Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in?Vitro and in Silico

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔH_ind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔS_ind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function.     

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔH_ind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔS_ind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Sampling Protein Form and Function with the Atomic Force Microscope

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments.To understand biological processes at the molecular level it is essential to identify the involved proteins and proteinaceous assemblies, to characterize their structure and function, and to unravel their interplay with other proteins and molecules (1). Techniques like x-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry have contributed massively to elucidate such protein properties. These techniques can easily sample the properties of a large ensemble of proteins; however, they require subjecting the sample to harsh treatments such as drying, crystallizing, or vaporizing in vacuum, thereby limiting the range of measurable dynamical properties of the sample. One powerful method that permits the investigation of molecules in their native physiological buffer condition is atomic force microscopy (AFM)¹ (2). An atomic force microscope is a microscope and force spectrometer at the same time. The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3, 4). In addition, its ability to “touch” the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5–8). However, AFM force spectroscopy is currently a technique with rather fast pulling and pushing speeds, thereby often operating out of equilibrium conditions. Improvements with ultrastable atomic force microscopes are underway to tackle this problem with promising results (9, 10). Furthermore, AFM is not well suited to apply and resolve forces at the single piconewton range due to large size tips and relatively stiff cantilevers. The issue of nonspecificity of the tip interaction with the sample is also of concern, especially in pulling experiments that require the capability to accurately recognize and select the appropriate molecule or point of interest. The current introduction of carbon nanotube tips can address the former issue (11, 12), whereas techniques in chemical functionalization can provide directed tip specificity and recognition capability (13–18), thereby further improving and widening the applicability of AFM in the future. In addition, the coupling of the atomic force microscope to fluorescence microscopes further enhances its versatility by adding (single molecule) fluorescence imaging to the AFM imaging capability (19–21), and the development of high speed systems makes it possible for AFM to probe fast dynamics of various biological processes (22–26).The applicability of AFM in proteomics is diverse and includes the characterization of the cell surface proteome (for a recent review, see Ref. 27), label-free detection and counting of single proteins (28, 29), and force spectroscopy measurements of binding and unbinding events (30, 31). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3, 6) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32, 33). These experiments show the potential of AFM to study both “form” and “function” of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously. In the following, we will explain the principles of atomic force microscopy and its different operation modes and finally discuss examples of imaging, nanoindentation, and protein (un)binding and unfolding studies using AFM.     

Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca²⁺ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.