Sucrose Treatment Enhances the Electrotransfer of DNA by Activating Phospholipase A2.

Our previous study discovered that sucrose and other non-reducing sugars (e.g., trehalose and raffinose) could be used to improve the electrotransfer (ET) of molecular cargo, including DNA, mRNA, and ribonucleoprotein in various cell lines and primary human cells in vitro and in vivo. To understand the molecular mechanisms of this improvement, we used RNA sequencing technology to analyze changes in the cell transcriptome after sucrose treatment. The results from our analysis demonstrated that the sucrose treatment upregulated phospholipase A2 and V-ATPase gene families, which could potentially influence the acidity of intracellular vesicles through augmenting vesicle fusion and the influx of proton, respectively. To determine how this upregulation affects ET efficiency, we treated cells with pharmaceutical inhibitors of phospholipase A2 and V-ATPase. The data demonstrated that the treatment with the phospholipase A2 inhibitor could reverse the ET improvement elicited by the sucrose treatment. The V-ATPase inhibitor treatment either had little influence or further enhanced the effect of the sucrose treatment on the ET efficiency. These observations provide a molecular explanation for our previous findings, demonstrating that the sucrose treatment primarily enhanced the ET efficiency by promoting vesicle trafficking and fusion through the activation of phospholipase A2.

Crystal-Phase-Engineered High-Entropy Alloy Aerogels for Enhanced Ethylamine Electrosynthesis from Acetonitrile.

Crystal-phase engineering that promotes the rearrangement of active atoms to form new structural frameworks achieves excellent result in the field of electrocatalysis and optimizes the performance of various electrochemical reactions. Herein, for the first time, it is found that the different components in metallic aerogels will affect the crystal-phase transformation, especially in high-entropy alloy aerogels (HEAAs), whose crystal-phase transformation during annealing is more difficult than medium-entropy alloy aerogels (MEAAs), but they still show better electrochemical performance. Specifically, PdPtCuCoNi HEAAs with the parent phase of face-centered cubic (FCC) PdCu possess excellent 89.24% of selectivity, 746.82 mmol h-1 g-1 cat. of yield rate, and 90.75% of Faraday efficiency for ethylamine during acetonitrile reduction reaction (ARR); while, maintaining stability under 50 h of long-term testing and ten consecutive electrolysis cycles. The structure-activity relationship indicates that crystal-phase regulation from amorphous state to FCC phase promotes the atomic rearrangement in HEAAs, thereby optimizing the electronic structure and enhancing the adsorption strength of reaction intermediates, improving the catalytic performance. This study provides a new paradigm for developing novel ARR electrocatalysts and also expands the potential of crystal-phase engineering in other application areas.

Light-responsive MXenegel via interfacial host-guest supramolecular bridging.

Living in the global-changing era, intelligent and eco-friendly electronic components that can sense the environment and recycle or reprogram when needed are essential for sustainable development. Compared with solid-state electronics, composite hydrogels with multi-functionalities are promising candidates. By bridging the self-assembly of azobenzene-containing supramolecular complexes and MXene nanosheets, we fabricate a MXene-based composite gel, namely MXenegel, with reversible photo-modulated phase behavior. The MXenegel can undergo reversible liquefication and solidification under UV and visible light irradiations, respectively, while maintaining its conductive nature unchanged, which can be integrated into traditional solid-state circuits. The strategy presented in this work provides an example of light-responsive conducting material via supramolecular bridging and demonstrates an exciting platform for functional soft electronics.

MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

Antiphospholipid antibodies are enriched post-acute COVID-19 but do not modulate the thrombotic risk.

BACKGROUND AND OBJECTIVES: COVID-19-associated coagulopathy, shown to increase the risk for the occurrence of thromboses and microthromboses, displays phenotypic features of the antiphospholipid syndrome (APS), a prototype antibody-mediated autoimmune disease. Several groups have reported elevated levels of criteria and non-criteria antiphospholipid antibodies (aPL), assumed to cause APS, during acute or post-acute COVID-19. However, disease heterogeneity of COVID-19 is accompanied by heterogeneity in molecular signatures, including aberrant cytokine profiles and an increased occurrence of autoantibodies. Moreover, little is known about the association between autoantibodies and the clinical events. Here, we first aim to characterise the antiphospholipid antibody, anti-SARS-CoV-2 antibody, and the cytokine profiles in a diverse collective of COVID-19 patients (disease severity: asymptomatic to intensive care), using vaccinated individuals and influenza patients as comparisons. We then aim to assess whether the presence of aPL in COVID-19 is associated with an increased incidence of thrombotic events in COVID-19. METHODS AND RESULTS: We conducted anti-SARS-CoV-2 IgG and IgA microELISA and IgG, IgA, and IgM antiphospholipid line immunoassay (LIA) against 10 criteria and non-criteria antigens in 155 plasma samples of 124 individuals, and we measured 16 cytokines and chemokines in 112 plasma samples. We additionally employed clinical and demographic parameters to conduct multivariable regression analyses within multiple paradigms. In line with recent results, we find that IgM autoantibodies against annexin V (AnV), ß2-glycoprotein I (ß2GPI), and prothrombin (PT) are enriched upon infection with SARS-CoV-2. There was no evidence for seroconversion from IgM to IgG or IgA. PT, ß2GPI, and AnV IgM as well as cardiolipin (CL) IgG antiphospholipid levels were significantly elevated in the COVID-19 but not in the influenza or control groups. They were associated predominantly with the strength of the anti-SARS-CoV-2 antibody titres and the major correlate for thromboses was SARS-CoV-2 disease severity. CONCLUSION: While we have recapitulated previous findings, we conclude that the presence of the aPL, most notably PT, ß2GPI, AnV IgM, and CL IgG in COVID-19 are not associated with a higher incidence of thrombotic events.

