Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Šresolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

Nanocapsules Comprised of Purified Protein: Construction and Applications in Vaccine Research.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

Childhood Bacterial Meningitis Surveillance in Southern Vietnam: Trends and Vaccination Implications From 2012 to 2021.

Background: This retrospective hospital-based surveillance aimed to assess the epidemiology, causative pathogens trend, and serotypes distribution of pneumococcal meningitis among children aged under 5 years with bacterial meningitis in Southern Vietnam after the introduction of pentavalent vaccine in the Expanded Program on Immunization (EPI). Methods: From 2012 to 2021, cerebrospinal fluid samples were collected from children aged under 5 years with suspected bacterial meningitis at Children's Hospitals 1 and 2 in Ho Chi Minh City. Probable bacterial meningitis (PBM) cases were identified using biochemistry and cytology. Real-time polymerase chain reaction was used to confirm cases of confirmed bacterial meningitis (CBM) caused by Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis. Streptococcus pneumoniae serotyping was performed. Results: Of the 2560 PBM cases, 158 (6.2%) were laboratory-confirmed. The CBM proportion decreased during the 10-year study and was associated with age, seasonality, and permanent residence. Streptococcus pneumoniae was the most common pathogen causing bacterial meningitis (86.1%), followed by H influenzae (7.6%) and N meningitidis (6.3%). The case-fatality rate was 8.2% (95% confidence interval, 4.2%-12.2%). Pneumococcal serotypes 6A/B, 19F, 14, and 23F were the most prevalent, and the proportion of pneumococcal meningitis cases caused by the 10-valent pneumococcal conjugate vaccine (PCV) serotypes decreased from 96.2% to 57.1% during the PCV eras. Conclusions: Streptococcus pneumoniae is the most frequent causative agent of bacterial meningitis in children aged under 5 years in Southern Vietnam over the last decade. Policymakers may need to consider introducing PCVs into the EPI to effectively prevent and control bacterial meningitis.

Increased plasma lipocalin-2 levels correlate with disease severity and may be a marker of acute inflammatory response in patients with psoriasis.

More than a skin disease, psoriasis is also considered a systemic disorder. Lipocalin-2, an adipokine, may be a link between psoriasis and systemic inflammation. We conducted this study to measure the plasma level of lipocalin-2 and investigate its relationship with the clinical manifestations in patients with psoriasis. We assessed 62 patients with psoriasis and 31 healthy controls. Their demographic information and clinical characteristics were determined by physical examination and review of the recorded medical history. Plasma lipocalin-2 levels were measured using an enzyme-linked immunosorbent assay. Plasma lipocalin-2 concentration was significantly higher in patients with psoriasis than in the control group (P<0.001). Patients with acute psoriatic subgroups, including psoriatic erythroderma and pustular psoriasis, had significantly higher plasma lipocalin-2 levels than those with the chronic plaque type. In addition, plasma lipocalin-2 concentration positively correlates with the disease severity index, including the psoriasis area severity index, body surface area, high-sensitivity C-reactive protein, nail psoriasis severity index, and pustular severity index. In patients with psoriasis, increased plasma lipocalin-2 levels correlated with severity and indicated an active disease state. These findings suggest that lipocalin-2 may play an important role in determining the pathogenesis of acute psoriasis and may serve as a valuable clinical biomarker of this disease.

Long lasting anxiety following early life stress is dependent on glucocorticoid signaling in zebrafish.

Chronic adversity in early childhood is associated with increased anxiety and a propensity for substance abuse later in adulthood, yet the effects of early life stress (ELS) on brain development remain poorly understood. The zebrafish, Danio rerio, is a powerful model for studying neurodevelopment and stress. Here, we describe a zebrafish model of ELS and identify a role for glucocorticoid signaling during a critical window in development that leads to long-term changes in brain function. Larval fish subjected to chronic stress in early development exhibited increased anxiety-like behavior and elevated glucocorticoid levels later in life. Increased stress-like behavior was only observed when fish were subjected to ELS within a precise time window in early development, revealing a temporal critical window of sensitivity. Moreover, enhanced anxiety-like behavior only emerges after two months post-ELS, revealing a developmentally specified delay in the effects of ELS. ELS leads to increased levels of baseline cortisol, and resulted in a dysregulation of cortisol receptors' mRNA expression, suggesting long-term effects on cortisol signaling. Together, these findings reveal a 'critical window' for ELS to affect developmental reprogramming of the glucocorticoid receptor pathway, resulting in chronic elevated stress.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Synthesis, binding and bioactivity of gamma-methylene gamma-lactam ecdysone receptor ligands: advantages of QSAR models for flexible receptors.

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or 'attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for gamma-methylene gamma-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.

Telomeric G-quadruplexes are a substrate and site of localization for human telomerase.

It has been hypothesized that G-quadruplexes can sequester the 3' end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures implies that parallel telomeric G-quadruplexes may play an important biological role.

Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus.

Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.

A human monoclonal antibody against insulin-like growth factor-II blocks the growth of human hepatocellular carcinoma cell lines in vitro and in vivo.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.