to E. canis. According to their owners, only 5 (8.3%) of the 60 seroreactive animals had a history of tick-borne disease. The most common profile was that of mixed breed animals living with their owners, older than 7 years, who had not been treated preventatively with specific drugs against ectoparasites. Laboratory tests showed significant differences between groups in terms of total protein (TP), and calcium and urinary protein-creatinine ratio (UPC). TP and UPC were elevated in the non-reactive group, while the only significant change in the reactive group was mild hypocalcemia. In this study, 30% (18/60) of the bitches were seroreactive to E. canis and had hypocalcemia. Of these, 50% (9/18) had a UPC above 0.5. Furthermore, 66.7% (12/18) of this group with hypocalcemia also showed urine density (UD) of less than 1024. Among these 18 bitches, 5 had both alterations, i.e., UPC > 0.5 and UD < 1024.Discussion: In this study, a high prevalence of bitches seroreactive to Ehrlichia canis was observed, despite the absence of clinical and/or laboratory signs indicative of the disease. In the investigation of IgG class antibodies, it is not possible to determine the exact time of infection, and titers may remain high for a period of more than 11 months, even after treatment and elimination of the bacterium. The fact that most seroreactive bitches showed no symptoms compatible with the disease either before or during the study suggests that they were in the subclinical phase of ehrlichiosis. The main reason for calcium metabolism disorders is a phosphorus imbalance, a condition that occurs in kidney diseases. Isosthenuria reflects the kidney’s inability to concentrate urine. This finding may be one of the first clinical manifestations of chronic kidney disease (CKD), especially in dogs. On the other hand, the UPC ratio may increase with the progression of CKD. The presence of hypocalcemia, isosthenuria and increased UPC associated with seroreactivity suggests that infection by E. canis may be associated with the onset of CKD. Veterinarians should keep in mind the complexity of the pathophysiology of ehrlichiosis to ensure the disease is not underdiagnosed in any of its phases, thereby ensuring the correct treatment is provided. Such awareness is expected to reduce the chronicity of the disease and underlying sequelae among dogs.Keywords: Ehrlichia canis, serology, tick, clinic, renal.Descritores: Ehrlichia canis, sorologia, carrapato, clínica, renal.Título: Perfil clínico e laboratorial de cadelas sororeativas para erliquiose tratadas em um Hospital Veterinário Universitário em Niterói, Estado do Rio de Janeiro, Brasil

Immune Modulation and CMV Reactivation In Sepsis-Induced Immunosuppression

Introduction/Objective Sepsis is a global health priority and is often accompanied by a transient immune paralysis, associated with impairment in innate and adaptive immunity leading to progressive immunosuppression and higher susceptibility to secondary infections. Reactivation of Cytomegalovirus (CMV) occurs frequently and has been associated with adverse outcomes even in immunocompetent patients, with sepsis. Study objective was to evaluate the association between incidence of CMV reactivation and immune alteration in sepsis-induced immunosuppression in patients with prolonged sepsis. Methods Prospective observational study, which included consecutive patients admitted to hospital ICU, with severe sepsis and length of stay &gt; 48 hours. Patients with other causes of immune-suppression and anti-CMV treatment were excluded. Blood samples were collected on enrolment and further weekly until 21 days or death/discharge. Quantification of CMV viremia was done using RT-PCR (qPCR). Markers used to evaluate immune suppression using Flow Cytometry were i) lymphocyte subsets (CD3+,CD19+,CD16+CD56+,CD4+,CD8+ and regulatory T cells - CD25+ CD127-), ii) surface receptor expression of HLA-DR on monocytes, and Programmed Death marker expression (PD-1) on T lymphocyte, iii) Measurement of pro-inflammatory(IL-6,TNF-a,IFN-g) and anti-inflammatory cytokines(IL-4,IL-10) by Cytometric Bead Array (CBA) assay. Results A total of 25 CMV IgG positive patients and 11 healthy controls were analyzed. CMV reactivation occurred in 20 patients. Median time for reactivation was 7 days. Patients with CMV reactivation had significant T-cell lymphopenia (p&lt;0.01). PD-1 expression on both CD4+ and CD8+ T cells in these patients was markedly elevated as compared to non-reactive group. HLA-DR expression was significantly low on monocytes in all sepsis patients (p&lt;0.01) vs healthy controls; however it did not show any significant correlation. Levels of IL-6 showed marked elevation from day 7 while, IL-10 was observed to be significantly higher from day 0 in CMV reactivated group as compared to the CMV non-reactive group of patients. Conclusion Our study evidence suggests that monitoring lymphocyte subsets, PD-1expression on T lymphocyte, and levels of IL-6/IL-10 using flow cytometry, may serve as indicators for reactivation of CMV. Individualized immune therapy such as PD-1 receptor blockade drugs can be used to optimize treatment of patients with severe sepsis.

An In-Depth Analysis Approach Enabling Precision Single Chain Nanoparticle Design

The formation of single chain nanoparticles (SCNPs) is typically characterized by a shift towards higher elution volumes in SEC due to size decrease upon folding. However, a step-change in SCNP analysis is required for understanding of the nature of intramolecular SCNP folding. Herein, we exploit a unique combination of SANS, 19F NMR spectroscopy, and multidetection SEC to generate a systematic view of the folded morphology of poly(butyl acrylate) based-SCNPs as a function of their reactive group density and molar mass. In addition to detailed morphological insights, we establish that the primary factor dictating the compaction of SCNPs is their reactive group density, which is ineffective below 5 mol%, reaching maximum compaction close to 30 mol%. Above 20 kDa the molar mass of the precursor polymers has a minor impact on how an SCNP compacts.

An In-Depth Analysis Approach Enabling Precision Single Chain Nanoparticle Design

The formation of single chain nanoparticles (SCNPs) is typically characterized by a shift towards higher elution volumes in SEC due to size decrease upon folding. However, a step-change in SCNP analysis is required for understanding of the nature of intramolecular SCNP folding. Herein, we exploit a unique combination of SANS, 19F NMR spectroscopy, and multidetection SEC to generate a systematic view of the folded morphology of poly(butyl acrylate) based-SCNPs as a function of their reactive group density and molar mass. In addition to detailed morphological insights, we establish that the primary factor dictating the compaction of SCNPs is their reactive group density, which is ineffective below 5 mol%, reaching maximum compaction close to 30 mol%. Above 20 kDa the molar mass of the precursor polymers has a minor impact on how an SCNP compacts.

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug Carmofur

The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 μM) and it is a promising lead compound to develop new antiviral treatment for COVID-19.