Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

Can Inhaled Nitric Oxide Response Predict Tolerance to Therapies and Survival in Patients With Combined Precapillary and Postcapillary Pulmonary Hypertension?

Inhaled nitric oxide (iNO) relaxes the pulmonary circulation and variably increases the left ventricular preload and pulmonary artery wedge pressure (PAWP)-hemodynamic information that may help guide treatment decisions and assess prognosis in patients with combined precapillary and postcapillary pulmonary hypertension (PH). We included consecutive patients with combined precapillary and postcapillary PH (mean pulmonary artery pressure >20 mm Hg, PAWP >15 mm Hg, and pulmonary vascular resistance [PVR] >2 Woods unit [WU]) who underwent right-sided cardiac catheterization with iNO at the Cleveland Clinic Pulmonary Vascular Disease program between 2017 and 2022. We included 104 patients with baseline PAWP and PVR of 22.2 ± 4.2 mm Hg and 6.1 ± 3.2 WU, respectively. Pulmonary arterial hypertension (PAH) with postcapillary component and PH left heart disease with precapillary component were identified in 27 (26%) and 77 patients (74%), respectively. No side effects were noted during the administration of iNO. During iNO, the PVR decreased 1.1 ± 1.4 WU and the PAWP increased 1.3 ± 3.7 mm Hg. A more pronounced increase in PAWP with iNO was associated with a decrease in PVR (R -0.35, p <0.001) and increase in stroke volume (R 0.20, p = 0.046). Tolerance to PAH-specific medications, overall survival, and heart failure hospitalizations were not significantly associated with the change in PAWP or PVR with iNO. In conclusion, in patients with combined precapillary and postcapillary PH, iNO challenge is safe and caused a significant decrease in PVR, with an increase in PAWP. The changes in PAWP and PVR during iNO administration were not associated with tolerance to PAH-specific medications, heart failure-related hospitalization, or survival.

Impact on Pulmonary Hypertension Hemodynamic Classification Based on the Methodology Used to Measure Pulmonary Artery Wedge Pressure and Cardiac Output.

Rationale: Guidelines recommend using end-expiration pulmonary pressure measurements to determine the hemodynamic subgroups in pulmonary hypertension. Pulmonary artery wedge pressure (PAWP) determinations averaged across the respiratory cycle (PAWPav) instead of PAWP at end-expiration (PAWPee) and cardiac output (CO) measured by Fick (COFick) instead of thermodilution (COTD) may affect the hemodynamic classification of pulmonary hypertension. Objectives: To assess the impact on the pulmonary hypertension hemodynamic classification of the use of PAWPee versus PAWPav as well as COFick versus COTD. Methods: This was a single-center retrospective study of consecutive patients (n = 151) who underwent right heart catheterization with COTD, COFick, PAWPee, and PAWPav. A secondary cohort consisted of consecutive patients (n = 71) who had mean pulmonary artery pressure at end-expiration (mPAPee) and mPAP averaged across the respiratory cycle (mPAPav) measured, as well as PAWPee and PAWPav. Results: The PAWPee and PAWPav were 16.8 ± 6.4 and 15.1 ± 6.8 mm Hg, respectively, with a mean difference of 1.7 ± 2.1 mm Hg. The COTD and COFick determinations were 5.0 ± 2.4 and 5.3 ± 2.5 L/min, respectively, with a mean difference of -0.4 ± 1.3 L/min. The hemodynamic group distribution was significantly different when using PAWPee versus PAWPav, when using either COTD or COFick (P < 0.001 for both comparisons), and these results were consistent in our secondary cohort. The pulmonary hypertension hemodynamic group distribution was not significantly different between COTD and COFick when using either PAWPee or PAWPav. Conclusions: The methodology used to measure PAWP, either at end-expiration or averaged across the respiratory cycle, significantly impacts the hemodynamic classification of pulmonary hypertension.

Does veno-arterial carbon dioxide gradient provide an adequate estimation of cardiac index in pulmonary hypertension?

