Cystathionine-gamma-lyase overexpression in T cells enhances antitumor effect independently of cysteine autonomy.

T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long-lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine-gamma-lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8+ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH-expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.

Suspected heparin-induced thrombocytopenia in a COVID-19 patient on extracorporeal membrane oxygenation support: a case report.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) support can be life-saving in critically ill COVID-19 patients. However, there are many complications associated with this procedure, including Heparin-induced thrombocytopenia (HIT.) Despite its rarity in ECMO cases, HIT can lead to devastating consequences and is difficult to manage. CASE PRESENTATION: In this report, we present a case of a COVID-19 patient on ECMO support who was diagnosed with HIT and required intensive treatment. Initially, HIT was only suspected due to newly-developed thrombocytopenia and oxygenator dysfunction, with thrombi observed later. Regarding his treatment, since there was no recommended replacement to heparin available to us at the time of diagnosis, we decided to use rivaroxaban temporarily. No adverse events were recorded during that period. The patient was able to make a full recovery. CONCLUSION: HIT may jeopardize patient's care during ECMO. As COVID-19 may bring about a surge in the number of patients requiring ECMO support, we need consented guidance to optimize treatment in this specific situation.

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells.

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

Cyclotides derived from Viola dalatensis Gagnep: A novel approach for enrichment and evaluation of antimicrobial activity.

Cyclotides, plant-derived cysteine-rich peptides, exhibit a wide range of beneficial biological activities and possess exceptional structural stability. Cyclotides are commonly distributed throughout the Violaceae family. Viola dalatensis Gagnep, a Vietnamese species, has not been well studied, especially for cyclotides. This pioneering research explores cyclotides from V. dalatensis as antimicrobials. This study used a novel approach to enhance cyclotides after extraction. The approach combined 30% ammonium sulfate salt precipitation and RP-HPLC. A comprehensive analysis was performed to ascertain the overall protein content, flavonoids content, polyphenol content, and free radical scavenging capacity of compounds derived from V. dalatensis. Six known cyclotides were sequenced utilizing MS tandem. Semi-purified cyclotide mixtures (M1, M2, and M3) exhibited antibacterial efficacy against Bacillus subtilis (inhibitory diameters: 19.67-23.50 mm), Pseudomonas aeruginosa (22.17-23.50 mm), and Aspergillus flavus (14.67-21.33 mm). The enriched cyclotide precipitate from the stem extract demonstrated a minimum inhibitory concentration (MIC) of 0.08 mg/mL against P. aeruginosa, showcasing significant antibacterial effectiveness compared to the stem extract (MIC: 12.50 mg/mL). Considerable advancements have been achieved in the realm of cyclotides, specifically in their application as antimicrobial agents.

Machine Learning-Based Quantitative Structure-Property Relationships for the Electronic Properties of Cyano Polycyclic Aromatic Hydrocarbons.

In this study, quantitative structure-property relationships (QSPR) based on a machine learning (ML) methodology and the truncated degree of π-orbital overlap (DPO) to predict the electronic properties, namely, the bandgaps, electron affinities, and ionization potentials of the cyano polycyclic aromatic hydrocarbon (CN-PAH) chemical class were developed. The level of theory B3LYP/6-31+G(d) of density functional theory (DFT) was used to calculate a total of 926 data points for the development of the QSPR model. To include the substituents effects, a new descriptor was added to the DPO model. Consequently, the new ML-DPO model yields excellent linear correlations to predict the desired electronic properties with high accuracy to within 0.2 eV for all multi-CN-substituted PAHs and 0.1 eV for the mono-CN-substituted PAH subclass.

Atom-Based Machine Learning Model for Quantitative Property-Structure Relationship of Electronic Properties of Fusenes and Substituted Fusenes.

This study presents the development of machine-learning-based quantitative structure-property relationship (QSPR) models for predicting electron affinity, ionization potential, and band gap of fusenes from different chemical classes. Three variants of the atom-based Weisfeiler-Lehman (WL) graph kernel method and the machine learning model Gaussian process regressor (GPR) were used. The data pool comprises polycyclic aromatic hydrocarbons (PAHs), thienoacenes, cyano-substituted PAHs, and nitro-substituted PAHs computed with density functional theory (DFT) at the B3LYP-D3/6-31+G(d) level of theory. The results demonstrate that the GPR/WL kernel methods can accurately predict the electronic properties of PAHs and their derivatives with root-mean-square deviations of 0.15 eV. Additionally, we also demonstrate the effectiveness of the active learning protocol for the GPR/WL kernel methods pipeline, particularly for data sets with greater diversity. The interpretation of the model for contributions of individual atoms to the predicted electronic properties provides reasons for the success of our previous degree of π-orbital overlap model.

