Deeper Insights into Mixed Crowding through Enzyme Activity, Dynamics, and Crowder Diffusion.

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Concentration Dependence of Elastic and Viscoelastic Properties of Aqueous Solutions of Ficoll and Bovine Serum Albumin by Brillouin Light Scattering Spectroscopy.

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule and the globular protein bovine serum albumin (BSA). Recent studies have indicated that a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 to 35 wt % and BSA with concentrations of 1 to 27 wt %. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. The existing theory that ignores intermolecular interactions can capture only the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semidilute regime. In the Ficoll solutions (but not BSA), we see evidence for a central mode, which is indicative of relaxation in the hydration shell of Ficoll.

Preserving frozen stallion sperm on dry ice using polymers that modulate ice crystalization kinetics.

Cryopreserved semen is routinely shipped in liquid nitrogen. Dry ice could serve as an alternative coolant, however, frozen storage above liquid nitrogen temperatures (LN2, -196 °C) may negatively affect shelf-life and cryosurvival. In this study, we determined critical temperatures for storage of cryopreserved stallion sperm. We evaluated: (i) effects of cooling samples to different subzero temperatures (-10 °C to -80 °C) prior to storing in LN2, (ii) stability at different storage temperatures (i.e., in LN2, dry ice, -80 °C and -20 °C freezers, 5 °C refrigerator), and (iii) sperm cryosurvival during storage on dry ice (i.e., when kept below -70 °C and during warming). Furthermore, (iv) we analyzed if addition of synthetic polymers (PVP-40, Ficoll-70) modulates ice crystallization kinetics and improves stability of cryopreserved specimens. Sperm motility and membrane intactness were taken as measures of cryosurvival, and an artificial insemination trial was performed to confirm fertilizing capacity. We found that adding PVP-40 or Ficoll-70 to formulations containing glycerol reduced ice crystal sizes and growth during annealing. Post-thaw sperm viability data indicated that samples need to be cooled below -40 °C before they can be safely plunged and stored in LN2. No negative effects of relocating specimens from dry ice to LN2 and vice versa became apparent. However, sample warming above -50 °C during transport in dry ice should be avoided to ensure preservation of viability and fertility. Moreover, addition of PVP-40 or Ficoll-70 was found to increase sperm cryosurvival, especially under non-ideal storage conditions where ice recrystallization may occur.

Two-photon excited-state dynamics of mEGFP-linker-mScarlet-I crowding biosensor in controlled environments.

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L-1) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet-I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

Minimal Coarse-Grained Model for Immunoglobulin G: Diffusion and Binding under Crowding.

Immunoglobulin G (IgG) is the most common type of antibody found in blood and extracellular fluids and plays an essential role in our immune response. However, studies of the dynamics and reaction kinetics of IgG-antigen binding under physiological crowding conditions are scarce. Herein, we develop a coarse-grained model of IgG consisting of only six beads that we find minimal for a coarse representation of IgG's shape and a decent reproduction of its flexibility and diffusion properties measured experimentally. Using this model in Brownian dynamics simulations, we find that macromolecular crowding affects only slightly the IgG's flexibility, as described by the distribution of angles between the IgG's arms and stem. Our simulations indicate that, contrary to expectations, crowders slow down the translational diffusion of an IgG less strongly than they do for a smaller Ficoll 70, which we relate to the IgG's conformational size changes induced by crowding. We also find that crowders affect the binding kinetics by decreasing the rate of the first binding step and enhancing the second binding step.

Comparison of Haemonetics Cell Saver 5+ and manual density separation for optimum depletion of red blood cells and preservation of CD34+ cells in major ABO-incompatible bone marrow grafts.

