Extended characterization of anti-CD19 CAR T cell products manufactured at the point of care using the CliniMACS Prodigy system: comparison of donor sources and process duration.

BACKGROUND AIMS: The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. METHODS: Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. RESULTS: The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells-median for long process [LP] and 1.6 × 109-for short process [SP]), transduction efficiency (43.5%-median for LP and 41.0%-for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. CONCLUSIONS: This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays.

Noise Filtering Algorithm Using Gaussian Mixture Models for High-Resolution Mass Spectra of Natural Organic Matter.

High-resolution mass spectra of natural organic matter (NOM) contain a large number of noise signals. These signals interfere with the correct molecular composition estimation during nontargeted analysis because formula-assignment programs find empirical formulas for such peaks as well. Previously proposed noise filtering methods that utilize the profile of the intensity distribution of mass spectrum peaks rely on a histogram to calculate the intensity threshold value. However, the histogram profile can vary depending on the user settings. In addition, these algorithms are not automated, so they are handled manually. To overcome the mentioned drawbacks, we propose a new algorithm for noise filtering in mass spectra. This filter is based on Gaussian Mixture Models (GMMs), a machine learning method to find the intensity threshold value. The algorithm is completely data-driven and eliminates the need to work with a histogram. It has no customizable parameters and automatically determines the noise level for each individual mass spectrum. The algorithm performance was tested on mass spectra of natural organic matter obtained by averaging a different number of microscans (transients), and the results were compared with other noise filters proposed in the literature. Finally, the effect of this noise filtering approach on the fraction of peaks with assigned formulas was investigated. It was shown that there is always an increase in the identification rate, but the magnitude of the effect changes with the number of microscans averaged. The increase can be as high as 15%.

Humic Polyelectrolytes Facilitate Rapid Microwave Synthesis of Silver Nanoparticles Suitable for Wound-Healing Applications.

This article describes the one-pot microwave synthesis of silver nanoparticles (AgNPs) assisted with natural polyelectrolytes-humic substances (HS). The humic polyelectrolytes served both as chemical reductants for silver ions and as end-capping agents for AgNPs. Three commercially available sodium humates extracted from lignites and leonardite and one sodium fulvate isolated from natural brown water seeped through peat deposits were used in this study. The dynamics of the growth rate of AgNPs was characterised by UV-VIS spectroscopy by measuring the intensity of surface plasmon resonance at 420 nm. Transmission electron microscopy was used to characterise the size and morphology of AgNPs. Dynamic light scattering was used to determine size distributions of the synthesised AgNPs in the solutions. It was established that both conventional and microwave syntheses assisted with the coal humates produced small-size AgNPs in the range from 4 to 14 nm, with the maximum share of particles with sizes of (6 ± 2) nm by TEM estimates. The peat fulvate yielded much larger NPs with sizes from 10 to 50 nm by TEM estimates. DLS measurements revealed multimodal distributions of AgNPs stabilised with HS, which included both single NPs with the sizes from 5 to 15 nm, as well as their dominating aggregates with sizes from 20 to 200 nm and a smaller portion of extra-large aggregates up to 1000 nm. The given aggregates were loosely bound by humic polyelectrolyte, which prevented the coalescence of AgNPs into larger particles, as can be seen in the TEM images. The significant acceleration in the reaction time-a factor of 60 to 70-was achieved with the use of MW irradiation: from 240 min down to 210-240 s. The coal humate stabilised AgNPs showed antimicrobial properties in relation to S. aureus. A conclusion was made regarding the substantial advantages of microwave synthesis in the context of time and scaling up for the large-scale production of AgNP-HS preparations with antimicrobial properties suitable for external wound-healing applications.

Yedoma Permafrost Releases Organic Matter with Lesser Affinity for Cu2+ and Ni2+ as Compared to Peat from the Non-Permafrost Area: Risk of Rising Toxicity of Potentially Toxic Elements in the Arctic Ocean.

