Ensemble deep learning of embeddings for clustering multimodal single-cell omics data.

MOTIVATION: Recent advances in multimodal single-cell omics technologies enable multiple modalities of molecular attributes, such as gene expression, chromatin accessibility, and protein abundance, to be profiled simultaneously at a global level in individual cells. While the increasing availability of multiple data modalities is expected to provide a more accurate clustering and characterization of cells, the development of computational methods that are capable of extracting information embedded across data modalities is still in its infancy. RESULTS: We propose SnapCCESS for clustering cells by integrating data modalities in multimodal single-cell omics data using an unsupervised ensemble deep learning framework. By creating snapshots of embeddings of multimodality using variational autoencoders, SnapCCESS can be coupled with various clustering algorithms for generating consensus clustering of cells. We applied SnapCCESS with several clustering algorithms to various datasets generated from popular multimodal single-cell omics technologies. Our results demonstrate that SnapCCESS is effective and more efficient than conventional ensemble deep learning-based clustering methods and outperforms other state-of-the-art multimodal embedding generation methods in integrating data modalities for clustering cells. The improved clustering of cells from SnapCCESS will pave the way for more accurate characterization of cell identity and types, an essential step for various downstream analyses of multimodal single-cell omics data. AVAILABILITY AND IMPLEMENTATION: SnapCCESS is implemented as a Python package and is freely available from https://github.com/PYangLab/SnapCCESS under the open-source license of GPL-3. The data used in this study are publicly available (see section 'Data availability').

KARS Mutations Impair Brain Myelination by Inducing Oligodendrocyte Deficiency: One Potential Mechanism and Improvement by Melatonin.

It is very crucial to investigate key molecules that are involved in myelination to gain an understanding of brain development and injury. We have reported for the first time that pathogenic variants p.R477H and p.P505S in KARS, which encodes lysyl-tRNA synthetase (LysRS), cause leukoencephalopathy with progressive cognitive impairment in humans. The role and action mechanisms of KARS in brain myelination during development are unknown. Here, we first generated Kars knock-in mouse models through the CRISPR-Cas9 system. Kars knock-in mice displayed significant cognitive deficits. These mice also showed significantly reduced myelin density and content, as well as significantly decreased myelin thickness during development. In addition, Kars mutations significantly induced oligodendrocyte differentiation arrest and reduction in the brain white matter of mice. Mechanically, oligodendrocytes' significantly imbalanced expression of differentiation regulators and increased capase-3-mediated apoptosis were observed in the brain white matter of Kars knock-in mice. Furthermore, Kars mutations significantly reduced the aminoacylation and steady-state level of mitochondrial tRNALys and decreased the protein expression of subunits of oxidative phosphorylation complexes in the brain white matter. Kars knock-in mice showed decreased activity of complex IV and significantly reduced ATP production and increased reactive oxygen species in the brain white matter. Significantly increased percentages of abnormal mitochondria and mitochondrion area were observed in the oligodendrocytes of Kars knock-in mouse brain. Finally, melatonin (a mitochondrion protectant) significantly attenuated mitochondrion and oligodendrocyte deficiency in the brain white matter of KarsR504H/P532S mice. The mice treated with melatonin also showed significantly restored myelination and cognitive function. Our study first establishes Kars knock-in mammal models of leukoencephalopathy and cognitive impairment and indicates important roles of KARS in the regulation of mitochondria, oligodendrocyte differentiation and survival, and myelination during brain development and application prospects of melatonin in KARS (or even aaRS)-related diseases.

Analysis of serum metabolome of laborers exposure to welding fume.

