Reduced serotonergic transmission alters sensitivity to cost and reward via 5-HT1A and 5-HT1B receptors in monkeys.

Serotonin (5-HT) deficiency is a core biological pathology underlying depression and other psychiatric disorders whose key symptoms include decreased motivation. However, the exact role of 5-HT in motivation remains controversial and elusive. Here, we pharmacologically manipulated the 5-HT system in macaque monkeys and quantified the effects on motivation for goal-directed actions in terms of incentives and costs. Reversible inhibition of 5-HT synthesis increased errors and reaction times on goal-directed tasks, indicating reduced motivation. Analysis found incentive-dependent and cost-dependent components of this reduction. To identify the receptor subtypes that mediate cost and incentive, we systemically administered antagonists specific to 4 major 5-HT receptor subtypes: 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4. Positron emission tomography (PET) visualized the unique distribution of each subtype in limbic brain regions and determined the systemic dosage for antagonists that would achieve approximately 30% occupancy. Only blockade of 5-HT1A decreased motivation through changes in both expected cost and incentive; sensitivity to future workload and time delay to reward increased (cost) and reward value decreased (incentive). Blocking the 5-HT1B receptor also reduced motivation through decreased incentive, although it did not affect expected cost. These results suggest that 5-HT deficiency disrupts 2 processes, the subjective valuation of costs and rewards, via 5-HT1A and 5-HT1B receptors, thus leading to reduced motivation.

Nerve transcriptomes in autoimmune and genetic demyelinating neuropathies: Pathogenic pathway assessment of nerve demyelination.

The pathogenesis of autoimmune demyelinating neuropathies is poorly understood compared to inherited demyelinating forms. We performed whole transcriptome (RNA-Seq) using nerve biopsy tissues of patients with different autoimmune and inherited demyelinating neuropathies (CIDP n = 10, POEMS n = 18, DADS n = 3, CMT1 n = 3) versus healthy controls (n = 6). A limited number of differentially expressed genes compared to healthy controls were identified (POEMS = 125, DADS = 15, CMT = 14, CIDP = 5). Divergent pathogenic pathways including inflammatory, demyelinating and neurite regeneration such as with the triggering receptor expressed on myeloid cells (TREM1) part of the immunoglobulin superfamily and RhoGD1 are found. Shared and discordant pathogenic injury are discovered between autoimmune and inherited forms.

Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages.

Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. IMPORTANCE Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii's avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.

Histochemical assessment of osteoclast-like giant cells in Rankl-/- mice.

OBJECTIVES: We examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. METHODS: The tibiae and femora of ten-week-old male wild-type, c-fos-/-, and Rankl-/- mice were used for immunohistochemistry and transmission electron microscopy (TEM). RESULTS: In Rankl-/- mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl-/- mice. Unlike Rankl-/- mice, c-fos-/- mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl-/- mice. CONCLUSIONS: It seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.

Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Heme oxygenase-1 ameliorates endotoxin-induced acute lung injury by modulating macrophage polarization via inhibiting TXNIP/NLRP3 inflammasome activation.

Acute lung injury (ALI) remains a global public health issue without specific and effective treatment options available in the clinic. Alveolar macrophage polarization is involved in the initiation, development and progression of ALI; however, the underlying mechanism remains poorly understood. Heme oxygenase-1 (HO-1) acts as an antioxidant in pulmonary inflammation and has been demonstrated to be linked with the severity and prognosis of ALI. In this study, the therapeutic effects of HO-1 were examined, along with the mechanisms involved, mainly focusing on alveolar macrophage polarization. HO-1 depletion induced higher iNOS and CD86 (M1 phenotype) expression but was significantly decreased in Arg-1 and CD206 (M2 phenotype) expression in BALF alveolar macrophages after equivalent LPS stimulation. We also found that HO-1 deletion distinctly accelerated the expression of inflammasome-associated components NLRP3, ASC and caspase-1 in vivo and in vivo and in vitro. Moreover, on the basis of LPS for MH-S cells, levels of TXNIP, NLRP3, ASC and caspase-1 were increased and HO-1 depletion exacerbated these changes, whereas double depletion of HO-1 and TXNIP partially mitigated these elevations. Also, HO-1 knockdown induced more M1 phenotype and less M2 phenotype compared with LPS alone, whereas double silence of HO-1 and TXNIP partially changed the polarization state. Taken together, we demonstrated that HO-1 could modulate macrophage polarization via TXNIP/NLRP3 signaling pathway, which could be a potential therapeutic target for ALI treatment.

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium.

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper.

Chemical Replacement of Noggin with Dorsomorphin Homolog 1 for Cost-Effective Direct Neuronal Conversion.

