Cellular dynamics in pig-to-human kidney xenotransplantation.

BACKGROUND: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING: This work was supported by NIH RM1HG009491 and DP5OD033430.

Sf3b4 mutation in Xenopus tropicalis causes RNA splicing defects followed by massive gene dysregulation that disrupt cranial neural crest development.

Nager syndrome is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4) a component of the pre-mRNA spliceosomal machinery. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, most spliceosomopathies - including Nager syndrome - are cell/tissue-specific in their pathology. In Nager syndrome patients, it is the neural crest (NC)-derived craniofacial skeletal structures that are primarily affected. To understand the pathomechanism underlying this condition, we generated a Xenopus tropicalis sf3b4 mutant line using the CRISPR/Cas9 gene editing technology. Here we describe the sf3b4 mutant phenotype at neurula, tail bud, and tadpole stages, and performed temporal RNA-sequencing analysis to characterize the splicing events and transcriptional changes underlying this phenotype. Our data show that while loss of one copy of sf3b4 is largely inconsequential in Xenopus tropicalis, homozygous deletion of sf3b4 causes major splicing defects and massive gene dysregulation, which disrupt cranial NC cell migration and survival, thereby pointing at an essential role of Sf3b4 in craniofacial development.

Loss of Notch signaling in skeletal stem cells enhances bone formation with aging.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

BACKGROUND: Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS: We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy.

Non-small lung cancers (NSCLC) frequently (∼30%) harbor KRAS driver mutations, half of which are KRASG12C. KRAS-mutant NSCLC with comutated STK11 and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapy. Development of KRASG12C inhibitors (G12Ci) provided a major therapeutic advance, but resistance still limits their efficacy. To identify genes whose deletion augments efficacy of the G12Cis adagrasib (MRTX-849) or adagrasib plus TNO155 (SHP2i), we performed genome-wide CRISPR/Cas9 screens on KRAS/STK11-mutant NSCLC lines. Recurrent, potentially targetable, synthetic lethal (SL) genes were identified, including serine-threonine kinases, tRNA-modifying and proteoglycan synthesis enzymes, and YAP/TAZ/TEAD pathway components. Several SL genes were confirmed by siRNA/shRNA experiments, and the YAP/TAZ/TEAD pathway was extensively validated in vitro and in mice. Mechanistic studies showed that G12Ci treatment induced gene expression of RHO paralogs and activators, increased RHOA activation, and evoked ROCK-dependent nuclear translocation of YAP. Mice and patients with acquired G12Ci- or G12Ci/SHP2i-resistant tumors showed strong overlap with SL pathways, arguing for the relevance of the screen results. These findings provide a landscape of potential targets for future combination strategies, some of which can be tested rapidly in the clinic. SIGNIFICANCE: Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005.

Identification and Surveys of Promoting Plant Growth VOCs from Biocontrol Bacteria Paenibacillus peoriae GXUN15128.

The role of microbial volatile organic compounds (MVOCs) in promoting plant growth has received much attention. We isolated Paenibacillus peoriae from mangrove rhizosphere soil, which can produce VOCs to promote the growth of Arabidopsis thaliana seedlings, increase the aboveground biomass of A. thaliana, and increase the number of lateral roots of A. thaliana. The effects of different inoculation amounts and different media on the composition of MVOCs were studied by solid-phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) and headspace sampler/GC-MS. We found that the growth medium influences the function and composition of MVOCs. To survey the growth-promoting functions, the transcriptome of the receptor A. thaliana was then determined. We also verified the inhibitory effect of the soluble compounds produced by P. peoriae on the growth of 10 pathogenic fungi. The ability of P. peoriae to produce volatile and soluble compounds to promote plant growth and disease resistance has shown great potential for application in the sustainability of agricultural production. IMPORTANCE Microbial volatile organic compounds (MVOCs) have great potential as "gas fertilizers" for agricultural applications, and it is a promising research direction for the utilization of microbial resources. This study is part of the field of interactions between microorganisms and plants. To study the function and application of microorganisms from the perspective of VOCs is helpful to break the bottleneck of traditional microbial application. At present, the study of MVOCs is lacking; there is a lack of functional strains, especially with plant-protective functions and nonpathogenic application value. The significance of this study is that it provides Paenibacillus peoriae, which produces VOCs with plant growth-promoting effects and broad-spectrum antifungal activity against plant-pathogenic fungi. Our study provides a more comprehensive, new VOC component analysis method and explains how MVOCs promote plant growth through transcriptome analysis. This will greatly increase our understanding of MVOC applications as a model for other MVOC research.