Pollution of the Arctic Ocean by potentially toxic elements (PTEs) is a current environmental problem. Humic acids (HAs) play an important role in the regulation of PTE mobility in soil and water. The permafrost thaw releases ancient organic matter (OM) with a specific molecular composition into the Arctic watersheds. This could affect the mobility of PTEs in the region. In our study, we isolated HAs from two types of permafrost deposits: the Yedoma ice complex, which contains pristine buried OM, and the alas formed in the course of multiple thaw-refreezing cycles with the most altered OM. We also used peat from the non-permafrost region as the recent environmental endmember for the evolution of Arctic OM. The HAs were characterized using 13C NMR and elemental analysis. Adsorption experiments were conducted to assess the affinity of HAs for binding Cu2+ and Ni2+. It was found that Yedoma HAs were enriched with aliphatic and N-containing structures as compared to the much more aromatic and oxidized alas and peat HAs. The adsorption experiments have revealed that the peat and alas HAs have a higher affinity for binding both ions as compared to the Yedoma HAs. The obtained data suggest that a substantial release of the OM from the Yedoma deposits due to a rapid thaw of the permafrost might increase the mobility of PTEs and their toxicity in the Arctic Ocean because of much lesser "neutralization potential".

Infrared Spectroscopy in Aqueous Solutions: Capabilities and Challenges.

Fourier-transform infrared (FTIR) spectroscopy provides rapid, reliable, quantitative, and qualitative analysis of samples in different aggregation states, i.e., gases, thin films, solids, liquids, etc. However, when analyzing aqueous solutions, particular issues associated with the rather pronounced IR absorption characteristics of water appear to interfere with the solute determination. In this review, Fourier-transform infrared spectroscopic techniques and their analytical capabilities for analyzing aqueous solutions are reviewed, and highlight examples are discussed.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

FTIR Photoacoustic and ATR Spectroscopies of Soils with Aggregate Size Fractionation by Dry Sieving.

Granulometric fractionation as a source of additional information on organic-matter and inorganic matrix components of soils using FTIR-photoacoustic spectroscopy (FTIR-PAS) supported by attenuated-total reflection FTIR spectroscopy (ATR-FTIR) for a wide range of aggregate fractions (10-5000 µm) was used to compare the sensitivity, reproducibility, information contents, and representativity of fractionated samples. For chernozem and sod-podzolic soils and different agricultural-use chernozem samples, differences in the composition were found, manifested in normalized spectra of microaggregate fractions, with the range of 10-100 µm bearing the complete information. Most changes are observed in the soil organic matter range (1900-1340 cm-1), although these changes are slight, and in the soil-matrix region (550-300 cm-1). The latter region increases the intensity of bands corresponding to amorphous silica and clay minerals in fine fractions, while the intensity of bands attributed to quartz lattice vibrations decreases. FTIR-PAS spectra do not differ considerably at high interferometer modulation frequencies as the signal-penetration depth is comparable with particle sizes. The soil fractions below 20 µm result in the maximum sensitivity, reproducibility, and signal-to-noise ratio, showing no changes from coarser fractions by the information content and, thus, providing representative samples for analysis. The fractionation shows more differences in the sod-podzolic and chernozem soil fractions than the whole soil spectra. FTIR-PAS provides better sensitivity and reproducibility in the 4000-2000 cm-1 region and ATR-FTIR in the 2000-100 cm-1 region.

Thermal Conductivity of Detonation Nanodiamond Hydrogels and Hydrosols by Direct Heat Flux Measurements.

The methodology and results of thermal conductivity measurements by the heat-flow technique for the detonation nanodiamond suspension gels, sols, and powders of several brands in the range of nanoparticle concentrations of 2-100% w/w are discussed. The conditions of assessing the thermal conductivity of the fluids and gels (a FOX 50 heat-flow meter) with the reproducibility (relative standard deviation) of 1% are proposed. The maximum increase of 13% was recorded for the nanodiamond gels (140 mg mL-1 or 4% v/v) of the RDDM brand, at 0.687 ± 0.005 W m-1 K-1. The thermal conductivity of the nanodiamond powders is estimated as 0.26 ± 0.03 and 0.35 ± 0.04 W m-1 K-1 for the RUDDM and RDDM brands, respectively. The thermal conductivity for the aqueous pastes containing 26% v/v RUDDM is 0.85 ± 0.04 W m-1 K-1. The dignities, shortcomings, and limitations of this approach are discussed and compared with the determining of the thermal conductivity with photothermal-lens spectrometry.

A Use of Tritium-Labeled Peat Fulvic Acids and Polyphenolic Derivatives for Designing Pharmacokinetic Experiments on Mice.

