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Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1 , HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56 bright and CD56 dim NK cells. Finally, a donor with GATA2 T354M mutation exhibits reduced percentage of CD56 bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells. Natural killer (NK) cells are important innate immune cells with diverse functions. Here the authors use single-cell RNA-sequencing of purified human bone marrow and peripheral blood NK cells to define five populations of NK cells with distinct transcriptomic profile to further our understanding of NK development and heterogeneity.

Chronic inflammation plays a key tumor-promoting role in lung cancer. Our previous studies in mice demonstrated that neutrophils are critical mediators of tumor promotion in methylcholanthrene (MCA)-initiated, butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated the role of neutrophil myeloperoxidase (MPO) activity in this inflammation promoted model. Increased levels of MPO protein and activity were present in the lungs of mice administered BHT. Treatment of mice with N-acetyl lysyltyrosylcysteine amide (KYC), a novel tripeptide inhibitor of MPO, during the inflammatory stage reduced tumor burden. In a separate tumor model, KYC treatment of a Lewis Lung Carcinoma (LLC) tumor graft in mice had no effect on tumor growth, however, mice genetically deficient in MPO had significantly reduced LLC tumor growth. Our observations suggest that MPO catalytic activity is critical during the early stages of tumor development. However, during the later stages of tumor progression, MPO expression independent of catalytic activity appears to be required. Our studies advocate for the use of MPO inhibitors in a lung cancer prevention setting.

Purpose A subset of common variable immunodeficiency (CVID) patients either presents with or develops autoimmune and lymphoproliferative complications, such as granulomatous lymphocytic interstitial lung disease (GLILD), a major cause of morbidity and mortality in CVID. While a myriad of phenotypic lymphocyte derangements has been associated with and described in GLILD, defects in T and B cell antigen receptor (TCR/BCR) signaling in CVID and CVID with GLILD (CVID/GLILD) remain undefined, hindering discovery of biomarkers for disease monitoring, prognostic prediction, and personalized medicine approaches. Methods To identify perturbations of immune cell subsets and TCR/BCR signal transduction, we applied mass cytometry analysis to peripheral blood mononuclear cells (PBMCs) from healthy control participants (HC), CVID, and CVID/GLILD patients. Results Patients with CVID, regardless of GLILD status, had increased frequency of HLADR + CD4 + T cells, CD57 + CD8 + T cells, and CD21 lo B cells when compared to healthy controls. Within these cellular populations in CVID/GLILD patients only, engagement of T or B cell antigen receptors resulted in discordant downstream signaling responses compared to CVID. In CVID/GLILD patients, CD21 lo B cells showed perturbed BCR-mediated phospholipase C gamma and extracellular signal-regulated kinase activation, while HLADR + CD4 + T cells and CD57 + CD8 + T cells displayed disrupted TCR-mediated activation of kinases most proximal to the receptor. Conclusion Both CVID and CVID/GLILD patients demonstrate an activated T and B cell phenotype compared to HC. However, only CVID/GLILD patients exhibit altered TCR/BCR signaling in the activated lymphocyte subsets. These findings contribute to our understanding of the mechanisms of immune dysregulation in CVID with GLILD.

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The \"floral organ-building\" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how \"floral organ identity\" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