Metabolomic, proteomic, and transcriptomic changes in adults with epilepsy on modified Atkins diet.

OBJECTIVE: High-fat and low-carbohydrate diets can reduce seizure frequency in some treatment-resistant epilepsy patients, including the more flexible modified Atkins diet (MAD), which is more palatable, mimicking fasting and inducing high ketone body levels. Low-carbohydrate diets may shift brain energy production, particularly impacting neuron- and astrocyte-linked metabolism. METHODS: We evaluated the effect of short-term MAD on molecular mechanisms in adult epilepsy patients from surgical brain tissue and plasma compared to control participants consuming a nonmodified higher carbohydrate diet (n = 6 MAD, mean age = 43.7 years, range = 21-53, diet for average 10 days; n = 10 control, mean age = 41.9 years, range = 28-64). RESULTS: By metabolomics, there were 13 increased metabolites in plasma (n = 15 participants with available specimens), which included 4.10-fold increased ketone body 3-hydroxybutyric acid, decreased palmitic acid in cortex (n = 16), and 11 decreased metabolites in hippocampus (n = 6), which had top associations with mitochondrial functions. Cortex and plasma 3-hydroxybutyric acid levels had a positive correlation (p = .0088, R2  = .48). Brain proteomics and RNAseq identified few differences, including 2.75-fold increased hippocampal MT-ND3 and trends (p < .01, false discovery rate > 5%) in hippocampal nicotinamide adenine dinucleotide (NADH)-related signaling pathways (activated oxidative phosphorylation and inhibited sirtuin signaling). SIGNIFICANCE: Short-term MAD was associated with metabolic differences in plasma and resected epilepsy brain tissue when compared to control participants, in combination with trending expression changes observed in hippocampal NADH-related signaling pathways. Future studies should evaluate how brain molecular mechanisms are altered with long-term MAD in a larger cohort of epilepsy patients, with correlations to seizure frequency, epilepsy syndrome, and other clinical variables. [Clinicaltrials.gov NCT02565966.].

CXCR6 promotes dermal CD8+ T cell survival and transition to long-term tissue residence.

Tissue resident memory T cells (TRM) provide important protection against infection, and yet the interstitial signals necessary for their formation and persistence remain incompletely understood. Here we show that antigen-dependent induction of the chemokine receptor, CXCR6, is a conserved requirement for TRM formation in peripheral tissue after viral infection. CXCR6 was dispensable for the early accumulation of antigen-specific CD8+ T cells in skin and did not restrain their exit. Single cell sequencing indicated that CXCR6-/- CD8+ T cells were also competent to acquire a transcriptional program of residence but exhibited deficiency in multiple pathways that converged on survival and metabolic signals necessary for memory. As such, CXCR6-/- CD8+ T cells exhibited increased rates of apoptosis relative to controls in the dermis, leading to inefficient TRM formation. CXCR6 expression may therefore represent a common mechanism across peripheral non-lymphoid tissues and inflammatory states that increases the probability of long-term residence.

Hedgehog and Platelet-derived Growth Factor Signaling Intersect during Postnatal Lung Development.

Normal lung development critically depends on HH (Hedgehog) and PDGF (platelet-derived growth factor) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH signaling and PDGF signaling and their impact on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of Pdgfa (platelet-derived growth factor subunit A) and Pdgfra (platelet-derived growth factor receptor alpha) knockouts during secondary alveolar septation. Using a dual signaling reporter, Gli1lZ;PdgfraEGFP, we show that HH and PDGF pathway intermediates are concurrently expressed during alveolar septal myofibroblast accumulation, suggesting pathway convergence in the generation of lung myofibroblasts. Consistent with this hypothesis, HH inhibition reduces Pdgfra expression and diminishes the number of Pdgfra-positive and Pdgfra-lineage cells in postnatal lungs. Bulk RNA sequencing data of Pdgfra-expressing cells from Postnatal Day 8 (P8) lungs show that HH inhibition alters the expression not only of well-established HH targets but also of several putative PDGF target genes. This, together with the presence of Gli-binding sites in PDGF target genes, suggests HH input into PDGF signaling. We identified these HH/PDGF targets in several postnatal lung mesenchymal cell populations, including myofibroblasts, using single-cell transcriptomic analysis. Collectively, our data indicate that HH signaling and PDGF signaling intersect to support myofibroblast/fibroblast function during secondary alveolar septum formation. Moreover, they provide a molecular foundation relevant to perinatal lung diseases associated with impaired alveolarization.

