The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. The synthesis is shown to be highly sensitive to conditions, Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications. The use of silica as a structural support for industrial catalysts is well established, allowing for the improved catalyst activity, stability and processability,234Enzymes are typically attached via physisorption or covalent bonding, in addition to encapsulation within a porous material.6in situ encapsulation of biomolecules and other active species during the material synthesis.78711Recent developments in the field of milder (often dubbed 'bioinspired') silica syntheses have established the The goal of the method described herein is to prepare a porous silica with controllable properties, under ambient conditions, by using a bioinspired organic additive. The method can be easily modified to include encapsulation of either inorganic or bioorganic molecules, a selection of which shall be shown. We further show a facile method for modifying the as-synthesised materials to achieve desired bulk properties and purification by removing the organic template through acid elution.e.g.,silica materials templated through supramolecular surfactant assemblies like MCM-41 or SBA-15)in situ encapsulation without the need for numerous immobilization steps and laborious purification. Furthermore, the use of acid elution rather than calcination opens the possibility of organic surface functionalization.Compared to the traditional synthesis of templated porous silica supports , and dissolve in 20 mL of deionized water.e.g.,diethylenetriamine (DETA) or triethylenetetraamine (TETA), ensure that the total Si:N mole ratio remains constant at 1 When using alternative amine-containing compounds e.g.,poly(ethyleneimine) (PEI) or poly (PAH), maintain a concentration of 1 mg/mL When using polymeric amine additives Similarly, in a second container, measure 0.25 mmol of pentaethylene hexamine  and dissolve in 20 mL of deionized water. in situ encapsulation during synthesis, dissolve a pre-determined mass of protein  in 5 mL of deionized water. Subtract this amount of water from the volume of deionized water to be used for the dissolution of sodium silicate pentahydrate. To ease the protein dissolution without altering its structure, once mixed with deionized water, cap the container and store at 4 °C. Check occasionally on the dissolution progress, preferably without stirring.To perform in situ encapsulation is omitted).Combine the solutions of sodium silicate pentahydrate and PEHA in one of the 180 mL container and add sufficient deionized water to make the final solution volume 41 mL  as quickly as possible. NOTE: The final volume given these quantities is 50 mL of total reaction mixture, leading to Si and N concentrations of 30mM. This can be scaled as desired by multiplying all above quantities by a constant amount.Record the pH after 5 min to determine the reaction completion; ensure that the pH is 7 ± 0.05.Modify the composition of produced silica after the reaction has reached completion (either as an as-made coagulum or by resuspending a previous synthesized sample of silica) by the addition of further acid.If resuspending silica, mix approximately 150 mg as-prepared bioinspired silica with 100 mL of deionized water in a 180 mL plastic container, and place on the top of a stirrer plate.Once the suspension is well mixed, suspend a pH probe in the vessel.Titrate in further HCl until the desired pH (between 7 and 2) has been reached and allow to stabilize for ca. 1 min.Wait a further 5 min to ensure the system has fully equilibrated, and then proceed to isolate the solid silica.Decant the bioinspired silica suspension into 50 mL centrifuge tubes.Centrifuge the suspension at 5,000 g for 15 min.e.g., Bradford assay, see below). Refill the centrifuge tubes with deionized water, and re-suspend the silica using a vortex mixer.Remove the supernatant after centrifugation and store for further analysis  ammonium molybdate tetrahydrate in a fume cupboard.Dissolve this in 500 mL deionized water under stirring.Acidify the solution by carefully adding 60 mL of 10 M HCl solution.Adjust the final volume to 1 L.Place a 500 mL glass volumetric flask in a water bath at ambient temperature on a stirrer plate in a fume- cupboard.Add 111 mmol (10 g) of anhydrous oxalic acid, 19.5 mmol (3.35 g) of para-aminophenol sulfate, and 16 mmol (2 g) of sodium sulfite, and dissolve in 250 mL water.Carefully and slowly add 92 g (50 mL) of saturated sulfuric acid while stirring and wait for the solution to cool.Finally, dilute to 500 mL with deionized water.In a 5 mL plastic vial, dilute 300 µL of MBR produced in step 5.4 with 3 mL of deionized water.Add 10 µL of a silicic acid test solution and shake to mix. NOTE: This solution will slowly turn yellow.After exactly 15 min, add 1.6 mL of the reducing agent prepared from section 6 to reduce the yellow silicomolybdate complex to its blue isomer.Allow a blue color to develop for at least 2, but not more than 24 h.Measure sample absorbance at 810 nm in a UV-vis spectrophotometer and calculate [Si] against a calibration curve.To measure the concentration of polysilicate species using the molybdenum blue method, in a microcentrifuge tube, combine 750 µL of 2 M sodium hydroxide solution with 750 µL silica suspension.Ensure sufficient headspace is left in the tube to prevent bursting due to pressure build up. NOTE: A headspace of 500 µL is usually sufficient to avoid this. Alternatively, the procedure can be carried out in open vials so long as liquid loss due to evaporation is accounted for.Seal and place in a microcentrifuge float. Float the microcentrifuge tubes in a water bath heated to 80 °C and leave it to dissolve for 1 h.After 1 h has elapsed, remove the microcentrifuge tubes and wipe the outside dry.Once cooled, [Si] can be determined as described above as described in steps 7.2 to 7.5.Table 1 and Table 2 for specific volumes). Use disposable pipette tips for every cuvette to avoid volume alterations due to the nature of the reagent and repeat each point in triplicate.Insert a predetermined amount of (room temperature) Bradford reagent and sample in each assigned cuvette .Create a calibration curve for each set of experiments by plotting measured absorbance against concentration of BSA to avoid random fluctuations that might affect the assay's sensitivity.Although this protein assay is meant to use BSA as a standard to quantify any type of protein, create a calibration curve for each specific protein of interest for improved accuracy.If the protein content of the unknown sample is expected to be higher than the covered range of the calibration curve, dilute it as needed.Calculate the protein concentration of Unknown sample using a calibration curve . In caseDetermine protein content for each sample during re-suspension to monitor possible protein loss.The techniques described above are able to consistently and reproducibly precipitate silica. This is easiest to determine by the rapid onset of turbidity within the reaction vessel, which upon cessation of agitation will spontaneously settle into a thick coagulum of precipitated silica . The extFurther insight into the reaction progression can be generated by adapting the molybdenum blue spectroscopic method to detect the amount of unreacted monomeric silicate species as well as those species which have reacted to form polysilicates or 'oligomers', but have not managed to reach sufficient size to coagulate , there is a clear difference in the amount of oligomeric silica species which precipitate in each case. This is in agreement with traditional theory on sol-gel silicas - in the 'undershoot' case the pH is held higher for longer, allowing for individual particles to grow and hence aiding efficient coagulation; in the 'overshoot' case the coagulation is induced much faster due to the rapid titration, hence fewer of the silica species have grown to a sufficient size to coagulate and remain trapped in the colloid phase.16a priori knowledge of the appropriate titration volume is essential. Although not extractable from the reaction stoichiometry due to the complex protonation behavior of the amine additives and change in silica surface acidity on coagulation, highly reliable empirical relationships between system contents, concentrations and titer volumes are readily generated .Once coagulation has been completed, material surfaces can be readily modified through the use of acid elution, as has recently been reported by the authors elsewhere.In this study, BSA was used as an exemplar encapsulant enzyme, however, the techniques described here can be used for multiple enzymes17There are three types of assay format, depending on the sample volume, the expected amount of protein to be detected and the measurement method used. Herein, the followed format is specified for a spectrophotometer, requires disposable cuvettes of macro and of micro size and can detect from 10 µg/mL to 1.4 mg/mL of protein.In e.g.,BSA addition during mixing of the silicate and the additive solutions, prior to neutralization or enzyme added to the silicate or additive solution before their mixing and neutralization. Some of these possibilities were explored further and the immobilization efficiencies  and the amount of BSA in the final silica were measured , there are other possible variations Table 1: Macro Bradford assay set-up and calculated component volumes. Valid for determination range 0.1-1.4mg/mL (volumes for 1 replicate)Table 2: Micro Bradford assay set-up and calculated component volumes. Valid for determination range 1-10 µg/mL (volumes for 1 replicate): Required titer volume against silica concentration for reaction systems using either DETA or PEHA as the additive. Silica was synthesized at varying concentrations while maintaining an [N]:[Si] ratio of 1, for two different additive chemicals. Error bars are one standard deviation around the mean.: Photographs of silica coagulum in the reaction vessel (a) during and (b) after agitation, demonstrating the solution turbidity and settling that are indicative of an optimal reaction. : Exemplar calibration curve for Bradford macro assay. Supernatant from bioinspired silica synthesis in absence of BSA is mixed with a known amount of the protein, after which Bradford analysis is performed as described in step 9.1.: Final polymerization states of silica species for different reaction conditions. Silica is synthesized using optimal (baseline) conditions, as well as with over- or under-titration, after which relative silica concentration is quantified for monomeric or dimeric silicates (red), polysilicate 'oligomers' (blue) and unstable coagulating silica (yellow).:Progression of pH through reaction system as a function of initial titer volume. Acid is immediately dosed after ca. 38s of mixing, causing the pH to rapidly drop to below 8. Afterward, further quantities of acid are automatically dosed such that the pH was 7.0 ± 0.05 300s after initial addition. In the case of over-titrating, this was not achievable, as the initial dose was sufficient to drop the pH below 7, reaching pH 6.65 after 300s. Initial HCl volume added for 'undershoot,' 'baseline,' and 'overshoot' was 6.90, 7.05, and 7.20mL respectively. : Representative property changes upon acidification of coagulated silica material. (a) Change of additive concentration with respect to pH, and (b) change of silica porosity with respect to pH. Reproduced from Manning et al. : BSA concentration in bioinspired silica synthesis supernatants. Bradford assays were carried out on reaction supernatants after centrifugation, from which the relative amount remaining (therefore occluded from the synthesized silica) was determined. : FTIR analysis on bioinspired silica with and without active species encapsulation. Spectra showed: black line: bioinspired silica, gray line: pure BSA, blue line: bioinspired silica loaded with BSA. Vertical dashed lines indicate characteristic amide bands. :Immobilization efficiency and the amount of BSA in the composite for silica produced using PEHA. BSA was added (A) in the PEHA solution before mixing with silicate, (B) in the silicate solution before mixing with PEHA, (C) after initial mixing of PEHA and silicate solutions, and (D) after mixing PEHA and silicate solutions and neutralizing. Efficiency is measured as% BSA encapsulated from the reaction mixture as a proportion of total BSA added, while BSA in silica signifies% concentration of BSA in final silica composite by mass. Error bars are one standard deviation around the mean.In the current work, we present a method for rapidly precipitating bioinspired silica materials and encapsulation of biomolecules therein. We demonstrate critical steps within the procedure, namely the amount of reaction-initiating acid to be added, and timing of addition of the biomolecule encapsulant. We show the effect of acid addition amount on both reaction progression and yield , the polyfunctional nature of bioinspired additives cannot produce ordered pore structures, nor highly monodisperse particle-size distributions characteristic of Stöber-type silica.26On the other hand, this polyfunctional additive nature enables the use of shorter reaction times and milder temperature & pressure compared to other organic-mediated silica syntheses. This also leads to the possibility of room-temperature additive elution as described above, which has yet to be achieved for these other silica families due to the specifics of their surface chemistry.272814in situ encapsulation of an array of different organic, inorganic, or bio-organic materials. Although independent post-synthetic modification of bioinspired additive and encapsulant concentration has yet to be achieved, these methods represent a promising step towards environmentally benign chemical processes.In summary, bioinspired silica synthesis represents a rapid, facile method for producing active species supports or gas sorbent media. Through tight control of pH during and after the reaction, a wide array of silica-amine composites can be synthesized with varying properties, which is further complemented by the possibility of The authors declare no competing financial interest.


