Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

BACKGROUND: Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. METHODS: Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. RESULTS: Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. CONCLUSIONS: Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

ZWC complex-mediated SPT5 phosphorylation suppresses divergent antisense RNA transcription at active gene promoters.

The human genome encodes large numbers of non-coding RNAs, including divergent antisense transcripts at transcription start sites (TSSs). However, molecular mechanisms by which divergent antisense transcription is regulated have not been detailed. Here, we report a novel ZWC complex composed of ZC3H4, WDR82 and CK2 that suppresses divergent antisense transcription. The ZWC complex preferentially localizes at TSSs of active genes through direct interactions of ZC3H4 and WDR82 subunits with the S5p RNAPII C-terminal domain. ZC3H4 depletion leads to increased divergent antisense transcription, especially at genes that naturally produce divergent antisense transcripts. We further demonstrate that the ZWC complex phosphorylates the previously uncharacterized N-terminal acidic domain of SPT5, a subunit of the transcription-elongation factor DSIF, and that this phosphorylation is responsible for suppressing divergent antisense transcription. Our study provides evidence that the newly identified ZWC-DSIF axis regulates the direction of transcription during the transition from early to productive elongation.

Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

Application of the 3' mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue.

BACKGROUND: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3' mRNA-seq method sequences the 3' region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3' mRNA-Seq using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. RESULTS: The total mapped reads of QuantSeq 3' mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3' mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3' mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). CONCLUSION: In this study, QuantSeq 3' mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3' mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs.

BACKGROUND: There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 µg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. RESULTS: Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3' region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). CONCLUSION: In this study, our results showed that 1 µg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.

Donor protection: Iron supplementation for frequent blood donors in Korea.

OBJECTIVE: This study aimed to evaluate the effect of oral iron supplementation in frequent donors in Korea, based solely on donation history. STUDY DESIGN: The hemoglobin (Hb) level, ferritin level, soluble transferrin receptor (sTfR), total iron binding capacity (TIBC), and transferrin saturation of frequent donors at high risk for iron deficiency were compared to those of first donors. The frequent donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects. RESULT: A total of 53 male and 57 female frequent donors were recruited. After 4-week iron supplementation, among the men, the prevalence of a: low Hb level (<13.0 g/dL) decreased from 25% to 2%; low ferritin level (<15.0 ng/mL) decreased from 58% to 4%; iron deficient erythropoiesis (IDE) (log(sTfR/ferritin) ≥ 2.07) decreased from 77% to 33%. Among the women, the percentage of a: low Hb level (<12.0 g/dL) decreased from 44% to 9%; low ferritin level decreased from 79% to 11%; IDE decreased from 95% to 47%. In total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. No serious adverse events were reported. CONCLUSION: Ferritin level, a reliable indicator of iron status, increased and IDE decreased significantly after four-week iron supplementation in the female, but not in the male, donor group, compared to those of control donors. Four-week oral iron supplement was not enough to restore iron storage level in the male donor group.

Bacterial Single Cell Whole Transcriptome Amplification in Microfluidic Platform Shows Putative Gene Expression Heterogeneity.

Single cell RNA sequencing is a technology that provides the capability of analyzing the transcriptome of a single cell from a population. So far, single cell RNA sequencing has been focused mostly on human cells due to the larger starting amount of RNA template for subsequent amplification. One of the major challenges of applying single cell RNA sequencing to microbial cells is to amplify the femtograms of the RNA template to obtain sufficient material for downstream sequencing with minimal contamination. To achieve this goal, efforts have been focused on multiround RNA amplification, but would introduce additional contamination and bias. In this work, we for the first time coupled a microfluidic platform with multiple displacement amplification technology to perform single cell whole transcriptome amplification and sequencing of Porphyromonas somerae, a microbe of interest in endometrial cancer, as a proof-of-concept demonstration of using single cell RNA sequencing tool to unveil gene expression heterogeneity in single microbial cells. Our results show that the bacterial single-cell gene expression regulation is distinct across different cells, supporting widespread heterogeneity.

High frequency electrical stimulation promotes expression of extracellular matrix proteins from human astrocytes.

Therapeutic benefits of deep brain stimulation (DBS), a neurosurgical treatment for certain movement disorders and other neurologic conditions, are well documented, but DBS mechanisms remain largely unexplained. DBS is thought to modulate pathological neural activity. However, although astrocytes, the most numerous cell type in the brain, play a significant role in neurotransmission, chemical homeostasis and synaptic plasticity, their role in DBS has not been fully examined. To investigate astrocytic function in DBS, we applied DBS-like high frequency electrical stimulation for 24 h to human astrocytes in vitro and analyzed single cell transcriptome mRNA profile. We found that DBS-like high frequency stimulation negatively impacts astrocyte metabolism and promotes the release of extracellular matrix (matricellular) proteins, including IGFBP3, GREM1, IGFBP5, THBS1, and PAPPA. Our results suggest that astrocytes are involved in the long-term modulation of extra cellular matrix environments and that they may influence persistent cell-to-cell interaction and help maintain neuromodulation over time.