Gagones A-F: Six prenylated chalcones from the heartwood of Mansonia gagei.

Six undescribed prenylated chalcones gagones A-F were isolated from the acetone fraction of Mansonia gagei heartwood. Their structures were unambiguously established based on spectroscopic analysis (HRESIMS, 1D and 2D NMR), as well as comparison to literature data. Their absolute configurations were elucidated using DP4 and electronic circular dichroism calculations. Isolated compounds were evaluated for their inhibitory activity against α-glucosidase and DPPH assay. All of the tested compounds exhibited better activity than that of acarbose (IC50 93.6 ± 0.5 µM). Among them, gagone D exhibited the highest α-glucosidase inhibitory with the IC50 value of 3.6 ± 0.4 µM. For antioxidant activity, gagones A-C, and E showed more active than that of ascorbic acid (IC50 30.2 ± 0.5 µM) with the IC50 values of 13.2 ± 0.7, 20.1 ± 0.4, 19.3 ± 0.5 and 12.8 ± 0.2 µM, respectively.

Mechanisms underlying reshuffling of visual responses by optogenetic stimulation in mice and monkeys.

The ability to optogenetically perturb neural circuits opens an unprecedented window into mechanisms governing circuit function. We analyzed and theoretically modeled neuronal responses to visual and optogenetic inputs in mouse and monkey V1. In both species, optogenetic stimulation of excitatory neurons strongly modulated the activity of single neurons yet had weak or no effects on the distribution of firing rates across the population. Thus, the optogenetic inputs reshuffled firing rates across the network. Key statistics of mouse and monkey responses lay on a continuum, with mice/monkeys occupying the low-/high-rate regions, respectively. We show that neuronal reshuffling emerges generically in randomly connected excitatory/inhibitory networks, provided the coupling strength (combination of recurrent coupling and external input) is sufficient that powerful inhibitory feedback cancels the mean optogenetic input. A more realistic model, distinguishing tuned visual vs. untuned optogenetic input in a structured network, reduces the coupling strength needed to explain reshuffling.

Countering the future chemical weapons threat.

Shift focus from disarmament to preventing reemergence.

Application of Machine Learning in Developing Quantitative Structure-Property Relationship for Electronic Properties of Polyaromatic Compounds.

The degree of π orbital overlap (DPO) model has been demonstrated to be an excellent quantitative structure-property relationship (QSPR) that can map two-dimensional structural information of polycyclic aromatic hydrocarbons (PAHs) and thienoacenes to their electronic properties, namely, band gaps, electron affinities, and ionization potentials. However, the model suffers from significant limitations that narrow its applications due to inefficient manual procedures in parameter optimization and descriptor formulation. In this work, we developed a machine learning (ML)-based method for efficiently optimizing DPO parameters and proposed a truncated DPO descriptor, which is simple enough that can be automatically extracted from simplified molecular-input line-entry system strings of PAHs and thienoacenes. Compared with the result from our previous studies, the ML-based methodology can optimize DPO parameters with four times fewer data, while it can achieve the same level of accuracy in predictions of the mentioned electronic properties to within 0.1 eV. The truncated DPO model also has similar accuracy to the full DPO model. Consequently, the ML-based DPO approach coupled with the truncated DPO model enables new possibilities for developing automatic pipelines for high-throughput screening and investigating new QSPR for new chemical classes.

The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

Rotational thromboelastometry parameters as predicting factors for bleeding in immune thrombocytopenic purpura.