Single-cell RNA sequencing reveals distinct T cell populations in immune-related adverse events of checkpoint inhibitors.

PD-1 is an inhibitory receptor in T cells, and antibodies that block its interaction with ligands augment anti-tumor immune responses. The clinical potential of these agents is limited by the fact that half of all patients develop immune-related adverse events (irAEs). To generate insights into the cellular changes that occur during anti-PD-1 treatment, we performed single-cell RNA sequencing of circulating T cells collected from patients with cancer. Using the K-nearest-neighbor-based network graph-drawing layout, we show the involvement of distinctive genes and subpopulations of T cells. We identify that at baseline, patients with arthritis have fewer CD8 TCM cells, patients with pneumonitis have more CD4 TH2 cells, and patients with thyroiditis have more CD4 TH17 cells when compared with patients who do not develop irAEs. These data support the hypothesis that different populations of T cells are associated with different irAEs and that characterization of these cells' pre-treatment has the potential to serve as a toxicity-specific predictive biomarker.

Brain molecular mechanisms in Rasmussen encephalitis.

OBJECTIVE: This study was undertaken to identify molecular mechanisms in brain tissue of Rasmussen encephalitis (RE) when compared to people with non-RE epilepsy (PWE) and control cases using whole exome sequencing (WES), RNAseq, and proteomics. METHODS: Frozen brain tissue (ages = 2-19 years) was obtained from control autopsy (n = 14), surgical PWE (n = 10), and surgical RE cases (n = 27). We evaluated WES variants in RE associated with epilepsy, seizures, RE, and human leukocyte antigens (HLAs). Differential expression was evaluated by RNAseq (adjusted p < .05) and label-free quantitative mass spectrometry (false discovery rate < 5%) in the three groups. RESULTS: WES revealed no common pathogenic variants in RE, but several rare and likely deleterious variants of unknown significance (VUS; ANGPTL7/MTOR, SCN1A, FCGR3B, MTOR) and more common HLA VUS in >25% of RE cases (HLA-DRB1, HLA-DQA2), all with allele frequency < 5% in the general population. RNAseq in RE versus PWE (1516 altered transcripts) revealed significant activation of crosstalk between dendritic and natural killer cells (p = 7.94 × 10-6 , z = 2.65), in RE versus control (7466 transcripts) neuroinflammation signaling activation (p = 6.31 × 10-13 , z = 5.07), and in PWE versus control (945 transcripts) phagosome formation activation (p = 2.00 × 10-13 , z = 5.61). Proteomics detected fewer altered targets. SIGNIFICANCE: In RE, we identified activated immune signaling pathways and immune cell type annotation enrichment that suggest roles of the innate and adaptive immune responses, as well as HLA variants that may increase vulnerability to RE. Follow-up studies could evaluate cell type density and subregional localization associated with top targets, clinical history (neuropathology, disease duration), and whether modulating crosstalk between dendritic and natural killer cells may limit disease progression.

Renal tubular epithelial cell necroptosis promotes tubulointerstitial fibrosis in patients with chronic kidney disease.

Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney disease (CKD), ultimately predisposes patients to end-stage renal disease. However, there is no effective therapy for renal fibrosis. Our earlier studies proved that RIP3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury. Under transmission electron microscopy (TEM), we found morphological changes in the necrosis of human renal tissue, and the percentage of necrotic cells increased significantly in patients with stages 2 and 3a CKD. Immunofluorescence analyses showed that the percentages of TUNEL+ /RIP3+ double-positive and TUNEL+ /MLKL+ double-positive tubular epithelial cells in renal tubules of patients with stages 2 and 3a CKD were significantly increased compared to those in control patients without renal disease. Immunohistochemistry analyses of renal biopsy specimens from patients with CKD revealed RIP3, MLKL, and p-MLKL upregulation in patients with stages 2 and 3a CKD, suggesting that necroptosis of renal tubular epithelial cells in CKD patients occurs, and the peak of necroptosis was in stages 2 and 3a CKD. We showed that profibrotic factor proteins (TGF-ß1, Smad2 and Smad3) and fibroblast activation markers (α-SMA and Vimentin) were specifically upregulated in stage 2 and 3a CKD patients. In addition, Pearson correlation analysis showed that the percentage of necroptotic renal tubular epithelial cells was positively correlated with TGF-ß1 and collagen-I. We also showed that RIP1/3 or MLKL inhibitors decreased the expression of RIP3, MLKL, TGF-ß1, and Smad3 in HK-2 cells treated with TNF-α. FGF-2, α-SMA, Vimentin and FN were overexpressed in the hRIFs cultured with the supernatant of necroptotic HK-2 cells, whereas necroptosis blockers (Nec-1s, GSK'872 and NSA) and TGF-ß1/Smad3 pathway antagonists (LY364947 and SIS3) reduced FGF-2, α-SMA, Vimentin and FN levels. Collectively, necroptosis of renal tubular epithelial cells in CKD patients occurs, and the peak of necroptosis was in stages 2 and 3a CKD. Renal tubular epithelial cell necroptosis mediates renal tubulointerstitial fibrosis in patients with chronic kidney disease, which is related to the TGF-ß1/Smad3 signaling pathway.