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection.

IMPORTANCE: The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

Boosting Ion Conductivities: Light-Modulated Azobenzene-Based Ionic Liquids in Vertical Nanochannels.

Exploring stimuli-responsive ion-conductive materials is a challenging task, but it has gained increasing attention because of their enormous potential applications in actuators, sensors, and smart electronics. Here, we demonstrate a distinctive photoresponsive ion-conductive device that utilizes azobenzene-based ionic liquids ([AzoCnMIM][Br], where n = 2, 6, and 10), confined in nanochannels of anodic aluminum oxide (AAO) templates for photoisomerization. The structure of [AzoCnMIM][Br] comprises photoresponsive and hydrophobic azobenzene moieties, hydrophilic imidazolium cations, and negatively charged bromide ions. Therefore, [AzoCnMIM][Br] can form micelles and exhibit photoresponsive ion conductivities. The nanochannels of AAO templates exhibit a confinement effect on the formation of azobenzene-based ionic liquid micelles due to the pore size, thereby preventing the formation of larger micelles that could lead to a decrease in conductivity. Consequently, the ion conductivities of the azobenzene-based ionic liquids are higher in the nanochannels of the AAO templates. The effects of the length of carbon chains on the azobezene-based ionic liquids and the pore size of the AAO templates have also been investigated. Additionally, through irradiation with UV/vis light, [AzoCnMIM][Br] can undergo reversible isomerization, thereby reversibly changing the sizes of the micelles and subsequently altering the ion conductivities.

Copy number of naked DNA delivered into nucleus of mammalian cells by electrotransfection.

Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Hyperinflammatory environment drives dysfunctional myeloid cell effector response to bacterial challenge in COVID-19.

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients.

Blunted sFasL signalling exacerbates TNF-driven neutrophil necroptosis in critically ill COVID-19 patients.

OBJECTIVES: Critically ill coronavirus disease 2019 (COVID-19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. METHODS: Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients. RESULTS: COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage-associated molecular patterns (DAMPs), increased receptor-interacting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. CONCLUSION: Our results suggest a novel role for sFasL signalling in the TNF-α-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

Exploring the factors related to adolescent health literacy, health-promoting lifestyle profile, and health status.

BACKGROUND: Health literacy has been concerned a key factor for determining the use of health information and promoting health. The study aimed to explore adolescent health literacy, health-promoting lifestyle profile, and health status and related factors. METHODS: A cross-sectional study design was used; 918 first year junior college students were recruited in Taiwan. The measurements were the Chinese Health Literacy Survey Questionnaire (HLS-C-Q), the Chinese Health-Promoting Lifestyle Profile (HPLP-S), and the Health Status Questionnaire. RESULTS: The mean score for health literacy was 36.15 (±6.21), with 30.17% of the participants having insufficient or problematic health literacy. Further, 19.9% of participants were obese and 11.2% experienced emotional instability. Health literacy and health-promoting lifestyle profile showed significant positive and negative correlations with perceived health status and depression, respectively (p < 0.05). An exercise frequency of ≥3 times/week was a predictor of health literacy, health-promoting lifestyle profile, and emotional stability. CONCLUSIONS: Adolescent health literacy, health-promoting lifestyle profile, and health status require careful consideration. In adolescents, developing regular exercise may increase health literacy, thereby developing healthy lifestyle profiles and ameliorating obesity and depression-related issues.

Lactobacillus rhamnosus JB3 inhibits Helicobacter pylori infection through multiple molecular actions.