The direct conversion of adult human skin fibroblasts (FBs) into induced neurons (iNs) represents a useful technology to generate donor-specific adult-like human neurons. Disease modeling studies rely on the consistently efficient conversion of relatively large cohorts of FBs. Despite the identification of several small molecular enhancers, high-yield protocols still demand addition of recombinant Noggin. To identify a replacement to circumvent the technical and economic challenges associated with Noggin, we assessed dynamic gene expression trajectories of transforming growth factor-ß signaling during FB-to-iN conversion. We identified ALK2 (ACVR1) of the bone morphogenic protein branch to possess the highest initial transcript abundance in FBs and the steepest decline during successful neuronal conversion. We thus assessed the efficacy of dorsomorphin homolog 1 (DMH1), a highly selective ALK2-inhibitor, for its potential to replace Noggin. Conversion media containing DMH1 (+DMH1) indeed enhanced conversion efficiencies over basic SMAD inhibition (tSMADi), yielding similar ßIII-tubulin (TUBB3) purities as conversion media containing Noggin (+Noggin). Furthermore, +DMH1 induced high yields of iNs with clear neuronal morphologies that are positive for the mature neuronal marker NeuN. Validation of +DMH1 for iN conversion of FBs from 15 adult human donors further demonstrates that Noggin-free conversion consistently yields iN cultures that display high ßIII-tubulin numbers with synaptic structures and basic spontaneous neuronal activity at a third of the cost.

Epinephrine Auto-Injector Prescription and Use: A Retrospective Analysis and Clinical Risk Assessment of Adult Patients Sensitized to Lipid Transfer Protein.

Lipid transfer proteins (LTPs) are widely widespread plant food allergens which represents the main cause of food allergy in adults living in the Mediterranean basin. The purpose of this study was to investigate in LTP patients the actual use of prescribed epinephrine auto-injector and appropriateness of its prescription. In addition, we investigated in these patients: (1) occurrence of new food reaction in the following three years after to diagnosis; (2) need and number of access to emergency services; (3) presence of possible predictive factors to further food reactions. One-hundred sixty-five adult patients sensitized to LTPs have been included. During follow-up, we recorded 68 further reactions, most of them (77.9%) characterized by local symptoms; rarely the patients required an emergency-department visits (16.1%) and only one patient (1.7%) used the epinephrine auto-injector. The patients with a previous history of anaphylaxis at baseline turned back to access to emergency services also during the follow-up (p = 0.006). The majority of patients with recorded systemic reactions (p = 0.004) and treated in an emergency room (p = 0.028) did not have any co-factor-enhanced at diagnosis. We noted an association between platanus pollen sensitization and severity of further reactions during the follow-up (p = 0.026). Epinephrine auto-injector were prescribed to 108/165 patients (65.5%) with an over-prescription rate of 25%. The unforeseeable clinical presentation of LTP allergic reactions and the eventual role played by the cofactor make necessary schedule a follow-up to monitor the patients over time and to assess the actual use of epinephrine auto injectors prescribed.

Identification of Phage Receptor-Binding Protein Sequences with Hidden Markov Models and an Extreme Gradient Boosting Classifier.

Receptor-binding proteins (RBPs) of bacteriophages initiate the infection of their corresponding bacterial host and act as the primary determinant for host specificity. The ever-increasing amount of sequence data enables the development of predictive models for the automated identification of RBP sequences. However, the development of such models is challenged by the inconsistent or missing annotation of many phage proteins. Recently developed tools have started to bridge this gap but are not specifically focused on RBP sequences, for which many different annotations are available. We have developed two parallel approaches to alleviate the complex identification of RBP sequences in phage genomic data. The first combines known RBP-related hidden Markov models (HMMs) from the Pfam database with custom-built HMMs to identify phage RBPs based on protein domains. The second approach consists of training an extreme gradient boosting classifier that can accurately discriminate between RBPs and other phage proteins. We explained how these complementary approaches can reinforce each other in identifying RBP sequences. In addition, we benchmarked our methods against the recently developed PhANNs tool. Our best performing model reached a precision-recall area-under-the-curve of 93.8% and outperformed PhANNs on an independent test set, reaching an F1-score of 84.0% compared to 69.8%.

Objective assessment of adrenocortical carcinoma driver genes and their correlation with tumor pyruvate kinase M2.

Glandular cancers have a significant share of the total cancer patients all over the world. In the case of adrenocortical carcinomas (ACCs), although the benign form is more frequent and common, the malignant form provides a very less percentage of patients with five or more than five years of survival rate. There are gene alterations that are involved as a crucial factor behind the occurrence of ACCs. Out of these, the most prominent genetic alterations (PRKAR-1A, CTNNB1, ZNRF3, TP53, CCNE1 and TERF2 genes) are linked with a glycolytic enzyme pyruvate kinase M2 (PKM2), which converts phosphoenolpyruvate (PEP) to pyruvate in the glycolytic pathway. The involvementof PKM2 renders a cumulative effect through different pathways that may result in the onset of ACCs. Thus, this review aims to establish a link between ACCs, alterations of specific genes and PKM2.

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature.

BACKGROUND: Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically. METHODS: We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes. RESULTS: The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes. CONCLUSION AND REMARKS: MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V.

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein-protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0-C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

Applying Bioinformatic Platforms, In Vitro, and In Vivo Functional Assays in the Characterization of Genetic Variants in the GH/IGF Pathway Affecting Growth and Development.