Perioperative Fresh Red Blood Cell Transfusion May Negatively Affect Recipient Survival After Liver Transplantation.

OBJECTIVE: The aim of this study is to evaluate the association between fresh red blood cell (RBC) transfusion and recipient survival after liver transplantation. BACKGROUND: Fresh RBC products contain many viable leukocytes. Allogeneic leukocytes are responsible for adverse transfusion reactions in the immunocompromised host. METHODS: Among 343 liver transplant recipients who underwent perioperative RBC transfusion, 91 of 226 who did not receive fresh RBCs were matched with 91 of 117 who received fresh RBCs with 1:1 matching ratio using the propensity score based on the amount of transfused blood products and others. Survival analysis was performed using the Cox model. RESULTS: All transfused 3230 RBCs were leukoreduced and irradiated. Before matching, recipients in fresh RBC group received 3 U (2-6 U) of fresh RBCs. After a median follow-up of 60 months, 60 of 343 recipients (17.5%) died. Survival probability at 1/2/5 years after transplantation was 94.7%/92.0%/85.8% for nonfresh RBC group and 82.9%/76.0%/72.0% for fresh RBC group [death hazard ratio (HR) = 2.37 (1.43-3.94), P = 0.001]. In multivariable analysis, fresh RBC transfusion was significantly associated with increased death risk [HR = 2.33 (1.35-4.01), P = 0.002]. After matching, recipients in fresh RBC group received 3 U (2-5 U) of fresh RBCs. After a median follow-up of 56 months, 35 of 182 recipients (19.2%) died. Survival probability at 1/2/5 years was 95.6%/93.2%/86.0% for nonfresh RBC group and 85.7%/78.0%/73.0% for fresh RBC group [HR = 2.23 (1.43-3.94), P = 0.028]. Multivariable analysis confirmed a significance of fresh RBC transfusion [HR = 3.20 (1.51-6.78), P = 0.002]. CONCLUSION: Our findings suggest a potential negative impact of fresh RBC transfusion on the survival of patients undergoing liver transplantation.

Composite hemangioendothelioma with neuroendocrine marker expression: an aggressive variant.

Aberrant expression of neuroendocrine markers is extremely rare in endothelial neoplasms, with only a single report describing three cases. Although originally classified as conventional angiosarcoma, further assessment of these tumors revealed a strikingly composite morphology composed of retiform and epithelioid elements reminiscent of composite hemangioendothelioma, a rare subtype of hemangioendothelioma. To further investigate these findings, available materials from 11 morphologically distinctive endothelial tumors showing neuroendocrine marker expression were retrieved from our archives. Immunohistochemistry for CD31, CD34, FLI-1, synaptophysin, chromogranin, D2-40, ERG, keratin (OSCAR), and CAMTA1 was performed. Total RNA from five cases were extracted and subjected to whole transcriptome sequencing. Clinical follow-up was obtained. These tumors were found to arise in five males and six females in patients from 9 to 55 years in age (median 47 years). They arose both in superficial (wrist, ankle, scalp, hip, and foot) and deep (periaortic tissues, C5 vertebra, pulmonary vein, and liver) locations. All contained elongated, retiform vascular channels lined by hyperchromatic 'hobnail' endothelial cells and a solid growth of uniform epithelioid cells reminiscent of epithelioid hemangioendothelioma. Hemangioma-like foci also lined by hobnail endothelial cells were frequently present. Mitotic activity was typically <1/10 HPF, and necrosis or areas of conventional angiosarcoma was absent. The results of immunohistochemistry were: CD31 (10/10), FLI-1 (10/10), ERG (9/9), CD34 (5/10), D2-40 (7/10), synaptophysin (11/11), chromogranin A (1/11), CD56 (5/11), keratin (0/11), and CAMTA1 (0/6). Sequencing analysis showed one case with PTBP1-MAML2 and one case with EPC1-PHC2 fusion transcripts; fusion transcripts were not identified in the remaining cases. Follow-up (8 cases) revealed local recurrence in one patient and metastatic spread in four individuals (bone, lung, liver, and brain). One person died of disease. Although the morphological features of these tumors are characteristic of composite hemangioendothelioma, this distinctive subset with neuroendocrine differentiation more often involves deep locations and displays more aggressive behavior than typically described in other cases of composite hemangioendothelioma.

Composite hemangioendothelioma with neuroendocrine marker expression: an aggressive variant.

This corrects the article DOI: 10.1038/modpathol.2017.83.

