Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 â€‹min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P â€‹< â€‹0.05, CI: 78.2-93.8%) and specificity of 93.9% (P â€‹< â€‹0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

Dual Inhibition of Bruton's Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat Non-Hodgkin's B Cell Malignancies.

Although new targeted therapies, such as ibrutinib and idelalisib, have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies, or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors, possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We discovered a single molecule, MDVN1003 (1-(5-amino-2,3-dihydro-1H-inden-2-yl)-3-(8-fluoro-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), that inhibits Bruton's tyrosine kinase and phosphatidylinositol-3-kinase δ, two proteins regulated by the B cell receptor that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2, two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together, these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

Discovery of Pyrazolopyrimidine Derivatives as Novel Dual Inhibitors of BTK and PI3Kδ.

The aberrant activation of B-cells has been implicated in several types of cancers and hematological disorders. BTK and PI3Kδ are kinases responsible for B-cell signal transduction, and inhibitors of these enzymes have demonstrated clinical benefit in certain types of lymphoma. Simultaneous inhibition of these pathways could result in more robust responses or overcome resistance as observed in single agent use. We report a series of novel compounds that have low nanomolar potency against both BTK and PI3Kδ as well as acceptable PK properties that could be useful in the development of treatments against B-cell related diseases.

Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.

Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.

Modulation of the Akt pathway reveals a novel link with PERK/eIF2α, which is relevant during hypoxia.

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.

Dendritic and stromal cells from the spleen of lupic mice present phenotypic and functional abnormalities.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increase in the percentage of autoreactive B and T lymphocytes. Since dendritic cells (DCs) are essential for B cell and T cell function, we hypothesized that changes in DC biology may play a critical role in the pathogenesis of the disease. We analyzed the phenotype and distribution of two main DC subsets, conventional (cDC) and plasmacytoid (pDC), in lupus prone (NZW × NZB)F1 (BWF1) mice and age-matched NZW × BALB/c control mice. Our results show that both subsets of lupic DCs displayed an abnormal phenotype, characterized by an over-expression of the co-stimulatory molecules CD80, CD86, PD-L1 and PD-L2 compared with control mice. Accordingly, spleen CD4(+) T cells from lupic mice exhibit an activated phenotype characterized by a higher expression of PD-1, CD25, CD69 and increased secretion of IFN-γ and IL-10. Interestingly, lupic mice also present an increase in the percentage of cDC in peripheral blood and an increase in the percentage of pDCs in spleen and mesenteric lymph nodes (MLNs) compared with control and pre-lupic mice. Homing experiments demonstrate that lupic and pre-lupic DCs migrate preferentially to the spleen compared to DCs from control mice. This preferential recruitment and retention of DCs in the spleen is related to an altered expression of different chemokine and chemokine receptors on both, DCs and stromal cells from lupic mice. Our results suggest that this altered phenotype and migratory behavior shown by DCs from lupic mice may account for the abnormal T cell and B cell responses in lupus.

Development of murine lupus involves the combined genetic contribution of the SLAM and FcgammaR intervals within the Nba2 autoimmune susceptibility locus.

Autoantibodies are of central importance in the pathogenesis of Ab-mediated autoimmune disorders. The murine lupus susceptibility locus Nba2 on chromosome 1 and the syntenic human locus are associated with a loss of immune tolerance that leads to antinuclear Ab production. To identify gene intervals within Nba2 that control the development of autoantibody-producing B cells and to determine the cellular components through which Nba2 genes accomplish this, we generated congenic mice expressing various Nba2 intervals where genes for the FcgammaR, SLAM, and IFN-inducible families are encoded. Analysis of congenic strains demonstrated that the FcgammaR and SLAM intervals independently controlled the severity of autoantibody production and renal disease, yet are both required for lupus susceptibility. Deregulated homeostasis of terminally differentiated B cells was found to be controlled by the FcgammaR interval where FcgammaRIIb-mediated apoptosis of germinal center B cells and plasma cells was impaired. Increased numbers of activated plasmacytoid dendritic cells that were distinctly CD19+ and promoted plasma cell differentiation via the proinflammatory cytokines IL-10 and IFNalpha were linked to the SLAM interval. These findings suggest that SLAM and FcgammaR intervals act cooperatively to influence the clinical course of disease through supporting the differentiation and survival of autoantibody-producing cells.

Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2.

Binding of MgATP to the allosteric site of phosphofructokinase-2 promotes a dimer to tetramer conversion. In the presence of Fru-6-P the enzyme remains as a dimer. Limited proteolysis in the presence of MgATP completely protects the enzyme against inactivation and cleavage, while Fru-6-P provides a partial protection. A 28-kDa proteolytic fragment containing the N-terminus of the protein is inactive, but retains the ability to bind Fru-6-P and the allosteric effector MgATP. The fragment remains as a dimer but does not form a tetramer in the presence of MgATP. The results suggest major conformational changes of the enzyme upon ligand binding that confer a higher degree of compactness to the monomers in the dimer and in the tetramer, demonstrate the presence of the active and allosteric sites in this N-terminus fragment, and stress the importance of the C-terminus region of the protein for catalytic activity and ligand-induced oligomerization.