Photoluminescence Spectroscopy Sheds New Light on Silicon Microchip Functional Properties.

In this study, we demonstrate that photoluminescence spectroscopy probing local interaction and dynamics at the fundamental level is a versatile tool for testing and evaluation of semiconducting materials as well as microscale chips based on them. Monocrystalline silicon, which is still an undisputed leader among semiconductors in microelectronics, exhibits very low photoluminescence emission additionally shielded by the metallization and passivating layers at the integrated circuit level. To unleash the full potential of photoluminescence spectroscopy for advanced testing and evaluation of the functional properties of the silicon microchips, new essential conceptually built approaches are required that overcome this problem. Here, we report on the first fundamental research-based application of a potentiometric dye to sense the electric field of operational silicon chips. Furthermore, we demonstrate high sensitivity of crystalline silicon phonon-assisted photoluminescence to local temperature in the 27-64 °C range. Our results show that sensing and mapping of thermal and electric field distributions using photoluminescence can enable precision testing of the structure, function, operation, and security, not only at the component level but also at the level of the entire integrated circuit.

Evaluation of pulse-jet baghouse dust collectors' contribution to CO2 emissions.

Dust cleaning systems are mandatory for use almost in any manufacturing process. Their market size is expected at US$10.77 billion by 2030 growing from US$7.28 billion in 2022. Removing dust particles is the main purpose of these systems and they make an invaluable contribution to environmental safety. However, while cleaning the air from solid particles, industrial pulse-jet baghouse collectors have an additional impact on the environment that usually is not considered. An analysis of energy consumption at the manufacturing and operation stages of the baghouse dust collectors allows for the evaluation of CO2 emissions. The analysis shows that, given the current state of affairs in the industry, by 2030 manufacturing and operation of baghouse dust collectors over the world will emit 70+ million tons of carbon dioxide additionally to the levels of 2021. To reduce the CO2-related environmental impact of industrial pulse-jet baghouse collectors, among all scientific and technical measures, it is recommended to simply scale up the dust collection system, which involves replacing several low-capacity collectors with one general-capacity collector within one industrial enterprise. This allows for a reduction in energy consumption at the collector manufacturing stage from 3 to 10 times and also ensures a significant reduction in operation energy consumption of the dust collector during its service life.

Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity.

BACKGROUND: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism. OBJECTIVES: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets. METHODS: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied. RESULTS: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold. CONCLUSION: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.

Plasticizer-Induced Enhancement of Mesoscale Dissymmetry in Thin Films of Chiral Polymers with Variable Chain Length.

Conjugated polymers with chiral side chains are of interest in areas including chiral photonics, optoelectronics, and chemical and biological sensing. However, the low dissymmetry factors of most neat polymer thin films have limited their practical application. Here, a robust method to increase the absorption dissymmetry factor in a poly-fluorene-thiophene (PF8TS series) system is demonstrated by varying molecular weight and introducing an achiral plasticizer, polyethylene mono alcohol (PEM-OH). Extending chain length within the optimal range and adding this long-chain alcohol significantly enhance the chiroptical properties of spin-coated and annealed thin films. Mueller matrix spectroscopic ellipsometry (MMSE) analysis shows good agreement with the steady-state transmission measurements confirming a strong chiral response (circular dichroism (CD) and circular birefringence (CB)), ruling out linear dichroism, birefringence, and specific reflection effects. Solid-state NMR studies of annealed hybrid chiral polymer systems show enhancement of signals associated with aromatic π-stacked backbone and the ordered side-chain conformations. Further studies using Raman spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), atomic force microscopy (AFM), and polarized optical microscopy (POM) indicate that PEM-OH facilitates mesoscopic crystal domain ordering upon annealing. This provides new insights into routes for tuning optical activity in conjugated polymers.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Upconversion Nanoparticles as Imaging Agents for Dental Caries.

Dental caries (cavities) is the most prevalent disease worldwide; however, current detection methods suffer from issues associated with sensitivity, subjective interpretations, and false positive identification of carious lesions. Therefore, there is a great need for the development of more sensitive, noninvasive imaging methods. The 30 nm core@shell NaYF4; Yb20%, Er2%@NaYF4 upconversion nanoparticles (UCNPs), exhibiting strong upconversion emission from erbium upon excitation at 975 nm, were used in the imaging of locations of demineralized enamel and oral biofilm formation for the detection of dental caries. UCNPs were modified with poly(acrylic acid) (PAA) or poly-d-lysine (PDL), and targeting peptides were conjugated to their surface with affinity for either hydroxyapatite (HA), the material dentin is composed of, or the caries causing bacteria Streptococcus mutans. A statistical difference in the binding of targeted vs nontargeted UCNPs to HA was observed after 15 min, using both upconversion fluorescence of UCNP (p < 0.001) and elemental analysis (p = 0.0091). Additionally, using the HA targeted UCNPs, holes drilled in the enamel of bovine teeth with diameters of 1.0 and 0.5 mm were visible by the green emission after a 20 min incubation with no observable nonspecific binding. A statistical difference was also observed in the binding of targeted versus nontargeted UCNPs to S. mutans biofilms. This difference was observed after 15 min, using the fluorescence measurements (p = 0.0125), and only 10 min (p < 0.001) using elemental analysis via ICP-OES measurements of Y3+ concentration present in the biofilms. These results highlight the potential of these UCNPs for use in noninvasive imaging diagnosis of oral disease.