BACKGROUND AIMS: The current approach for preventing hemolysis of red blood cells (RBCs) in major ABO-incompatible bone marrow (BM) grafts after infusion is to deplete RBCs from BM products before transplantation. Traditionally, manual density separation (MDS) using Ficoll-Hypaque (Cytiva Sweden AB, Uppsala, Sweden has been used to accomplish RBC depletion. This process yields good CD34+ cell recovery, but it requires open manipulation and is labor-intensive and time-consuming. We hypothesized that an alternative automated method using Haemonetics Cell Saver 5+ (Haemonetics Corporation, Boston, MA, USA) would offer equivalent RBC depletion and CD34+ cell recovery. Small marrow volumes from pediatric donors can be processed using Cell Saver (CS) without adding the third-party RBCs necessary for other automated methods. METHODS: This retrospective analysis comprised data from 58 allogeneic BM grafts. RBC depletion and CD34+ cell recovery from BM using MDS (35 grafts) were compared with CS (14 grafts). Nine products underwent RBC depletion using CS with Ficoll (CS-F) when RBC volume was less than 125 mL. RESULTS: Linear regression analysis of log transformation of CD34+ cell recovery adjusted for log transformation of both baseline CD34+ cell content and baseline total volume showed no significant difference between MDS and CS (estimated coefficient, -0.121, P = 0.096). All products contained an RBC volume of less than 0.25 mL/kg post-processing. CD34+ cell recovery with CS-F was comparable to MDS and CS and suitable for pediatric recipients of allogeneic hematopoietic cell transplantation. CONCLUSIONS: We provide evidence that an automated method using Haemonetics Cell Saver 5+ achieves RBC depletion and CD34+ cell recovery comparable to MDS when adjusting for baseline factors.

Chemical interactions modulate λ6-85 stability in cells.

Because steric crowding is most effective when the crowding agent is similar in size to the molecule that it acts upon and the average macromolecule inside cells is much larger than a small protein or peptide, steric crowding is not predicted to affect their folding inside cells. On the other hand, chemical interactions should perturb in-cell structure and stability because they arise from interactions between the surface of the small protein or peptide and its environment. Indeed, previous in vitro measurements of the λ-repressor fragment, λ6-85 , in crowding matrices comprised of Ficoll or protein crowders support these predictions. Here, we directly quantify the in-cell stability of λ6-85 and distinguish the contribution of steric crowding and chemical interactions to its stability. Using a FRET-labeled λ6-85 construct, we find that the fragment is stabilized by 5°C in-cells compared to in vitro. We demonstrate that this stabilization cannot be explained by steric crowding because, as anticipated, Ficoll has no effect on λ6-85 stability. We find that the in-cell stabilization arises from chemical interactions, mimicked in vitro by mammalian protein extraction reagent (M-PER™). Comparison between FRET values in-cell and in Ficoll confirms that U-2 OS cytosolic crowding is reproduced at macromolecule concentrations of 15% w/v. Our measurements validate the cytomimetic of 15% Ficoll and 20% M-PER™ that we previously developed for protein and RNA folding studies. However, because the in-cell stability of λ6-85 is reproduced by 20% v/v M-PER™ alone, we predict that this simplified mixture could be a useful tool to predict the in-cell behaviors of other small proteins and peptides.

Investigating the folding dynamics of NS2B protein of Zika virus.

NS2B protein of the Zika virus acts as a co-factor for NS3 protease and also involves in remodeling NS3 protease structure. Therefore, we investigated the overall dynamics of NS2B protein. We find surprising similarities between selected flavivirus NS2B model structures predicted from Alphafold2. Further, the simulated ZIKV NS2B protein structure shows a disordered cytosolic domain (residues 45-95) as a part of a full-length protein. Since only the cytosolic domain of NS2B is sufficient for the protease activity, we also investigated the conformational dynamics of only ZIKV NS2B cytosolic domain (residues 49-95) in the presence of TFE, SDS, Ficoll, and PEG using simulation and spectroscopy. The presence of TFE induces α-helix in NS2B cytosolic domain (residues 49-95). On the other hand, the presence of SDS, ficoll, and PEG does not induce secondary structural change. This dynamics study could have implications for some unknown folds of the NS2B protein.

Diffusion NMR and Rheology of a Model Polymer in Bacterial Cell Lysate Crowders.

The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (Rg ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.