Inflammation of the tumor microenvironment is linked to cancer progression, promoting cell survival, proliferation and migration of transformed cells. The immune system’s main role is to protect the body against foreign pathogens and remove them via a variety of mechanisms. Cancer has evolved to harness the immune system’s basic function for its own growth, survival, and metastasis. Immune cells, including neutrophils and macrophages, which are part of the innate immune system are the body’s first responders to infection. These players in the innate immune system have recently come under scrutiny as possible promoters of cancer growth. This thesis will look at both sides of the issue: how the tumor microenvironment can initiate and promote cancer growth and how the loss of a tumor suppressor mediates tumor growth and survival. The first project will look at how the microenvironment can help initiate cancer in a model of inflammation-promoted lung carcinogenesis. First, I examined chronic inflammation and lung tumorigenesis focusing on the role of neutrophils, specifically, in lung cancer progression. Using a two-step carcinogenesis model with methylcholanthrene (MCA), a tobacco-related carcinogen and butylated hydroxytoluene (BHT) as the inflammatory agent, a once common food preservative, I demonstrate that chronic inflammation increases the level of neutrophils in the bronchoaveolar lavage fluid (BAL) resulting in increased tumor burden. Neutrophils generate reactive oxygen species (ROS) via several enzymes including myeloperoxidase (MPO) to oxidize various proteins on invading pathogens. In the twostep carcinogenesis model, levels of MPO in the BAL were significantly increased. Treatment of mice with N-acetyl-lysyltyrosylcysteine-amide (KYC), a novel tripeptide 3 inhibitor of MPO, during the BHT-promoted inflammatory model of lung cancer reduced tumor burden by 50%. For the second part of this thesis, I will look at the role of a tumor suppressor in promoting malignancy. Glioblastoma multiform (GB) is an aggressive cancer with poor survival rates even with aggressive treatment. Small GTPases are a family of proteins that interact with various regulators and effectors to control a number of signal transduction pathways in normal cells, as well as in cancer cells. Small GTPases switch between a GTP-bound active form and a GDP-bound inactive form, thus acting as a molecular switch. The most commonly studied small GTPase is K-Ras, a wellknown proto-oncogene whose constitutive activation can lead to malignant cell transformation. A unique family of small GTPases, Distinct Subgroup of Ras (DiRas), consists of three proteins: DiRas1, DiRas2, and DiRas3, which are tumor suppressive rather than tumor promoting. Expression of the DiRas family of proteins is lost in glioblastoma, though the role this family of proteins in mediating tumor progression is not fully determined. My studies showed that DiRas expression reduces proliferation and migration in glioblastoma cell lines. DiRas also reduced the transcriptional activity of NF-κB, a key transcription factor in mediation inflammation, resulting in reduced levels of IL-8, a chemokine that has a significant role in cancer progression. Little is understood about the bind partners of the DiRas family, we demonstrate that DiRas binds to p110β, a member of the phosphatidylinositol 3-kinase (PI3K) pathway, and SmgGDS a guanine nucleotide exchange factor for RhoA. We further demonstrate that re-expression of the DiRas family of proteins in vivo reduced tumor volume, further demonstrating its function as a tumor suppressor. Understanding the tumor microenvironment and the tumor itself allows for the development of new and novel therapeutics targeting this dynamic interaction.

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The \"floral organ-building\" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how \"floral organ identity\" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

Relatively little is known about the range of RNA levels in human blood. This report provides assessment of peripheral blood RNA level and its inter-individual differences in a group of 35 healthy humans consisting of 25 females and 10 males ranging in age from 50 to 89 years. In this group, the average total RNA level was 14.59 μg/ml of blood, with no statistically significant difference between females and males. The individual RNA level ranged from 6.7 to 22.7 μg/ml of blood. In healthy subjects, the repeated sampling of an individual's blood showed that RNA level, whether high or low, was stable. The inter-individual differences in RNA level in blood can be attributed to both, differences in cell number and the amount of RNA per cell. The 3.4-fold range of inter-individual differences in total RNA levels, documented herein, should be taken into account when evaluating the results of quantitative RT-PCR and/or RNA sequencing studies of human blood. Based on the presented results, a comprehensive assessment of gene expression in blood should involve determination of both the amount of mRNA per unit of total RNA (U / ng RNA) and the amount of mRNA per unit of blood (U / ml blood) to assure a thorough interpretation of physiological or pathological relevance of study results.

Previously, we introduced an advanced version of the single-step method for RNA extraction. We have now adapted this methodology for the isolation of total RNA, mRNA, small RNA and microRNA from whole blood, plasma or serum of human and animal origin. This new reagent, RNAzol® BD, provides the highest yield and purity of blood-derived RNA, and it also allows for the simultaneous isolation of DNA from samples used for RNA isolation.