Resurgence of Respiratory Syncytial Virus Infection During COVID-19 Pandemic Among Children in Shanghai, China.

Respiratory syncytial virus (RSV) is the most common pathogen causing acute lower respiratory tract infection (LRTI) in children. RSV usually peaks in winter and declines by early spring in China. The outbreak of coronavirus disease 2019 (COVID-19) was reported to bring changes to the transmission pattern of respiratory pathogens including RSV. Here in this paper, we analyzed RSV-positive nasopharyngeal aspirates from inpatients in the Children's Hospital of Fudan University from October 2019 to October 2021 and compared the clinical features of the RSV-positive patients before and during COVID-19. We found an atypical upsurge of RSV infection in the late summer of 2021 after a major suppression in 2020. RSV B was the main subtype spreading among children throughout the study. Phylogenetic analysis revealed that all RSV A strains belonged to ON1 genotype and all RSV B strains were BA9 genotype. Deduced amino acid analysis displayed different substitutions in the RSV strains observed before and during COVID-19. Demographic analysis suggested that males and infants aged under 5 months were the main populations infected with RSV by gender and age, respectively. Less severe clinical outcomes were observed in patients during COVID-19 than before the pandemic, especially in RSV B-positive patients. Our findings described the epidemiological changes in RSV infection brought by COVID-19, which further underscored the importance of continuous surveillance of RSV in the shadow of COVID-19 at both local and global scales.

Investigation of Global Gene Expression of Human Blastocysts Diagnosed as Mosaic using Next-generation Sequencing.

Embryos are diagnosed as mosaic if their chromosomal copy number falls between euploid and aneuploid. The purpose of this study was to investigate the impact of mosaicism on global gene expression. This study included 42 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) and were donated for IRB approved research. Fourteen blastocysts were diagnosed as mosaic with Next-generation Sequencing (NGS). Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 array Comparative Genomic Hybridization, 2 Single Nucleotide Polymorphism array) were used as comparisons. RNA-sequencing was performed on all of the blastocysts. Differentially expressed genes (DEGs) were calculated using DESeq2/3.5 (R Bioconductor Package) with p < 0.05 considered significantly differentially expressed. Pathway analysis was performed on mosaic embryos using EnrichR with p < 0.05 considered significant. With euploid embryo gene expression used as a control, 12 of 14 mosaic embryos had fewer DEGs compared to aneuploid embryos involving the same chromosome. On principal component analysis (PCA), mosaic embryos mapped separately from aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted within mosaic embryos. Mosaic embryos have decreased disruption of global gene expression compared to aneuploid embryos. This study was limited by the small sample size, lack of replicate samples for each mosaic abnormality, and use of multiple different PGT-A platforms for the diagnosis of aneuploid embryos.

Gpr125 is a unifying hallmark of multiple mammary progenitors coupled to tumor latency.

Gpr125 is an orphan G-protein coupled receptor, with homology to cell adhesion and axonal guidance factors, that is implicated in planar polarity and control of cell movements. By lineage tracing we demonstrate that Gpr125 is a highly specific marker of bipotent mammary stem cells in the embryo and of multiple long-lived unipotent basal mammary progenitors in perinatal and postnatal glands. Nipple-proximal Gpr125+ cells express a transcriptomic profile indicative of chemo-repulsion and cell movement, whereas Gpr125+ cells concentrated at invasive ductal tips display a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix. Gpr125 progenitors acquire bipotency in the context of transplantation and cancer and are greatly expanded and massed at the pushing margins of short latency MMTV-Wnt1 tumors. High Gpr125 expression identifies patients with particularly poor outcome within the basal breast cancer subtype highlighting its potential utility as a factor to stratify risk.