OBJECTIVE: Welding fume exposure is inevitable of welding workers and poses a severe hazard to their health since welding is a necessary industrial process. Thus, preclinical diagnostic symptoms of worker exposure are of great importance. The aim of this study was to screen serum differential metabolites of welding fume exposure based on UPLC-QTOF-MS/MS. METHODS: In 2019, 49 participants were recruited at a machinery manufacturing factory. The non-target metabolomics technique was used to clarify serum metabolic signatures in people exposed to welding fume. Differential metabolites were screened by OPLS-DA analysis and Student's t-test. The receiver operating characteristic curve evaluated the discriminatory power of differential metabolites. And the correlations between differential metabolites and metal concentrations in urine and whole blood were analyzed utilizing Pearson correlation analysis. RESULTS: Thirty metabolites were increased significantly, and 5 metabolites were decreased. The differential metabolites are mainly enriched in the metabolism of arachidonic acid, glycero phospholipid, linoleic acid, and thiamine. These results observed that lysophosphatidylcholine (20:1/0:0) and phosphatidylglycerol(PGF1α/16:0) had a tremendous anticipating power with relatively increased AUC values (AUC > 0.9), and they also presented a significant correlation of Mo concentrations in whole blood and Cu concentrations in urine, respectively. CONCLUSION: The serum metabolism was changed significantly after exposure to welding fume. Lysophosphatidylcholine (20:1/0:0) and phosphatidylglycerol (PGF1α/16:0) may be a potential biological mediator and biomarker for laborers exposure to welding fume.

Grammar of protein domain architectures.

From an abstract, informational perspective, protein domains appear analogous to words in natural languages in which the rules of word association are dictated by linguistic rules, or grammar. Such rules exist for protein domains as well, because only a small fraction of all possible domain combinations is viable in evolution. We employ a popular linguistic technique, n-gram analysis, to probe the "proteome grammar"-that is, the rules of association of domains that generate various domain architectures of proteins. Comparison of the complexity measures of "protein languages" in major branches of life shows that the relative entropy difference (information gain) between the observed domain architectures and random domain combinations is highly conserved in evolution and is close to being a universal constant, at ∼1.2 bits. Substantial deviations from this constant are observed in only two major groups of organisms: a subset of Archaea that appears to be cells simplified to the limit, and animals that display extreme complexity. We also identify the n-grams that represent signatures of the major branches of cellular life. The results of this analysis bolster the analogy between genomes and natural language and show that a "quasi-universal grammar" underlies the evolution of domain architectures in all divisions of cellular life. The nearly universal value of information gain by the domain architectures could reflect the minimum complexity of signal processing that is required to maintain a functioning cell.

Photoregulation of Gene Expression with Ligand-Modified Caged siRNAs through Host/Guest Interaction.

Small interfering RNA (siRNA) can effectively silence target genes through Argonate 2 (Ago2)-induced RNA interference (RNAi). It is very important to control siRNA activity in both spatial and temporal modes. Among different masking strategies, photocaging can be used to regulate gene expression through light irradiation with spatiotemporal and dose-dependent resolution. Many different caging strategies and caging groups have been reported for light-activated siRNA gene silencing. Herein, we describe a novel caging strategy that increases the blocking effect of RISC complex formation/process through host/guest (including ligand/receptor) interactions, thereby enhancing the inhibition of caged siRNA activity until light activation. This strategy can be used as a general approach to design caged siRNAs for the photomodulation of gene silencing of exogenous and endogenous genes.

Lipopolysaccharide-Induced Microglial Neuroinflammation: Attenuation by FK866.

Alleviating microglia-mediated neuroinflammation bears great promise to reduce neurodegeneration. Nicotinamide phosphoribosyltransferase (NAMPT) may exert cytokine-like effect in the brain. However, it remains unclear about role of NAMPT in microglial inflammation. Also, it remains unknown about effect of NAMPT inhibition on microglial inflammation. In the present study, we observed that FK866 (a specific noncompetitive NAMPT inhibitor) dose-dependently inhibited lipopolysaccharide (LPS)-induced proinflammatory mediator (interleukin (IL)-6, IL-1ß, inducible nitric oxide synthase, nitric oxide and reactive species) level increase in BV2 microglia cultures. FK866 also significantly inhibited LPS-induced polarization change in microglia. Furthermore, LPS significantly increased NAMPT expression and nuclear factor kappa B (NF-κB) phosphorylation in microglia. FK866 significantly decreased NAMPT expression and NF-κB phosphorylation in LPS-treated microglia. Finally, conditioned medium from microglia cultures co-treated with FK866 and LPS significantly increased SH-SY5Y and PC12 cell viability compared with conditioned medium from microglia cultures treated with LPS alone. Our study strongly indicates that NAMPT may be a promising target for microglia modulation and NAMPT inhibition may attenuate microglial inflammation.