Ion-releasing polymeric restorative materials seem to be promising solutions, due to their possible anticaries effect. However, acid functional groups (monomers) and glass filler increase hydrophilicity and, supposedly, water sorption. The purpose of the study was to evaluate the influence of water sorption of polymeric materials on the stress state at the restoration-tooth interface. Beautifil Bulk Fill Flow, Beautifil Flow Plus F00, Beautifil Flow F02, Dyract eXtra, Compoglass Flow, Ionosit, Glasiosite, TwinkiStar, Ionolux and Fuji II LC were used for the study. The stress state was measured using photoelastic analysis after: 0.5, 24, 72, 96, 168, 240, 336, 504, 672, 1344 and 2016 h. Moreover, water sorption, solubility and absorption dynamic were assessed. The water sorption, solubility and absorption dynamic of ion-releasing restorative materials are material dependent properties. The overall results indicated that the tested restorative materials showed significant stress decrease. The total reduction in contraction stress and water expansion stress was not observed for materials with low value of water sorption . The photoelastic method turned out to be inadequate to evaluate stress changes of resin modified glass-ionomer cement . Development in dentistry is driven by the desire to create a restorative material exhibiting high aesthetics, biocompatibility, durability and permanent adhesion to tooth structure . Many prThe polymerization shrinkage is an integral feature of all currently available resin-based/polymer materials ,11. PrevThe composition of investigated materials and bonding systems is presented in 2, as stated by the manufacturer. The detailed procedure for determination of absorption dynamic has also been described in our earlier works ,14. In oAbsorption is a physical or chemical phenomenon or a process in which atoms, molecules or ions enter some bulk phase—gas, liquid or solid material. This is a different process from adsorption, since molecules undergoing absorption are taken up by the volume, not by the surface (as in the case for adsorption). A more general term is sorption, which covers absorption, adsorption, and ion exchange. The main phenomenon observed in the present study is the absorption. The samples were weighted  immediately after preparation and then daily for 30 days, and after 1344 h (56 days) and 2016 h (84 days).A—water absorption, m0—the mass of the sample in dry condition [g] and im—mass of the sample after storage in water for a specified (i) period of time.Water absorption was calculated according to following equations:®, Mainz, Germany) at a temperature of 37 ± 1 °C for 23 h, transferred to a second desiccator at the temperature of 25 ± 1 °C for 1 h, and then weighed in the balance. This cycle was repeated until a constant mass was obtained (m1). Upon stabilization, specimens were immersed in distilled water at a temperature of 37 ± 1 °C for 7 days. Specimens were removed, gently dried with absorbent paper, and weighed again to obtain m2. Using the same protocol as for m1, specimens were then reconditioned in the desiccators until a constant mass was obtained (m3). Water sorption and solubility was investigated according to ISO 4049 . Five saspW—water sorption, slW—water solubility, m1—initial constant mass [μg], m2—mass after 7 days of water immersion [μg], m3—final constant mass [μg] and V—specimen volume [mm3].Water sorption and solubility were calculated according to following equations: The modified photoelastic analysis method enables analysis of the relationship between water sorption and the change of stress state (contraction or expansion) of materials with resin matrix. This test was extensively described ,14. Phot3.Mean values of water sorption and solubility were presented in The polymerization shrinkage stress for RMGI (Fuji II LC and Ionolux) was observed after 30 min. Based on photoelastic images, additional isochromes around restoration appeared after 24 h . They ocIn contrast, the changes in the stress state resulting from the water sorption in compomers and giomers was feasible. The results were presented in All ion-releasing restorative materials shrank during hardening process and exhibited the associated contraction stress. The significant decrease in the stress state was observed due to hygroscopic expansion of materials . The absIon-releasing restorative materials are a substantial part of modern restorative dentistry. Released fluoride may be incorporated into the surrounding enamel or dentin with anti-cariogenic effect. These materials complement their fluoride reservoir from the oral cavity by building in fluoride delivered via rinses, toothpastes or topical application of fluoride preparations. Thus, the filling is a local fluoride storage that is used when the decrease in pH level occurs ,18. Howe3) due to the addition of UDMA and bis-MEPEPP resins. Both resins exhibited lower water sorption than bis-GMA and TEGDMA [3. In the present study, the overall results indicated that the ion-releasing restorative materials showed greater stress decrease as compared with resin composites . It mighd TEGDMA ,22. The The water absorption of any material was controlled by a variety of mechanisms, but the osmotic effect led to an increased radial pressure. Since the Beautifil Flow F02 (giomer) demonstrated significant swelling along with the greatest radial pressure, it might be assumed that this material - more likely than any other studied material - was able to generate a significant osmotic effect. The main difference in microstructure between the giomers and compomers was the presence of pre-reacted glass-polyacid zones in the giomer structure (as a part of the filler) . It seemMost compomers and giomers showed an extended process of water sorption. UDMA and a high degree of filler content were used to limit water sorption . The higThe photoelastic method turned out to be inadequate to evaluate stress changes of RMGI (Fuji II LC and Ionolux). Consequently, a different method allowing for observation of the state stress development in RMGI during water aging is highly needed. An alternative measurement technique might be a tensiometer ,28, straA major drawback associated with resin dental materials was polymerization shrinkage. It could cause a marginal gap formation. Water sorption might help compensate the hygroscopic expansion . MoreoveThe water sorption, solubility and absorption dynamic of ion-releasing restorative materials were material dependent properties. The overall results indicated that the tested restorative materials showed significant stress decrease. The total reduction in contraction stress or water expansion stress was not observed for materials with low value of water sorption . The photoelastic method turned out to be inadequate to evaluate stress changes of RMGI.