AIMS: Pulmonary hypertension (PH) management is dependent on cardiac output (CO) assessment. The gold standard Fick method for CO and cardiac index (CI) measurement is not widely available. An accessible and reliable method for CO/CI estimation is needed not only in catheterization labs but also in other environments such as the intensive care unit, where pulmonary artery catheters are less likely to be used. We hypothesized that veno-arterial carbon dioxide gradient (PvaCO2) is a reliable surrogate for Fick CI in patients with PH. METHODS AND RESULTS: A single-centre retrospective analysis of patients with PH who underwent direct Fick CI (DFCI) measurement during right heart catheterization. The primary outcome was correlation between PvaCO2 and DFCI. To assess the agreement between central and mixed venous CO2 values, a separate prospective cohort of patients was analysed. Data from 186 patients with all haemodynamic types of PH were analysed. PvaCO2 moderately correlated with Fick CI, R = -0.51 [95% confidence interval (CI): -0.61, -0.39]. A higher PvaCO2 was associated with an increased risk of CI < 2.5 L/min/m2 (odds ratio: 1.88, 95% CI: 1.55, 2.35). Low thermodilution CI with normal veno-arterial carbon dioxide gradient values was associated with a thermodilution underestimation of Fick CI. In the prospective analysis of 32 patients, central venous CO2 overestimated mixed venous values (mean difference 3.3, 95% CI: 2.5, 4.0) and there was poor agreement overall (limits of agreement -1.10, 7.59). CONCLUSION: Veno-arterial carbon dioxide gradient moderately correlates with Fick CI and may be useful to identify patients with low CI. Central and mixed venous CO2 values should not be used interchangeably in PH.

The lifetime of the oxygen-evolving complex subunit PSBO depends on light intensity and carbon availability in Chlamydomonas.

PSBO is essential for the assembly of the oxygen-evolving complex in plants and green algae. Despite its importance, we lack essential information on its lifetime and how it depends on the environmental conditions. We have generated nitrate-inducible PSBO amiRNA lines in the green alga Chlamydomonas reinhardtii. Transgenic strains grew normally under non-inducing conditions, and their photosynthetic performance was comparable to the control strain. Upon induction of the PSBO amiRNA constructs, cell division halted. In acetate-containing medium, cellular PSBO protein levels decreased by 60% within 24 h in the dark, by 75% in moderate light, and in high light, the protein completely degraded. Consequently, the photosynthetic apparatus became strongly damaged, probably due to 'donor-side-induced photoinhibition', and cellular ultrastructure was also severely affected. However, in the absence of acetate during induction, PSBO was remarkably stable at all light intensities and less substantial changes occurred in photosynthesis. Our results demonstrate that the lifetime of PSBO strongly depends on the light intensity and carbon availability, and thus, on the metabolic status of the cells. We also confirm that PSBO is required for photosystem II stability in C. reinhardtii and demonstrate that its specific loss also entails substantial changes in cell morphology and cell cycle.

Ion-driven nanograin formation in early-stage degradation of tri-cation perovskite films.

The operational stability of organic-inorganic halide perovskite based solar cells is a challenge for widespread commercial adoption. The mobility of ionic species is a key contributor to perovskite instability since ion migration can lead to unfavourable changes in the crystal lattice and ultimately destabilisation of the perovskite phase. Here we study the nanoscale early-stage degradation of mixed-halide mixed-cation perovskite films under operation-like conditions using electrical scanning probe microscopy to investigate the formation of surface nanograin defects. We identify the nanograins as lead iodide and study their formation in ambient and inert environments with various optical, thermal, and electrical stress conditions in order to elucidate the different underlying degradation mechanisms. We find that the intrinsic instability is related to the polycrystalline morphology, where electrical bias stress leads to the build-up of charge at grain boundaries and lateral space charge gradients that destabilise the local perovskite lattice facilitating escape of the organic cation. This mechanism is accelerated by enhanced ionic mobility under optical excitation. Our findings highlight the importance of inhibiting the formation of local charge imbalance, either through compositions preventing ionic redistribution or local grain boundary passivation, in order to extend operational stability in perovskite photovoltaics.