Electronic Cigarette Use Promotes a Unique Periodontal Microbiome.

Electronic cigarettes (e-cigs) have become prevalent as an alternative to conventional cigarette smoking, particularly in youth. E-cig aerosols contain unique chemicals which alter the oral microbiome and promote dysbiosis in ways we are just beginning to investigate. We conducted a 6-month longitudinal study involving 84 subjects who were either e-cig users, conventional smokers, or nonsmokers. Periodontal condition, cytokine levels, and subgingival microbial community composition were assessed, with periodontal, clinical, and cytokine measures reflecting cohort habit and positively correlating with pathogenic taxa (e.g., Treponema, Saccharibacteria, and Porphyromonas). α-Diversity increased similarly across cohorts longitudinally, yet each cohort maintained a unique microbiome. The e-cig microbiome shared many characteristics with the microbiome of conventional smokers and some with nonsmokers, yet it maintained a unique subgingival microbial community enriched in Fusobacterium and Bacteroidales (G-2). Our data suggest that e-cig use promotes a unique periodontal microbiome, existing as a stable heterogeneous state between those of conventional smokers and nonsmokers and presenting unique oral health challenges. IMPORTANCE Electronic cigarette (e-cig) use is gaining in popularity and is often perceived as a healthier alternative to conventional smoking. Yet there is little evidence of the effects of long-term use of e-cigs on oral health. Conventional cigarette smoking is a prominent risk factor for the development of periodontitis, an oral disease affecting nearly half of adults over 30 years of age in the United States. Periodontitis is initiated through a disturbance in the microbial biofilm communities inhabiting the unique space between teeth and gingival tissues. This disturbance instigates host inflammatory and immune responses and, if left untreated, leads to tooth and bone loss and systemic diseases. We found that the e-cig user's periodontal microbiome is unique, eliciting unique host responses. Yet some similarities to the microbiomes of both conventional smokers and nonsmokers exist, with strikingly more in common with that of cigarette smokers, suggesting that there is a unique periodontal risk associated with e-cig use.

Electronic cigarette use enriches periodontal pathogens.

The effect of electronic cigarette (e-cigarette) smoking, especially its long-term impact on oral health, is poorly understood. Here, we conducted a longitudinal clinical study with two study visits, 6 months apart, to investigate the effect of e-cigarette use on the bacterial community structure in the saliva of 101 periodontitis patients. Our data demonstrated that e-cigarette use altered the oral microbiome in periodontitis patients, enriching members of the Filifactor, Treponema, and Fusobacterium taxa. For patients at the same periodontal disease stage, cigarette smokers and e-cigarette smokers shared more similarities in their oral bacterial composition. E-cigarette smoking may have a similar potential as cigarette smoking at altering the bacterial composition of saliva over time, leading to an increase in the relative abundance of periodontal disease-associated pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum. The correlation analysis showed that certain genera, such as Dialister, Selenomonas, and Leptotrichia in the e-cigarette smoking group, were positively correlated with the levels of proinflammatory cytokines, including IFN-γ, IL-1ß, and TNF-α. E-cigarette use was also associated with elevated levels of proinflammatory cytokines such as IFN-γ and TNF-α, which contribute to oral microbiome dysbiosis and advanced disease state.

Potent and Targeted Sindbis Virus Platform for Immunotherapy of Ovarian Cancer.

Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of ovarian and other types of cancers. In this study we show that SV.IL-12 combined with an agonistic OX40 antibody can eliminate ovarian cancer in a Mouse Ovarian Surface Epithelial Cell Line (MOSEC) model and further prevent tumors in mice rechallenged with tumor cells after approximately 5 months. Treatment efficacy is shown to be dependent upon T-cells that are transcriptionally and metabolically reprogramed. An influx of immune cells to the tumor microenvironment occurs. Combination of sequences encoding both IL-12 and anti-OX40 into a single SV vector, SV.IgGOX40.IL-12, facilitates the local delivery of immunoregulatory agents to tumors enhancing the anti-tumor response. We promote SV.IgGOX40.IL-12 as a safe and effective therapy for multiple types of cancer.

Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.