Interface optimization in preparing natural fiber based biocomposite becomes a key factor that determines overall properties, especially mechanical performance. The solution for upgrading interfacial adhesion stemmed from polar fiber and nonpolar polymer remains unclear. Here, a kind of pulp fiber/acrylonitrile-butadiene-styrene (ABS) composite with content ratio of 1:1 was fabricated by functionalizing the cellulose fiber to coordinate interaction between fiber and ABS. With addition of 5 wt % polyacrylamide (PAM) there existed an interlinked three-element structure in composite. Three types of treatment to cellulose fiber, including alkali immersion, pivaloyl chloride grafting for 10 h and 20 h were conducted. Pulp fiber that was treated with alkali for one hour, followed by pivaloyl chloride reaction for ten hours, proved to be effective for interfacial adhesion. X-ray Photoelectron Spectroscopy (XPS) analysis reveals 21.9% of carbonyl and 12.1% of ester function in this fiber, which corresponds to oxidation and grafting. For its composite SEM picture displays that most of cellulose fiber are rooted in ABS and evident traces of tearing or fracture can be observed after tension test. DMA test indicates that this modified pulp fiber/ABS composite exhibits great compatibility, because of combined loss modulus peak ranging from 80 °C to 100 °C. Moreover, the well miscible composite has a tensile strength of 58.1 MPa and elastic modulus of 2515 MPa, increasing by nearly 50% and 60% from those of pure ABS, respectively. Acrylonitrile-butadiene-styrene (ABS) synthesized from acrylonitrile, butadiene, and styrene is a wide-used engineering polymer. It is well known for anti-chemical erosion, excellent flexibility, and appropriate rigidity. As a kind of thermoplastic material, ABS can be molded into various shapes or compounded with other polymers for further application. For instance, ABS/PC composite that was prepared by twin screw extruder is considered as crust of electronic device for impact resistance and insulation. PA added in ABS increases the tenacity of composite and in automobile ABS/PA is manufactured into panels. Most countries have replaced general plastics, such as polypropylene and polyvinyl chloride for glass fiber reinforced ABS in construction industry, as well because of its outstanding flexural strength. To some extent, the blending of ABS and other polymers expands the application of ABS in packaging industry.Although ABS composites have been extensively popularized in many sections, some drawbacks also exist. Those petroleum-based plastics produces much pollution during raw synthesis. In melt compounding, high temperature and pressure are applied, which is labelled as an energy intensive process. More importantly, these non-degradable composites bring about obstacles in recycling and violate the principle of environmentally friendly development. By contrast, when comparing to traditional polymers, natural fiber derived from plants and trees consists of cellulose, hemicellulose, and little glucide. A great aspect ratio of cellulose fiber, together with high crystallinity induced by hydrogen bonding, renders supreme mechanical properties. As a reinforcing filler, natural fiber has light weight and low processing temperature. Its blending with ABS provides degradability for composite. In turn, ABS makes up the shortage of chemical resistance of natural fiber. It seems that natural fiber reinforcing ABS has the potential to take place of conventional ABS composites.Natural fiber/ABS composites possess poor compatibility between polar fiber and nonpolar ABS when compared to composites that were constituted by several nonpolar resins. The research core of natural fiber reinforcing ABS is to optimize interface by manipulating the interaction of different molecules in blenders, so as to increase the miscibility. It means that in the manner of adjusting the type or distribution of characteristic groups phase interface can be converted from discrete state to successive interwoven state. Two strategies, including modification of cellulose and addition of compatibilizers, are adopted to consolidate interface. Yano et al.  reportedHowever, it is noticed that the efficiency of chemical modification is limited and the promotion of compatibility can only be realized in composite with low filling fraction of natural fiber. The high filling of natural fiber in plastics is seldom reported for difficulty in optimizing interfaces and related poor mechanical properties. There is no denying that composites incorporating a high content of natural fiber enlarge the advantages of biomaterial, such as easy degradability, green sustainability, and low carbon emission. We investigated interfacial improvement of ABS/pulp fiber composite at 1:1 ratio in order to prepare ABS composite with high filling natural fiber.In this article, the modification on pulp fiber was carried out for accommodating the features of ABS. Research in effects of conditions of chemical treatment upon functional groups of cellulose fiber was conducted based on the molecular structure of cellulose. PAM was added in the blender to stabilize the compounding system. Subsequently, compatibility between modified fiber and ABS was evaluated for exploring potential mechanism of interface consolidation. The critical technique for improving miscibility was to functionalize cellulose fiber. By oxidating and esterifying cellulose the changed functional groups triggered hydrogen bonding among polymers, thus strengthening the interfacial adhesion of phases. The major concern of research was linkage of oxidation and esterification level to the properties of composite. Distinguished from previous modification , our proN-methyl pyrrolidone (NMP) that was used as solvent with purity above 99% . Pyridine (P111513-100 mL) with purity of 99.8% was purchased from Aladdin  and used as catalyst. Anionic polyacrylamide (APAM) with molecular weight between three-million and 25-million and solid content above 90% was obtained from Teng Long water purification corporation .ABS (trade name: HI100) with melt flow rate of 11 g/10 min. under load of 10 kg at 220 °C (ASTM D1238) was provided by LG Chem Corporation . Its tensile strength and tensile elongation at yield in 23 °C were 37 MPa and 5%, respectively (ASTM D638). Yang Run trade limited corporation  supplied pulp fiber that was derived from needle leave wood. Water content of pulp was lower than 10%. Pivaloyl chloride (T109597-100 mL) with purity of 98% was bought from Aladdin . Ya Tai Union Chemistry Co. Ltd produced N-methyl pyrrolidone (NMP) solvent followed by continuous stirring. Prior to adding pyridine and pivaloyl chloride in NMP suspension temperature of system was raised to 90–100 °C for the removal of residue water in the case that pivaloyl chloride reacted with water. At the end of reaction pulp fiber was filtered and washed by glacial acetic acid, ethanol, and distilled water [Dry pulp was put into distilled water for preparing 2 wt % suspension. Subsequently, 16 wt % sodium hydroxide solution was formulated and poured in suspension. Before stirring the mixed suspension temperature remained constant at 40 °C. Alkali treatment was performed for about one hour. After immersion in alkali solution, pulp fiber was washed by distilled water and dried in vacuum at 60 °C. Subsequently, the dried fiber was picked in Tension Test: A sample in shape of dumb bell with size of 25.4 mm × 3.5 mm × 3.2 mm in length, width, and height was obtained from injection molding. According to method of ASTM D638 V tensile clamp with BSS-100 kg AT and 2.9998 mv/V was selected in tension test by a multi-functional mechanical test machine . Each kind of sample was tested five times. Stress-strain curve of each sample was plotted after testing.Scanning Electron Microscopy (SEM): The cross section of fractured dumb-bell shaped samples was observed after the tensile test. Cross section had been gold-sputtered and the surface morphology was investigated by SEM instrument , with an acceleration voltage of 20 kV.−1 at resolution of 3 cm−1 by Nicolet IS10 spectrometer . Each kind of sample was observed four times. The changes of peaks attached to specific groups were identified.Fourier Transform Infrared Spectroscopy (FTIR): Different types of chemical groups on cellulose fiber under specific treatment were analysed. The FTIR spectra of the modified and unmodified cellulose were collected over the wavenumber range of 4000–600 cmX-ray Photoelectron Spectroscopy (XPS): XPS can classify the main elements of cellulose fiber and characterize the types of bonds corresponding to main elements in detail. The C1s accurate spectra of modified and unmodified cellulose fiber were obtained over the binding energy range of 278–296 eV via AXIS UltraDLD instrument  equipped with a 165 mm hemispherical electron energy analyser and a monochromatized Al Kα (600W) source. Analysis was done with a depth of 2–3 nm.Tg) and compatibility between cellulose fiber and ABS. Samples measuring 16 mm × 4.5 mm × 3.5 mm were tested with maximum dynamic force of 4N and heated from 0 to 150 °C at a rate of 3 °C/min. The DMA test was carried out in single cantilever mode with an amplitude of 50 μm and frequency of 1,2,5 Hz via DMA 242D instrument . Each kind of sample was observed five times.Dynamic Mechanical Analysis (DMA): DMA was used to discuss the changing process of storage modulus (E’) and loss modulus (E’’) of composite samples, so as to compare the softening points  0.4, which was reported as an optimal trial according to comparative discussion and substitution of alkenyl succinic anhydride (ASA) by Yano et al. , this prIn this part Type1, Type2, and Type3 mentioned before were abbreviated to T1, T2, and T3. We denoted those pulp fibers that were treated by T1, T2, and T3 as pulp1(P1), P2, and P3, respectively. Similarly, we denoted those composite samples containing P1, P2, and P3 as sample1(S1), S2, and S3.The mechanical performance of three sorts of composite samples was displayed with that of pure ABS in From the Stress-Strain curve, S2 exhibited the highest tensile strength (58.1 MPa). When compared to pure ABS, the tensile strength of S2 almost increased by 50%. While S1 and S3 had similar tensile strength, 45.8 and 43.1 MPa, respectively. They raised slightly from 39.3 MPa of pure sample. When it comes to tensile modulus, it can be seen that pure ABS had lower elastic modulus than any other because before reaching the yielding point the tensile stress of pure ABS did not increase as sharply by strain as others. S3, with the highest elastic modulus of 2640 MPa, presented rigidity in the tensile test. Slightly decreasing from the modulus of S3, the elastic modulus of S1, S2 remained the same level of 2567 and 2515 MPa. Moreover, apart from pure ABS whose curve had a yielding stage and showed characteristics of ductile fracture, the rupture performance of other samples exhibited brittleness owing to stiff natural fiber filling in ABS. All three kinds of composites improved their tensile strength despite inflexibility brought about by high-filling cellulose fiber. However, the increasing in tensile strength differed among these composites. It was mainly attributed to distinction in interfacial adhesion between fiber and ABS, which was vital to load transfer .SEM observation for surface morphology of fracture cross section was conducted to verify this assumption.As shown in Functionalized cellulose was prepared and three types of treatment for cellulose fiber were investigated in order to strengthen interfacial adhesion. −1 with similar absorption intensity among four spectra is attached to hydrogen bonds and free hydroxyl groups on cellulose chain. The peak at 2898 cm−1 is related to stretch vibrations of CH. For a wavelength below 1500 cm−1, the band around 1000 cm−1 is attributed to COC (1160 cm−1) and coexistence of peaks at 1052 cm−1, 1104 cm−1 is evidence of primary and secondary alcohol [−1, while this peak is intensified and it appears at 1645 cm−1 in the other three spectra. Associating carbonyl groups existing in a small amount of hemicellulose or adsorbed water might be responsible for the peak at 1640 cm−1 [−1 [−1 are connected with degree of conjugate action. The lowest peak intensity of P3 implies a high level of conjugation and, hence, more cleavage of cellulose chain as guided by Hosoya’s model [−1 is indistinctively detected in spectrum of P2, as shown by In  alcohol ,15,16. T640 cm−1 . After a640 cm−1 . This pr640 cm−1 . As a recm−1 [−1 . Differe’s model . This caXPS analysis aims to identify types of bonds in cellulose and calculate relative content of each bond. Two principal peaks around 533 and 286 eV correspond to oxygen and carbon, respectively, in the spectrum of unmodified cellulose. A small peak at 1072 eV is related to sodium, because sodium hydroxide was adsorbed on the hydroxyl of cellulose during alkali treatment. However, only oxygen and carbon were detected in modified cellulose. Sodium hydroxide was removed from cellulose chain due to the penetration of pivaloyl chloride and solvent added during chemical modification. One big advantage of XPS analysis is access to specific type of bond via identifying an accurate element spectrum. As illustrated by (i). The C1 peak at 284.9 eV usually represents alkane-type carbon atoms. Cellulose chain includes this type of carbon partly and some of them exist in impurities, like lignin, extractives, and fatty acids .(ii). The C2 peak at 286.4 eV is the contribution of the presence of ether groups (C–O–C). C–OH of the unmodified glucose ring is also responsible for this signal. Most of carbon atoms on cellulose are concerned with this peak .(iii). The C3 peak at 287.7 eV is mainly attributed to carbonyl groups produced by oxidation. Another fractional contribution comes from a few carbon atoms of the AGU unit in cellulose. Here, the change of this signal is connected with the degree of oxidation.(iv). The C4 peak at 288.9 eV is derived from ester groups, which is absent in unmodified cellulose. Its presence in P1 might be related to formation of carboxyl via oxidation in the alkali condition.Here, the content of four types of carbon in P1 is set as reference and variation of content of C3 reflects total of carbonyl from surface oxidation and that within newly formed ester during modification. For content of C4, its change related to substitution determines degree of grafting. In this way, the ratio of oxidation to grafting can be roughly measured with equations 1–3, as presented below:In It is worth noticing that the degree of oxidation increases with the extension of modification time, which is indicated by the increase in C3 from P1 to P3. It results from alkaline condition induced by rid of sodium hydroxide that was adsorbed on cellulose during alkali immersion. The hydroxyl groups of cellulose tend to be oxidated in alkaline environment over 40 °C. In addition, the proportion of C4 in P2 reaches up to 12%, but it drops when prolonging reaction time to 20 h. This can be ascribed to the hydrolysis of ester. What is found in C4 also applies to the change of C1. Tertiary butyl of pivaloyl chloride grafted on cellulose accounts for the increase in C1 from P1 to P2. After the grafting reaction came to a certain degree hydrolysis became the principal reaction in last period of T3. Therefore, the reduction of tertiary butyl owing to hydrolysed ester leads to a decrease in C1 of P3.In general, the degree of grafting is not high. For the ideal or higher degree of substitution, one had to add plenty of reagent, which produced considerable waste and caused difficulty in recycling. When compared to T3, T2 modification maintains a balance between esterification and oxidation. Great interfacial adhesion in P2 and ABS implies the effectiveness of T2 modification. It means that moderate surface oxidation on cellulose and slight grafting with branched tertiary butyl structure has synergic effects on improving the interface. For questing mechanism of interfacial reinforcement interaction among components needs to be examined. Herein, further investigation in compatibility between fiber and ABS was conducted.Tg) and loss performance can be obtained from the DMA test. By comparing the trends of mechanical behaviour internal relations of composites are evaluated. DMA was used for thermal and mechanical properties of composite under dynamic force and the changed frequency. Glass transition temperature  printing with increased diffusion and entanglement of chains , interfaAs illustrated in Additional information about morphology and sketch map of interlinked structure can be obtained from In our work, a kind of natural fiber reinforced ABS composite is prepared by melt compounding. Functionalized modification on cellulose fiber, including moderate surface oxidation and slight grafting with tertiary butyl, was carried out. A favourable procedure starting from alkali immersion for one hour and ending up with ten-hour pivaloyl chloride reaction was adopted by comparing three types of treatment to cellulose fiber. It was an economic modification marked by limited modifier, whose mass ratio to pulp fiber only came to 1:1.6 as compared to Agustin’s method , and lesThis project is centered on interfacial reinforcement in natural fiber-based ABS composite. The results of chemical treatment revealed inhibited grafting reaction, but mild controllable surface oxidation. Microscopic morphology and thermal mechanical performance of the composite verify that surface oxidation and slight grafting have synergic effects on improving compatibility. Ascribed to great miscibility, the interfaces between fiber and ABS are finally enhanced. Overall, interfacial optimization was realized by hydrogen bonding, owing to stepwise treatment on pulp fiber with low-volume and recycled reagents in expedient time. Melt compounding via twin screw extruder and injection moulding ensured continuous processing. High filling natural fiber reinforced ABS biocomposite is desired to be utilized based on this study.


We assessed the prevalence of adherence to the American College of Obstetricians and Gynecologists (ACOG) recommendations regarding physical activity during pregnancy among Sri Lankan women and explored the relationship between physical activity during pregnancy and neonatal birth weight. In total, 141 pregnant women  were included from October to December 2015 and followed up until delivery. A validated questionnaire regarding physical activity during pregnancy was administered in the second and third trimesters. Activities were grouped by type  and intensity {sedentary [<1.5 metabolic equivalents (METs)], light intensity [1.5–2.9 METs], moderate intensity [3.0–6.0 METs], and vigorous intensity [>6.0 METs]}. Women were categorised as active or inactive based on the ACOG recommendations. In total, 79.1% and 45.2% of women met the guidelines in the second and third trimesters, respectively. The overall time spent and total energy expenditure was significantly higher in the second trimester (p < 0.001). We found no relationship between physical activity during pregnancy and neonatal birth weight. This study indicates that a considerable reduction of time and total energy expenditure occur as pregnancy progresses. Physical activity during pregnancy does not appear to significantly affect neonatal birth weight. Despite this recommendation, the worldwide prevalence of PA during pregnancy is reportedly low because many women choose to reduce their level of PA and increase relaxation during pregnancy2. The performance of moderate to vigorous PA in early pregnancy is particularly low among women of South Asian and Middle East origin3. The main reasons for reduced levels of PA during pregnancy are physical discomfort, complications associated with pregnancy, growth of the woman’s body, and a sense of insecurity with PA4.There is growing evidence that physical activity (PA) during pregnancy is beneficial for both the woman and foetus. The American College of Obstetricians and Gynecologists (ACOG) recommends that pregnant women who are free of medical or obstetrical complications engage in ≥30 minutes of moderate PA per day on most if not all days of the weeket al.5 provided new information on the effects of moderate PA on the pregnant woman and foetus, showing that PA has no adverse effects on either maternal or foetal health. Another study showed evidence of a protective effect of mild PA during pregnancy in the second trimester on low birth weight (LBW), preterm birth, and intrauterine growth restriction6. A systematic review and meta-analysis of observational studies reported an inverted U-shaped curve; moderate PA levels were associated with increased birth weight but high levels were associated with decreased birth weight7.In 2003, Davies 8. Several factors have been found to be associated with LBW of neonates. Some of these factors include pregnancy-induced hypertension9, underweight pre-pregnancy body mass index (BMI) and inadequate gestational weight gain11, maternal malnutrition13, active or passive tobacco smoking16 and exposure to biomass fuel smoke during cooking18, and anaemia20.Sri Lanka is a developing country in southern Asia, in which LBW  constitutes a significant public health problem22. These findings may reflect the negative birth outcomes associated with increased PA during pregnancy. The prevalence of adherence to the ACOG guidelines regarding PA among pregnant women in Sri Lanka remains unknown. However, the culturally adopted pattern of PA during pregnancy among Sri Lankans is characterised by avoidance of strenuous activities, heavy lifting, exercise, and even fast walking. To our knowledge, the relationship between PA during pregnancy and neonatal birth weight has not been explored specifically in Sri Lankan women. Considering the unknown impact of PA on LBW in Sri Lankan context, this prospective longitudinal study was performed to evaluate patterns of PA during pregnancy and the relationship between PA and neonatal birth weight.The effect of PA during pregnancy on pregnancy outcomes was recently established as a research interest. The districts in Sri Lanka with a LBW prevalence of >18% are predominantly those where >40% of the population is engaged in agriculture, and >30% of these individuals are women23. Initially, 150 pregnant women at a gestational age of 18 to 24 weeks were included in the study and they were followed-up until the delivery. Women with multiple foetuses, a history of miscarriage/abortion, pregnancy-induced hypertension, gestational diabetes mellitus, and chronic medical diseases  were excluded at the time of recruitment, or whenever such a condition was identified during pregnancy. Neonates with a low Apgar score of <5 at 5 minutes after birth were also excluded from the final sample.The present study was part of a larger study designed to assess the maternal factors associated with neonatal birth weight. The study was conducted in a tertiary-care hospital in Sri Lanka during the 9-month period from October 2015 to June 2016. A detailed description of the patient recruitment procedure has been published elsewhereThe study was approved by the ethical review committees of the Graduate School of Health Sciences, Niigata University, Japan (No. 125); Faculty of Allied Health Sciences, University of Peradeniya; and the Institutional Ethical Review Committee of the Teaching Hospital, Kurunegala, Sri Lanka (ERC/2015/06). It was performed in compliance with the ethical principles outlined in the Declaration of Helsinki. Furthermore, prior to data collection, informed written consent was obtained from all participants.24. The PPAQ was a semi-quantitative questionnaire that asked participants to report the average time spent in 33 activities (31 multiple-choice plus 2 open-ended questions). The women were asked to choose the best approximate amount of time spent in each activity per day or week during the on-going trimester. The provided time durations ranged from 0 to ≥6 hours per day and from 0 to ≥3 hours per week. Self-administration of the PPAQ took approximately 7 to 10 minutes. The total gestational weight gain and neonatal data were collected from the hospital records after all participating women had delivered their babies.Socio-economic and demographic data were collected using a pre-designed and pre-tested interviewer-administered questionnaire. Maternal height and weight were recorded by a trained research assistant. Height was measured at the time of recruitment, whereas body weight was measured using a Tanita Digital Scale at each clinic visit prior to delivery. Body weight measurements taken prior to recruitment and at the time of delivery were directly obtained from pregnancy cards and hospital records respectively. Maternal PA was assessed once during the second trimester (approximately 22 weeks of gestation) and once during the third trimester (approximately 34weeks of gestation) using a validated pregnancy PA questionnaire (PPAQ)25. The MET is a unit used to estimate the metabolic cost of PA. The value of 1− MET is approximately equal to a person’s resting energy expenditure. The self-reported time spent in each activity was then multiplied by the corresponding intensity to obtain the average energy expenditure per week (MET-hours/week). When the reported time was in hours per day, it was multiplied by 7 to determine the average weekly energy expenditure. Activities were classified into 5 categories by type: household/caregiving (13 activities), occupational (5 activities), transportation (3 activities), sports/exercises (7 activities plus 2 open-ended questions), and inactivity (3 activities). In addition, each activity was classified into 4 categories based on its intensity: sedentary (<1.5 METs), lightintensity (1.5–2.9 METs), moderate intensity (3.0–6.0 METs), and vigorous intensity (>6.0 METs). The women were categorised into two groups based on the ACOG recommendations as active (≥30 min of moderate PA per day) and inactive (<30 min of moderate PA per day). The women were also categorised into four groups: those who stayed active (active at both 22 and 34 weeks), stayed inactive (inactive at both 22 and 34 weeks), became active (inactive at 22 weeks and active at 34 weeks), and became inactive (active at 22 weeks and inactive at 34 weeks). The outcome variables were the total number of hours per week and total number of MET hours per week.Each activity was assigned an intensity using the metabolic equivalent (MET) tableAll statistical analyses were performed using Minitab version 17 . Mean with standard deviation (SD) of time spent in PA (hours/week) and energy expenditure (MET-hours/week) are reported for 22 and 34 weeks of gestation separately. Categorical data are expressed as percentages. All variables were first assessed using numerical and graphical techniques to determine whether they met the distributional assumptions of the statistical tests used to analyse them. The Anderson-Darling test was used to explore whether outcome variables were normally distributed; and it was used with the normal probability plot. Participants’ characteristics were compared between active and inactive women using one-way analysis of variance (ANOVA) and the paired t-test. The levels of PA at 22 and 34 weeks were compared using a paired t-test for each type and intensity of PA. Pregnancy outcomes were first assessed based on the categories in the ACOG recommendations using a two-sample t-test and based on the change in the pattern of PA during pregnancy using one-way ANOVA. Neonatal birth weight was first adjusted for the gestational age and thereafter a regression analysis was used to estimate the effect of PA during pregnancy second trimester (active/inactive) on adjusted neonatal birth weight. The covariates in the initial model were maternal age, pre-pregnancy BMI, and gestational age, while the factors were PA during pregnancy , level of education , monthly household income , area of residence , history of LBW , and parity . Variables with the highest p value were removed from the initial model one variable at a time until the final model contained only the variables with a p value of <0.1. A p value of <0.05 was considered statistically significant.In total, 150 patients were recruited into the study. Nine women were excluded from the study because of spontaneous abortions (n = 2), multiple foetuses identified by 20 weeks using ultrasound (n = 2), maternal desire to deliver at another hospital (n = 3), and personal withdrawal (n = 2). Of the remaining 141 patients, data regarding total gestational weight were missing for 22 patients. The researcher failed to find neonatal data for 14 neonates from the hospital delivery registry records because of confusion due to many similar names, and one neonate with a 5-minute Apgar score of <5 was excluded at the end of the study, resulted for 126 neonatal data set.23. Final sample consisted of 126 healthy new-borns and among them 22(17.4%) were LBW babies. Among the 141 women, only 139 (98.6%) at 22 weeks of gestation and 62 (44.0%) at 34 weeks of gestation responded to the PPAQ. At 22 weeks of gestation, 110 (79.1%) women were above the ACOG guidelines regarding PA during pregnancy, whereas only 28 (45.2%) women met the guidelines at 34 weeks of gestation. Table The overall background characteristics of the participants have been published separatelyThe number of hours spent for all modes of activity per week was significantly higher in the second than third trimester (p < 0.001). When the activities were divided into groups according to their intensity, a significant reduction in time spent in PA during the third trimester compared with the second trimester was observed in terms of sedentary activities (p < 0.05), light intensity (p < 0.001), and moderateintensity (p < 0.05). The mean time spent in vigorous-intensity activity was very low in both the second and third trimesters, and the difference did not reach statistical significance. A similar pattern was observed for the total energy expenditure (MET-hours/week) except for sedentary activities. The average energy expenditure corresponding to sedentary activities was significantly higher in the third than second trimester (p < 0.001). Division of the activities by type revealed significant reduction of time (hours/week) spent in household/caregiving activities (p < 0.01), transportation (p < 0.001), and inactivity (p < 0.05) during the third trimester Table . A similTable No difference was observed in the total gestational weight gain, gestational age, or neonatal birth weight based on either the ACOG recommendations or the changing pattern of PA during pregnancy (p > 0.05) Table .Table 4PAssessment using the regression analysis was limited to the PA level during the second trimester because of the low PPAQ response rate during the third trimester. The regression analysis revealed significant impacts of gestational age on neonatal birth weight p < 0.05). We found no significant impact of moderate PA during the second trimester on neonatal birth weight (p = 0.718) (Table . We foun26 (11.1%), Ireland27 (21.5%), and the United States28 (22.9%). However, the average energy expenditure in the second trimester among the women in the current study was lower than that reported in previous studies in Australia29 and France30. Moreover, in the current study, there was a significant reduction in the time spent in PA and the total energy expenditure during the third trimester. In addition to physical discomfort and bodily growth, this reduction may be related to the traditional Sri Lankan culture, which defines pregnancy as a vulnerable period that requires rest and protection. Therefore, pregnant Sri Lankan women tend to obey the traditional beliefs of no lifting heavy objects, no fast walking, and no strenuous activities during pregnancy. As a result, vigorous activity during the second trimester was very low in the present study, and no women reported vigorous activities during the third trimester. In addition, nearly half of the total energy expenditure during the third trimester was covered by sedentary activities, highlighting the increase in low activity as pregnancy progresses. A major proportion of time spent in PA and the total energy expenditure among the pregnant women in the current study was covered by household/caregiving activities.Of all participants, 98.6% responded at 22 weeks of gestation, but only 44% responded at 34 weeks of gestation, reflecting loss to follow-up. In Sri Lanka, maternal care is free throughout the ante-, intra- and postnatal periods. Antenatal care is provided by maternal and child health clinics, both in the field and in medical institutions. Most of the women in the current study attended both types of clinics. As institutional clinics are usually very crowded, the women sometimes tended to attend only field clinics, which do not perform advanced procedures such as blood evaluations or ultrasound on women with low-risk pregnancies at 34 weeks of gestation. In total, 79.1% and 45.2% of the pregnant women who participated in this study met the ACOG recommendations for PA during pregnancy in the second and third trimesters, respectively. This rate is significantly higher than the rates recorded in Chinaet al.31 found that pregnant women engaging in high-level sports/ exercise during the first trimester delivered infants of lower birth weight. Similarly, a cohort study performed in Iran reported that sporting activities increased the risk of an LBW infant32. We found that most Sri Lankan women did not engage in sport/exercise, rendering comparisons difficult. This is a common feature of many Asian countries; women of reproductive age carry the most substantial burden of the household and caregiving activities. Although it did not reach statistical significance, most women in the lowest income group (92.9%) were active during the second trimester, indicating an association between household income and energy expenditure in Sri Lanka. The analysis of the changing pattern of PA during pregnancy revealed that 41% of women stayed active throughout the pregnancy while 42.6% women became inactive during the third trimester.In 2017, Bisson 34, which indicated no significant association between maternal PA and gestational weight gain, gestational age, or neonatal birth weight. In contrast, some studies have shown a protective effect of mild PA against LBW and preterm birth6, whereas others have shown an increased risk of intrauterine growth restriction with moderate or vigorous occupational PA35.Our univariate analyses showed no relationships between PA during pregnancy and gestational weight gain, gestational age, and neonatal birth weight. These findings are in agreement with the results of previous studieset al.36 indicated that pregnant women who engaged in purposeful walking routines associated with activities of daily living achieved healthy weekly weight gains. However, in the current study, we did not focus on weekly gestational weight gains or the relationships between various PAs and birth outcomes. Moreover, Cohen et al.36 showed that moderate-intensity sports/exercise was not associated with appropriate gestational weight gain37 and stressed that sedentary pregnant women should not be encouraged to engage in vigorous activity to achieve the recommended weight gain37. However, Cohen et al. focused on prevention of overweight; most enrolled subjects exceeded the recommended gestational weight gains37. Therefore, the meaning of “appropriate weight gain” in the cited work may differ from ours; most of the women we enrolled exhibited less weight gain than the recommended total gestational weight gain. The resultsof the current study showed that women who were below the ACOG recommendations of PA during pregnancy delivered neonates with a higher mean birth weight than did women who did not meet the ACOG recommendations, although the difference was not statistically significant. On the contrary, a systematic review7 suggested that moderate PA during pregnancy was associated with increased birth weight, but high PA was associated with reduced birth weight. However, these associations remained when leisure time PA alone was considered. Moreover, more than one-third of all studies included in the cited review were not adjusted for covariates known or thought to influence both PA and foetal growth7. The initial sample size of our current study was chosen to afford a power of 0.8 at an alpha level of 0.05. However, the loss to follow-up experienced at 34 weeks of gestation reduced the statistical power of our tests and rendered it difficult to draw robust conclusions; our data lost at least some significance. One hypothesis states that an inverse relationship exists between PA during pregnancy and utero-placental blood flow, ultimately leading to a reduced foetal oxygen and glucose supply. However, the proportion of women who engaged in vigorous PA in our study was very low, rending it difficult to draw robust conclusions on any association between increased PA and birth weight. In addition, our sample size was likely insufficient to detect the significance of such a relationship. Therefore, the results regarding observed association between PA and Neonatal birth weight may require further inquiry and verification before firm conclusion can be reached. Although no relationship between PA during pregnancy and pregnancy outcomes was observed in this group of women, the associations between postpartum weight retention and other medical/obstetric complications were not assessed. Large-scale studies are warranted to assess the factors influencing PA during pregnancy and the effects of PA during pregnancy on the mother and infant even after childbirth. The findings regarding PA during the second trimester and neonatal birth weight need to be confirmed in further large-scale studies.In 2003, Cohen 24. The fact that the PPAQ is brief and user-friendly renders it attractive to researchers who wish to examine changes in PA during pregnancy, and also to prenatal healthcare providers who wish to address PA in pregnant women39. Despite these strengths, our study had certain limitations. First, although we used a validated and reliable instrument, the reliability of our data may have been affected by recall bias and the cognitive burden associated with instrument self-administration. Many women were lost to follow-up; the PPAQ response rate was very low in the third trimester, rendering the sample size much smaller than expected. Also, we did not evaluate the use of medications during pregnancy, intra-uterine infection status, placental abnormalities, or genetic factors possibly associated with LBW. Another limitation of this study is that administration of the PPAQ once at around 22 and 34 weeks of gestation may not be a reliable measure of PA during the second and third trimesters.The strengths of our study included the use of a reliable questionnaire affording reasonably accurate measures of a variety of PAs performed during pregnancy. This is the first validated questionnaire that can be employed to determine the lower and upper extent of PA, in terms of frequency, duration, and intensity, during pregnancyMore than half of women achieved the recommended PA level during the second trimester of pregnancy, whereas only 45.2% achieved the recommended PA level during the third trimester. Significant reductions in the time spent in PA and the total energy expenditure were observed as pregnancy progressed. No significant association between PA during pregnancy and neonatal birth weight was found.