In-frame multi-exon deletion of SMC1A in a severely affected female with Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a genetically heterogeneous disorder characterized by dysmorphic facial features, cleft palate, limb defects, growth retardation, and developmental delay. Approximately 60% of patients with CdLS have an identifiable mutation in the NIPBL gene at 5p13.2. Recently, an X-linked form of CdLS with a generally milder phenotype was attributed to mutation of the structural maintenance of chromosomes 1A gene (SMC1A) at Xp11.22. Relatively few CdLS patients with mutations in SMC1A are known; female carriers have minor facial dysmorphism and cognitive deficiency without major structural abnormalities. To date, all mutations identified in SMC1A are missense or small in-frame deletions that preserve the open reading frame of the gene and likely have a dominant-negative effect. We report on a female with monosomy X mosaicism and a phenotype suggestive of a severe form of CdLS who presented with growth and mental retardation, multiple congenital anomalies, and facial dysmorphism. Array CGH confirmed mosaic monosomy X and identified a novel deletion of SMC1A spanning multiple exons, suggesting a possible loss-of-function effect. Sequencing of both genomic and cDNA demonstrated an 8,152 bp deletion of genomic DNA from exon 13 to intron 16. Although a loss-of-function effect cannot be excluded, the resulting mRNA remains in-frame and is expressed in peripheral blood lymphocytes, suggesting a dominant-negative effect. We hypothesize that the size of this deletion compared to previously reported mutations may account for this patient's severe CdLS phenotype. The presence of mosaic monosomy X may also modify the phenotype.

Global Transcriptomic Profiling Identifies Differential Gene Expression Signatures Between Inflammatory and Noninflammatory Aortic Aneurysms.

OBJECTIVE: To identify hallmark genes and biomolecular processes in aortitis using high-throughput gene expression profiling, and to provide a range of potentially new drug targets (genes) and therapeutics from a pharmacogenomic network analysis. METHODS: Bulk RNA sequencing was performed on surgically resected ascending aortic tissues from inflammatory aneurysms (giant cell arteritis [GCA] with or without polymyalgia rheumatica, n = 8; clinically isolated aortitis [CIA], n = 17) and noninflammatory aneurysms (n = 25) undergoing surgical aortic repair. Differentially expressed genes (DEGs) between the 2 patient groups were identified while controlling for clinical covariates. A protein-protein interaction model, drug-gene target information, and the DEGs were used to construct a pharmacogenomic network for identifying promising drug targets and potentially new treatment strategies in aortitis. RESULTS: Overall, tissue gene expression patterns were the most associated with disease state than with any other clinical characteristic. We identified 159 and 93 genes that were significantly up-regulated and down-regulated, respectively, in inflammatory aortic aneurysms compared to noninflammatory aortic aneurysms. We found that the up-regulated genes were enriched in immune-related functions, whereas the down-regulated genes were enriched in neuronal processes. Notably, gene expression profiles of inflammatory aortic aneurysms from patients with GCA were no different than those from patients with CIA. Finally, our pharmacogenomic network analysis identified genes that could potentially be targeted by immunosuppressive drugs currently approved for other inflammatory diseases. CONCLUSION: We performed the first global transcriptomics analysis in inflammatory aortic aneurysms from surgically resected aortic tissues. We identified signature genes and biomolecular processes, while finding that CIA may be a limited presentation of GCA. Moreover, our computational network analysis revealed potential novel strategies for pharmacologic interventions and suggests future biomarker discovery directions for the precise diagnosis and treatment of aortitis.

Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.

BACKGROUND: MicroRNAs (miRNAs) represent a growing class of small non-coding RNAs that are important regulators of gene expression in both plants and animals. Studies have shown that miRNAs play a critical role in human cancer and they can influence the level of cell proliferation and apoptosis by modulating gene expression. Currently, methods for the detection and measurement of miRNA expression include small and moderate-throughput technologies, such as standard quantitative PCR and microarray based analysis. However, these methods have several limitations when used in large clinical studies where a high-throughput and highly quantitative technology needed for the efficient characterization of a large number of miRNA transcripts in clinical samples. Furthermore, archival formalin fixed, paraffin embedded (FFPE) samples are increasingly becoming the primary resource for gene expression studies because fresh frozen (FF) samples are often difficult to obtain and requires special storage conditions. In this study, we evaluated the miRNA expression levels in FFPE and FF samples as well as several lung cancer cell lines employing a high throughput qPCR-based microfluidic technology. The results were compared to standard qPCR and hybridization-based microarray platforms using the same samples. RESULTS: We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array. CONCLUSION: The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.

Characteristics of porous carbon nano-fibers synthesized by selective catalytic gasification.