Varying molecular interactions explain aspects of crowder-dependent enzyme function of a viral protease.

Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasm's volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders also affect the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic H57 but Ficoll crowders increase substrate binding more than PEG molecules.

Contrasting effect of ficoll on apo and holo forms of bacterial chemotaxis protein Y: Selective destabilization of the conformationally altered holo form.

Chemotaxis Y (CheY), upon metal binding, displays a drastic alteration in its structure and stability. This premise prompted us to study the effect of crowding on the two conformationally distinct states of the same test protein. A comparative analysis on the structure and thermal stability in the presence and absence of the macromolecular crowder, ficoll, and its monomeric unit, sucrose, revealed a contrasting effect of ficoll on the apo and holo forms. In the presence of ficoll while the thermal stability (Tm) of the apo form is enhanced, the thermal stability of the holo form is reduced. The selective lowering of Tm for the holo form in the combined presence of ficoll and sucrose and not in sucrose alone suggests that the contrasting effect is due to the macromolecular nature of ficoll. Since metal-protein interaction remains unperturbed in the presence of ficoll and Mg2+ sequestration is ruled out in a systematic manner the alternative possibility for the exclusive reduction in the thermal stability of the holo form is the ficoll-induced modulation of the relative population of apo and holo forms of CheY.

Manufacturing of CD34 + HPC-enriched, high-purity mononuclear cell products from umbilical cord blood.

The purpose of this study was to explore methods of selectively enriching CD34 + haematopoietic progenitor cells (HPC) in mononuclear cell (MNC) preparations, and to outline a procedure for cryopreservation and thawing of manufactured material. Density gradient centrifugation of umbilical cord blood was achieved using Ficoll-Paque™ media at 1.077 g/mL and 1.065 g/mL densities and Leucosep preparation tubes. Post-process samples were analysed for CD34 + and MNC content. Finally, MNCs were frozen down at a concentration of 8.5 × 106 cells/mL in CryoStor CS10 using an Asymptote VIAFreeze controlled rate freezer at a rate of - 2 °C per minute, then thawed and analysed for viability and recovery. Processing with 1.065 g/mL media selectively depleted non-HPC cell types, producing an approximately fourfold increase in CD34 + frequency (M ± 1SD = 1.4 ± 1.3%, P < 0.01) relative to the pre-process sample (M ± 1SD = 0.4 ± 0.3%), whereas 1.077 g/mL media produced only a twofold enrichment (0.7 ± 0.6, P < 0.01). This was not accompanied by any significant forfeit of CD34 + recovery (79 ± 32% vs. 78 ± 32% respectively; P = 0.87). The MNCs generated by the 1.065 g/mL procedure were of greater purity (96 ± 2%) than in the 1.077 g/mL procedure (80 ± 7%, P < 0.01). Post-thaw, MNC viability was 95 ± 1% and CD34 + viability was 98 ± 1%. Ultra-pure MNCs rich in CD34 + HPCs can be generated with a simple, inexpensive modification to Ficoll-Paque™ media. These products can be easily cryopreserved using a simple controlled rate freezing procedure.

Brain Cancer Cell-Derived Matrices and Effects on Astrocyte Migration.

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

Phenotyping of M1 and M2a Macrophages and Differential Expression of ACE-2 on Monocytes by Flow Cytometry: Impact of Cell Culture Conditions and Sample Processing.

Macrophages are ubiquitously distributed throughout the various tissues of the body and perform many functions including the orchestration of inflammatory responses against pathogens by classically activated M1 macrophages and the regulation of wound healing and tissue remodeling by anti-inflammatory, alternatively activated M2 macrophages. The responsibility for these pleiotropic functions lies in the expression of a myriad of surface receptors unique to given subsets of macrophages. Much of what we know about the function of human macrophage subsets has been gleaned by studying in vitro generated macrophages matured in the presence of GM-CSF or M-CSF and polarized with different cytokines. Oftentimes, culture conditions, such as the type of serum used, the duration of the culture, and the use of polarizing cytokines, vary between studies making direct comparisons difficult. Sample preparation and processing (e.g., Ficoll® enrichment of leukocytes from whole blood) can also influence gene expression on human monocytes. Furthermore, overlap in surface marker expression can make it difficult to distinguish between different macrophage subsets.We directly compared the expression of over 20 different surface markers on M1 and M2a macrophages cultured in either serum-free media or in the presence of fetal bovine serum or human AB serum and found that the presence or type of serum used affected the expression of several markers such as CD200R1 and CD32. Moreover, we compared the expression of these surface markers on polarized and unpolarized macrophages and determined that polarization was critical to the expression of several of these markers including CD38 and SLAM F7. Differences in sample processing can alter the expression of surface markers, such as ACE-2, on monocytes. We observe that ACE-2 expression is higher on human whole blood CD14+ monocytes versus Ficoll®-enriched CD14+ monocytes derived from PBMCs (peripheral blood mononuclear cells), where expression can be reduced by up to 50%. These results indicate that differences in serum, culture media, and sample processing can alter gene expression in both human macrophages and monocytes. Importantly, the results of these studies significantly expand our knowledge of the phenotypic differences between human M1 and M2a macrophages and demonstrate the importance of culture conditions in generating these phenotypes.

E6020, a TLR4 Agonist Adjuvant, Enhances Both Antibody Titers and Isotype Switching in Response to Immunization with Hapten-Protein Antigens and Is Diminished in Mice with TLR4 Signaling Insufficiency.

The mechanisms by which TLR4-based adjuvants enhance immunogenicity are not fully understood. We have taken advantage of a novel knock-in mouse strain that homozygously expresses two single-nucleotide polymorphisms (SNPs) that are homologous to human TLR4 (rs4986790 and rs4986791) and have been associated with LPS hyporesponsiveness in vivo and in vitro. TLR4-SNP (coexpressing mutations D298G/N397I in TLR4) mice that recapitulate the human phenotype were compared with wild-type (WT) mice for their hapten-specific Ab responses after immunization with hapten 4-hydroxy-3-nitrophenyl acetyl (NP) NP-Ficoll or NP-OVA in the absence or presence of a water-soluble TLR4 analog adjuvant, E6020. IgM and IgG anti-NP responses were comparable in WT and TLR4-SNP mice after immunization with either NP-Ficoll or NP-OVA only. E6020 significantly yet transiently improved the IgM and IgG anti-NP responses of both WT and TLR4-SNP mice to NP-Ficoll (T-independent), with modestly enhanced Ab production in WT mice. In contrast, T-dependent (NP-OVA), adjuvant-enhanced responses showed sustained elevation of NP-specific Ab titers in WT mice, intermediate responses in TLR4-SNP mice, and negligible enhancement in TLR4-/- mice. E6020-enhanced early humoral responses in WT and TLR4-SNP mice to NP-OVA favored an IgG1 response. After a second immunization, however, the immune responses of TLR4-SNP mice remained IgG1 dominant, whereas WT mice reimmunized with NP-OVA and E6020 exhibited increased anti-NP IgG2c titers and a sustained increase in the IgG1 and IgG2c production by splenocytes. These findings indicate that E6020 increases and sustains Ab titers and promotes isotype class switching, as evidenced by reduced titers and IgG1-dominant immune responses in mice with TLR4 insufficiency.

Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress.

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

Macromolecular crowding agents enhance the sensitivity of lateral flow immunoassays.

Lateral flow immunoassays (LFIA) have a plethora of applications in health, environmental and food sectors for low-cost, simple, and rapid point-of-need testing. Typically, the user only needs to add the sample without any other intervention from sample application to results. A compelling challenge, and a constant pursuit in LFIA is to improve the assay sensitivity without compromising the simplicity and practicality. We report that the addition of water-soluble macromolecular crowding agents leads to an enhancement of the sensitivity, which is attributed to the fact that the exposure of antibodies and micro/nanoparticle conjugates to macromolecularly crowded environment, while migrating through the confining pores of the strip-pads by capillary forces, promotes the interactions that are responsible for analyte recognition and signal generation. The effect was shown by using two of the most widely established LFIA tests worldwide, that is, detection of nucleocapsid protein from SARS-CoV-2 associated with COVID-19 and detection of Strep-A antigen from Streptococcus pyogenes associated with pharyngitis. For immediate demonstration of the sensitivity enhancement, we worked directly on commercially available devices already optimized in terms of reagents and conditions. Of the crowders used, ficoll, Mr 400000, and ficoll, Mr 70000, gave a 5-10-fold improvement of the signal without affecting the background. Because the addition of macromolecular crowding agents is complementary to other strategies of sensitivity enhancement, such as the design of novel labels and the introduction of signal amplification, we anticipate that the proposed modulation will be extended to numerous analytes with a variety of reporters and LFIA configurations.

IFNγ-IL-17-IL-22+CD4+ subset and IL-22-producing cells in tumor draining lymph nodes of patients with breast cancer.

BACKGROUND: A recently introduced CD4+ T subset that mainly secretes interleukin (IL-) 22 has been reported to be associated with a variety of tumors, including colon, gastric, hepatocellular, and small- and large-cell lung carcinoma. Both tumor-promoting and - suppressing roles have been suggested for these cells. In the present study, we aimed to investigate the frequency of IL-22-producing subsets in tumor-draining lymph nodes (TDLNs) of the patients with breast cancer and determine their association with the clinicopathological characterizations of the disease. METHODS: Thirty untreated women diagnosed with breast cancer were enrolled and their axillary lymph nodes were dissected during surgery. Mononuclear cells were isolated using Ficoll density gradient, activated, permeabilized, and stained by fluorochrome-conjugated antibodies against CD4, IL-22, IL-17, and IFNγ. The cells were then acquired on the FACSCalibur flow cytometer, and raw data was analyzed by the FlowJo software package (V10). RESULTS: Our results demonstrated that 2.39% ± 0.39 of CD4+ lymphocytes in TDLNs of patients with breast cancer produced IL-22. Among them, 0.64% ± 0.8 just produced IL-22 but were negative for IFNγ and IL-17. Statistical analysis indicated that the frequency of CD4+IL-22+ cells was significantly higher in the patients with stage III and the ones with 3-9 tumor involved lymph nodes (N2) compared to those with stage II and those having 1-3 tumor involved lymph nodes (N1) (P = 0.008 and P = 0.004, respectively). CONCLUSION: The higher frequency of IL-22-producing cells in draining lymph nodes of patients with more advanced tumors (higher stage (stage III) and more involved lymph nodes) suggests a role for IL-22-producing cells in the tumor progression and invasion. However, further studies with larger sample size and more functional studies are needed to clarify the role of IL-22-producing cells in breast cancer pathogenesis.

Determination of reference intervals for neutrophil granular enzymes is affected by cell isolation techniques.

OBJECTIVES: Neutrophils and their granular enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO) play important roles in inflammatory diseases, and might be utilized as biomarkers for disease severity and progression. The aim of this study was to determine reference intervals for NE and MPO activity in healthy volunteers comparing two methods of neutrophil isolation. METHODS: Neutrophils were isolated using ficoll density gradient centrifugation or immunomagnetic negative selection in two separate volunteers' cohorts. Subsequently, cells were lysed and incubated with chromogens for NE and MPO activity measurements, then measured with a microplate reader at 415 or 450 nm respectively. RESULTS: The enzymatic activity of NE and MPO depended on the neutrophil isolation technique. Both enzymatic activities were significantly higher (P < 0.001) after isolating neutrophils with ficoll density gradient centrifugation than using the immunomagnetic negative selection. CONCLUSIONS: We demonstrated that neutrophil isolation is an important factor that influences the outcome of enzymatic activity measurements. Techniques based on immunomagnetic negative selection are favorable, specifically for investigations related to NE and MPO activity. When using NE and MPO activity measurements in clinical practice, care must be taken to interpret the data depending on the applied cell isolation technique.