The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform (“hook shaped”) sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult.We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample.Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology. Subtle morphological differences in sperm nuclei can be detected with a new analysis technique; in mice, C57Bl6 and CBA crosses are intermediate to their parental shapes, and the direction of the cross matters. Cell nuclei are complex, dynamic structures that can adopt a wide range of shapes beyond simply spherical . One of Mouse sperm shape analysis has proven useful in three interrelated areas: evolutionary biology (including speciation), infertility, and toxicology. In evolutionary biology, the questions of how evolutionary forces such as sperm competition and cryptic female choice affect sperm form and function are active fields of research , 8, whilWhile much sperm analysis still relies on time-consuming and subjective manual scoring, various efforts have been directed towards the development of automated morphometric analyses in an effort to improve both reproducibility and predictive value. To date, these approaches have fallen into three main groups: the measurement of basic parameters such as lengths, widths, and areas of objects; the use of elliptic Fourier analysis to investigate the two dimensional outlines of sperm; and the use of Procrustes analyses to examine differences in fixed landmarks within sperm heads. Each has advantages and disadvantages.Basic measures such as area (A), length (L), width (W), and perimeter (P) were the first statistics recorded describing sperm morphology , and stiMore powerful elliptic Fourier descriptors  allow anThe third major method, Procrustes-based geometric morphometric analysis, uses landmarks and semilandmarks within the object to align individual samples to consistent size, position, and orientation , 30. PriTo address the unmet need for rapid, unbiased measurement, analysis, and categorization of nuclear morphologies, we have developed a new image analysis tool that automates object finding, alignment, landmark discovery, and sample comparison. This generates quantitative information on the underlying regions of the nucleus that differ within and between samples, independent of nuclear size. Here, we demonstrate the use of this software for each of these approaches by comparing a range of different inbred, outbred, and wild-derived mouse strains, quantifying the morphological variation in highly pleomorphic BALB/c sperm samples, and tracing the genetic influences on sperm morphology in a reciprocal F1 cross between CB57Bl6 and CBA strains.2 followed by cervical dislocation (UM) or only cervical dislocation and tissues collected post mortem for analysis.All animal procedures were in accordance with the United Kingdom Animal Scientific Procedures Act 1986 and the University of Montana Institute for Animal Care and Use Committee (protocol 002-13) and were subject to local ethical review. Animals were sourced either from an approved supplier , bred at Cambridge University Central Biomedical Services (Home Office licenses 80/2451 and 70/8925 held by PE), or bred at the University of Montana . Sperm were transferred to a microfuge tube, and tissue clumps were allowed to settle. Sperm were transferred to a new tube and pelleted at 500 g for 5min. The supernatant was removed, and sperm fixed dropwise with either 3:1 methanol-acetic acid or 2% paraformaldehyde (PFA) in PBS. Sperm were pelleted at 500 g for 5min, washed in fixative twice more, then stored at –20°C (methanol-acetic acid) or 4°C (PFA).Samples were diluted in fixative as required to obtain an evenly spread preparation, and 8 μl of sample dropped onto a slide and allowed to air dry. Slides were stained with 16 μl VectorShield with DAPI (Vector Labs) under a 22 × 50 mm cover slip and imaged using an Olympus UPFLN100XOI2 100× oil immersion plan semiapochromat objective (NA 1.30) on an Olympus BX-61 epifluorescence microscope equipped with a Hamamatsu Orca-ER C4742-80 cooled CCD camera and appropriate filters. Images were captured using Smart-Capture 3 . To validate the reproducibility of the software, sample images were also gathered on three other microscopes: (1) an Olympus BX61 with a Hamamatsu C10600 orca r² camera, (2) an Olympus BX61 with a Hamamatsu Orca-03G camera (both (1) and (2) using an Olympus UPFLN100 × 100× oil immersion plan semiapochromat objective [NA 1.30]), and (3) a Nikon Microphot-SA epifluorescence microscope using a Nikon 100× oil immersion plan apochromat objective (NA 1.40) with a Photometrics Metachrome II CH250 cooled CCD camera.http://bitbucket.org/bmskinner/nuclear_morphology/wiki/Home/ together with full installation instructions, an online wiki user manual, and example testing images. Analyses were conducted using software version 1.14.1. The software allows a user to select a folder of TIFF images previously captured using a fluorescence microscope, and interactively define the nucleus detection parameters. The program then automatically detects and analyses the nuclei in the images.Image analysis was performed using a custom program designed as a plugin for the freely available image analysis program ImageJ . The sofOnce nuclei are acquired from a set of images, they are consistently oriented and aligned. Landmarks are automatically identified using a modification of the Zahn-Roskies (ZR) transform  to generThe angle profiles from each nucleus are interpolated to a consistent length and aligned against other. A median profile is constructed by taking the median of the angles at each point along the interpolated profile length. The median profile is segmented at local mimima and local maxima below or above 180 degrees respectively, automatically defining landmarks at convex or concave corners in the shape. The landmark locations in each nucleus are then identified via the best fit of the nucleus profile to the median at each landmark. The flat region below the hook is used to allow consistent vertical orientation of the nuclei. A diagram of the full analysis pipeline is provided as Following segmentation, standard nuclear parameters are automatically measured: area, perimeter, and ellipticity, the width of the nuclear body versus the length of the hook as described in other papers , and theData were exported for further processing in R. Differences between mouse strains were tested using a pairwise Wilcoxon rank sum test, with Bonferroni multiple testing correction. The coefficient of variability (standard deviation/mean) was also calculated for each of the other measured parameters.The “average shape” of the nuclei was calculated by averaging the x and y coordinates at consistent semilandmarks spaced every 1% of the perimeter across all nuclei, vertically aligned and with their centers of mass at . This yielded a “consensus nucleus” visualizing the overall shape of the population. Clustering was implemented via the WEKA data mining software library .Before investigating the biological differences between samples, we needed to be confident that our analyses were reproducible and not biased by small differences in data gathering—for example, the camera and microscope used to capture images, and the exposure time during image capture. The choice of fixative (3:1 methanol:acetic acid [MeAc] vs 2% PFA) did not affect overall shape , but hadCBA and C57Bl6 sperm are distinguishable to the trained eye, and make a useful demonstration of the software, as the angle profiles generated are distinct for each genotype Figure . CBA speCBA and C57Bl6 have previously been characterized by Wyrobek et al. , who meaHaving demonstrated the software can distinguish differences between two genotypes, we carried out a preliminary investigation of the extent to which sperm shape variability in classical laboratory strains is affected by two factors: inbreeding and the complex inter-subspecific mosaic origin of these strains. To do this, we compared a panel of inbred laboratory strains to (a) outbred laboratory strains, and (b) wild-derived inbred strains Table . BiologiSome of the automatically identified landmarks were consistently found across all strains, such as the tip of the apical hook and the point of maximum curvature at the base of the sperm head, while others  were variable between strains. Of all the strains studied, only five showed a clear dorsal angle, with the others having a smoother profile posterior to the acrosome. The distance from the rear reference point to the dorsal angle was characteristic for each of these five strains, as was the variability in this measurement, with BALB/c mice showing highest variability. RIII both showed slightly higher intra-sample variability. This may reflect the fact that these samples were pooled samples derived from multiple genetically unique individuals. Of the wild-derived strains, all three lineages analyzed  had lower variability than any of the standard laboratory strains, despite that fact that these wild-derived strains are inbred.Overall, sperm shape variability within each strain was assessed using a new measure based on the similarity of each cell's angle profile to the median for that strain. This correlated well with other population measures of variability such as the coefficients of variation for area, bounding height and perimeter . The BALWe investigated the impact of strain background and genetic interactions using one specific reciprocal cross, between C57Bl6 and CBAs. The use of F1 animals means the resulting animals are no longer inbred, but still yields a uniform population of genetically identical males from each cross. B6CBA mice are the F1 offspring of a female B6 with a male CBA and CBAB6 mice are the reciprocal cross. Sperm morphology for both directions of the cross matches the CBA parental strain closely, indicating a dominant effect of the CBA genotype Figure , and bot2, P < 0.001) and the region around the posterior of the nucleus is widened in the CBAB6s, intermediate to CBA and C57Bl6 , as are C57Bl6 and CBA (P = 1.27E-12), but there is no significant difference between C57Bl6 and CBAB6 (P = 0.18) or between CBA and B6CBA (P = 0.095). This suggests that this aspect of sperm shape may be influenced either by sex chromosome or mitochondrial background or by autosomal imprinted loci.The reciprocal cross data allows us to look for parent-of-origin effects on sperm shape. We found two differences, in sperm cross-sectional area and in bounding width. CBAB6s have a slightly larger sperm area than the B6CBAs  as experienced assessors (97% accuracy), and substantially better than novice assessors (75% accuracy) at distinguishing between these two strains .Next, we looked at using the clustering for novel shape discovery in BALB/c, the strain with highest within-sample variability. Clustering revealed four major groups of sperm shape, from mostly normal sperm to severely shrunken and misshapen sperm Figure . Each ofWe present a novel tool for nuclear morphometry, which quantitatively measures a range of nuclear and