Carbon nanofibers (CNFs) with uniquely oriented channels were prepared via selective catalytic gasification in air at 450 and 500 degrees C, using Pt or Ru nano particles as catalysts. Catalytic gasification was chosen because it can selectively generate channels in the vicinity of the catalyst particles at relatively low temperatures, where thermal oxidation does not intensively occur. The structures and surface properties of the CNFs were examined via X-ray diffraction, analysis of the nitrogen adsorption-desorption isotherms, and high-resolution transmission electron microscopy. The effects of the catalyst species and loading amount on the formation of pores (channels) were investigated. The gasification mechanism, especially the channeling direction, throught the selection of the gasification catalysts, is discussed based on the results. This process can be effectively utilized for preparation of porous carbons, which have a well-aligned graphitic structure, and also channel-type pores can be designed by selection of gasification catalysts and conditions. The present porous CNF can be applied for catalyst support in fuel cells, without further treatment (e.g., acid treatment for the removal of metallic components).

Maternal obesity is associated with phenotypic alterations in fetal immune cells by single-cell mass cytometry.

PROBLEM: Prenatal exposure to metabolic dysregulation arising from maternal obesity can have negative health consequences in post-natal life. To date, the specific effects of maternal obesity on fetal immunity at a cellular level have not been well characterized. METHOD OF STUDY: Using cord blood mononuclear cells (CBMCs) and cord plasma (n = 9/group) isolated from infants born to women with a high body mass index (BMI>25kg/m2 ) compared to women with a normal BMI (18-25kg/m2 ), we evaluated differences in immune cell populations using single-cell mass cytometry (CyTOF). CBMCs were matched according to potentially confounding variables, such as maternal and gestational age, ethnicity, smoking status, and gravidity. Statistical results were adjusted for fetal sex. Data were analyzed by viSNE and FlowSOM softwares in Cytobank™ . RESULTS: In newborn CBMCs from women with high BMI, we observed changes in frequency and phenotype of immune cell populations, including significant increases in CD4+ T cells and decreases in myeloid cell populations. IL-12p40 and MDC concentrations were significantly elevated in the high BMI group compared to control. CONCLUSION: This study demonstrates an association between maternal obesity and fetal immunity. Our results warrant following long-term immunologic outcomes and associated clinical risks in children born to women with a high pre-pregnancy BMI.

Decrease in the Risk of Posttransplant Hepatocellular Carcinoma Recurrence After the Conversion to Prestorage Leukoreduction for Transfused Red Blood Cells.

BACKGROUND: Prestorage leukoreduction has the advantage over poststorage leukoreduction in reducing leukocyte-derived molecules in red blood cells (RBC) unit, which induce immunomodulation. Our institution newly introduced prestorage leukoreduction, instead of conventional poststorage leukoreduction, for liver transplant recipients since March 2012. In this study, we aimed to evaluate the risk of posttransplant hepatocellular carcinoma (HCC) recurrence after the conversion of poststorage leukoreduction into prestorage leukoreduction for transfused allogeneic RBCs. METHODS: Among 220 patients who underwent living-donor liver transplantation for HCC, 83 of 113 who received only poststorage-leukoreduced RBCs were matched with 83 of 107 who received only prestorage-leukoreduced RBCs using 1:1 propensity score matching based on factors like tumor biology. The primary outcome was overall HCC recurrence. Survival analysis was performed with death as a competing risk event. RESULTS: In the matched cohort, recurrence probability at 1, 2, and 5 years posttransplant was 9.6%, 15.6%, and 18.1% in prestorage group and 15.6%, 21.6%, and 33.7% in poststorage group (hazard ratio [HR], 0.52; 0.28-0.97; P = 0.040). Multivariable analysis confirmed a significance of prestorage leukoreduction (HR, 0.29; 0.15-0.59; P < 0.001). Overall death risk was also lower with prestorage leukoreduction (HR, 0.51; 0.26-0.99; P = 0.049). In subgroup analysis for the unmatched cohort, recurrence risk was significantly lower in prestorage group within the patients who underwent surgery 2 years (HR, 0.24; 0.10-0.61; P = 0.002), 1 year (HR, 0.16; 0.03-0.92; P = 0.040), and 6 months (HR, 0.13; 0.02-0.85; P = 0.034), respectively, before and after the conversion to prestorage leukoreduction. CONCLUSIONS: Our findings suggest a potential benefit of prestorage leukoreduction in reducing the risk of HCC recurrence in liver transplant recipients who received allogeneic RBCs during the perioperative period.

In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells.

BACKGROUND: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. METHODS: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. RESULTS: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. CONCLUSION: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. TRIAL REGISTRATION: NCT03384108 and NCT03119883.

TBK1 regulates regeneration of pancreatic ß-cells.

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate ß-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in ß-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat ß-cells. Conversely, TBK1 overexpression decreased sensitivity of ß-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of ß-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of ß-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived ß-cells and human islets. TBK1 expression was increased in ß-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and ß-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional ß-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a ß-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional ß-cells.