Natural products (e.g., polyphenols) have been used as biologically active compounds for centuries. Still, the mechanisms of biological activity of these multicomponent systems are poorly understood due to a lack of appropriate experimental techniques. The method of tritium thermal bombardment allows for non-selective labeling and tracking of all components of complex natural systems. In this study, we applied it to label two well-characterized polyphenolic compounds, peat fulvic acid (FA-Vi18) and oxidized lignin derivative (BP-Cx-1), of predominantly hydrophilic and hydrophobic character, respectively. The identity of the labeled samples was confirmed using size exclusion chromatography. Using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), key differences in the molecular composition of BP-Cx-1 and FA-Vi18 were revealed. The labeled samples ([3H]-FA-Vi18 (10 mg/kg) and [3H]-BP-Cx-1 (100 mg/kg)) were administered to female BALB/c mice intravenously (i.v.) and orally. The label distribution was assessed in blood, liver, kidneys, brain, spleen, thymus, ovaries, and heart using liquid scintillation counting. Tritium label was found in all organs studied at different concentrations. For the fulvic acid sample, the largest accumulation was observed in the kidney (Cmax 28.5 mg/kg and 5.6 mg/kg, respectively) for both routes. The organs of preferential accumulation of the lignin derivative were the liver (Cmax accounted for 396.7 and 16.13 mg/kg for i.v. and p.o. routes, respectively) and kidney (Cmax accounted for 343.3 and 17.73 mg/kg for i.v. and p.o. routes, respectively). Our results demonstrate that using the tritium labeling technique enabled successful pharmacokinetic studies on polyphenolic drugs with very different molecular compositions. It proved to be efficient for tissue distribution studies. It was also shown that the dosage of the polyphenolic drug might be lower than 10 mg/kg due to the sensitivity of the 3H detection technique.

Speciation of organosulfur compounds in carbonaceous chondrites.

Despite broad application of different analytical techniques for studies on organic matter of chondrite meteorites, information about composition and structure of individual compounds is still very limited due to extreme molecular diversity of extraterrestrial organic matter. Here we present the first application of isotopic exchange assisted Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for analysis of alkali extractable fraction of insoluble organic matter (IOM) of the Murchison and Allende meteorites. This allowed us to determine the individual S-containing ions with different types of sulfur atoms in IOM. Thiols, thiophenes, sulfoxides, sulfonyls and sulfonates were identified in both samples but with different proportions, which contribution corroborated with the hydrothermal and thermal history of the meteorites. The results were supported by XPS and thermogravimetric analysis coupled to FTICR MS. The latter was applied for the first time for analysis of chondritic IOM. To emphasize the peculiar extraterrestrial origin of IOM we have compared it with coal kerogen, which is characterized by the comparable complexity of molecular composition but its aromatic nature and low oxygen content can be ascribed almost exclusively to degradation of biomacromolecules.

Green and rapid preparation of long-term stable aqueous dispersions of fullerenes and endohedral fullerenes: The pros and cons of an ultrasonic probe.

A green, scalable, and sustainable approach to prepare aqueous fullerene dispersions (AFD) C60, C70, endohedral metallofullerene Gd@C82, and their derivatives C60Cl6, C70Cl10, and supramolecular and ester-like derivatives, 10 fullerene species total, is proposed. For the first time, an immersed ultrasonic probe was used to preparing dispersions for pristine fullerenes without addends. Both ultrasound-assisted solvent-exchange and direct sonication techniques for AFD preparation using an immersed probe were tested. The average time for AFD preparation decreases 10-15 times compared to an ultrasound-bath-assisted technique, while final fullerene concentrations in AFDs remained at tens of ppm (up to 80 ppm). The aqueous dispersions showed long-term stability, a negatively charged surface with a zeta potential up to -32 mV with an average nanocluster diameter of no more than 180 nm. The total anionic and cationic compositions of samples were found by inductively coupled plasma atomic emission spectroscopy and chromatographic techniques. The highlights and challenges of using an ultrasound probe for AFD production are discussed.

Taphonomic experiments imply a possible link between the evolution of multicellularity and the fossilization potential of soft-bodied organisms.

The reliability of evolutionary reconstructions based on the fossil record critically depends on our knowledge of the factors affecting the fossilization of soft-bodied organisms. Despite considerable research effort, these factors are still poorly understood. In order to elucidate the main prerequisites for the preservation of soft-bodied organisms, we conducted long-term (1-5 years) taphonomic experiments with the model crustacean Artemia salina buried in five different sediments. The subsequent analysis of the carcasses and sediments revealed that, in our experimental settings, better preservation was associated with the fast deposition of aluminum and silicon on organic tissues. Other elements such as calcium, magnesium, and iron, which can also accumulate quickly on the carcasses, appear to be much less efficient in preventing decay. Next, we asked if the carcasses of uni- and multicellular organisms differ in their ability to accumulate aluminum ions on their surface. The experiments with the flagellate Euglena gracilis and the sponge Spongilla lacustris showed that aluminum ions are more readily deposited onto a multicellular body. This was further confirmed by the experiments with uni- and multicellular stages of the social ameba Dictyostelium discoideum. The results lead us to speculate that the evolution of cell adhesion molecules, which provide efficient cell-cell and cell-substrate binding, probably can explain the rich fossil record of soft-bodied animals, the comparatively poor fossil record of nonskeletal unicellular eukaryotes, and the explosive emergence of the Cambrian diversity of soft-bodied fossils.

Poly(Ethylene Glycol)-b-Poly(D,L-Lactide) Nanoparticles as Potential Carriers for Anticancer Drug Oxaliplatin.

Nanoparticles based on biocompatible methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG113-b-P(D,L)LAn) copolymers as potential vehicles for the anticancer agent oxaliplatin were prepared by a nanoprecipitation technique. It was demonstrated that an increase in the hydrophobic PLA block length from 62 to 173 monomer units leads to an increase of the size of nanoparticles from 32 to 56 nm. Small-angle X-ray scattering studies confirmed the "core-corona" structure of mPEG113-b-P(D,L)LAn nanoparticles and oxaliplatin loading. It was suggested that hydrophilic oxaliplatin is adsorbed on the core-corona interface of the nanoparticles during the nanoprecipitation process. The oxaliplatin loading content decreased from 3.8 to 1.5% wt./wt. (with initial loading of 5% wt./wt.) with increasing PLA block length. Thus, the highest loading content of the anticancer drug oxaliplatin with its encapsulation efficiency of 76% in mPEG113-b-P(D,L)LAn nanoparticles can be achieved for block copolymer with short hydrophobic block.

Detonation Nanodiamonds: A Comparison Study by Photoacoustic, Diffuse Reflectance, and Attenuated Total Reflection FTIR Spectroscopies.

The qualitative analysis of nanodiamonds by FTIR spectrometry as photoacoustic (FTIR-PAS), diffuse-reflectance (DRIFT), and attenuated total reflection (ATR) modalities was evaluated for rapid and nondestructive analysis and comparison of nanodiamonds. The reproducibility and signal-gathering depth of spectra was compared. The assignment of characteristic bands showed that only six groups of bands were present in spectra of all the modalities with appropriate sensitivity: 1760 (C=O stretch, isolated carboxyl groups); 1640-1632 (H-O-H bend, liquid water); 1400-1370 (non-carboxyl C-O-H in-plane bend and CH2 deformation); 1103 (non-carboxyl C-O stretch); 1060 (in-plane C-H bend, non-aromatic hydrocarbons and carbohydrates); 940 cm-1 (out-of-plane carboxyl C-O-H bend). DRIFT provides the maximum number of bands and is capable of measuring hydrogen-bonded bands and CHx groups. ATR provides the good sensitivity for water and C-H/C-C bands in the range 2000-400 cm-1. FTIR-PAS reveals less bands than DRIFT but more intense bands than ATR-FTIR and shows the maximum sensitivity for absorption bands that do not appear in ATR-IR spectra and are expedient for supporting either DRIFT or FTIR-PAS along with depth-profiling. Thus, all three modalities are required for the full characterization of nanodiamonds surface functional groups.

La, Ce and Nd in the soil-plant system in a vegetation experiment with barley (Hordeum vulgare L.).

Rare earth elements (REEs) have received enormous attention in recent years. However, there are many gaps in the understanding of their behavior in the soil-plant system. The aim of this study is to investigate the behavior of three most common REEs (La, Ce, Nd) in the soil-plant system directly on soil samples using barley (Hordeum vulgare L.) in a vegetation experiment. We attribute the absence of significant changes in plant biomass and photosynthetic pigment content to the reduced availability of REEs in soil samples. The concentration of water-soluble forms of La, Ce and Nd didn't exceed 1 mg/kg, while the concentration of exchangeable forms varied and decreased in a row La > Ce > Nd. The transfer factor (TF) from soil to above-ground biomass was low for all three elements (<1). The stem-to-leaf TF increased with the increase in REEs concentration in soil. The concentration in plant material increased in the row Ce < Nd < La. REEs concentrations in barley leaves didn't exceed 1-3% of the corresponding element concentration in soil samples. REEs concentration in plant tissues is in close direct correlation with the REEs total concentration in soil, water-soluble and exchange forms. REEs concentration in barley leaves is 3-4 times higher than in the stems and for the group with extraneous concentration of 200 mg/kg for La, Ce and Nd was 6.20 ± 1.48, 2.10 ± 0.51, 6.90 ± 3.00 mg/kg, respectively. We show that there were no major changes in barley plants, but further study is needed of the relationship between the absorption of lanthanides by plants and the content of various forms of lanthanides in the soil.

FTIR photoacoustic spectroscopy for identification and assessment of soil components: Chernozems and their size fractions.

FTIR photoacoustic spectroscopy was used to approach inorganic matrix components and organic-matter constituents of chernozem size fractions (1-5000 µm, by dry sieving) with a different history of use (from intact steppe to permanent bare fallow, a continuous long-term field experiment). The conditions of FTIR photoacoustic measurements in continuous-scan modes were compared with attenuated total reflection measurements, the advantages of photoacoustic measurements resulting from a higher intensity of the incident radiation and signal-generating volume were discussed. Overtone peaks of quartz as a soil matrix component at 2000-1700 cm-1 were selected as a possible internal-standard (guide) bands for the comparison of photoacoustic spectra. For different land-use samples, differences in the composition were found, which are differently manifested in normalized spectra of size fractions, with millimeter-size, 20-100 µm, and silt fraction bearing the maximum information.

Photoacoustic and photothermal methods in spectroscopy and characterization of soils and soil organic matter.

Review sums up the application of photoacoustic and photothermal spectroscopies for the analysis and characterization of soils and soil organic matter and discusses the outlooks in this area.

Application of copper plates for frequency tuning of surface wired and wireless MRI coils.

This study shows how a copper plate could be used for frequency tuning of surface wired and wireless MRI coils. For this purpose, it is proposed to place the copper plate directly on their conducting circuit. This leads to increase in the resonance frequency of coils. The effect is most perceptible if the copper plate is comparable in size to the conducting circuit of radiofrequency (RF) coil. The experimental work was performed on a 7.05 T MR scanner using surface MRI coils operating on different resonance frequencies: 1H (300 MHz), 31P (121 MHz), 23Na (79 MHz), 13C (75 MHz). Application of copper plate for frequency tuning of wireless multi-turn multi-gap transmission line resonator (MTMG-TLR) was considered for the first time. The proposed method can be claimed if the nominal variable inductance or capacitance is not enough for tuning the resonance frequency of the MRI coil to a higher frequency range.

Titanium Oxide Microspheres with Tunable Size and Phase Composition.

Due to their unique physical and chemical properties, monodisperse titanium oxide microspheres can be used in dye-sensitized solar cells, as cosmetic pigments, and for other applications. However, the synthesis of microspheres with narrow size distribution, desired phase composition, and porosity is still a challenge. In this work, spherical titania particles with controllable size, crystallinity, and pore size were obtained by Ti(OnBu)4 hydrolysis in ethanol. The influence of NaOH addition on the particles' size and morphology was investigated for the first time. Particle diameter can be tailored from 300 nm to 1.5 µm by changing water and NaOH concentrations. Particle size was analyzed by the statistical processing of scanning electron microscopy (SEM) images and differential centrifugal sedimentation (DCS) measurements. Optical properties of the microspheres were studied by diffuse reflectance UV-Vis spectroscopy. Thermal and hydrothermal treatment allowed transforming amorphous phase in as-prepared particles into nanocrystalline anatase and/or rutile. Transmission electron microscopy (TEM) study of the lamellae, cut out from spherical particles using focused ion beam (FIB), revealed that as-synthesized microspheres are non-hollow, homogeneous, and crystallize throughout the whole volume of the particle. The spherical particles possess photoprotective properties; the highest sun protection factor (SPF) was observed for amorphous microspheres.