Homogeneous nuclei-induced, secondary nuclei-induced, and spontaneous whey protein concentrate nanofibril formation through different pathways.

The addition of homogeneous nuclei (HN) or secondary nuclei (SN) could lead to different kinetics and thermodynamics as the nucleation energy barrier decreases and the lag time is shortened to different degrees compared with spontaneous fibrillation. To explain these differences, we monitored the formation and depletion of HN during fibril formation and found that both SN-induced fibrils and HN-induced fibrils follow the same nucleated growth pathway as spontaneously formed WPC fibrils. Moreover, there were also other paths, which were confirmed by X-ray diffraction, transmission electron microscopy, and atomic force microscopy. The surfaces of the SN could recruit monomers and resulted in stronger intersheet stacking and a larger fibril height and periodicity. The HN incorporation led to a propensity for hydrogen-bonding interactions and a longer fibril. Fibrillation by the addition HN and SN followed both common and distinct pathways, as spontaneous fibrillation and led to different capacities to induce fibrillation.

Investigation of morphological changes of HPS membrane caused by cecropin B through scanning electron microscopy and atomic force microscopy.

BACKGROUND: Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. OBJECTIVES: This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. RESULTS: Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 µg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. CONCLUSIONS: Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Influences of bioapatite mineral and fibril structure on the mechanical properties of chicken bone during the laying period.

Laying hens suffer from osteoporosis during their laying period, which causes bone fragility and susceptibility to fracture. This study evaluated the changes of mechanical properties of their bones during the laying period (from 18 to 77 wk) by using nano-indentation, atomic force microscope, X-Ray diffraction, and Raman spectroscopy. Results indicated that the crystallite sizes of bioapatite in femur decreased significantly from 34.45 to 29.26 nm during aging from 18 to 49 wk. Then, the value increased to 37.79 nm at 77 wk. Despite the abundance in bone (usually >50 wt.%), bioapatite mineral content showed no continuous enhancement during aging. The fibrils demonstrated more regular and organized structure during the laying period. Meanwhile the elastic moduli (E) and hardness (H) of femur increased from 10.84 to 18.39 GPa and 43.79 to 97.21 Vickers respectively during this period. The changes in mechanical properties are hence tightly related to the structure of bone (composed of both collagen and mineral), rather than directly related to the mineralogical properties of bone bioapatite. This study addressed the importance of the interaction between collagen and bioapatite mineral during the laying period of hens by microscopic, physicochemical, and mechanical analysis.

Nanostructural basis for the gloss of chicken eggshells.

The nanostructure greatly contributes to eggshell formation, the mechanical properties of eggshells, and mineral dissolution during incubation. In this study, to investigate the effect of the nanostructure on the gloss of eggs, the gloss and eggshell quality (cuticle coverage, color, and thickness) of 105 eggs were measured. According to the order of the gloss, the surface roughness of 30 high-gloss and 30 low-gloss eggs was compared. The gloss had no significant correlation with the eggshell color and thickness (P > 0.05) and a significant relationship with the cuticle coverage (r = 0.19, P < 0.01). The surface roughness significantly differed between the high- and low-gloss eggs (P < 0.001), and the gloss was negatively correlated with the surface roughness (r(high-group) = -0.61, r(low-group) = -0.56, P < 0.01). The shell gloss of 30 oiled eggs with mineral oil and 30 normal eggs from commercial brown-egg layers was also compared. The oil coating increased the eggshell gloss, but the roughness was unchanged. This is the first report to establish the contribution of nanostructure for the gloss of chicken eggshell. The surface roughness can be used as an indicator of the gloss, which could be helpful for selective breeding to improve the eggshell brightness. Our research also provides the foundation for further investigation of the effect of non-pigmentary contributors on the chicken eggshell appearance.

Descriptions of Acanthocephalus parallelcementglandatus (Echinorhynchidae) and Neoechinorhynchus (N.) pennahia (Neoechinorhynchidae) (Acanthocephala) from amphibians and fish in Central and Pacific coast of Vietnam, with notes on N. (N.) longnucleatus.

