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Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets., Author summary Acute myeloid leukemia or AML is a cancer of blood cells. Despite significant progress in recent years, a majority of afflicted individuals still succumbs to the disease. A variety of genetic defects have been associated to AML. Among these are chromosomal translocations, which entail the fusion of two genes, leading to the production of cancer-inducing chimeric proteins. A representative example is the NUP98-HOXA9 oncoprotein, which results from the fusion of the NUP98 and HOXA9 genes. The mechanism of action of NUP98-HOXA9 remains poorly understood. Given the evolutionarily conservation of NUP98 and HOXA9 as well as basic cellular processes across multicellular organisms, we took advantage of Drosophila fruit flies as a genetic tool to identify genes that impinge on the activity of human NUP98-HOXA9. Surprisingly, this approach identified a relatively large spectrum of conserved genes that engaged in functional interplay with NUP98-HOXA9, which indicated the pervasive effects that this oncogene has on basic cellular events. While some genes have been previously linked to NUP98-HOXA9, thus validating our experimental approach, several others are novel and as such represent potentially new avenues for therapeutic intervention.

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the I&kappa, B kinase &beta, (IKBKB). Here, specific IKBKB domains responsible for ORF3&ndash, IKBKB interaction were identified. Dysregulation of NF-kB and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-&kappa, B promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-&beta, promoter and expression of IFN-&beta, mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus&ndash, host interaction.

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host's first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution.

A total of 95 isolates of Burkholderia pseudomallei from 53 sporadic cases in Thailand were examined by pulsed-field gel electrophoresis. Digestion of genomic DNA of all isolates by NcoI generated a macrorestriction pattern similar to that of B. pseudomallei which cannot assimilate L-arabinose. Analysis using restriction enzymes SpeI and AvrII demonstrated greater sensitivity than NcoI digestion in the differentiation of B. pseudomallei and could be used for epidemiological groupings. Four cluster groups were evident among 37 isolates tested and the majority of isolates within each cluster displayed more than 65% similarity. Furthermore, multiple isolates from 18 and 35 patients with single and recurrent episodes of melioidosis, respectively, were examined. All patients with a single episode yielded genetically identical isolates and four of 35 patients with recurrent episodes were infected with strains of different genotypic patterns from the primary isolate(s). Hence, most repeated episodes of infection in melioidosis are as a result of the original infecting strains.

The ability of the transmembrane adaptor protein linker for activation of T cells (LAT) to regulate T cell development, activation, survival, and homeostasis depends upon phosphorylation of its multiple tyrosine residues. The mutation of tyrosine 136 on LAT abrogates its interaction with phospholipase C-γ1, causing severe ramifications on TCR-mediated signaling. Mice harboring this mutation, LATY136F mice, have significantly impaired thymocyte development; however, they rapidly develop a fatal lymphoproliferative disease marked by the uncontrolled expansion of Th2-skewed CD4+ T cells, high levels of IgE and IgG1, and autoantibody production. In this study, we assessed the contribution of multiple signaling pathways in LATY136F disease development. The deletion of the critical signaling proteins Gads and RasGRP1 caused a further block in thymocyte development, but, over time, could not prevent CD4+ T cell hyperproliferation. Also, restoring signaling through the NF-κB and NFAT pathways was unable to halt the development of disease. However, expression of a constitutively active Raf transgene enhanced lymphoproliferation, indicating a role for the Ras–MAPK pathway in LAT-mediated disease.

Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-gamma1-binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LAT(Y136F) mice. This defect was not a result of the autoimmune environment as LAT(Y136F) T reg cells also failed to develop in healthy LAT-/- mice that received mixed wild-type and LAT(Y136F) bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LAT(Y136F) mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LAT(Y136F) mice likely via granzymes and/or TGF-beta-mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LAT(Y136F) T cells. Our data indicate that the LAT-PLC-gamma1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells.

NTAL (non-T cell activation linker)/LAB (linker for activation of B cells) is a LAT (linker for activation of T cells)-like molecule that is expressed in B cells, mast cells, natural killer cells, and monocytes. Upon engagement of the B cell receptor or Fc receptors, it is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT(-/-) mice. In this study, we utilized various LAB Tyr to Phe mutants to map the phosphorylation and Grb2-binding sites of LAB. We also examined the function of these mutants by investigating their ability to rescue signaling defects in LAT-deficient Jurkat cells and thymocyte development in LAT(-/-) mice. Our results indicated that human LAB was primarily phosphorylated on three membrane-distal tyrosines, Tyr(136), Tyr(193), and Tyr(233). Mutation of these three tyrosines abolished Grb2 binding and LAB function. Our data suggested that these tyrosines are the most important tyrosines for LAB function.

Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.

The adaptor molecule, linker for activation of T cells (LAT), is essential in T cell activation and development; a similar molecule in B cells has not yet been identified. Here, we report the identification of a new adaptor protein, linker for activation of B cells (LAB). Like LAT, LAB was localized to lipid rafts. Upon activation via the B cell receptor (BCR), LAB was phosphorylated and interacted with the adaptor protein Grb2. Decreased LAB expression led to a reduction in BCR-mediated calcium flux and Erk activation. LAB rescued thymocyte development but not normal T cell activation in LAT(-/-) mice. Our data suggest that LAB links BCR engagement to downstream signaling pathways.

Roughly three-quarter of the genes associated with human diseases have fly counterparts. This high degree of conservation, combined to a wide range of genetic tools, makes Drosophila an attractive model to study basic mechanisms lying at the heart of various human disorders. Several oncogenes mediate their effects by interfering with specific cell machinery components common to all eukaryotes. The systematic identification of cell components influencing the activity of oncogenes should therefore accelerate the characterization of those oncogenes. Toward this goal, we take advantage of Drosophila molecular genetics to identify conserved genes that functionally interact with oncogenes. Our effort currently focuses on the t(7;11)(p15;p15) translocation associated with acute myeloid leukemia (AML) and which fuses the N-terminal part of Nucleoporin 98 (NUP98) to the C-terminal part of the transcription factor HOXA9. As homologues of NUP98 and HOXA9 are present in flies, we hypothesized that expression of NUP98-HOXA9 during development will affect some of the same protein networks that are perturbed in human hematopoietic cells. We successfully conducted several modifier screens in the past by exploiting dosage-sensitive phenotypes specifically induced in the eyes. To that end, we expressed NUP98-HOXA9 during eye development, which interestingly phenocopied Homothorax (HTH) overexpression in its ability to block eye development and promoted head cuticle formation. HTH is the homologue of MEIS1; a DNA-binding co-factor for HOXA9 that functions with a third partner, PBX, and which together form a ternary complex that regulates gene expression. Importantly, we found that the NUP98-HOXA9 eye phenotype was suppressed by mutations in the hth and exd (Drosophila pbx) genes, thus lending support to the specificity of the phenotype. In agreement with this, a structure/function analysis of NUP98-HOXA9 conducted in the fly eye narrowed down the same functional domains/motifs as those that had been identified using mouse models, namely, the HOXA9 homeodomain, the HOXA9 ANW motif (a PBX-interaction site) and the NUP98 portion. Remarkably, we also found that NUP98-HOXA9 and HTH/MEIS synergistically induced cell proliferation when coexpressed in the developing eye. As a result, large tissue overgrowths were produced. The cooperation observed in this experimental setting is reminiscent of the ability of MEIS1 to accelerate AML onset when co-expressed with NUP98-HOXA9 in mouse models. Moreover, we found that the collaboration strictly depends on endogenous EXD/PBX as its depletion by RNAi completely prevents overgrowth formation. Together, these findings provide compelling evidence that the NUP98-HOXA9 fly model recapitulates several of the key functional features that had been established in mammals for this oncogene and thus should prove useful to further delineate the immediate events disturbed by NUP98-HOXA9 expression. Based on these premises, we conducted a genetic screen to isolate dominant modifiers of the NUP98-HOXA9 eye phenotype. Approximately 100,000 fly progeny have been screened, which led to the isolation of a few hundred mutations acting either as suppressors or enhancers. Several complementation groups have now been uncovered and their molecular identification is currently underway. Validation of relevant genes in mouse leukemia models will be conducted to confirm their significance with respect to HOX-dependent leukemia. The NUP98-HOXA9 fly model as well as the early findings of the screen will be presented.

Summary LAB (linker for activation of B cells), also known as NTAL (non-T cell activation linker), is a LAT (linker for activation of T cells)-like adaptor protein that is expressed in B, NK, and mast cells. Its role in lymphocytes has not been clearly demonstrated. Here, we showed that aged LAB-deficient ( Lat2 −/− ) mice developed an autoimmune syndrome. Lat2 −/− T cells were hyperactivated and produced more cytokines than Lat2 +/+ T cells. Even though LAB was absent in naive T cells, LAB could be detected in activated Lat2 +/+ T cells. LAT-mediated signaling events were enhanced in Lat2 −/− T cells; however, they were suppressed in T cells that overexpressed LAB. Mice with the Lat2 gene conditionally deleted from T cells also developed the autoimmune syndrome like Lat2 −/− mice. Together, these data demonstrated an important role of LAB in limiting autoimmune response and exposed a mechanism regulating T cell activation.