sub-nuclear size and shape parameters. While we have chosen mouse sperm to demonstrate the software, the analysis steps will work on many symmetric or asymmetric shapes of nuclei including, but not limited to sperm from other species .At the object detection stage, we use an edge detection algorithm that is markedly more effective than fixed-threshold detection. At the shape decomposition step, we introduce a modification of the ZR transform  that senOur observations support previous studies of mouse sperm morphological variation , 20, andThe greatest sperm shape variability was observed in the BALB/c animals, a strain with poor sperm morphology and high levels of sperm aneuploidy. Kishikawa et al.  observedM. m. musculus, M. m. domesticus and M. spretus respectively. Since these three strains are also inbred, this suggests that the variety of sperm shapes in laboratory strains, and the elevated level of intra-individual variability in all the laboratory strains is not solely a consequence of inbreeding. Instead, this is potentially linked to the status of the laboratory mouse as a hybrid between several mouse subspecies—a factor that may have disrupted regulatory interactions throughout the genome, particularly interactions involving the sex chromosomes [musculus origin, nevertheless has substantial introgression of domesticus DNA, of the order of ∼6%–7% of the genome [Consistent with , we founomosomes . Againste genome , 41. TheAn alternative but not mutually exclusive explanation for the difference between classical laboratory inbred strains and wild-derived inbred strains is that the classical strains have been selected over multiple generations for their ability to breed well in captivity—indeed FVB is particularly known for its fecundity . Under lSperm morphology is of interest from an evolutionary perspective; sperm are under intense selection, sperm morphology has been found to be an important criterion influencing male fertility in many species . AlteredFertility rate and IVF efficiency has been correlated with the genetic background of sperm among inbred mouse strains . FurtherIn toxicological analysis, rodent sperm are conventionally manually classified into classes of predefined morphological abnormality , 48. TheSupplementary Methods and Results: Detailed description of the analysis methods and software validation.Supplementary Table S1. The mean measured parameters by strain, with coefficient of variability, standard error, and standard deviation per parameterSupplementary Table S2: The mean measured parameters by individual sample, with coefficient of variability, standard error, and standard deviation per parameterSupplementary Table S3: The default mean segment lengths by strain, with coefficient of variability, standard error, and standard deviation per segmentioz013_Supplemental_FilesClick here for additional data file.


The aim of this study was to determine the HPV positivity rate in patients with laryngeal cancer, and to determine the effect of HPV positivity on survival. An additional aim was to determine if patients with HPV positive laryngeal cancer are more sensitive to chemotherapy and if such sensitivity differs according to chemotherapy protocol.The study included laryngeal specimens obtained from 82 laryngeal cancer patients and 11 laryngeal specimens with normal laryngeal mucosa that were obtained from our hospital’s paraffin block archives between 1995 and 2013. HPV was detected via chromogenic in situ hybridization (cISH) and confirmed via genotyping.HPV was not detected in any of the 82 laryngeal cancer patients’ laryngeal specimens, nor in any of the 11 archived laryngeal specimens with normal laryngeal mucosa via cISH. Genotyping confirmed these findings; none of the HPV types studied were detected in any of the specimens. As none of the study samples were HPV positive, it was not possible to compare survival, recurrence, or chemotherapy sensitivity.HPV infection is not a leading cause of laryngeal cancer; however, additional research on HPV positivity in patients with laryngeal cancer and its effect on recurrence, survival, and chemotherapy sensitivity is warranted. Laryngeal cancer is the 20th most common cancer in Europe, with around 39,900 new cases diagnosed in 2012  . LaryngeAlthough the reported incidence of HPV positivity in laryngeal cancer patients varies –12 fewerRetrospective data analysis was performed to include patients with laryngeal cancer between 1995 and 2013. The patients who had laryngeal squamous cell carcinoma, who had a biopsy spesimen available in the Pathology Paraffin Block Archives, who received chemoradiotherapy as the first line treatment were included. All patients underwent chemoradiotherapy post diagnosis, and all chemotherapy protocols included an alkylating agent. Patients that received any treatment before direct laryngoscopy and biopsy, had surgery as the primary treatment procedure, received only radiotherapy, and those with follow-up < 2 years were excluded. 82 patients met the criteria and the formalin-fixed paraffin-embedded tissue specimens (FFPE) and slides of tumors stained with H&E for each patient were retrieved. All patient slides were re-examined for confirmation of the diagnosis.The retrospective data analysis included demographic and clinical findings, as well as the histopathological characteristics of the tumor. Grade of differentiation, primary tumor extension, and nodal status, were recorded. Treatment regimens and the recurrence status were also assessed. Disease-free survival (DFS) was defined as the time from the date chemoradiotherapy was initiated to the time recurrence was detected.The control group comprised of 11 laryngeal specimens reported as normal laryngeal mucosa obtained from the Department of Pathology Paraffin Block Archives. These pathological specimens included 6 male and 5 female patients. The mean age of these subjects was 47.45 years, ranging from 35 to 62 years. These 11 patients had undergone endoscopic laryngeal surgery for benign lesions. These lesions were resected and biopsied during the direct laryngoscopy procedure. These biopsy specimens were reported to be normal laryngeal mucosa without evidence of a premalignant or a malignant lesion.HPV was detected via chromogenic in situ hybridization (cISH) and was confirmed via genotyping. The study was approved by the University Ethics Committee and was conducted in accordance with the Declaration of Helsinki.High-risk (HR)-HPV DNA in-situ hybridization (ISH) was done using proprietary reagents , which can detect high risk HPV genotypes  . HR-HPV in accordance to the manufacturer’s instructions. The HPV GenoArray helps the identification of 21 HPV genotypes  and CP8304. Of the 21 genotypes, 5 were classified as low risk  and the remaining HPV genotypes are classified as high-risk .HPV positivity and all clinicopathological data, including age, gender, tumor stage, differentiation, tumor localization, and tobacco and alcohol use, underwent descriptive statistical analysis using SPSS v.10.0 for Windows .n = 58 [70.7%]) and glottis (n = 24 [29.3%]). In all, 5 (6.1%) patients were clinically stage II, 38 (46.3%) were stage III, and 39 (47.6%) were stage IV. In total, 46 (56.1%) patients had no evidence of nodal involvement at the time of diagnosis. Tumors were histopathologically classified as well differentiated carcinoma (n = 33 [40.2%]), moderately differentiated carcinoma (n = 44 [53.7%]), and poorly differentiated carcinoma (n = 5 [6.1%]). Table The study included laryngeal specimens obtained from 82 patients with laryngeal cancer and 11 laryngeal specimens reported as normal laryngeal mucosa. Among the 82 laryngeal cancer patients, 78 were male and 4 were female; mean age was 56.6 years (range: 40–75 years). Tumor localization included the supraglottis (Chromogenic In-Situ Hybridization (cISH) was performed to assess HR-HPV positivity. cISH findings were evaluated by 2 independent observers. None of the 82 laryngeal specimens obtained from patients with squamous cell carcinoma and none of the 11 laryngeal specimens with normal laryngeal mucosa exhibited a positive cISH staining pattern. Genotyping confirmed the cISH findings; none of the HPV types studied were present in any of the specimens.HPV infection has been shown to cause oropharyngeal cancer. However, the clinical significance of HPV infection in head and neck squamous cell cancer other than oropharygeal cancer is yet to be determined. The prevalence of laryngeal and hypopharyngeal squamous cell carcinomas that are HPV positive varies from 0% , 15, 16 PCR and ISH are widely used HPV detection systems. ISH is commonly used to detect HPV in clinical biopsy specimens. Some studies report that PCR-based methods are more sensitive than ISH. Besides, PCR-based methods can yield false positive results, because they may not differentiate biologically irrelevant HPV from clinically significant HPV, whereas ISH correlates with biologically and transcriptionally active HPV, differentiating between clinically significant and non-tumorigenic HPV DNA –21. HPV In the current study we used HR-HPV chromogenic ISH for detection of HR-HPV; however, none of the 82 laryngeal specimens of squamous cell carcinoma and none of 11 laryngeal specimens of normal laryngeal mucosa exhibited a positive cISH staining pattern. Although the reported prevalence of HPV positive squamous cell cancer of the larynx and hypopharynx varies widely, the literature generally indicates that the prevalence is higher than that observed in the present study, but some studies have also reported no HPV positivity or a very low prevalence of HPV positivity , 23–25. The effect of tobacco use on laryngeal cancer is well-known, and we also reviewed the data and/or asked the patients about their tobacco use. Although the data were not very reliable because they were collected via self-report and the patients did not remember precisely how long they smoked or how much they smoked, all our patients had a history of tobacco use. Gheit et al.  reportedSome studies regarding head and neck cancer found out that HPV positivity was observed in younger patients compared to patients with HPV negative cancer , 4. The Earlier studies on HPV positivity in laryngeal cancer in Turkey reported that 7.4%  and 10.6The present study is among the few from Turkey to investigate HPV infection in patients with laryngeal cancer, and compare it to HPV positivity in normal laryngeal tissue. The cISH technique used in the present study is not widely used for studying laryngeal cancer. A strength of the present study is that HPV negativity based on cISH was confirmed via genotyping. As some earlier studies reported, HPV positivity was not observed in any of the present study’s laryngeal specimens  , 15, 16 The present findings suggest that HPV infection does not play a major role in laryngeal cancer; however, additional research is required to increase our understanding of the prevalence of HPV positivity in laryngeal cancer patients and the effect of HPV positivity on recurrence, survival, and chemotherapy sensitivity.