Heritability accounts for over 80% of adult human height, indicating that genetic variability is the main determinant of stature. The rapid technological development of Next-Generation Sequencing (NGS), particularly Whole Exome Sequencing (WES), has resulted in the characterization of several genetic conditions affecting growth and development. The greatest challenge of NGS remains the high number of candidate variants identified. In silico bioinformatic tools represent the first approach for classifying these variants. However, solving the complicated problem of variant interpretation requires the use of experimental approaches such as in vitro and, when needed, in vivo functional assays. In this review, we will discuss a rational approach to apply to the gene variants identified in children with growth and developmental defects including: (i) bioinformatic tools; (ii) in silico modeling tools; (iii) in vitro functional assays; and (iv) the development of in vivo models. While bioinformatic tools are useful for a preliminary selection of potentially pathogenic variants, in vitro-and sometimes also in vivo-functional assays are further required to unequivocally determine the pathogenicity of a novel genetic variant. This long, time-consuming, and expensive process is the only scientifically proven method to determine causality between a genetic variant and a human genetic disease.

Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico, in vitro, and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro, to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro. The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Fitness Costs of Chlorantraniliprole Resistance Related to the SeNPF Overexpression in the Spodoptera exigua (Lepidoptera: Noctuidae).

Spodopteraexigua, a multifeeding insect pest, has developed a high level of resistance to chlorantraniliprole, which is a benzoylurea insecticide that targets the ryanodine receptors (RyRs). Herein, the resistant strain (SE-Sel) and sensitive strain (SE-Sus) were obtained by bidirectional screening for six generations. The potential oviposited eggs and oviposition rate of the SE-Sel strain were dramatically lower than those of the SE-Sus strain; on the contrary, the weights of prepupae and preadult were significantly increased. As a post-mating response, the higher number of non-oviposited eggs in the SE-Sel strain was caused by a lower mating rate. In addition, the expression levels of vitellogenin (SeVg) and its receptor (SeVgR) in the SE-Sel strain were consistently lower than those in the SE-Sus strain. An RyRI4743M mutation, contributing to the resistance to chlorantraniliprole, was located in the S3 transmembrane segments and might have affected the release of calcium ions; it led to the upregulated expression of the neuropeptide SeNPF and its receptor SeNPFR, and the mating and oviposition rate were significantly recovered when the SeNPF was knocked down though RNA interference (RNAi) in the male adult of the SE-Sel strain. Moreover, the expression of the juvenile hormone-binding proteins SeJHBWDS3 and SeJHBAN in the male adult of the SE-Sel strain was significantly decreased, which proved the existence of a fitness cost from another angle. Therefore, these results indicate that the fitness cost accompanied by chlorantraniliprole resistance in S. exigua may be related to the decrease in mating desire due to SeNPF overexpression.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD-thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 µg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 µg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD-thanatin interaction.

Dysfunction of estrogen-related receptor alpha-dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH development.

Rationale: Men and postmenopausal women are more prone to developing non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) than premenopausal women. However, the pathological links and underlying mechanisms of this disparity are still elusive. The sex-difference in hepatic very low-density lipoprotein (VLDL) assembly and secretion may contribute to NAFLD development. Estrogen-related receptor alpha (ERRα) is a key regulator of several metabolic processes. We hypothesized that ERRα plays a role contributing to the sex-difference in hepatic VLDL assembly and secretion. Methods: VLDL secretion and essential genes governing said process were assessed in male and female mice. Liver-specific ERRα-deficient (ERRαLKO) mice were generated to assess the rate of hepatic VLDL secretion and alteration in target gene expression. Overexpression of either microsomal triglyceride transfer protein (Mttp) or phospholipase A2 G12B (Pla2g12b) by adenovirus was performed to test if the fatty liver phenotype in male ERRαLKO mice was due to defects in hepatic VLDL secretion. Female ERRαLKO mice were put on a diet high in saturated fat, fructose and cholesterol (HFHC) to promote NASH development. Wild type female mice were either ovariectomized or treated with tamoxifen to induce a state of estrogen deficiency or disruption in estrogen signaling. Adenovirus was used to overexpress ERRα in these mice to test if ERRα was sufficient to rescue the suppressed VLDL secretion due to estrogen dysfunction. Finally, wild type male mice on a high-fat diet (HFD) were treated with an ERRα inverse agonist to assess if suppressing ERRα activity pharmacologically would lead to fatty liver development. Results: ERRα is an indispensable mediator modulating hepatic triglyceride-rich very low-density lipoprotein (VLDL-TG) assembly and secretion through coordinately controlling target genes apolipoprotein B (Apob), Mttp and Pla2g12b in a sex-different manner. Hepatic VLDL-TG secretion is blunted in ERRαLKO mice, leading to hepatosteatosis which exacerbates endoplasmic reticulum stress and inflammation paving ways for NASH development. Importantly, ERRα acts downstream of estrogen/ERα signaling in contributing to the sex-difference in hepatic VLDL secretion effecting hepatic lipid homeostasis. Conclusions: Our results highlight ERRα as a key mediator which contributes to the sex disparity in NAFLD development, suggesting that selectively restoring ERRα activity in the liver may be a novel strategy for treating NAFLD/NASH.

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining.

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities.

Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.