The EG95 recombinant vaccine is protective against cystic echinococcus in animal intermediate hosts. Preparation of the existing, registered EG95 vaccines involves semi-purification of the vaccine protein, adding to the cost of production. Truncation of the EG95 cDNA, shortening both the amino and carboxy-termini of the protein, leads to high levels of recombinant protein expression. The recombinant EG95 protein was prepared as a bacterin from clarified, whole bacterial lysate, and used in a vaccine trial in sheep against an experimental challenge infection with Echinococcus granulosus eggs. The EG95 bacterin was found to induce 98% protection. Use of this in a new generation EG95 vaccine would simplify production, facilitate new sources of the vaccine and potentially enhance uptake of vaccination in control of E. granulosus transmission.

Phosphatase PPM1F is a regulator of cell adhesion by fine-tuning integrin activity and actin cytoskeleton structures. Elevated expression of this enzyme in human tumors is associated with high invasiveness, enhanced metastasis, and poor prognosis. Thus, PPM1F is a target for pharmacological intervention, yet inhibitors of this enzyme are lacking. Here, we use high-throughput screening to identify Lockdown, a reversible and non-competitive PPM1F inhibitor. Lockdown is selective for PPM1F, because this compound does not inhibit other protein phosphatases in vitro and does not induce additional phenotypes in PPM1F knockout cells. Importantly, Lockdown-treated glioblastoma cells fully re-capitulate the phenotype of PPM1F-deficient cells as assessed by increased phosphorylation of PPM1F substrates and corruption of integrin-dependent cellular processes. Ester modification yields LockdownPro with increased membrane permeability and prodrug-like properties. LockdownPro suppresses tissue invasion by PPM1F-overexpressing human cancer cells, validating PPM1F as a therapeutic target and providing an access point to control tumor cell dissemination.

Fungi infect over a billion people worldwide and contribute substantially to human morbidity and mortality despite all available therapies. New antifungal drugs are urgently needed. Decades of study have revealed numerous protein targets of potential therapeutic interest for which potent, fungal-selective ligands remain to be discovered and developed. To measure the binding of diverse small molecule ligands to their larger protein targets, fluorescence polarization (FP) can provide a robust, inexpensive approach. The protocols in this article provide detailed guidance for developing FP-based assays capable of measuring binding affinity in whole cell lysates without the need for purification of the target protein. Applications include screening of libraries to identify novel ligands and the definition of structure-activity relationships to aid development of compounds with improved target affinity and fungal selectivity. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Use of saturation binding curves to optimize tracer and lysate protein concentrations Basic Protocol 2: Establishment of competition binding experiments Support Protocol 1: Preparation of fungal cell lysates Support Protocol 2: Preparation of human HepG2 cell lysate.

Immunoprecipitation is a technique which enables a macromolecule of interest to be isolated from heterogenous mixtures (particularly cell lysates). However, the immunoprecipitation of protein(s) can be challenging, with multiple variations of the basic technique required for successful antigenic pull-down. This depends on the target of interest, cell source, and localization. Here, immunoprecipitation of MIF from mouse and human macrophage cell lysates is described, which is both reliable and replicable, derived from multiple optimization experiments.

Known for its tumor suppressor activity in breast and ovarian cancers, the breast cancer 1 susceptibility gene (Brca1) is involved in a variety of cellular pathways including DNA repair, antioxidant signaling, apoptosis, and cell cycle regulation. BRCA1 can translocate between the cytoplasm and nucleus to perform its various roles. Herein is a procedure for measuring BRCA1 protein levels in the whole cell lysate (WCL), as well as in the nuclear (N) and cytoplasmic (C) fractions of mouse tissues at different gestational ages. The method employs multiple loading controls to ensure proper separation of fractions and a total protein stain for more consistent comparisons of dissimilar samples. This method is useful for identifying BRCA1 deficiencies and localization in a variety of research fields, including development, neurodegeneration, and cancer.