BACKGROUND: Eradication of Helicobacter pylori infection is the most direct and effective way for preventing gastric cancer. Lactic acid bacteria are considered as alternative therapeutic agents against H. pylori infection. METHODS: Effects of Lactobacillus rhamnosus JB3 (LR-JB3) on the virulence gene expression of H. pylori and infection-induced cellular responses of AGS cells were investigated by co-cultivating infected AGS cells with different multiplicity of infections (MOIs) of LR-JB3. RESULTS: LR-JB3, specifically at a MOI of 25, suppressed the association ability of H. pylori and its induced IL-8 levels, as well as the mRNA levels of vacA, sabA, and fucT of H. pylori, infection-induced Lewis (Le)x antigen and Toll-like receptor 4 (TLR4) expressions in AGS cells. However, the apoptosis mediated by infection was inhibited by LR-JB3 in a dose-dependent manner. In addition, autoinducer (AI)-2 was observed to have increased the association ability and fucT expression of H. pylori, and Lex antigen and TLR4 expression of AGS cells. Interestingly, an unknown bioactive cue was hypothesized to have been secreted from LR-JB3 at a MOI of 25 to act as an antagonist of AI-2. CONCLUSIONS: LR-JB3 possesses various means to interfere with H. pylori pathogenesis and infection-induced cellular responses of AGS cells to fight against infection.

Novel Virulence Role of Pneumococcal NanA in Host Inflammation and Cell Death Through the Activation of Inflammasome and the Caspase Pathway.

Streptococcus pneumoniae is one of most deadly Gram-positive bacterium that causes significant mortality and morbidity worldwide. Intense inflammation and cytotoxicity is a hallmark of invasive pneumococcal disease. Pneumococcal NanA has been shown to exaggerate the production of inflammatory cytokines via unmasking of inhibitory Siglec-5 from its sialyl cis-ligands. To further investigate the mechanistic role of NanA and Siglec-5 in pneumococccal diseases, we systemically analyzed genes and signaling pathways differentially regulated in macrophages infected with wild type and NanA-deficient pneumococcus. We found that NanA-mediated desialylation impairs the Siglec-5-TLR-2 interaction and reduces the recruitment of phosphatase SHP-1 to Siglec-5. This dysregulated crosstalk between TLR-2 and inhibitory Siglec-5 exaggerated multiple inflammatory and death signaling pathways and consequently caused excessive inflammation and cytotoxicity in the infected macrophage. Collectively, our results reveal a novel virulence role of NanA in pneumococcal pathogenesis and suggest that targeting NanA activity may ameliorate the pneumococcus-mediated inflammation and cytotoxicity in severe invasive pneumococcal diseases.

A statistical framework for determination of minimal plasmid copy number required for transgene expression in mammalian cells.

Plasmid DNA (pDNA) has been widely used for non-viral gene delivery. After pDNA molecules enter a mammalian cell, they may be trapped in subcellular structures or degraded by nucleases. Only a fraction of them can function as templates for transcription in the nucleus. Thus, an important question is, what is the minimal amount of pDNA molecules that need to be delivered into a cell for transgene expression? At present, it is technically a challenge to experimentally answer the question. To this end, we developed a statistical framework to establish the relationship between two experimentally quantifiable factors - average copy number of pDNA per cell among a group of cells after transfection and percent of the cells with transgene expression. The framework was applied to the analysis of electrotransfection under different experimental conditions in vitro. We experimentally varied the average copy number per cell and the electrotransfection efficiency through changes in extracellular pDNA dose, electric field strength, and pulse number. The experimental data could be explained or predicted quantitatively by the statistical framework. Based on the data and the framework, we could predict that the minimal number of pDNA molecules in the nucleus for transgene expression was on the order of 10. Although the prediction was dependent on the cell and experimental conditions used in the study, the framework may be generally applied to analysis of non-viral gene delivery.

Enhancing Cell Viability and Efficiency of Plasmid DNA Electrotransfer Through Reducing Plasma Membrane Permeabilization.

BACKGROUND: Pulsed electric field has been widely used to facilitate molecular cargo transfer into cells. However, the cell viability is often decreased when trying to increase the electrotransfer efficiency. We hypothesize that the decrease is due to electropermeabilization of cell membrane that disrupts homeostasis of intracellular microenvironment. Thus, a reduction in the membrane permeabilization may increase the cell viability. MATERIALS AND METHODS: Different compounds were supplemented into the pulsing buffer prior to electrotransfer for reduction of cell membrane damage. Extent of the damage was quantified by leakiness of the membrane to a fluorescent dye, calcein, preloaded into cells. At 24 hours post electrotransfer, cell viability and electrotransfer efficiency were quantified with flow cytometry. RESULTS: The cell viability could be substantially increased by supplementation of either type B gelatin or bovine serum albumin (BSA), without compromising the electrotransfer efficiency. The supplementation also decreased the amount of calcein leaking out of the cells, suggesting that the improvement in cell viability was due to the reduction in electrotransfer-induced membrane damage. CONCLUSION: Data from the study demonstrate that type B gelatin and BSA can be used as inexpensive supplements for improving cell viability in electrotransfer.