Mitochondrial Dysfunction and Apoptosis in Brain Microvascular Endothelial Cells Following Blast Traumatic Brain Injury.

Blood brain barrier (BBB) breakdown is a key driver of traumatic brain injury (TBI), contributing to prolonged neurological deficits and increased risk of death in TBI patients. Strikingly, the role of endothelium in the progression of BBB breakdown has not been sufficiently investigated, even though it constitutes the bulk of BBB structure. In the current study, we investigate TBI-induced changes in the brain endothelium at the subcellular level, particularly focusing on mitochondrial dysfunction, using a combination of confocal imaging, gene expression analysis, and molecular profiling by Raman spectrometry. Herein, we developed and applied an in-vitro blast-TBI (bTBI) model that employs an acoustic shock tube to deliver injury to cultured human brain microvascular endothelial cells (HBMVEC). We found that this injury results in aberrant expression of mitochondrial genes, as well as cytokines/ inflammasomes, and regulators of apoptosis. Furthermore, injured cells exhibit a significant increase in reactive oxygen species (ROS) and in Ca2+ levels. These changes are accompanied by overall reduction of intracellular proteins levels as well as profound transformations in mitochondrial proteome and lipidome. Finally, blast injury leads to a reduction in HBMVEC cell viability, with up to 50% of cells exhibiting signs of apoptosis following 24 h after injury. These findings led us to hypothesize that mitochondrial dysfunction in HBMVEC is a key component of BBB breakdown and TBI progression.

Tryptophan Hydroxylase-2-Mediated Serotonin Biosynthesis Suppresses Cell Reprogramming into Pluripotent State.

The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.

Decoupled Rare-Earth Nanoparticles for On-Demand Upconversion Photodynamic Therapy and High-Contrast Near Infrared Imaging in NIR IIb.

Rare-earth doped multi-shell nanoparticles slated for theranostic applications produce a variety of emission bands upon near-infrared (NIR) excitation. Their downshifting emission is useful for high-contrast NIR imaging, while the upconversion light can induce photodynamic therapy (PDT). Unfortunately, integration of imaging and therapy is challenging. These modalities are better to be controlled independently so that, with the help of imaging, selective delivery of a theranostic agent at the site of interest could be ensured prior to on-demand PDT initiation. We introduce here multi-shell rare-earth doped nanoparticles (RENPs) arranged in a manner to produce only downshifting emission for NIR imaging when excited at one NIR wavelength and upconversion emission for therapeutic action by using a different excitation wavelength. In this work, multi-shell RENPs with a surface-bound sensitizer have been synthesized for decoupled 1550 nm downshifting emission upon 800 nm excitation and 550 nm upconversion emission caused by 980 nm irradiation. The independently controlled emission bands allow for high-contrast NIR imaging in NIR-IIb of optical transparency that gives high-contrast images due to significantly reduced light scattering. This can be conducted prior to PDT using 980 nm to produce upconverted light at 550 nm that excites the RENP surface-bound photosensitizer, Rose Bengal (RB), to effect photodynamic therapy with high specificity and safer theranostics.

Degradable Poly(3-hydroxybutyrate)-The Basis of Slow-Release Fungicide Formulations for Suppressing Potato Pathogens.

Three-component slow-release fungicide formulations with different modes of action of the active ingredients for suppressing potato pathogens were constructed for the first time. The difenoconazole, mefenoxam, prothioconazole, and azoxystrobin fungicides were embedded in the degradable polymer P(3HB)/birch wood flour blend and examined using SEM, IR spectroscopy, X-ray analysis, DTA, and DSC. Results showed that no chemical bonds were established between the components and that they were physical mixtures that had a lower degree of crystallinity compared to the initial P(3HB), which suggested different crystallization kinetics in the mixtures. The degradation behavior of the experimental formulations was investigated in laboratory micro-ecosystems with pre-characterized field soil. The slow-release fungicide formulations were prolonged-action forms with a half-life of at least 50-60 d, enabling gradual and sustained delivery of the active ingredients to plants. All slow-release fungicide formulations had a strong inhibitory effect on the most common and harmful potato pathogens (Phytophthorainfestans, Alternarialongipes, Rhizoctoniasolani, and Fusariumsolani).

Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction.

AIMS: Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. METHODS AND RESULTS: Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. CONCLUSIONS: These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.

Mitochondrial Dysfunction: A Prelude to Neuropathogenesis of SARS-CoV-2.