Campylobacter, Prevotella and Sphaerochaeta were different in the duodenum (P < 0.05); Sanguibacter, Kingella and Anaeroplasma in jejunum; Anaeroplasma, Arthrobacter, Kingella, Megasphaera and SMB53 in the ileum; Butyricicoccus, Campylobacter, Mitsuokella, and Coprobacillus in the cecum; Lactococcus and Peptococcus in the colon; Staphylococcus in the rectum; and Rothia in feces. The prevalence of microbial genera in certain locations could potentially be used as biomarkers to distinguish between high and low FCR. Functional prediction clustering analysis suggested that bacteria in the hindgut mainly participated in carbohydrate metabolism and amino acid metabolism, and different in the relative abundance of metabolic pathways, as predicted from the microbial taxa present, were identified by comparing the high and low groups of each location. The results may provide insights for the alteration of the intestinal microbial communities to improve the growth rate of pigs.The goal of this study was to evaluate the microbial communities in the gut and feces from female finishing Landrace pigs with high and low feed conversion ratio (FCR) by 16S rRNA gene amplicon sequencing. Many potential biomarkers can distinguish between high and low FCR groups in the duodenum, ileum, cecum, colon, and rectum, according to linear discriminant analysis effect sizes. The relative abundance of microbes were tested by Mann–Whitney test between the high and low FCR groups in different organs: The online version of this article (10.1007/s10482-018-1057-1) contains supplementary material, which is available to authorized users. Because of the rapid development of metagenomic studies, the gut microbiota from a variety of animals have been studied extensively in recent years for their role in disease causation and gut health maintenance, which has a marked influence on the health and performance of domestic livestocks. Shaped by genetic and environmental factors, especially diet, gut microbial diversity in pigs is also affected by the breed  of FCR is 0.13–0.31  of the high and low FCR groups. We then determined whether the presence of certain bacteria is correlated with pig production performance.120 female Landrace pigs were housed in an environmentally controlled room (ten pigs in each pen), and given the same corn-soybean commodity diet without antibiotics or medicines. Clean water was provided ad libitum throughout the experiment. FCR was determined from feed intake and body weight, which was recorded from 120 to 165 days of age using a Velos  automated individual feeding system that recognized an electronic ear mark. Individuals were ranked by FCR, and there was a significant difference between the high and low end . We defined the L group as individuals with low FE and high FCR values, and the H group as individuals with high FE and low FCR values . Two full-sibling pairs and two half-sibling pairs were selected, such that the siblings within each pair had opposite FCR phenotypes (Supplementary Table 1).Fresh fecal materials were collected from each individual on day 165 and kept frozen in liquid nitrogen. The chosen pigs were euthanized on day 166, and digesta samples were collected from the duodenum, jejunum (middle section), ileum , cecum, colon (middle section), and rectum  within 30 min of euthanizing. All methods were in accordance with the guidelines approved by the Quality Supervision, Inspection, and Quarantine of the People’s Republic of China (GB/T 17236–2008). The Animal Welfare Committee of China Agricultural University approved all experimental protocols (permit number: DK996).All samples were collected in sterile tubes, and then stored in liquid nitrogen until analysis. DNA was extracted and purified using a QIAamp DNA Stool Mini Kit  following the manufacturer’s instructions. Adequate quantities of high-quality genomic DNA were extracted, and the concentration of DNA was measured using a UV–Vis spectrophotometer .® High-Fidelity PCR Master Mix (New England Biolabs). Mixed PCR products were purified using a GeneJET Gel Extraction Kit (Thermo Scientific) following the manufacturer’s instructions. Sequencing libraries were generated using an NEB Next® Ultra™ DNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. The library was sequenced on an Illumina MiSeq platform, and 250 bp paired-end reads were generated.The V3–V4 region of the 16S rRNA gene was amplified (341F–806R) by polymerase chain reaction (PCR)  . Non-parametric tests (Mann–Whitney) were carried out to identify differences in microbial communities between the two groups. The data were deposited in the National Center for Biotechnology Information’s Short Read Archive under Accession No. SRR5038273.To confirm differences in the abundances of individual taxonomies between the two groups, we carried out statistical analyses using R . The number of OTUs with a 97% identity cut-off was determined for each group. The average number of sequences in each group ranged from 31,463 to 37,355, with the number of OTUs from 1214 to 2352. Bacterial diversities were compared among each location (intestines and feces) using diversity and richness estimators ; the candidate genus SMB53 was the most abundant in Ljejunum, Hileum, and Lileum. Prevotella was the predominant genus in both the high and low FCR groups in the cecum, colon, rectum, and Lfeces locations.At the genus level, unclassified bacteria in all locations accounted for 41.95–59.28% of the total reads , Firmicutes, Bacteroidetes, and Proteobacteria, were the three most abundant phyla. They constituted more than 90% of all phyla detected. Firmicutes accounted for over 60% in every group.Lactobacillus, Streptococcus, Prevotella, SMB53, and Oscillospira  were 1.55, 2.34, and 1.78, respectively. There was a higher abundance of Campylobacter and Sphaerochaeta in the Hduodenum microbiota (P < 0.05) than in the Lduodenum . Sanguibacter was the only significantly different genus between the two groups  were 0.23 and 0.53, respectively. The abundance of Kingella and SMB53 was higher in Hileum than Lileum (P < 0.05). Anaeroplasma, Arthrobacter, and Megasphaera were less abundant in the Hileum group (P < 0.05) , the four most abundant phyla,  constituted more than 95% of all the phyla detected. Firmicutes accounted for over 50% in every group and Bacteroidetes accounted for more than 20%. her Fig. .In the cecum, 13 genera were potential biomarkers for distinguishing between high and low FCR groups by LEfSe analysis; there were 7 genera unique to Lcecum and 6 genera unique to Hcecum Fig. c.Campylobacter and Butyricicoccus were more abundant in the cecum microbiota of the high FCR group (P < 0.05) compared to the low FCR group. Megasphaera, Mitsuokella, and Rhodoplanes were more abundant in the cecum microbiota of the lower FCR (P < 0.05) (Table 5) Table .Coprobacillus and Lactococcus, and a lower abundance of Peptococcus in the Hcolon microbiota (P < 0.05) than in the Lcolon microbiota , were verified based on the structure of the gastrointestinal microbiota established, accroding to PICRUSt Fig. . Other tDiet is the main factor that determines intestinal microflora in animals. Several other factors also influence animal performance  and average daily gain (ADG)  components between groups in the different locations . Except in the duodenum, the pathway differences were analyzed by LEfSe. These different pathways could be explained by different microorganisms between high and low groups in certain positions of the intestine.Prevotella and Bacteroides. By functional comparison of the high and low FCR groups in cecal and colonic microbiota, we found the microorganisms that differed in abundance were mainly related to carbohydrate metabolism. These organisms may affect the growth of the host. The cecum of individuals with high FE contained differentially abundant genes that affect the host energy absorption, mainly through the pyruvate-related metabolism pathway, such as phenylalanine metabolism, synthesis and degradation of ketone bodies, arginine and ornithine metabolism, etc. , which convert carbohydrates to lactic acid by homofermentation or heterofermentation, or to acetic acid by heterofermentation  were found in different locations of the complete intestinal tract in the high and low FCR groups, which could be of potential use to distinguish individuals for growth efficiency. Functional prediction and cluster analysis confirmed bacteria in the hindgut mainly participated in nutrient metabolism. Metabolic pathways in different locations were different between the high and low groups because of the presence of different microbes. As only four pairs of pigs were used in this study, these results need be validated using a larger cohort in the future. It will benefit pork production by facilitating the detection and alteration of the intestinal microbial community, potentially improving the growth rate of pigs.Supplementary Table 1Individuals selected for this study and their pedigrees. Supplementary material 1 (DOCX 14 kb)Supplementary Fig. 1Feed Conversion Ratio (FCR) calculated in high and low groups. Significant difference was tested of twenty individuals at the ends of the high and low FCR respectively by one way variance analysis. Supplementary material 2 (TIFF 181 kb)Supplementary Fig. 2Linear discriminant analysis (LDA) effect size (LEfSe) results for KEGG pathways for differentially abundant microbial features of intestinal locations. Histogram of the LDA scores computed for features differentially abundant in the jejunum (A), ileum (B), cecum (C), colon (D), rectum (E) and feces (F) among high and low groups (only genera LDA scores above 2 are shown). Supplementary material 3 (TIFF 1924 kb)Below is the link to the electronic supplementary material.