Wet spinning of silk polymer. II. Effect of drawing on the structural characteristics and properties of filament

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The rheological behavior of the SF dope solution prepared with formic acid was examined and the drawing effect on the structural characteristics and mechanical properties of SF filament was comparatively studied with those of natural silk fiber. SF dope exhibited shear thinning, but, as the dope concentration increased, the effect of shear thinning decreased, an indication that a higher concentration of dope solution will result in good spinnability. Wet-spun SF filaments exhibited a uniform and circular cross-sectional shape and dense morphology under SEM observation. X-ray diffraction (XRD) results revealed that the crystallinity of wet-spun regenerated filaments was hardly affected by the draw ratio, whereas the crystalline and amorphous orientation of regenerated SF filament showed different features depending on the drawing. The crystalline orientation of regenerated filaments increased with an increase of draw ratio and was lower than that of natural silk fiber. On the contrary, the amorphous orientation was constant throughout 1X-5X draw ratios, after an abrupt increase at 1X, and was higher than that of natural silk fiber. These differences in the orientation behaviors are attributed to the different spinning mechanisms involved. The tensile property was strongly dependent on the draw ratio. The breaking strength and elongation of the regenerated filament at 5X draw ratio were 2.2 g/day and 17%, respectively.     

Preparation and characterization of wet spun silk fibroin/poly(vinyl alcohol) blend filaments

Silk fibroin (SF)/poly(vinyl alcohol) (PVA) blend filaments were prepared by a wet spinning process. Regenerated SF and PVA were dissolved in formic acid and the dope solution exhibited good fiber formation in a methanol coagulation bath. Due to the miscibility of SF/PVA in formic acid, the filament had a smooth surface and dense structure with a circular cross-section. The crystalline structure and thermal properties were varied with different SF/PVA ratios. The mechanical properties of the filament were also controlled by blending PVA with SF. Especially, the knot strength of the SF filament, which is a very important suture property, could be significantly improved by blending with PVA.     

Porosity-geometry interaction in the conservation of bone strength

Automated image analysis of a small sample of femoral cross-section radiographs has revealed a consistent difference in bone porosity relative to geometry. Two sub-periosteal fields were assessed microscopically, with field location determined with reference to the principal moment axes (Imax, Imin). The data indicate that: (1) porosity is greatest in the direction of maximum geometric resistance to bending, along the Imin axis; and (2) porosity differences between the Imax and Imin fields decrease as the bone becomes more circular in cross-sectional shape.     

Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 degrees C and a cold crystallization peak appeared at 35 degrees C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 degrees C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 degrees C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19mum, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E=8.7 GPa, sigma(b)=120 MPa and epsilon(b)=35%.     

Effect of increased gas density on pulmonary gas exchange in man.

Pulmonary gas exchange was measured in seven resting supine subjects breathing air or a dense gas mixture containing 21% O2 in sulfur hexafluoride (SF6). The mean value of the alveolar-arterial oxygen difference (AaDO2) decreased from 12.4 on air to 7.0 on SF6 (P less than 0.01), and increased again to 13.4 when air breathing resumed (P less than 0.01). No differences occurred between gas mixtures for O2 consumption, respiratory quotient, minute ventilation, breathing frequency, heart rate, or blood pressure, and the improved oxygen transfer could not be attributed to changes in cardiac output or mixed venous oxygen content in the one subject in which they were measured. These results are best explained by an altered distribution of ventilation during dense gas breathing, so that the ventilation-perfusion ratio (VA/Q) variance was reduced. Of several considered mechanisms, we favor one in which SF6 promotes cardiogenic gas mixing between peripheral parallel units having different alveolar gas concentrations. This mechanism allows for observed increases in arterial carbon dioxide tension and dead space-to-tidal volume ratio during dense gas breathing, and suggests that intraregional VA/Q variance accounts for at least one-half of the resting AaDO2 in healthy supine young men.     

Pre-exposure to UV irradiation increases the transfer frequency of the IncJ conjugative transposon-like elements R391, R392, R705, R706, R997 and pMERPH and is recA+ dependent

The enteric conjugative transposon-like IncJ elements R391, R392, R705, R706 and pMERPH, all demonstrated increased conjugative transfer upon UV irradiation. The transfer frequency increased on average from its basal rate of 10(-5) to 10(-3) per recipient, upon pre-exposure to UV irradiation. However, the transfer frequency of R997, which was higher than the other IncJ elements at 10(-3) per donor, showed a smaller increase. This effect was shown to be recA+ dependent in all cases. Using PCR primers directed outwards from the ends of the integrated R391 element it was observed that a circular intermediate of the element forms within the host, which has been proposed to be a transfer intermediate. Using real-time PCR, it was determined that the amount of the circular intermediate produced increased substantially upon UV irradiation.     

Separation of Alcaligenes eutrophus cells containing Poly(3-hydroxybutyrate) from fermentation broth with pretreatment using Al- and Fe-based coagulants

Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants. Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6-5.6 for the Al-based coagulants and about 5-8 for the Fe-based coagulants. Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration. In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also. The coagulant requirement increased with the cell concentration regardless of coagulant type. The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate. The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5-210 g/L. It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation.     

Role of stress fibers in the association of intermediate filaments with microtubules in fibroblast cells.

The association between intermediate filaments (IF) and microtubules (MT) has been demonstrated by several experiments using MT inhibitors and by microinjecting specific antibodies. The actin cytoskeleton has recently been assigned a role in this process of drug induced IF collapse. However, this was not found to be true in large cells with irregular morphology. For instance, in early passage diploid fibroblasts of human origin and in armadillo cell lines, where the cells are large, irregular in shape and exhibit prominent stress fibers ( SF ), depolymerization of MT with nocodazole did not lead to collapse of IF . Instead, the IF formed bundles of coils that seemed to associate with the SF . Disintegration of the SF with cytochalasin B led to the collapse of the IF . It appears that the actin organization in such large cells with extensive SF , is not as contractile as in typical spindle shaped fibroblasts which have relatively less stable actin organization. The stable SF may actually prevent IF collapse.     

Direct Observation of Subunit Exchange along Mature Vimentin Intermediate Filaments

Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament.     

Determination of very thin pili by the bacterial drug resistance plasmid R485.

The IncX bacterial drug resistance plasmid R485 was found by electron microscopy to determine numerous very thin filaments (designated 485 pili) only 5.0 nm thick. They exhibited a characteristic helical structure (pitch, 4.6 nm), and were able to form large pseudocrystals when detached from the cell. The concomitant transfer of both pili and the sulfonamide resistance determinant of R485 to RecA strains of Escherichia coli confirmed that the pilus determinant was part of the plasmid and had not been mobilized from the chromosome of the host strain. An extensive examination failed to reveal any similar pili on strains carrying the IncX type plasmid R6K.     

The effect of swimming activity on bone architecture in growing rats

The effect of non-habitual physical activity on bone architecture in the rat humeral shaft was examined. Two groups of rats were trained to swim for 1 h a day, for 20 weeks, at two training levels. The control group consisted of sedentary rats. Parameters of cross-sectional bone morphology (cross-section areas, principal area moments of inertia and their ratio) were used to evaluate the response of bone architecture to mechanical loading. The strength of bone was assessed by measuring the ultimate compressive force and stress. The cortical cross-section area and principal moments of inertia were found to be significantly higher in the swimming groups than in the controls. Examination of the ratio between the major and minor moments of inertia revealed a pronounced change in the shape of the bone cross-section which became more rounded following swimming training. The ultimate compressive force was significantly higher in the swimming rats while the changes in ultimate stress were not significant. Our results indicate a gain of bone strength due to increased periosteal apposition and modified bone tissue distribution. The marked changes in bone morphology are attributed to the different nature of the forces and moments exerted on the humerus during swimming compared to those prevailing during normal locomotion.     

