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.

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.

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.

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.

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.

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.

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.

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.

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.

Cytokine Secretion Dynamics of Isolated PBMC after Cladribine Exposure in RRMS Patients.

Cladribine (CLD) treats multiple sclerosis (MS) by selectively and transiently depleting B and T cells with a secondary long-term reconstruction of the immune system. This study provides evidence of CLD's immunomodulatory role in peripheral blood mononuclear cells (PBMCs) harvested from 40 patients with untreated relapsing-remitting MS (RRMS) exposed to CLD. We quantified cytokine secretion from PBMCs isolated by density gradient centrifugation with Ficoll−Paque using xMAP technology on a FlexMap 3D analyzer with a highly sensitive multiplex immunoassay kit. The PBMC secretory profile was evaluated with and without CLD exposure. PBMCs isolated from patients with RRMS for ≤12 months had significantly higher IL-4 but significantly lower IFN-γ and TNF-α secretion after CLD exposure. PBMCs isolated from patients with RRMS for >12 months had altered inflammatory ratios toward an anti-inflammatory profile and increased IL-4 but decreased TNF-α secretion after CLD exposure. CLD induced nonsignificant changes in IL-17 secretion in both RRMS groups. Our findings reaffirm CLD's immunomodulatory effect that induces an anti-inflammatory phenotype.

Ficoll density gradient sedimentation isolation of pelage hair follicle mesenchymal stem cells from adult mouse back skin: a novel method for hair follicle mesenchymal stem cells isolation.

BACKGROUND: Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring. METHODS: Two-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining. RESULTS: Isolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes. CONCLUSION: A large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle.

Correlating the Local and Global Dynamics of an Enzyme in the Crowded Milieu.

Enzymes are dynamic biological macromolecules, with their catalytic function(s) being largely influenced by the changes in local fluctuations of amino acid side chains as well as global structural modulations that the enzyme undergoes. Such local and global motions can be highly affected inside the crowded physiological interior of the cell. Here, we have addressed the role of dynamic structural flexibility in affecting the activation energy barrier of a flexible multidomain enzyme adenylate kinase (AK3L1, UniProtKB: Q9UIJ7). Activation energy profiles of both local (at three different sites along the polypeptide backbone) and global dynamics of the enzyme have been monitored using solvation studies on the subnanosecond time scale and tryptophan quenching studies over the temperature range of 278-323 K, respectively, under crowded conditions (Ficoll 70, Dextran 40, Dextran 70, and PEG 8). This study not only provides the site-specific mapping of dynamics but reveals that the activation energies associated with these local motions undergo a significant decrease in the presence of macromolecular crowders, providing new insights into how crowding affects internal protein dynamics. The crowded scenario also aids in enhancing the coupling between the local and global motions of the enzyme. Moreover, select portions/regions of the enzyme when taken together can well mirror the overall dynamics of the biomolecule, showing possible energy hotspots along the polypeptide backbone.

The kinetics of islet amyloid polypeptide phase-separated system and hydrogel formation are critically influenced by macromolecular crowding.

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid-liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic-hydrophilic interfaces (e.g. air-water interface and/or phospholipids-water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Effects of macromolecular crowding on the folding and aggregation of glycosylated MUC5AC.

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

Production of quail (Coturnix japonica) germline chimeras by transfer of Ficoll-enriched spermatogonial stem cells.

Due to the absence of long-term in vitro germline competent stem cell maintenance systems and efficient methods for germline transmission, efforts to develop an effective transgenic system in quail has remained limited. To overcome this limitation, here we produced germline chimeric quails through transplantation of spermatogonial stem cells (SSCs) enriched by density gradient methods utilizing Ficoll-Paque PLUS (Ficoll), Percoll and sucrose solution as a practical strategy for germline transmission in quail. For all gradient methods, testicular cells were separated as two fractions, and the expression levels of SSC-specific genes (GFRA1, ITGA6, ITGB1) and pluripotency genes (NANOG, POUV) were examined. As a result, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA probe hybridization analysis revealed that the upper fraction that was separated by Ficoll showed the highest expression of SSC-specific and pluripotency genes among all fractions. Cells in the upper Ficoll gradient fraction also displayed reduced heterochromatin distribution, as observed in differentiated spermatogonia using transmission electron microscopy (TEM). These results indicate that SSCs were enriched in the upper fraction by Ficoll density gradient centrifugation. Subsequent transplantation experiments revealed that the efficiency of germline transmission to donor-derived gametes in the germline chimeras with transplanted SSCs and whole testicular cells was 0-13.2% and 0-4.4%, respectively. Collectively, these results demonstrate that quail SSCs were easily enriched with a density gradient method and that this method is a feasible and practical way to preserve the germplasm of quail. Furthermore, we can expect to apply this method in research examining the production of transgenic quail and preservation of avian species.

Effects of anticoagulants and Ficoll on human serum antibody reactivities and functions against Mycobacterium tuberculosis.

The ability to utilize leftover samples containing anticoagulants or Ficoll would provide substantial opportunities for future antibody and biomarker studies. Some anticoagulants might influence antibody reactivity against pathogens, but comprehensive studies investigating effects in the context of TB are lacking. We enrolled 24 individuals with and without history of M. tuberculosis and/or HIV-infection and investigated TB antibody reactivities, function, and other host protein biomarkers in simultaneously obtained serum and plasma from serum separation, EDTA, heparin, acid citrate dextrose (ACD), or mononuclear cell preparation (CPT™) tubes which contain heparin and Ficoll. Antibody isotype reactivities to two mycobacterial antigens, as well as phagocytosis of M. tuberculosis, correlated strongly and significantly between serum and plasma, irrespective of type of anticoagulant or Ficoll present (r ≥ 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of containing anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies.

Macromolecular Crowding Increases the Affinity of the PHD of ING4 for the Histone H3K4me3 Mark.

The five members of the family of tumor suppressors ING contain a Plant Homeodomain (PHD) that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3) with an affinity in the low micromolar range. Here, we use NMR to show that in the presence of 15% Ficoll 70, an inert macromolecular crowding agent, the mode of binding does not change but the affinity increases by one order of magnitude. The affinity increases also for unmethylated histone H3 tail, but the difference with H3K4me3 is larger in the presence of Ficoll. These results indicate that in the cellular milieu, the affinity of the ING proteins for their chromatin target is larger than previously thought.

Ras isoforms selectively regulate antigen-specific immune response.

H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.

Discarded portions of bone marrow aspirates are a new resource for patient-derived mesenchymal stem and stromal cells.

Bone marrow-derived mesenchymal stem and stromal cells (BM-MSCs) protect malignant cells from chemotherapy and are important potential therapeutic targets. Isolating primary BM-MSCs for research traditionally requires the sacrifice of valuable cell populations from within the same sample. To avoid this, we report here a resource for isolating patient-derived BM-MSCs from the red blood cell layer of ficoll gradients of bone marrow aspirates, a resource that has until now been universally discarded. This resource yields BM-MSCs nearly identical to those obtained conventionally and includes cells with a more stem-cell like nature. Obtaining primary BM-MSCs in this way will likely expand opportunities to study this important cell population.