Three species of acanthocephalans are described from fishes caught in the Pacific coast off eastern Vietnam and from amphibians in the midlands in 2016: (1) Acanthocephalus parallelcementglandatus Amin, Heckmann, Ha, 2014 (Echinorhynchidae), described from 1 male specimen is now fully described from males and females collected from 2 species of amphibians, the similar frog Hylarana attigua Inger, Orlov, Darevsky and the odorous frog Odorrana sp. Fei, Ye, Huang (Ranidae) in Huong Thuy, Hue City and Chu Yang Sin Park, central Vietnam, respectively, as well as from the needlefish Tylosurus sp. Cocco (Belonidae) in Binh Thuân in the Pacific South. The allotype female is designated. Neoechinorhynchus (N.) pennahia Amin, Ha, Ha, 2011 described from 1 female specimen is now fully described from males and females collected from the Toli shad (Chinese herring), Tenualosa toli (Valenciennes) (Clupeidae) in the Pacific north coast off Haiphong. The allotype male is designated. One specimen of Neoechinorhynchus (Neoechinorhynchus) longnucleatus Amin, Ha, Ha, 2011 is also reported from the common ponyfish, Leiognathus equulus (Forssskål) (Leiognathidae) in the Pacific south coast of Nha Trang and its ecology briefly discussed.

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm.

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.

Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy.

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Investigation of techniques for the measurement of articular cartilage surface roughness.

Articular cartilage is the bearing surface of synovial joints and plays a crucial role in the tribology to enable low friction joint movement. A detailed understanding of the surface roughness of articular cartilage is important to understand how natural joints behave and the parameters required for future joint replacement materials. Bovine articular cartilage on bone samples was prepared and the surface roughness was measured using scanning electron microscopy stereoscopic imaging at magnifications in the range 500× to 2000×. The surface roughness (two-dimensional, R(a), and three-dimensional, S(a)) of each sample was then measured using atomic force microscopy (AFM). For stereoscopic imaging the surface roughness was found to linearly increase with increasing magnification. At a magnification of 500× the mean surface roughness, R(a), was in the range 165.4±5.2 nm to 174±39.3 nm; total surface roughness S(a) was in the range 183-261 nm. The surface roughness measurements made using AFM showed R(a) in the range 82.6±4.6 nm to 114.4±44.9 nm and S(a) in the range 86-136 nm. Values obtained using SEM stereo imaging were always larger than those obtained using AFM. Stereoscopic imaging can be used to investigate the surface roughness of articular cartilage. The variations seen between measurement techniques show that when making comparisons between the surface roughness of articular cartilage it is important that the same technique is used.

Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China.

BACKGROUND: Orf is a zoonotic and epitheliotrophic contagious disease that mainly affects sheep, goats, wild ruminants, and humans with a worldwide distribution. To date, there is little information on the characterization of ORFV strains that are endemic in Mainland China. In addition, the relationship between the severity of disease and the molecular profile of ORFV strains has not been fully elucidated. RESULTS: From the recent outbreak of a sheep herd in Nongan, northeast of China, the novel orf virus (ORFV) strain NA1/11 was successfully isolated. Western blot analysis indicated that the NA1/11 strain cross reacts with monoclonal antibody A3 and infected sheep ORFV antiserum. The purified virions revealed the typical ovoid shape when observed by atomic force microscopy. To determine the genetic characteristics of the NA1/11 strain, the sequences of ORFV011 (B2L), ORFV059 (F1L), ORFV109, ORFV110 and ORFv132 (VEGF) genes were amplified and compared with reference parapoxvirus strains. Non-metric multidimensional scaling (nMDS) was performed to analyze the nucleotide similarities between different ORFV strains. CONCLUSIONS: Phylogenetic analysis based on ORFV 011 nucleotide sequences showed that the NA1/11strain was closely related to Xinjiang and Gansu strains. ORFV110 and ORFV132 genes are highly variable. The results revealed that precise phylogenetic analysis might provide evidence for genetic variation and movement of circulating ORFV strains in Northeast China. In addition, nMDS analysis showed that geographic isolation and animal host are likely major factors resulting in genetic differences between ORFV strains.

Structure and composition of the septal nacreous layer of Nautilus macromphalus L. (Mollusca, Cephalopoda).

The nacreous layer of Mollusca is the best-known aragonitic structure and is the usual model for biomineralization. However, data are based on less than 10 species. In situ observations of the septal nacreous layer of the cephalopod Nautilus shell has revealed that the tablets are composed of acicular laths. These laths are composed of round nanograins surrounded by an organic sheet. No hole has been observed in the decalcified interlamellar membranes. A set of combined analytical data shows that the organic matrices extracted from the nacreous layer are glycoproteins. In both soluble and insoluble matrices, S amino acids are rare and the soluble organic matrices have a higher sulfated sugar content than the insoluble matrices. It is possible that the observed differences in the structure and composition of the nacreous layers of the outer wall and septa of the Nautilus shell have a dual origin: evolution and functional adaptation. However, we have no appropriate data as yet to answer this question.