Substantia nigra Smad3 signaling deficiency: relevance to aging and Parkinson's disease and roles of microglia, proinflammatory factors, and MAPK.

BACKGROUND: Smad3 signaling is indicated to regulate microglia activity. Parkinson's disease (PD) neurodegeneration is shown to be associated with aging and neuroinflammation. However, it remains unclear about the relationship among Smad3 signaling, aging, neuroinflammation, and PD. METHODS: Rats were treated with SIS3 (a specific inhibitor of Smad3, intranigal injection) and/or lipopolysaccharide (intraperitoneal injection). We investigated the effect of SIS3 and lipopolysaccharide and their mechanism of action on motor behavior and nigrostriatal dopaminergic system in the rats. Furthermore, we explored the effect of SIS3 and LPS and their potential signaling mechanism of action on inflammatory response by using primary microglial cultures. Finally, we investigated the relationship among aging, Smad3 signaling, and neuroinflammation using animals of different ages. RESULTS: Both SIS3 and lipopolysaccharide induced significant behavior deficits and nigrostriatal dopaminergic neurodegeneration in the rats compared with the vehicle-treated (control) rats. Significantly increased behavior deficits and nigrostriatal dopaminergic neurodegeneration were observed in the rats co-treated with SIS3 and lipopolysaccharide compared with the rats treated with vehicle, SIS3, or lipopolysaccharide. Furthermore, both SIS3 and lipopolysaccharide induced significant microglia activation and proinflammatory factor (IL-1ß, IL-6, iNOS, and ROS) level increase in the SN of rats compared with the control rats. Significantly enhanced microglial inflammatory response was observed in the rats co-treated with SIS3 and lipopolysaccharide compared with the other three groups. For our in vitro study, both SIS3 and lipopolysaccharide induced significant proinflammatory factor level increase in primary microglia cultures compared with the control cultures. Significantly increased inflammatory response was observed in the cultures co-treated with SIS3 and lipopolysaccharide compared with the other three groups. MAPK (ERK/p38) contributed to microglial inflammatory response induced by co-treatment with SIS3 and lipopolysaccharide. Interestingly, there was decrease in Smad3 and pSmad3 expression (protein) and enhancement of neuroinflammation in the mouse SN with aging. Proinflammatory factor levels were significantly inversely correlated with Smad3 and pSmad3 expression. CONCLUSION: Our study strongly indicates the involvement of SN Smad3 signaling deficiency in aging and PD neurodegeneration and provides a novel molecular mechanism underlying the participation of aging in PD and helps to elucidate the mechanisms for the combined effect of multiple factors in PD.

The QuantiFERON Monitor® assay is predictive of infection post allogeneic hematopoietic cell transplantation.

INTRODUCTION: Following allogeneic hematopoietic stem cell transplantation (alloHCT), excessive immunosuppression can be complicated by infection, while inadequate immunosuppression can result in graft-vs-host disease (GVHD). An accurate method to assess overall immune status post HCT is lacking. The QuantiFERON Monitor® (QFM) assay measures interferon gamma (IFN-γ) release from whole blood following incubation with both innate (Toll-like receptor 7, TLR7) and adaptive (CD3 antibody) stimulants and may result in a more complete assessment of the immune system. METHODS: Whole blood samples were prospectively collected from alloHCT recipients at conditioning followed by days 10, 30, 60, 90, 120, and 180 post-transplant and assayed by the QFM test. IFN-γ levels were correlated to time post HCT and episodes of infection and GVHD. RESULTS: Forty patients were enrolled in the study (68% male; median age 47 years; 58% matched related donors, 42% unrelated; 33% myeloablative). Post-stimulation IFN-γ levels rose steadily over the first 180 days post transplantation. IFN-γ levels were significantly lower in those with active infection compared to those without during the neutropenic period (P < .001). The assay was predictive of CMV reactivation (VL > 1000 copies/mL) post alloHCT (P = .001). CONCLUSION: This is a promising assay to demonstrate immune recovery and predict risk of infection after alloHCT and may allow tailoring of immunosuppression, antimicrobial treatment, and prophylaxis.

Disassembly of Dimeric Cyanine Dye Supramolecular Assembly by Tetramolecular G-quadruplex Dependence on Linker Length and Layers of G-quartet.

Cyanine dyes have been widely applied in various biological systems owing to their specific photochemical properties. Assembly and disassembly process of cyanine dyes were constructed and regulated by special biomolecules. In this paper, dimeric cyanine dyes with different repeat units (oligo-oxyethylene) in linker (TC-Pn) (n = 3-6) were found to form H-aggregates or mixture aggregates in PBS. These aggregates could be disassembled into dimer and/or monomer by (TGnT) tetramolecular G-quadruplexes (n = 3-6, 8), which were affected by the linker length of dimeric cyanine dyes and layers of G-quartets. The 1H-NMR titration results suggest that the binding mode of dimeric cyanine dye with TGnT might be on both ends-stacking like a clip. This binding mode could clearly explain that matching structures between dimeric cyanine dyes and TGnT quadruplexes could regulate the disassembly properties of aggregates. These results could provide clues for the development of highly specific G-quadruplex probes.

ESCRT-0 complex modulates Rbf-mutant cell survival by regulating Rhomboid endosomal trafficking and EGFR signaling.

The Rb tumor suppressor is conserved in Drosophila, and its inactivation can lead to cell proliferation or death depending on the specific cellular context. Therefore, identifying genes that affect the survival of Rb-mutant cells can potentially identify novel targets for therapeutic intervention in cancer. From a genetic screen in Drosophila, we identified synthetic lethal interactions between mutations of fly Rb (rbf) and the ESCRT-0 components stam and hrs We show that inactivation of ESCRT-0 sensitizes rbf-mutant cells to undergo apoptosis through inhibition of EGFR signaling and accumulation of Hid protein. Mutation of stam inhibits EGFR signaling upstream of secreted Spi and downstream of Rhomboid expression, and causes Rhomboid protein to accumulate in the abnormal endosomes labeled with both the early and late endosomal markers Rab5 and Rab7. These results reveal that ESCRT-0 mutants inhibit EGFR signaling by disrupting Rhomboid endosomal trafficking in the ligand-producing cells. Because ESCRT-0 also plays crucial roles in EGFR downregulation after ligand binding, this study provides new insights into how loss of ESCRT-0 function can either increase or decrease EGFR signaling.

Caged siRNAs with Single cRGD Modification for Photoregulation of Exogenous and Endogenous Gene Expression in Cells and Mice.

RNA interference (RNAi) mediated gene silencing holds significant promise in gene therapy. It is very important to manually regulate the activity of small interference RNAs (siRNAs) in the controllable mode. Here, we designed and synthesized a series of caged siRNAs through bioconjugation of cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptide to the 5' end of siRNA through a photolabile linker. These cRGD modified caged siRNAs allowed for precise light-regulation of gene expression of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and an endogenous gene (the mitosis motor protein, Eg5) in the integrin αvß3 positive cells. This kind of bioconjugate further enabled photochemical activation of siRNA activity, and the target gene silencing was successfully achieved in tumor-bearing mice by intratumoral injection. This study also suggested that photomodulation of target gene expression using single cRGD caged siRNA at the 5' end of antisense strand RNA inhibited siRNA activity probably due to three factors: (1) trapping of cRGD modified siRNA in endosome and lysosome, (2) the steric hindrance of cRGD, (3) the binding of cRGD to its corresponding receptor.

Caged siRNAs with single folic acid modification of antisense RNA for photomodulation of exogenous and endogenous gene expression in cells.

Manually controlling siRNA activity is an essentially important way to spatiotemporally investigate gene expression and function. Owing to ease of operation and precise manipulation, light can be used for controlled regulation of siRNA-induced gene silencing. Here, we developed a series of caged siRNAs with folic acid modification at the 5' terminus of the antisense strand of the siRNA through a photolabile linker. The attachment of the folic acid moiety temporarily masked the corresponding siRNA activity. Upon illumination, these caged siRNAs were activated, and their gene silencing activities were restored. Based on this strategy, we successfully photomodulated gene expression of both an exogenous gene (for green fluorescent protein, GFP) and an endogenous gene (for mototic kinesin-5, Eg5) in cells.

Mutations in KARS cause early-onset hearing loss and leukoencephalopathy: Potential pathogenic mechanism.

Leukoencephalopathies are a broad class of common neurologic deterioration for which the etiology remains unsolved in many cases. In a Chinese Han family segregated with sensorineural hearing loss and leukoencephalopathy, candidate pathogenic variants were identified by targeted next-generation sequencing of 144 genes associated with deafness and 108 genes with leukoencephalopathy. Novel compound heterozygous mutations p.R477H and p.P505S were identified in KARS, which encodes lysyl-tRNA synthetase (LysRS), as the only candidate causative variants. These two mutations were functionally characterized by enzymatic assays, immunofluorescence, circular dichroism analysis, and gel filtration chromatography. Despite no alteration in the dimer-tetramer oligomerization and cellular distribution by either mutation, the protein structure was notably influenced by the R477H mutation, which subsequently released the protein from the multiple-synthetase complex (MSC). Mutant LysRSs with the R477H and P505S mutations had decreased tRNALys aminoacylation and displayed a cumulative effect when introduced simultaneously. Our studies showed that mutations in KARS lead to a newly defined subtype of leukoencephalopathy associated with sensorineural hearing impairment. The combined effect of reduced aminoacylation and release of LysRS from the MSC likely underlies the pathogenesis of the KARS mutations identified in this study.

A novel immune function biomarker identifies patients at risk of clinical events early following liver transplantation.

Balancing immunosuppression after liver transplant is difficult, with clinical events common. We investigate whether a novel immune biomarker based on a laboratory platform with widespread availability that measures interferon γ (IFNγ) after stimulation with a lyophilized ball containing an adaptive and innate immune stimulant can predict events following transplantation. A total of 75 adult transplant recipients were prospectively monitored in a blinded, observational study; 55/75 (73.3%) patients experienced a total of 89 clinical events. Most events occurred within the first month. Low week 1 results were significantly associated with risk of early infection (area under the receiver operating characteristic curve [AUROC], 0.74; P = 0.008). IFNγ ≤ 1.30 IU/mL (likelihood ratio positive, 1.93; sensitivity, 71.4%; specificity, 63.0%) was associated with the highest risk for infection with minimal rejection risk. Nearly half the cohort (27/60, 45.0%) expressed IFNγ ≤ 1.30 IU/mL. Moreover, an elevated week 1 result was significantly associated with the risk of rejection within the first month after transplant (AUROC, 0.77; P = 0.002), but no episodes of infection. On multivariate logistic regression, IFNγ ≥ 4.49 IU/mL (odds ratio, 4.75) may be an independent predictor of rejection (P = 0.05). In conclusion, low IFNγ suggesting oversuppression is associated with infections, whereas high IFNγ indicating undersuppression is associated with rejection. This assay offers the potential to allow individualization and optimization of immunosuppression that could fundamentally alter the way patients are managed following transplantation. Liver Transplantation 23 487-497 2017 AASLD.

Directly lighting up RNA G-quadruplexes from test tubes to living human cells.

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U(p)) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes successfully distinguished target RNA from single-mutated RNA analyte during an in vitro assay of RNA synthesis.

Oxygen modulates human embryonic stem cell metabolism in the absence of changes in self-renewal.

Human embryonic stem (ES) cells are routinely cultured under atmospheric oxygen (~20%), a concentration that is known to impair embryo development in vitro and is likely to be suboptimal for maintaining human ES cells compared with physiological (~5%) oxygen conditions. Conflicting reports exist on the effect of oxygen during human ES cell culture and studies have been largely limited to characterisation of typical stem cell markers or analysis of global expression changes. This study aimed to identify physiological markers that could be used to evaluate the metabolic impact of oxygen on the MEL-2 human ES cell line after adaptation to either 5% or 20% oxygen in extended culture. ES cells cultured under atmospheric oxygen displayed decreased glucose consumption and lactate production when compared with those cultured under 5% oxygen, indicating an overall higher flux of glucose through glycolysis under physiological conditions. Higher glucose utilisation at 5% oxygen was accompanied by significantly increased expression of all glycolytic genes analysed. Analysis of amino acid turnover highlighted differences in the consumption of glutamine and threonine and in the production of proline. The expression of pluripotency and differentiation markers was, however, unaltered by oxygen and no observable difference in proliferation between cells cultured in 5% and 20% oxygen was seen. Apoptosis was elevated under 5% oxygen conditions. Collectively these data suggest that culture conditions, including oxygen concentration, can significantly alter human ES cell physiology with coordinated changes in gene expression, in the absence of detectable alterations in undifferentiated marker expression.

Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin†E Modification.

Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5' terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6-fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE-modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE-binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.

A newly identified G-quadruplex as a potential target regulating Bcl-2 expression.

BACKGROUND: A new G-quadruplex structure located in the B-cell CLL/lymphoma 2 (Bcl-2) P1 promoter and its physiological function related to Bcl-2 transcription have been studied to find a potential anticancer therapeutic target. METHODS: Absorption, polyacrylamide gel electrophoresis, fluorescence, circular dichroism, and nuclear magnetic resonance spectra have been employed to determine G-quadruplex structure and the interaction between G-quadruplex and phenanthrolin-dicarboxylate. Real time polymerase chain reaction and luciferase assay were done to assess the physiological function of the G-quadruplex structure. RESULTS: The UV-melting and polyacrylamide gel electrophoresis studies show that the p32 DNA sequence forms an intramolecular G-quadruplex structure. Circular dichroism and nuclear magnetic resonance spectra indicate that the G-quadruplex is a hybrid-type structure with four G-tetrads. Fluorescence spectra show that a phenanthroline derivative has a higher binding affinity for p32 G-quadruplex than duplex. Further circular dichroism and nuclear magnetic resonance studies indicate that the phenanthroline derivative can regulate p32 G-quadruplex conformation. Real time polymerase chain reaction and luciferase assays show that the phenanthroline derivative has down-modulated Bcl-2 transcription activity in a concentration-dependent manner. However, no such effect was observed when p32 G-quadruplex was denatured through base mutation. CONCLUSION: The newly identified G-quadruplex located in the P1 promoter of Bcl-2 oncogene is intimately related with Bcl-2 transcription activity, which may be a promising anticancer therapeutic target. GENERAL SIGNIFICANCE: The newly identified G-quadruplex in the Bcl-2 P1 promoter may be a novel anticancer therapeutic target.

Targeted individual prophylaxis offers superior risk stratification for cytomegalovirus reactivation after liver transplantation.

Cytomegalovirus (CMV) can reactivate following liver transplantation. Management of patients currently considered low risk based on pretransplant serology remains contentious, with universal prophylaxis and preemptive strategies suffering from significant deficiencies. We hypothesized that a CMV-specific T cell assay performed early after transplant as part of a preemptive strategy could better stratify "low-risk" (recipient seropositive) patients. We conducted a prospective, blinded, observational study in 75 adult recipients. QuantiFERON-cytomegalovirus was performed both before and at multiple times after transplant. Low-risk patients (n = 58) were monitored as per unit protocol and treatment was commenced if CMV > 1000 copies/mL (DNAemia). Twenty patients needed antiviral treatment for other reasons and were censored (mainly for rejection or herpes simplex virus infection); 19/38 (50%) of the remaining low-risk patients developed DNAemia at mean 34.6 days after transplant. A week 2 result of <0.1 IU/mL was significantly associated with risk of subsequent DNAemia (hazard ratio [HR], 6.9; P = 0.002). The positive predictive value of 80% suggests these patients are inappropriately labeled low risk and are actually at high likelihood of CMV reactivation. A secondary cutoff of <0.2 IU/mL was associated with moderate risk (HR, 2.8; P = 0.01). In conclusion, a protocol based on a single early CMV-specific T cell based assay would offer improved risk stratification and individualization of patient management after transplant. This could offer improved drug and service utilization and potentially result in significant improvements over both currently used protocols to manage supposedly low-risk patients.