Influence of glottic aperture on the tracheal flow

The extra-thoracic mouth-throat area has a major influence on the aerosol delivery to the proximal or peripheral intra-thoracic airways. To characterize the particle deposition in this area, it is important to investigate first the flow structures in this crucial--in relation to the aerosol deposition--region. The glottis, which is delimited by the vocal cords and therefore has the narrowest passage, generates a laryngeal jet and a reverse flow downstream the glottis. It is generally assumed that the glottis has different shapes and cross-sectional areas at different moments during the respiratory cycle and also depends on the average inspiratory flow rate. Therefore, the influence of a circular glottal aperture, with a cross-sectional area of 90 mm2 and an elliptical and triangular shape, both with an area of 45 mm2, on the flow is investigated. However, the area of the circular aperture is twice as big as the area of the elliptical one, it has almost no influence on the flow structures. On the other hand, the triangular glottal aperture shifts the laryngeal jet in the direction of the posterior wall, and generates two pairs of counter rotating secondary vortices downstream the glottis, where the circular and elliptical only aperture generates one pair of vortices. The difference in pressure drop is more dominated by the cross-sectional area than by the shape of the glottis. This suggests the need for rendering geometry of future upper airway models even more realistic as the appropriate three-dimensional (3D) medical imaging techniques are becoming available.     

The elemental composition, the microfibril angle distribution and the shape of the cell cross-section in Norway spruce xylem

Relationships between the elemental composition, the microfibril angle (MFA) distribution and the average shape of the cell cross-section of irrigated-fertilised and untreated Norway spruce (Picea abies [L.] Karst.) earlywood were studied. Sample material was obtained from Flakaliden, Sweden. The elemental composition was studied by determining the relative mass fractions of the elements P, S, Cl, K, Ca and Mn by X-ray fluorescence and by determining the mass absorption coefficients for X-rays. X-ray diffraction was used to determine the MFA distribution and the average shape of the cell cross-section. The latter was also determined by light microscopy. In transition from juvenile wood to mature wood, a decrease of the mode of the MFA distribution from 13°–24° to 3°–6° was connected to a change in the shape of the cell cross-section from circular to rectangular. The irrigation-fertilisation treatment caused no change in the MFA distribution or in the shape of the cell cross-section, whereas the mass absorption coefficient was higher and the density was smaller in irrigated-fertilised wood. Larger proportion of the elements S, Cl and K, but smaller proportion of the element Mn, were observed due to the treatment. The results indicate that the shape of the cell cross-section or the MFA distribution are not directly linked to the growth rate of tracheids or to the nutrient-element content in the xylem and only show notable changes as a function of the cambial age.     

Interactive effects of serum ferritin and high sensitivity C-reactive protein on diabetes in hypertensive patients

BackgroundHypertensive patients, often characterized by chronic inflammation, are susceptible to diabetes. Evidence suggests that the positive association between serum ferritin (SF) and diabetes was affected by high-sensitivity C-reactive protein (hs-CRP), an inflammation marker. We investigate whether there was an interaction between SF and hs-CRP on diabetes in hypertensive patients.MethodsWe analysed data of 1,735 hypertensive people in this cross-sectional study. Diabetes was diagnosed when fasting blood glucose ≥ 7.0 mmol/L and/or a previous clinical diagnosis of diabetes. Logistic regression models were used to estimate the association of the SF and hs-CRP with diabetes. Multiplicative interaction was evaluated by incorporating a cross-product term for SF and hs-CRP to the logistic regression model. Additive interaction was assessed by calculating the relative excess risk of interaction (RERI) and attributed proportion due to interaction (AP).ResultsIn the adjusted analysis, SF (highest vs lowest tertile: odds ratio [OR], 1.61; 95 % confidence interval [CI], 1.20−2.16) was positively associated with diabetes. There was no multiplicative interaction between SF and hs-CRP, but evidence of additive interaction in regard to diabetes (RERI: 0.86; 95 % CI: 0.06−1.67). Compared to the patients with low SF (lower two thirds) and low hs-CRP (≤ 2 mg/L), those with high SF (upper one third) and high hs-CRP (> 2 mg/L) had increased OR for diabetes (adjusted OR: 2.33 [1.65−3.30]), with 37.0 % of the effects attributed to the additive interaction (AP: 0.37; 95 % CI: 0.09−0.65).ConclusionsWithin a cross-sectional study consisting of hypertensive patients, co-exposure to high SF and high hs-CRP was synergistically associated with diabetes. Dietary intervention or pharmacological treatment to lowering SF concentration may help to reduce diabetes morbidity in hypertensive patient with chronic inflammation.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