Redirecting Vesicular Transport to Improve Nonviral Delivery of Molecular Cargo.

Cell engineering relies heavily on viral vectors for the delivery of molecular cargo into cells due to their superior efficiency compared to nonviral ones. However, viruses are immunogenic and expensive to manufacture, and have limited delivery capacity. Nonviral delivery approaches avoid these limitations but are currently inefficient for clinical applications. This work demonstrates that the efficiency of nonviral delivery of plasmid DNA, mRNA, Sleeping Beauty transposon, and ribonucleoprotein can be significantly enhanced through pretreatment of cells with the nondegradable sugars (NDS), such as sucrose, trehalose, and raffinose. The enhancement is mediated by the incorporation of the NDS into cell membranes, causing enlargement of lysosomes and formation of large (>500 nm) amphisome-like bodies (ALBs). The changes in subcellular structures redirect transport of cargo to ALBs rather than to lysosomes, reducing cargo degradation in cells. The data indicate that pretreatment of cells with NDS is a promising approach to improve nonviral cargo delivery in biomedical applications.

Inhibition of Caspases Improves Non-Viral T Cell Receptor Editing.

T cell receptor (TCR) knockout is a critical step in producing universal chimeric antigen receptor T cells for cancer immunotherapy. A promising approach to achieving the knockout is to deliver the CRISPR/Cas9 system into cells using electrotransfer technology. However, clinical applications of the technology are currently limited by the low cell viability. In this study, we attempt to solve the problem by screening small molecule drugs with an immortalized human T cell line, Jurkat clone E6-1, for inhibition of apoptosis. The study identifies a few caspase inhibitors that could be used to simultaneously enhance the cell viability and the efficiency of plasmid DNA electrotransfer. Additionally, we show that the enhancement could be achieved through knockdown of caspase 3 expression in siRNA treated cells, suggesting that the cell death in electrotransfer experiments was caused mainly by caspase 3-dependent apoptosis. Finally, we investigated if the caspase inhibitors could improve TCR gene-editing with electrotransferred ribonucleoprotein, a complex of Cas9 protein and a T cell receptor-α constant (TRAC)-targeting single guide RNA (sgRNA). Our data showed that inhibition of caspases post electrotransfer could significantly increase cell viability without compromising the TCR disruption efficiency. These new findings can be used to improve non-viral T cell engineering.

Establishment of Guidelines for the Control of Glycosylation Reactions and Intermediates by Quantitative Assessment of Reactivity.

Stereocontrolled chemical glycosylation remains a major challenge despite vast efforts reported over many decades and so far still mainly relies on trial and error. Now it is shown that the relative reactivity value (RRV) of thioglycosides is an indicator for revealing stereoselectivities according to four types of acceptors. Mechanistic studies show that the reaction is dominated by two distinct intermediates: glycosyl triflates and glycosyl halides from N-halosuccinimide (NXS)/TfOH. The formation of glycosyl halide is highly correlated with the production of α-glycoside. These findings enable glycosylation reactions to be foreseen by using RRVs as an α/ß-selectivity indicator and guidelines and rules to be developed for stereocontrolled glycosylation.

Detection of Molecular Alterations in Taiwanese Patients with Medullary Thyroid Cancer Using Whole-Exome Sequencing.

Genetic and epigenetic alterations are associated with the progression and prognosis of medullary thyroid carcinoma (MTC). We performed whole-exome sequencing of tumor tissue from seven patients with sporadic MTC using an Illumina HiSeq 2000 sequencing system. We conducted Sanger sequencing to confirm the somatic mutations in both tumor and matched normal tissues. We applied Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis with the Database for Annotation, Visualization, and Integrated Discovery and STRING for pathway analysis. We detected new somatic mutations in the BICD2, DLG1, FSD2, IL17RD, KLHL25, PAPPA2, PRDM2, PSEN1, SCRN1, and TTC1 genes. We found a somatic mutation in the PDE4DIP gene that had previously been discovered mutated in other tumors but that had not been characterized in MTC. We investigated pathway deregulation in MTC. Data regarding 1152 MTCs were assembled from the Catalogue of Somatic Mutations in Cancer (COSMIC) and seven of our patients. Ontological analysis revealed that most of the variants aggregated in pathways that included the signaling pathways of thyroid cancer, central carbon metabolism, microRNAs in cancer, PI3K-Akt, ErbB, MAPK, mTOR, VEGF, and RAS. In conclusion, we conducted wide-ranging exome-wide analysis of the mutational spectrum of MTC in Taiwan's population and detected novel genes with potential associations with MTC tumorigenesis and irregularities in pathways that resulted in MTC pathogenesis.