Breast cancer impacts female population globally and is the second most common cancer for females. With various limitations and adverse effects of current therapies, several immunotherapies are being explored. Development of an effective breast cancer vaccine can be a groundbreaking immunotherapeutic approach. Such approaches are being evaluated by several clinical trials currently. On similar lines, our research study aims to evaluate a particulate breast cancer vaccine delivered via skin. This particulate breast cancer vaccine was prepared by spray drying technique and utilized murine breast cancer whole cell lysate as a source of tumor-associated antigens. The average size of the particulate vaccine was 1.5 μm, which resembled the pathogenic species, thereby assisting in phagocytosis and antigen presentation leading to further activation of the immune response. The particulate vaccine was delivered via skin using commercially available metal microneedles. Methylene blue staining and confocal microscopy were used to visualize the microchannels. The results showed that microneedles created aqueous conduits of 50 ± 10 μm to deliver the microparticulate vaccine to the skin layers. Further, an in vivo comparison of immune response depicted significantly higher concentration of serum IgG, IgG2a, and B and T cell (CD4+ and CD8+) populations in the vaccinated animals than the control animals (p < 0.001). Upon challenge with live murine breast cancer cells, the vaccinated animals showed five times more tumor suppression than the control animals confirming the immune response activation and protection (p < 0.001). This research paves a way for individualized immunotherapy following surgical tumor removal to prolong relapse episodes.

Staphylococcus aureus is a very important human pathogen that causes significant morbidity and mortality worldwide. Several vaccine clinical trials based on generating antibody against staphylococcal surface polysaccharides or proteins have been unsuccessful. A killed whole cell lysate preparation (SaWCA) was made by lysing a USA 300 strain with lysostaphin followed by sonication and harvest of the supernatant fraction. Immunization with SaWCA and cholera toxin (CT) generated robust IL-17A but relatively modest antibody responses, and provided protection in the skin abscess but not in the dermonecrosis or invasive infection model. In contrast, parenteral immunization with SaWCA and alum produced robust antibody and IL-17A responses and protected mice in all three models. Sera generated after immunization with SaWCA had measurable antibodies directed against six tested conserved surface proteins, and promoted opsonophagocytosis activity (OPA) against two S. aureus strains. Passive transfer of SaWCA-immune serum protected mice against dermonecrosis and invasive infection but provided no demonstrable effect against skin abscesses, suggesting that antibodies alone may not be sufficient for protection in this model. Thus, immunization with a SA lysate preparation generates potent antibody and T cell responses, and confers protection in systemic and cutaneous staphylococcal infection models.

The skin has been identified as a promising target to deliver vaccines. In this study, prostate cancer antigens were delivered in a spray-dried microparticulate carrier to a murine model via the transdermal route and the subcutaneous route. There was a significant increase in the humoral responses as determined by the total serum IgG titres (p < 0.05) and the cellular responses as determined by the T- and B-cells sub-population in spleen samples and delay in tumour growth till 8 weeks post-tumour challenge of both vaccinated groups when compared to the controls. The vaccine microparticles administered via the transdermal route induced a Th2-mediated immune response versus a mixed Th1- and Th2-mediated immune response via the subcutaneous route. Thus, the particulate vaccine delivery system proves to be a promising alternative for generation of a robust immune response against prostate cancer via the skin in a murine model.

P-selectin glycoprotein ligand-1 and β1 integrin play essential roles in T cell trafficking during inflammation. E-selectin and vascular cell adhesion molecule-1 are their ligands expressed on inflammation-activated endothelium. During the tethering and rolling of lymphocytes on endothelium, P-selectin glycoprotein ligand-1 binds E-selectin and induces signals. Subsequently, β1 integrin is activated and mediates stable adhesion. However, the intracellular signal pathways from PSGL-1 to β1 integrin have not yet been fully understood. Here, we find that p85, a regulatory subunit of phosphoinositide 3-kinase, forms a novel complex with Rho-GDP dissociation inhibitor-2, a lymphocyte-specific RhoGTPases dissociation inhibitor. Phosporylations of the p85-bound Rho-GDP dissociation inhibitor-2 on 130 and 153 tyrosine residues by c-Abl and Src were required for the complex to be recruited to P-selectin glycoprotein ligand-1 and thereby regulate β1 integrin-mediated T cell adhesion to vascular cell adhesion molecule-1. Both shRNAs to Rho-GDP dissociation inhibitor-2 and p85 and over-expression of Rho-GDP dissociation inhibitor-2 Y130F and Y153F significantly reduced the above-mentioned adhesion. Although Rho-GDP dissociation inhibitor-2 in the p85-Rho-GDP dissociation inhibitor-2 complex was also phosphorylated on 24 tyrosine residue by Syk, the phosphorylation is not required for the adhesion. Taken together, we find that specific phosphorylations on 130 and 153 tyrosine residues of p85-bound Rho-GDP dissociation inhibitor-2 are pivotal for P-selectin glycoprotein ligand-1-induced β1 integrin-mediated lymphocyte adhesion to vascular cell adhesion molecule-1. This will shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment.