Additional Pathways Research Articles

Metabolomic Effects of Folic Acid Supplementation in Adults: Evidence from the FACT Trial

BackgroundFolic acid (FA) is the oxidized form of folate found in supplements and FA-fortified foods. Most FA is reduced by dihydrofolate reductase to 5-methyltetrahydrofolate (5mTHF); the latter is the form of folate naturally found in foods. Ingestion of FA increases the plasma levels of both 5mTHF and unmetabolized FA (UMFA). Limited information is available on the downstream metabolic effects of FA supplementation, including potential effects associated with UMFA. ObjectiveWe aimed to assess the metabolic effects of FA-supplementation, and the associations of plasma 5mTHF and UMFA with the metabolome in FA-naïve Bangladeshi adults. MethodsSixty participants were selected from the Folic Acid and Creatine Trial; half received 800 μg FA/day for 12 weeks and half placebo. Plasma metabolome profiles were measured by high-resolution mass spectrometry, including 170 identified metabolites and 26,541 metabolic features. Penalized regression methods were used to assess the associations of targeted metabolites with FA-supplementation, plasma 5mTHF, and plasma UMFA. Pathway analyses were conducted using Mummichog. ResultsIn penalized models of identified metabolites, FA-supplementation was associated with higher choline. Changes in 5mTHF concentrations were positively associated with metabolites involved in amino acid metabolism (5-hydroxyindoleacetic acid, acetylmethionine, creatinine, guanidinoacetate, hydroxyproline/n-acetylalanine) and 2 fatty acids (docosahexaenoic acid and linoleic acid). Changes in 5mTHF concentrations were negatively associated with acetylglutamate, acetyllysine, carnitine, propionyl carnitine, cinnamic acid, homogentisate, arachidonic acid, and nicotine. UMFA concentrations were associated with lower levels of arachidonic acid. Together, metabolites selected across all models were related to lipids, aromatic amino acid metabolism, and the urea cycle. Analyses of nontargeted metabolic features identified additional pathways associated with FA supplementation. ConclusionIn addition to the recapitulation of several expected metabolic changes associated with 5mTHF, we observed additional metabolites/pathways associated with FA-supplementation and UMFA. Further studies are needed to confirm these associations and assess their potential implications for human health. Trial registration numberThis trial was registered at https://clinicaltrials.gov as NCT01050556.

Ru(II) Phenanthroline-Based Oligothienyl Complexes as Phototherapy Agents.

Ru(II) polypyridyl complexes have gained widespread attention as photosensitizers for photodynamic therapy (PDT). Herein, we systematically investigate a series of the type [Ru(phen)2(IP-nT)]2+, featuring 1,10-phenanthroline (phen) coligands and imidazo[4,5-f][1,10]phenanthroline ligands tethered to n = 0-4 thiophene rings (IP-nT). The complexes were characterized and investigated for their electrochemical, spectroscopic, and (photo)biological properties. The electrochemical oxidation of the nT unit shifted by -350 mV as n = 1 → 4 (+920 mV for Ru-1T, +570 mV for Ru-4T); nT reductions were observed in complexes Ru-3T (-2530 mV) and Ru-4T (-2300 mV). Singlet oxygen quantum yields ranged from 0.53 to 0.88, with Ru-3T and Ru-4T being equally efficient (∼0.88). Time-resolved absorption spectra of Ru-0T-1T were dominated by metal-to-ligand charge-transfer (3MLCT) states (τTA = 0.40-0.85 μs), but long-lived intraligand charge-transfer (3ILCT) states were observed in Ru-2T-4T (τTA = 25-148 μs). The 3ILCT energies of Ru-3T and Ru-4T were computed to be 1.6 and 1.4 eV, respectively. The phototherapeutic efficacy against melanoma cells (SK-MEL-28) under broad-band visible light (400-700 nm) increases as n = 0 → 4: Ru-0T was inactive up to 300 μM, Ru-1T-2T were moderately active (EC50 ∼ 600 nM, PI = 200), and Ru-3T (EC50 = 57 nM, PI > 1100) and Ru-4T (EC50 = 740 pM, PI = 114,000) were the most phototoxic. The activity diminishes with longer wavelengths of light and is completely suppressed for all complexes except Ru-3T and Ru-4T in hypoxia. Ru-4T is the more potent and robust PS in 1% O2 over seven biological replicates (avg EC50 = 1.3 μM, avg PI = 985). Ru-3T exhibited hypoxic activity in five of seven replicates, underscoring the need for biological replicates in compound evaluation. Singlet oxygen sensitization is likely responsible for phototoxic effects of the compounds in normoxia, but the presence of redox-active excited states may facilitate additional photoactive pathways for complexes with three or more thienyl groups. The 3ILCT state with its extended lifetime (30-40× longer than the 3MLCT state for Ru-3T and Ru-4T) implicates its predominant role in photocytotoxicity.

Redox-Active Thiocarbonyl Auxiliaries in Ni-Catalyzed Cross-Couplings of Aliphatic Alcohols.

ConspectusThe Barton-McCombie deoxygenation reaction first established the use of O-alkyl thiocarbonyl derivatives as powerful redox-active agents for C(sp3)-O reduction. In recent years, first-row transition metals capable of engaging with alkyl radical intermediates generated from O-alkyl thiocarbonyl derivatives using alternative stoichiometric radical precursors have been developed. Given the ability of select Ni catalysts to both participate in single-electron oxidative addition pathways and intercept alkyl radical intermediates, our group has investigated the use of O-alkyl thiocarbonyl derivatives as electrophiles in novel cross-coupling reactions. After describing related work in this area, this Account will first summarize our entry point into this field. Here, we used the cyclopropane ring as a reporter of leaving group reactivity to aid in the design and optimization of a novel redox-active O-thiocarbamate leaving group for C(sp3)-O arylation. Motivation for this pursuit was driven by the propensity of the cyclopropane ring to undergo ring opening under polar (2e) oxidative addition pathways or to be maintained under single-electron (1e) conditions. Using these guiding principles, we developed a method for the deoxygenative arylation of cyclopropanol derivatives using a Ni catalyst without the need for a stoichiometric external reductant or photocatalyst. We next summarize our evaluation of an alternative redox-active O-thiocarbonyl imidazole auxiliary in a related deoxygenative cross-coupling. This work demonstrated an extension of our initial approach to the deoxygenative arylation of primary and secondary aliphatic alcohol derivatives. A brief mechanistic investigation revealed that this reaction likely proceeds via a distinct mechanism involving direct homolytic C(sp3)-O bond cleavage. We conclude this Account with a summary of work aimed toward a unique approach for thiocarboxylic acid derivative synthesis. This project was inspired by the efficiency of thionoester generation under most of the reaction conditions evaluated in our prior investigations. Using alcohol, amine, or thiol starting materials, which were activated with convenient thiocarbonyl sources in a single step, we optimized for a Ni-catalyzed cross-coupling capable of providing access to a range of thionoester, thioamide, or dithioester products. In summary, our work has revealed the potential of redox-active thiocarbonyl auxiliaries in Ni-catalyzed cross-couplings with C(sp3)-O electrophiles. We anticipate that the continued investigation of aliphatic thiocarbonyl derivatives as radical precursors with alternative single-electron inputs will be an area of continued growth in the years to come.

A flexible methanol-to-methane thermochemical energy storage system (TCES) for gas turbine (GT) power production

This study introduces an innovative solution to address the challenges arising from the volatile natural gas market and the growing integration of renewable energy sources within the industrial sector. The research strives to confront this challenge by including renewable methanol (CH3OH) and converting it into methane (CH4), with an intermediate step involving synthesis gas (CO/H2) by using concentrating solar power. This approach provides a sustainable and adaptable solution to reduce dependence on natural gas. The process entails a methanol decomposition reaction at moderate temperatures (<350 °C). Subsequently, the synthesis gas is compressed to 40 bar, stored, and discharged through a methanation process that can be conducted at high temperatures (>500 °C). The resulting methane is used as fuel for gas turbines and can also serve as feedstock in the chemical industry. The simulations were conducted in ASPEN HYSYS and yielded overall system efficiencies exceeding 29% and roundtrip efficiencies of 44%. Through techno-economic optimisation of the reaction conditions, competitive levelized fuel costs (LCOF) of €172/MWh and future LCOE values of €145/MWh were achieved. These findings present an innovative strategy for integrating gas turbine cycles and additional conversion pathways for green methanol.

Enhanced electron transfer pathway of zero-valent iron particles immobilized on coconut shell derived carbon for prolonged Cr(VI) removal

Immobilizing zero-valent iron (ZVI) with modulated structure and good dispersibility is of great potential for the elimination of Cr(VI) in contaminated water or soil, but exploring how the support facilitate and prolong the removal of Cr(VI) by ZVI in terms of the electron transfer is inadequate. Coconut shell derived carbon could be promising for Cr(VI) removal due to the features of large specific surface area and porosity. Accordingly, ZVI particles are dispersed into low-cost and scalable coconut shell derived carbon (CSC) matrix uniformly in this study, with enhanced contact area and interaction of ZVI with CSC for effective elimination of Cr(VI). The optimal ZVI/CSC-0.65 demonstrates an equilibrium adsorption capacity of 307.8 mg/g and long-life Cr(VI) cleanup ability over 144 h. The interface reaction mechanism between ZVI/CSC and Cr contaminant have been systematically studied by various techniques including XRD, SEM/TEM and XPS, etc. ZVI’s aggregation and passivation in ZVI/CSC-0.65 have been greatly alleviated. Further analysis by Tafel polarization and DFT computation suggests ZVI/CSC-0.65 undergoes fast electron transfer, with a much higher adsorption energy (−2.90 eV) than that of pristine ZVI (−0.63 eV). The Bader charge analysis demonstrates that there is 4.40 |e| charge transferring the ZVI to the carbon, encouraging the Cr(VI) adsorption. A micro-battery based mechanism is proposed which provides an additional electron transfer pathway in ZVI/CSC for long-term Cr(VI) removal.

Prediction of Cancer Driver Genes through Integrated Analysis of Differentially Expressed Genes at the Individual Level

Introduction: It is expected that certain driver mutations may alter the gene expression of their associated or interacting partners, including cognate proteins. Methods: We introduced DEGdriver, a novel method that can discriminate between mutations in drivers and passengers by utilizing gene differential expression at the individual level. Results: After being tested on eleven TCGA cancer datasets, DEGdriver substantially outperformed cutting-edge approaches in distinguishing driver genes from passengers and exhibited robustness to varying parameters and protein-protein interaction networks. Conclusion: Through enrichment analysis, we prove that DEGdriver can identify functional modules or pathways in addition to novel driver genes.

Beyond Anatomy: Use of Sinus Propagation Mapping to Identify the Slow Pathway for Cryoablation in Pediatric Patients.

Slow pathway modification via cryoablation is a common treatment of atrioventricular nodal re-entrant tachycardia (AVNRT) in pediatric patients. Sinus propagation mapping (SPM) is a tool that has been used to augment identification of the AVNRT slow pathway. We hypothesize that the use of SPM will decrease the total number of ablations performed and decrease the number of ablations until the slow pathway is successfully modified without a significant increase in procedure time. We conducted a retrospective review of patients who underwent cryoablation for AVNRT from August 2016 through March 2021. We excluded patients >21 years of age, those who underwent radiofrequency ablation; those with prior AVNRT ablation, additional pathways, or arrhythmias; and those with congenital heart disease. Out of 122 patients identified by the IMPACT database query, 103 met the inclusion criteria. Fifty-two patients (50.5%) had SPM completed during their procedures. The median number of ablations needed until successful slow pathway modification was two ablations in patients who underwent SPM and four ablations in the non-SPM group (P = .03). There was no significant difference in the total number of ablations between groups. The median total procedural time was longer in the SPM group (152 vs. 125 min; P = .01). SPM can be utilized to further improve the successful treatment of AVNRT with cryotherapy by lowering the number of ablations needed until successful slow pathway modification. However, the technique requires some additional time to collect sufficient data points to create the sinus map.

Proteome Wide Association Studies of LRRK2 variants identify novel causal and druggable proteins for Parkinson’s disease

AbstractBackgroundLeucine‐rich repeat kinase 2 (LRRK2) common gene variants (tagged by rs76904798) are associated with Parkinson’s disease (PD) risk. In a previous study, we found that rs76904798 was associated with CSF GRN levels, but a comprehensive unbiased analysis of associated proteins remains incomplete due to prior focus on cis‐protein quantitative trait loci (pQTL). Using the largest aptamer‐based CSF proteomics study to date (7,006 aptamers (6,138 unique proteins) in 3,107 individuals), we performed a trans‐pQTL study to identify proteins associated with the LRRK2 variants and used additional mediation and pathway analyses to better understand downstream effects (Figure 1).MethodSNP selection using Conditional and Joint Association Analysis (GTCA‐COJO) and PLINK LD r2 scores identified 11 other independently associated SNPs. Fine mapping was performed between each significant aptamer and PD GWAS via proteome‐wide association studies (PWAS) and Mendelian randomization (MR). These aptamers were further analyzed based on PD risk association, brain cell‐type specificity, and gene pathway annotations.ResultOverall, 26 proteins passed FDR correction and eleven were implicated in PD prior like GRN and GPNMB. PWAS analyses using TWAS/FUSION also indicate 12 proteins were significant and positively associated with PD risk, such as novel proteins C1QTNF1 and SDCBP2, and half were validated by the PPMI data. MR analyses indicate that GRN, GPNMB, HLA‐DQA2, CD68, and LCT are also causal for PD risk (Table 1). Cell‐type enrichment analyses indicate microglia‐specific protein enrichment (Fold change: 7.35, P = 4.91×10−5). Enrichment analyses indicate enrichment for leukocyte activation (GO:0045321, P = 0.002) and microglial cell activation (GO:0001774, P = 0.003) pathways.ConclusionTrans‐pQTL can identify novel protein interactions in an unbiased manner. This study linked LRRK2 variants with both PD implicated (GRN, GPNMB) and novel (ITGB2, C1QTNF1) proteins that have support in our PWAS, MR, and PPMI differential protein level analyses. Microglia and lysosome enrichment support the previous observations regarding the importance of microglia and immune system function in the underlying pathobiology of PD (Figure 2). Our results suggest that the CSF concentrations of these proteins (such as GRN, GPNMB, C1QTNF1, and ITGB2) have potential as biomarkers for target engagement and disease progression modification in clinical trials that target LRRK2.

Abstract B083: Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in tumor types with a significant unmet clinical need, such as colorectal cancer (CRC). Activating mutations in the KRAS gene leading to amino acid exchange at position 12 from Glycine to Asparagine, G12D, occur in approximately 12.5% of CRC. The approval of mutant specific KRASG12C inhibitors MRTX849 and AMG510 by the FDA raised the question if KRAS targeted therapy approaches could provide benefit to cancer patients with other mutant alleles. Several KRASG12D inhibitors have recently been described and MRTX1133, a selective KRASG12D inhibitor from Mirati Therapeutics, has now entered clinical trial, recruiting patients with advanced solid tumors harboring a KRAS G12D mutation. Alongside the great hopes placed in KRAS targeting therapy, resistance is likely to occur, as it was observed in patients relapsing in response to KRASG12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against KRASG12D in the CRC setting. To this aim, the colorectal cancer cell line GP2d was continuously treated with MRTX1133 to generate resistance and individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s). These assays included analysis of gene expression changes, mutation calling, as well as qPCR and protein-based assays. Interestingly, we identified two individual secondary on-target mutations in the KRAS gene, which could affect binding of MRTX1133 to the pocket in KRAS. These findings were confirmed in an orthogonal assay, by screening for resistance in Ba/F3 cells transduced with the corresponding KRAS double mutant. Combination assays were then performed in parental and resistant cells to study whether additional vertical or alternative pathway inhibition could address the resistance. Collectively our pre-clinical study identified secondary KRAS mutations in the compound binding pocket as a mechanism of resistance to KRAS G12D inhibition by MRTX1133 in colorectal cancer cells. Citation Format: Simone Lieb, Sabine Jurado, Sergej Nowoshilow, Krzysztof M. Zak, Marco H. Hofmann. Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B083.

Abstract A020: Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting

Abstract Triple negative breast cancer (TNBC) is a heterogeneous molecular breast cancer (BC) subtype that still has a very poor prognosis. Genetic, molecular and clinical tumor heterogeneity represent major pathobiological factors involved in poor clinical outcome. The sequence specific transcriptional repressor and proto-oncogene B cell lymphoma protein 6 (BCL6) has emerged as a suitable therapeutic target as it plays broad and critical roles in many hematological and solid cancers. BCL6 exerts control over cellular proliferation and survival by binding to specific responsive elements located within the regulatory regions of target genes and recruiting of chromatin modifying co-repressor complexes such as silencing mediator for retinoid and thyroid receptors (SMRT), BCL6 corepressor (BCoR) and histone deacetylases (HDACs). Moreover, in hematological malignancies, BCL6 transcriptional activity is also associated to the recruitment of the chromatin remodeling factor, Enhancer of Zeste 2 (EZH2). The role of BCL6 in BC is still not well defined; however emerging studies report its involvement in tumorigenesis, disease progression and poor survival. We recently showed that BCL6 is expressed at significantly higher levels in TNBC mammospheres, 3D multicellular structures enriched in cancer stem cells (CSCs), than in their differentiated bulk cell counterparts. Additionally, we found that the functional role of BCL6 is crucial for mammosphere formation, as its loss of function decreased this process. Intriguingly, we uncovered a functional interplay occurring between BCL6 and EZH2, which triggers the BCL6/Notch stemness signaling axis by inhibiting Numb transcription in both SUM149 and SUM159 TNBC cell lines. We exploited the molecular mechanisms responsible for this specific interplay, taking advantage of two molecules, FX1, a small molecule inhibitor able to block BCL6 recruiting activity and EED226, a potent Polycomb Repressive Complex 2 (PRC2) inhibitor. In order to define the role of both BCL6, as epigenetic regulator, and NUMB, as inhibitor of NOTCH stemness pathway in TNBC, SUM149 and SUM159 cell lines were stably transduced with lentiviral shRNA to derive stable NUMB-silenced matched cells. The expression of Notch downstream target genes, such as HES1/2 and HEY1/2, was assessed by qRT PCR analysis in the engineered TNBC cell models and resulted only slightly increased following NUMB downmodulation. Upon FX1 or EED226 administration, a strongest effect was observed, indicating that NUMB is not the only player in regulating the Notch pathway. Further experiments are currently ongoing aiming at identify additional players and molecular pathway(s). Collectively, our preliminary results provide additional preclinical evidences on the role exerted by BCL6 as epigenetic regulator of TNBC and underlie its interplay with EZH2thus paving the way for a novel potential therapeutic strategy targeting BCL6/Notch signaling axis. Citation Format: Francesca De Santis, Francesca Putti, Giorgia Bravin, Carla Portulano, Federica Zonca, Lorenzo Castagnoli, Michele Magni, Filippo de Braud, Serenella M Pupa, Massimo Di Nicola. Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A020.

Poly(vinyl alcohol)-tragacanth gum/Titania bio-nanocomposite film for photodecomposition of malachite green

Concerned about the contamination as a consequence of untreated wastewater worldwide, nanomaterials have been utilized for purification. Nevertheless, their separation from water is difficult after the treatment. Herein, Titania nanomaterials were embedded in a poly(vinyl alcohol)-tragacanth gum (PVA-TG) matrix, due to easy separation. The polymer matrix was cross-linked by a green agent of citric acid, and the physicochemical properties of PVA-TG/Titania bio-nanocomposite (bio-NC) films were characterized, which displayed a high swelling ratio in the water because of the addition of TG. Further, Titania nanomaterials illustrated a cube-like shape in the PVA-TG/Titania bio-NC film confirming the complete dispersion. Afterward, the photodecomposition of malachite green (MG) over this bio-NC film was studied and optimized under ultraviolet (UV) irradiation. Scavenger agents determined that the electron was produced more than other radical agents. Ultimately, the photodegradation process of MG over bio-NC films was suggested by the hydroxyl addition, singlet oxygen, and UV irradiation transformation pathways.

Synchronous or stepwise Mechanism? a theoretical study on the Excited-State double proton transfer properties of shikonin and acetylshikosin

The excited state double proton transfer (ESDPT) mechanism of shikonin (Shk) and its derivative acetylshikosin (AcShk) were studied by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods. The potential energy curves scanned along the coordinates of proton transfer indicate a preference for the ESDPT reaction to occur step by step. The AcShk molecule possesses an additional reaction pathway in comparison to the Shk molecule. Furthermore, efforts have been made to compute the absorption and fluorescence peak, which exhibits favorable conformity with the experimental findings of the system investigated. The fluorescence spectra in cyclohexane and acetonitrile solvents indicate that the solvent polarity affects the location of the ESDPT fluorescence peak in both Shk and AcShk systems. The fluorescence spectra concentrated in the green light region (504 nm ∼ 550 nm) are obtained, which has the potential to promote human health through disinfection and boosting the immune system.

Structural and stratigraphic controls on reservoir architecture: A case study from the lower Oligocene Vicksburg Formation, Brooks County, Texas

A detailed structural and stratigraphic framework provides a precise understanding of the controls on the vertical and spatial distribution of reservoir units, as well as the identification of the most prospective hydrocarbon accumulation. In southern Brooks County, Texas, the Lower Oligocene Vicksburg Formation (LOVF), is being influenced by the Vicksburg Fault Zone (VFZ). This zone is characterized by listric-normal faults that have formed highly faulted rollover anticlines, which are valuable structural traps for hydrocarbon exploration. This study integrated 568 3D seismic lines, 39 well logs, and three velocity surveys to build a comprehensive conceptual model that explores how secondary synthetic (dipping east), antithetic (dipping west), and Texas Gulf Coast-perpendicular faults are influencing accumulation and spatial distribution of hydrocarbons within the LOVF (La Rucias Field). Results indicate that synthetic, antithetic, and coast-perpendicular faults affecting the V-102, V-17, and V-19 horizons provide conduits for hydrocarbon migration. Antithetic faults and coast-perpendicular faults within the rollover anticline are terminating beneath an overlying shale seal layer between the V-16 and V-17, influencing economic hydrocarbon accumulations through multiple reservoirs. While synthetic faults affect the overlying seal layer migrating hydrocarbon out of the V-102, V-17, and V-19, bidirectional faulting linking antithetic and coast-perpendicular faults provide additional pathways for hydrocarbon accumulation. Spatial distribution of hydrocarbons varies with the targeted horizon. V-102 reservoirs are situated on the western flank of the rollover anticline, whereas the V-17 and V-19 reservoirs are located on structural highs where overlying shale seal layer remains unaffected by the antithetic faults. V-17 and V-19 reservoirs are influenced by bidirectional faulting that terminates beneath the shale seal layer, enabling the accumulation within the rollover anticline. Petrophysical and lithological investigations of the seal layers within the V-102, V-17 and V-19 are highly recommended for better understanding of vertical compartmentalization of reservoir intervals and accumulation estimates within the La Rucias Field. Investigating the control of these fault systems enhances our understanding of subsurface fluid migration and accumulations (oil, gas, groundwater, and contaminants) in the expanded Vicksburg productivity trends. Furthermore, the insights gained from the examination of these fault systems not only contribute to our comprehension of subsurface fluid migrations within the Vicksburg trends but also serve as a valuable reference for similar geological systems and reservoirs worldwide, ultimately aiding in the development of more effective resource management.

Impact of photoinduced energy transfer and LSPR of Au nanoparticles prepared by pulsed laser ablation technique on nonlinear optical response of zinc tetraphenyl porphyrin

Nonlinear optical (NLO) response and optical limiting activity in zinctetraphenyporphyrine (ZnTPP)/Au nanocomposites are examined using z-scan experiment aiding pulsed laser that delivers 7 ns pulses at 532 nm wavelength. ZnTPP/Au nanocomposite is prepared by ablating Au target in ZnTPP solution using nanosecond laser pulses. Nanocomposite formation in ZnTPP with Au NPs is confirmed from the modification of absorption spectrum and quenching of fluorescent emission spectrum. When compared to the well-known pristine constituents, the nonlinear optical properties and optical limiting (OL) activity in ZnTPP/Au nanocomposites have been enhanced significantly, that accounts the photo-induced energy transference between ZnTPP and Au NPs along with the local field effect of Au NPs. The nanocomposite formation induces additional energy transfer pathways that are responsible for enhanced excited-state absorption. In addition, the augmentation will be supported by local field effects of the Au NPs, which significantly increase the interaction field strength on the surface of the NPs and in the surrounding medium. The excited state absorption is the major mechanism of NLA and optical limiting activity. The threshold value of the optical limiting of the compound is found to be superior to the values reported for many compounds under similar experimental conditions. We anticipate that the suggested method will be of great interest for the eventual implementation of optical limiting-based photonic devices suitable for integrated optics.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.

Deciphering Metabolomic Signatures Associated with Sickle Cell Disease Using Mouse Models

Introduction: Sickle cell disease (SCD) is debilitating and affects millions of people worldwide. Although SCD is caused by a single-point mutation of the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous, and there is a lack of well-characterized predictive biomarkers of disease progression. Metabolomics is a promising tool to interrogate disease pathophysiology and underlying molecular mechanisms. Although prior metabolomic studies have been performed on human and murine red blood cells (RBCs) in the context of SCD, no systematic investigation across commonly used mouse models of SCD has been published. In the present study, we examined the blood cell metabolome of 2 widely used SCD mouse models to study and compare the metabolomic changes in disease models. Methods: Blood samples were collected from 5 groups of mice (10-14 weeks old, n=6-7 per group): wild-type (WT) C57BL/6J mice; Townes AA, AS, and SS mice; and Berkeley SCD mice. Direct injection high-resolution mass spectrometry was used to generate untargeted metabolomics data. Three matrices (RBC, plasma, and whole blood) were isolated from each mouse group. The RBC fraction, containing peripheral blood mononuclear cells, was separated from plasma via centrifugation of whole blood. The current study focuses on the RBC dataset.Raw profile data were centroided, merged, and recalibrated using methods previously described (Fuhrer et al. Anal Chem 2011). Putative annotations were generated based on compounds contained in the Human Metabolome Database, KEGG, and ChEBI databases using accurate mass per charge (tolerance 0.001 m/z) and isotopic correlation patterns. Z-score normalization was applied across all samples for unsupervised clustering using principal component analysis (PCA) and to identify metabolites with significant changes between groups. Metabolites were mapped to central RBC metabolic pathways using MetaboAnalyst (Xia et al. Nat Protoc 2011) and manual curation. Results: Unsupervised PCA that used all detectable RBC metabolites resulted in clear separation of mouse groups by disease status (Figure 1), suggesting robust metabolomic changes in mouse models of SCD. Focusing on the top divergent metabolites across the mouse groups, several enriched pathways were identified, such as amino acid metabolism, pyrimidine and purine metabolism, and aminoacyl-tRNA biosynthesis, all potentially attributable to the buildup of RBC precursors. Metabolites characteristic of immature RBCs were further examined, with elevated hexokinase activity (hexose:hexose-6-phosphate ratio), ATP levels, and mitochondrial metabolites found in RBCs from SCD, in line with the significantly elevated reticulocyte content in SCD mice. Metabolites were curated from additional relevant RBC metabolic pathways; some showed significant differences among mouse groups, and others were significantly elevated in sickle mice (Townes SS and Berkeley SCD) compared with non-SCD controls (WT and Townes AA), including some glycolytic and pentose phosphate pathway (PPP) intermediates (Figure 2). The product-to-substrate ratios of metabolite levels from glycolytic and oxidative PPP (oxPPP) steps suggested glycolysis was favored and the later stage of the oxPPP was reduced in mouse models of SCD. There was also a higher metabolic turnover of glutathione, which is characteristic of SCD in humans, but we observed divergent metabolomic signatures between the disease models in our study (Figure 2). Conclusions: To our knowledge, this is the most comprehensive metabolomics study in mouse models of SCD. It provides a foundation to assess disease biology and response to treatment in future studies. Untargeted RBC metabolomics revealed metabolomic signatures that are consistent with human disease pathology in both murine SCD models, including reticulocytosis, hemolysis, and mitochondria retention in RBCs, and signatures that are unique to the murine models. Of the 2 main RBC metabolic pathways, glycolysis was favored over the oxPPP in SCD mice. This is the first step towards understanding the impact of SCD on RBC carbohydrate metabolism.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Stochastic nucleosome disassembly mediated by remodelers and histone fragmentation.

We construct and analyze monomeric and multimeric models of the stochastic disassembly of a single nucleosome. Our monomeric model predicts the time needed for a number of histone-DNA contacts to spontaneously break, leading to dissociation of a non-fragmented histone from DNA. The dissociation process can be facilitated by DNA binding proteins or processing molecular motors that compete with histones for histone-DNA contact sites. Eigenvalue analysis of the corresponding master equation allows us to evaluate histone detachment times under both spontaneous detachment and protein-facilitated processes. We find that competitive DNA binding of remodeling proteins can significantly reduce the typical detachment time but only if these remodelers have DNA-binding affinities comparable to those of histone-DNA contact sites. In the presence of processive motors, the histone detachment rate is shown to be proportional to the product of the histone single-bond dissociation constant and the speed of motor protein procession. Our simple intact-histone model is then extended to allow for multimeric nucleosome kinetics that reveal additional pathways of disassembly. In addition to a dependence of complete disassembly times on subunit-DNA contact energies, we show how histone subunit concentrations in bulk solutions can mediate the disassembly process by rescuing partially disassembled nucleosomes. Moreover, our kinetic model predicts that remodeler binding can also bias certain pathways of nucleosome disassembly, with higher remodeler binding rates favoring intact-histone detachment.

Simultaneous chelated heavy metals removal and sludge recovery through titanium coagulation: From waste to resource

Green methods for chelated heavy metals treatment and recovery are essential for coordinated development of resources and environment. Herein, a simple and competent method, titanium salt (TiCl4) coagulation was developed to remove and recycle chelated heavy metals. Our results revealed that this method proved to be effective for metals-citrate [Cu(II), Ni(II), Zn(II) and Cr(VI)], achieving removal efficiencies of 95 %, 92 %, 99 %, and 99 % within 30 min, surpassing direct alkaline precipitation and well-used Fe(III) coagulation. Whereafter, the copper-containing sludge was successfully transformed into copper-doped titanium dioxide (TiO2) photocatalysts by facile calcination. Through comprehensively investigating physicochemical properties by a suite of characterization techniques, we confirmed that doping of Cu induced bandgap narrowing, high specific surface area as well as the formation of oxygen vacancy. Accordingly, the recycling photocatalysts showed remarkable enhanced photocatalytic performance than the pristine TiO2, achieving improvement in the degradation efficiency of 82 %, 61 % and 67 % for carbamazepine(CBZ), bisphenol A (BPA) and methyl orange (MO). In addition, both radical (OH and O2−) and non-radical (1O2 and h+) pathways synergistically contributed to the removal of organic pollutants during photocatalysis. Ultimately, based on economic feasibility assessment and life cycle assessment (LCA), the copper-containing titanium coagulation sludge reuse for photocatalyst could bring lower carbon emissions, reduced environmental risks and higher economic benefits. The elucidation of this study provides new insights into the removal and recycle of chelated heavy metals from wastewater by using an environment-friendly and cost-effective method.

Theoretical study of reaction of urea with methylamine: nucleophilic addition pathway

Theoretical study of reaction of urea with methylamine: nucleophilic addition pathway