OBJECTIVE/BACKGROUND: Patients with immune thrombocytopenic purpura (ITP) often present with a severe reduction in platelet counts and suffer from an increased risk of bleeding. However, platelet counts do not accurately predict bleeding risk in these patients. METHODS: We thereby conducted a case series prospective study to compare the ability to predict hemorrhage in ITP patients between platelet counts and various rotational thromboelastometry (ROTEM) parameters. RESULTS: The inclusion criteria for patients diagnosed with acute, persistent, and chronic ITP were platelet counts of <30 × 109/L and no clinically significant bleeding (grade ≥ 2 according to the WHO Bleeding Scale) at the beginning of the study. After 24 hours of follow-up, of the 45 enrolled patients, 14 (31.1%) experienced clinically significant bleeding. The mean platelet counts of patients with and without clinically significant bleeding were not statistically different (p = .09). However, the mean EXTEM maximum clot firmness (MCF), EXTEM A10, EXTEM area under the curve (AUC), and platelet maximum clot elasticity (MCE) values of the two groups were statistically different (p < .05). There was also a significant difference in IPF values between these two groups (p < .05.) CONCLUSION: Results obtained from this preliminary study demonstrate that ROTEM parameters might be useful in predicting factors for hemorrhage in ITP patients. Future studies with a larger sample size is warranted to confirm our findings, which will allow prompt and effective bleeding management in ITP patients.

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach.

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing.

Maged1, a new regulator of skeletal myogenic differentiation and muscle regeneration.

BACKGROUND: In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers. RESULTS: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle. CONCLUSIONS: These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.

Survival and health status of DOTS tuberculosis patients in rural Lao PDR.

BACKGROUND: Contact tracing of tuberculosis (TB) patients is rarely performed in low-income countries. Our objective was to assess the outcome of and compliance with directly observed treatment (DOTS) of TB patients over a 3 year period in rural Lao PDR. METHODS: We performed a retrospective cohort study in which we enrolled TB patients who started DOTS treatment at Attapeu Provincial Hospital. We traced, through hospital records, all patients in their residential village. We conducted a standardized questionnaire with all TB patients and performed physical and anthropometric examinations as well as evaluations of compliance through counting of treatment pills at home and at the health facilities. RESULTS: Of 172 enrolled TB patients (sex ratio female/male: 0.52, mean age: 46.9 years ± 16.9), 26 (15.1%) died. These had a lower weight at the start (34.6 vs. 40.8 kg, p < 0.001) and were less compliant (91.6% vs. 19.2%, p < 0.001) than survivors. Low compliance was associated with poor accessibility to health care (p = 0.01) and symptomatic improvement (p = 0.02). Survivors had persistently poor health status. They were underweight (54.7%), and still had clinical symptoms (53.5%), including dyspnoea (28.8%) and haemoptysis (9.5%). CONCLUSION: Our study suggests a lower rate of survival than expected from official statistics. Additionally, it showed that follow-up of TB patients is feasible although the patients lived in very remote area of Laos. Follow-up should be strengthened as it can improve patient compliance, and allow contact tracing, detection of new cases and collection of accurate treatment outcome information.

Biosafety in ex vivo gene therapy and conditional ablation of lentivirally transduced hepatocytes in nonhuman primates.

Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand.

Risk of latent tuberculosis infection in children living in households with tuberculosis patients: a cross sectional survey in remote northern Lao People's Democratic Republic.

BACKGROUND: Tuberculosis is highly prevalent in Laos (289 per 100,000). We evaluated the risk of latent tuberculosis infection (LTBI) among children (0-15 years) living with tuberculosis patients in rural northern Laos. METHODS: In a cross sectional survey of 30 randomly selected villages, 72 tuberculosis patients were traced and their 317 contacts (148 were children) investigated using a questionnaire, a tuberculin skin tests (positive: > = 10 mm), a 3-day sputum examination for acid-fast bacilli (AFB), and chest radiography. RESULTS: None of the 148 contact-children received prophylaxis, one had cervical tuberculosis; the risk for LTBI was 31.0%. Awareness of the infectiousness of tuberculosis was low among patients (31%) and their contacts (31%), and risky behavior was common. After multivariate logistic analysis, increased LTBI was found in children with contact with sputum positive adults (OR: 3.3, 95% CI: 1.4-7.7), patients highly positive sputum prior to treatment (AFB >2+; OR: 4.7, 95% CI: 1.7-12.3), and living in ethnic minorities (OR: 5.4, 95% CI: 2.2-13.6). CONCLUSION: The study supports the importance of contact tracing in remote settings with high TB prevalence. Suggestions to improve the children's detection rate, the use of existing guidelines, chemoprophylaxis of contact-children and the available interventions in Laos are discussed. Improving education and awareness of the infectiousness of TB in patients is urgently needed to reduce TB transmission.