A Multi-Analytical Approach for Studying the Effect of New LED Lighting Systems on Modern Paints: Chemical Stability Investigations.

This study aims to investigate the chemical stability of some modern paint samples exposed to a new Light Emitting Diode (LED)-lighting system and a halogen lamp by using micro-attenuated total reflectance of Fourier transform infrared spectroscopy (µ-ATR-FTIR), µ-Raman, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermally assisted hydrolysis and methylation of GC/MS (THM-GC/MS). Those investigations were performed before and after the exposure of the samples to lightings for 1250, 2400, 3300, and 5000 h. The results obtained with µ-Raman spectroscopy show the high stability of the selected inorganic pigments after the exposure to the lighting systems; while similar to the UV/Vis/NIR results reported in a previous study, µ-ATR-FTIR and THM-GC/MS results evidence greater chemical changes occurring principally on the linseed oil binder-based mock-ups among the acrylic and alkyd-based samples. Moreover, principal component analyses (PCA) and hierarchical cluster analyses (HCA) of THM-GC/MS results highlight that those changes were mostly dependent on the exposure time and on the type of pigment, while being independent of the lighting system used. Finally, semi-quantitative µ-ATR-FTIR results show slight pigment enrichment at the paint surface due to the auto and photo-oxidative degradation of the linseed oil binder.

The effect of new LED lighting systems on the colour of modern paints.

This research focuses on the investigation of the effect of a new light emitting diode (LED)-lighting system which reproduces indoor museum conditions, on some self-made art paint colours (acrylic-, alkyd-, and linseed oil-based paints) often used in modern-contemporary art. A halogen lamp representing a traditional light source for museum lighting was also considered. Lighting-set-up and lighting optimization parameters were considered while Ultraviolet/Visible/Near Infrared (UV/Vis/NIR) spectrophotometry was used for investigating the colour change of the paint samples. Univariate analyses allowed determining the highest effect of the lighting systems on the linseed oil binder and ultramarine blue PB29 mixture upon ageing, according to the highest total colour change ΔE*ab. In a more specific and detailed way, variance analyses not only demonstrated the strong correlation between the type of binder and pigment used for the paint samples with the colour variation, but also showed that the short-wavelength blue LEDs influenced the change along the yellow-blue b* axis of the yellow and blue samples, whereas the halogen lamp mostly had an impact towards the red-green a* axis of mostly the green specimens.

The interplay between pathogens and Atg8 family proteins: thousand-faced interactions.

Autophagy is an intracellular degradation and recycling process that can also remove pathogenic intracellular bacteria and viruses from within cells (referred to as xenophagy) and activate the adaptive immune responses. But autophagy-especially Atg proteins including Atg8 family members-can also have proviral and probacterial effects. In this review, we summarize known interactions of bacterial, parasitic, and viral proteins with Atg8 family proteins and the outcome of these interactions on pathogen replication, autophagy, or mitophagy. We discuss the value of prediction software and the research methodology in the study of pathogen protein-Atg8 family protein interactions, with selected examples of potential LC3-interacting region motif-containing SARS-CoV-2 proteins.

Thin cell layer cultures of Chlamydomonas reinhardtii L159I-N230Y, pgrl1 and pgr5 mutants perform enhanced hydrogen production at sunlight intensity.

Photobiological hydrogen (H2) production is a promising renewable energy source. HydA hydrogenases of green algae are efficient but O2-sensitive and compete for electrons with CO2-fixation. Recently, we established a photoautotrophic H2 production system based on anaerobic induction, where the Calvin-Benson cycle is inactive and O2 scavenged by an absorbent. Here, we employed thin layer cultures, resulting in a three-fold increase in H2 production relative to bulk CC-124 cultures (50 µg chlorophyll/ml, 350 µmol photons m-2 s-1). Productivity was maintained when increasing the light intensity to 1000 µmol photons m-2s-1 and the cell density to 150 µg chlorophyll/ml. Remarkably, the L159I-N230Y photosystem II mutant and the pgrl1 photosystem I cyclic electron transport mutant produced 50% more H2 than CC-124, while the pgr5 mutant generated 250% more (1.2 ml H2/ml culture in six days). The photosynthetic apparatus of the pgr5 mutant and its in vitro HydA activity remained remarkably stable.