Effects of storage conditions of Moringa oleifera seeds on its performance in coagulation

Moringa oleifera is a plant whose seeds have coagulation properties for treating water and wastewater. In this study the coagulation efficiency of Moringa oleifera kept in different storage conditions were studied. The Moringa oleifera seeds were stored at different conditions and durations; open container and closed container at room temperature (28 degrees C) and refrigerator (3 degrees C) for durations of 1, 3 and 5 months. Comparison between turbidity removal efficiency of Moringa oleifera kept in refrigerator and room temperature revealed that there was no significant difference between them. The Moringa oleifera kept in refrigerator and room temperature for one month showed higher turbidity removal efficiency, compared to those kept for 3 and 5 months, at both containers. The coagulation efficiency of Moringa oleifera was found to be dependent on initial turbidity of water samples. Highest turbidity removals were obtained for water with very high initial turbidity. In summary coagulation efficiency of Moringa oleifera was found independent of storage temperature and container, however coagulation efficiency of Moringa oleifera decreased as storage duration increased. In addition, Moringa oleifera can be used as a potential coagulant especially for very high turbidity water.     

Colour removal from landfill leachate by coagulation and flocculation processes

A study was conducted to investigate the efficiency of coagulation and flocculation processes for removing colour from a semi-aerobic landfill leachate from one of the landfill sites in Malaysia. Four types of coagulant namely aluminium (III) sulphate (alum), ferric (III) chloride, ferrous (II) sulphate and ferric (III) sulphate were studied using standard jar test apparatus. Results indicated that ferric chloride was superior to the other coagulants and removed 94% of colour at an optimum dose of 800 mg/l at pH 4. The effect of coagulant dosages on colour removal showed similar trend as for COD, turbidity and suspended solids. This suggested that colour in landfill leachate was mainly contributed by organic matters with some insoluble forms that exhibited turbidity and suspended solids readings. The results from this study suggested that ferric chloride could be a viable coagulant in managing colour problems associated with landfill leachate.     

Characterization of distinct early assembly units of different intermediate filament proteins

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.     

Static suppression of tomato bacterial wilt by bacterial coagulation using a new functional polymer that coagulates bacterial cells and is highly biodegradable

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.     

Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid

Structural characteristics and thermal and solution properties of the regenerated silk fibroin (SF) prepared from formic acid (FU) were compared with those of SF from water (AU). According to the turbidity and shear viscosity measurement, SF formic acid solution was stable and transparent, no molecular aggregations occurred. The sample FU exhibited the beta-sheet structure, while AU random coil conformation using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry. The effects of methanol treatment on samples were also examined. According to the measurement of crystallinity (XRD) and crystallinity index (FTIR), the concept of long/short-range ordered structure formation was proposed. Long-range ordered crystallites are predominantly formed for methanol treated SF film while SF film cast from formic acid favors the formation of short-range ordered structure. The relaxation temperatures of SF films measured by dynamic thermomechanical analysis supported the above mechanism due to the sensitivity of relaxation temperature on the short-range order.