The SARS-CoV-2 virus is notorious for its neuroinvasive capability, causing multiple neurological conditions. The neuropathology of SARS-CoV-2 is increasingly attributed to mitochondrial dysfunction of brain microglia cells. However, the changes in biochemical content of mitochondria that drive the progression of neuro-COVID remain poorly understood. Here we introduce a Raman microspectrometry approach that enables the molecular profiling of single cellular organelles to characterize the mitochondrial molecular makeup in the infected microglia cells. We found that microglia treated with either spike protein or heat-inactivated SARS-CoV-2 trigger a dramatic reduction in mtDNA content and an increase in phospholipid saturation levels. At the same time, no significant changes were detected in Golgi apparatus and in lipid droplets, the organelles that accommodate biogenesis and storage of lipids. We hypothesize that transformations in mitochondria are caused by increased synthesis of reactive oxygen species in these organelles. Our findings call for the development of mitochondria-targeted therapeutic approaches to limit neuropathology associated with SARS-CoV-2.

Bacterial Cellulose (BC) and BC Composites: Production and Properties.

The synthesis of bacterial cellulose (BC) by Komagataeibacter xylinus strain B-12068 was investigated on various C-substrates, under submerged conditions with stirring and in static surface cultures. We implemented the synthesis of BC on glycerol, glucose, beet molasses, sprat oil, and a mixture of glucose with sunflower oil. The most productive process was obtained during the production of inoculum in submerged culture and subsequent growth of large BC films (up to 0.2 m2 and more) in a static surface culture. The highest productivity of the BC synthesis process was obtained with the growth of bacteria on molasses and glycerol, 1.20 and 1.45 g/L per day, respectively. We obtained BC composites with silver nanoparticles (BC/AgNPs) and antibacterial drugs (chlorhexidine, baneocin, cefotaxime, and doripenem), and investigated the structure, physicochemical, and mechanical properties of composites. The disc-diffusion method showed pronounced antibacterial activity of BC composites against E. coli ATCC 25922 and S. aureus ATCC 25923.

Building Blocks of Artificial CRISPR-Based Systems beyond Nucleases.

Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided.

4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Hot-band absorption of indocyanine green for advanced anti-stokes fluorescence bioimaging.

Bright anti-Stokes fluorescence (ASF) in the first near-infrared spectral region (NIR-I, 800 nm-900 nm) under the excitation of a 915 nm continuous wave (CW) laser, is observed in Indocyanine Green (ICG), a dye approved by the Food and Drug Administration for clinical use. The dependence of fluorescence intensity on excitation light power and temperature, together with fluorescence lifetime measurement, establish this ASF to be originated from absorption from a thermally excited vibrational level (hot-band absorption), as shown in our experiments, which is stronger than the upconversion fluorescence from widely-used rare-earth ion doped nanoparticles. To test the utility of this ASF NIR-I probe for advanced bioimaging, we successively apply it for biothermal sensing, cerebral blood vessel tomography and blood stream velocimetry. Moreover, in combination with L1057 nanoparticles, which absorb the ASF of ICG and emit beyond 1100 nm, these two probes generate multi-mode images in two fluorescent channels under the excitation of a single 915 nm CW laser. One channel is used to monitor two overlapping organs, urinary system & blood vessel of a live mouse, while the other shows urinary system only. Using in intraoperative real-time monitoring, such multi-mode imaging method can be beneficial for visual guiding in anatomy of the urinary system to avoid any accidental injury to the surrounding blood vessels during surgery.

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man.

Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol. In the recent two decades, a plethora of studies established that elevated ROS levels generated by oxidative eustress are crucial physiological mediators of many cellular and developmental processes. In this review, we discuss the mechanisms of ROS generation and regulation, current understanding of ROS functions in the maintenance of adult and embryonic stem cells, as well as in the process of cell reprogramming to a pluripotent state. Recently discovered cell-non-autonomous ROS functions mediated by growth factors are crucial for controlling cell differentiation and cellular immune response in Drosophila. Importantly, many physiological functions of ROS discovered in Drosophila may allow for deciphering and understanding analogous processes in human, which could potentially lead to the development of novel therapeutic approaches in ROS-associated diseases treatment.

A Single-Organelle Optical Omics Platform for Cell Science and Biomarker Discovery.

Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.

IDH1 mutations induce organelle defects via dysregulated phospholipids.

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

A dual mode nanophotonics concept for in situ activation of brain immune cells using a photoswitchable yolk-shell upconversion nanoformulation.

Here, we introduce a nanophotonics concept for optically triggered activation of microglia. Specifically, we synthesized a yolk-shell structured mesoporous silica coated core-shell upconverting nanoparticles (UCNP@ysSiO2). The nanoparticles are loaded with microglia activators-bacterial lipopolysaccharide (LPS) together with indocyanine green (ICG), and then capped with ß-cyclodextrin (CD) via selective affinity of this compound to photoswitchable azobenzene (Azo). Upon exposure to NIR light, and subsequent trans- to cis photoisomerization of the Azo group induced by the upconversion light, dissociation of ß-CD produces the release of LPS. The released LPS activates microglia through a toll-like receptor 4 mediated pathway, while ICG excited by its absorption of the 800 nm upconversion light, produces local heating, thus synergistically activating microglia through heat shock proteins. We propose that the controlled activation of microglia with deep tissue penetrating NIR triggered drug release, may provide a new strategy for in situ treatment of many brain diseases.