High-Sensitivity Dual Electrochemical QCM for Reliable Three-Electrode Measurements.

An electrochemical quartz crystal microbalance (EC-QCM) is a versatile gravimetric technique that allows for parallel characterization of mass deposition and electrochemical properties. Despite its broad applicability, simultaneous characterization of two electrodes remains challenging due to practical difficulties posed by the dampening from fixture parasitics and the dissipative medium. In this study, we present a dual electrochemical QCM (dual EC-QCM) that is employed in a three-electrode configuration to enable consequent monitoring of mass deposition and viscous loading on two crystals, the working electrode (WE) and the counter electrode (CE). A novel correction approach, along with a three standard complex impedance calibration, is employed to overcome the effect of dampening while keeping high spectral sensitivity. Separation of viscous loading and rigid mass deposition is achieved by robust characterization of the complex impedance at the resonance frequency. Validation of the presented system is done by cyclic voltammetry characterization of Ag underpotential deposition on gold. The results indicate mass deposition of 412.2 ng for the WE and 345.6 ng for the CE, reflecting a difference of the initially-present Ag adhered to the surface. We also performed higher harmonic measurements that further corroborate the sensitivity and reproducibility of the dual EC-QCM. The demonstrated approach is especially intriguing for electrochemical energy storage applications where mass detection with multiple electrodes is desired.

Ascorbate inactivates the oxygen-evolving complex in prolonged darkness.

Ascorbate (Asc, vitamin C) is an essential metabolite participating in multiple physiological processes of plants, including environmental stress management and development. In this study, we acquired knowledge on the role of Asc in dark-induced leaf senescence using Arabidopsis thaliana as a model organism. One of the earliest effects of prolonged darkness is the inactivation of oxygen-evolving complexes (OEC) as demonstrated here by fast chlorophyll a fluorescence and thermoluminescence measurements. We found that inactivation of OEC due to prolonged darkness was attenuated in the Asc-deficient vtc2-4 mutant. On the other hand, the severe photosynthetic phenotype of a psbo1 knockout mutant, lacking the major extrinsic OEC subunit PSBO1, was further aggravated upon a 24-h dark treatment. The psbr mutant, devoid of the PSBR subunit of OEC, performed only slightly disturbed photosynthetic activity under normal growth conditions, whereas it showed a strongly diminished B thermoluminescence band upon dark treatment. We have also generated a double psbo1 vtc2 mutant, and it showed a slightly milder photosynthetic phenotype than the single psbo1 mutant. Our results, therefore, suggest that Asc leads to the inactivation of OEC in prolonged darkness by over-reducing the Mn-complex that is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits. Our study is an example that Asc may negatively affect certain cellular processes and thus its concentration and localization need to be highly controlled.

Nanoscale Charge Accumulation and Its Effect on Carrier Dynamics in Tri-cation Perovskite Structures.

Nanoscale investigations by scanning probe microscopy have provided major contributions to the rapid development of organic-inorganic halide perovskites (OIHP) as optoelectronic devices. Further improvement of device level properties requires a deeper understanding of the performance-limiting mechanisms such as ion migration, phase segregation, and their effects on charge extraction both at the nano- and macroscale. Here, we have studied the dynamic electrical response of Cs0.05(FA0.83MA0.17)0.95PbI3-xBrx perovskite structures by employing conventional and microsecond time-resolved open-loop Kelvin probe force microscopy (KPFM). Our results indicate strong negative charge carrier trapping upon illumination and very slow (>1 s) relaxation of charges at the grain boundaries. The fast electronic recombination and transport dynamics on the microsecond scale probed by time-resolved open-loop KPFM show diffusion of charge carriers toward grain boundaries and indicate locally higher recombination rates because of intrinsic compositional heterogeneity. The nanoscale electrostatic effects revealed are summarized in a collective model for mixed-halide CsFAMA. Results on multilayer solar cell structures draw direct relations between nanoscale ionic transport, charge accumulation, recombination properties, and the final device performance. Our findings extend the current understanding of complex charge carrier dynamics in stable multication OIHP structures.

Ascorbate Deficiency Does Not Limit Nonphotochemical Quenching in Chlamydomonas reinhardtii.

Ascorbate (Asc; vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance, and photoprotection. In vascular plants, violaxanthin de-epoxidase requires Asc as a reductant; thereby, Asc is required for the energy-dependent component of nonphotochemical quenching (NPQ). To assess the role of Asc in NPQ in green algae, which are known to contain low amounts of Asc, we searched for an insertional Chlamydomonas reinhardtii mutant affected in theVTC2 gene encoding GDP-l-Gal phosphorylase, which catalyzes the first committed step in the biosynthesis of Asc. The Crvtc2-1 knockout mutant was viable and, depending on the growth conditions, contained 10% to 20% Asc relative to its wild type. When C. reinhardtii was grown photomixotrophically at moderate light, the zeaxanthin-dependent component of NPQ emerged upon strong red illumination both in the Crvtc2-1 mutant and in its wild type. Deepoxidation was unaffected by Asc deficiency, demonstrating that the Chlorophycean violaxanthin de-epoxidase found in C. reinhardtii does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component characteristic of photoautotrophic C. reinhardtii cultures grown at high light was not limited by Asc deficiency either. On the other hand, a reactive oxygen species-induced photoinhibitory NPQ component was greatly enhanced upon Asc deficiency, both under photomixotrophic and photoautotrophic conditions. These results demonstrate that Asc has distinct roles in NPQ formation in C. reinhardtii as compared to vascular plants.

Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.

Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.

Metadynamics for training neural network model chemistries: A competitive assessment.

Neural network model chemistries (NNMCs) promise to facilitate the accurate exploration of chemical space and simulation of large reactive systems. One important path to improving these models is to add layers of physical detail, especially long-range forces. At short range, however, these models are data driven and data limited. Little is systematically known about how data should be sampled, and "test data" chosen randomly from some sampling techniques can provide poor information about generality. If the sampling method is narrow, "test error" can appear encouragingly tiny while the model fails catastrophically elsewhere. In this manuscript, we competitively evaluate two common sampling methods: molecular dynamics (MD), normal-mode sampling, and one uncommon alternative, Metadynamics (MetaMD), for preparing training geometries. We show that MD is an inefficient sampling method in the sense that additional samples do not improve generality. We also show that MetaMD is easily implemented in any NNMC software package with cost that scales linearly with the number of atoms in a sample molecule. MetaMD is a black-box way to ensure samples always reach out to new regions of chemical space, while remaining relevant to chemistry near kbT. It is a cheap tool to address the issue of generalization.

The TensorMol-0.1 model chemistry: a neural network augmented with long-range physics.

Traditional force fields cannot model chemical reactivity, and suffer from low generality without re-fitting. Neural network potentials promise to address these problems, offering energies and forces with near ab initio accuracy at low cost. However a data-driven approach is naturally inefficient for long-range interatomic forces that have simple physical formulas. In this manuscript we construct a hybrid model chemistry consisting of a nearsighted neural network potential with screened long-range electrostatic and van der Waals physics. This trained potential, simply dubbed "TensorMol-0.1", is offered in an open-source Python package capable of many of the simulation types commonly used to study chemistry: geometry optimizations, harmonic spectra, open or periodic molecular dynamics, Monte Carlo, and nudged elastic band calculations. We describe the robustness and speed of the package, demonstrating its millihartree accuracy and scalability to tens-of-thousands of atoms on ordinary laptops. We demonstrate the performance of the model by reproducing vibrational spectra, and simulating the molecular dynamics of a protein. Our comparisons with electronic structure theory and experimental data demonstrate that neural network molecular dynamics is poised to become an important tool for molecular simulation, lowering the resource barrier to simulating chemistry.