Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch.Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries.A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.The online version of this article (10.1186/s13068-019-1432-9) contains supplementary material, which is available to authorized users. As one of the most abundant storage of carbohydrates, starch has been extensively employed as an inexpensive and renewable raw material for food, pharmaceutical, and bio-fuel industries . Due to Bacillus acidicola and Bacillus amyloliquefaciens exhibited 793 U/mg and 45 U/mg toward raw corn starch [Geobacillus thermoleovorans [Rhizopus oryzae [Thermomyces dupontii [α-Amylase EC 3.2.1.1) is one of the oldest and most important industrial enzymes used for starch processing .1 is one. Howevern starch , 11. Otheovorans , Rhizopus oryzae , and Thedupontii  possessedupontii .Bacillus licheniformis [B. amyloliquefaciens [Bacillus subtilis [G. thermoleovorans [Another factor that hampers the application of α-amylases is the low hydrolyzation efficiency towards high concentration raw starches. Typically, starch-processing industries employ 20–30% (w/v) starch slurries . Systemaniformis , 17, B. efaciens , Bacillusubtilis , 17, andeovorans , 18 haveeovorans . An alteeovorans . HoweverBacillus aquimaris MKSC 6.2 [Geobacillus sp. 4j [B. subtilis S8–18 [Halothermothrix orenii [Aeromonas salmonicida [Pontibacillus sp. ZY. A novel α-amylase coding gene, designated as AmyZ1, was successfully cloned from Pontibacillus sp. ZY and heterologously expressed in Escherichia coli. The recombinant enzyme AmyZ1 exhibited high specific activity and broad substrate specificity towards raw starches. Furthermore, AmyZ1 could efficiently hydrolyze high concentration raw starches at temperatures significantly lower than that used in current starch processing.Due to the complexity and diversity of the marine environment, the microbes in oceans are recognized as a tremendous treasure for the discovery of novel enzymes with unique properties. Several α-amylases have been identified from the marine bacteria, including MKSC 6.2 , Geobacis sp. 4j , B. subtx orenii , Aeromonmonicida , and a mAfter incubation at 15 °C for 3 days, approximately 3600 colonies grew on the screen plates containing soluble starch. About 200 strains showed a halo around the colonies when the plates were stained with Lugol’s iodine solution and were recognized as positive clones. One strain named ZY was chosen for further research because of its larger halo than other colonies.Pontibacillus halophilus JSM 076056. Thus, this positive strain was named as Pontibacillus sp. ZY. Pontibacillus sp., implicating “Bacillus affiliated with the marine”, was a novel genus identified for the first time in 2005 [http://www.ezbiocloud.net/). Several novel enzymes have been identified and characterized from Pontibacillus sp., including protease and cytochrome P450 [Pontibacillus sp. [Phylogenetic analysis suggested that the 16S rRNA gene of strain ZY showed 99% sequence identity to the marine bacteria  in 2005 –25. By 2ome P450 . In compllus sp. . In thisAmyZ1, was cloned from Pontibacillus sp. ZY. The deduced sequence encoded by AmyZ1 contained a signal peptide comprising of 21 amino acid residues as predicted by SignalP and simple modular architecture research tool (SMART). The occurrence of a secretion signal in the deduced sequence was in agreement with the fact that AmyZ1 was secreted as an extracellular soluble protein in Pontibacillus sp. ZY. AmyZ1 showed the highest sequence identity of 99% (99% similarity) with the α-amylase from P. halophilus JSM 076056, followed by 78% identity (88% similarity) with that from Pontibacillus chungwhensis. Both the enzymes were deduced from the whole-genome sequencing and have not been biochemically characterized previously.A gene of 1521 bp, named Pontibacillus. The GH13_5 mainly contains liquefying α-amylases from different marine bacterial sources, such as Bacillus sp. YX-1, B. amyloliquefaciens, Bacillus cereus and B. licheniformis NH1 [B. licheniformis isolated from soil. The AmyZ1 structure was obtained by homology modeling using the α-amylase from B. licheniformis  as the template. Results showed that AmyZ1 was comprised of three domains, including catalytic domain A, followed by domain B and C    concentration, induction time, and incubation temperature. Fortunately, AmyZ1 can be easily refolded to its active form following the protocol described in “The recombinant enzyme AmyZ1 was expressed as inclusion bodies even after expression condition optimization, including the initial induction 2HPO4 and Na2HPO4–KH2PO4 buffers  of reducing sugars were produced after 4 h incubation at 35 °C with an enzyme dosage of 5 U/mg biomass . When using AmyZ1 paired with the commercial glucoamylase, about 75% of raw starch was hydrolyzed within 4.5 h at 30 °C, without the need of gelatinization step. As a result, the combination of AmyZ1 and glucoamylase will sharply decrease the energy cost and eventually the production cost.To further explore the action pattern of AmyZ1 on raw starches, digested raw starch granules were visualized with a Scanning Electron Microscope (SEM) . The commercial glucoamylase of A. niger was purchased from Aladdin Biological Technology Co., Ltd. . The commercial α-amylase from B. licheniformis  was obtained from Sigma Chemical Co. . Other chemicals and reagents were of analytical grade.The sediment sample from Yongxing island in the South China Sea  was collected in Sep 2016 and stored at − 20 °C until use. Soluble starch, amylose, amylopectin, α-cyclodextrin, and β-cyclodextrin were obtained from Sigma Chemical Co. . Rice, corn, and wheat were purchased from the local supermarket . They were crushed using food grinder and milled with mortar. The algae powder of −6. Aliquots of 100 μL dilutions were spread on the screening agar plates  and incubated at 15 °C for 1–3 days. Then the clones were flooded with Lugol’s iodine solution. Positive clones were screened out by the formation of a halo around the clones against the purple background.One gram of wet sediment was mixed with 9 mL of sterilized seawater, and was shaken at 200 rpm, 15 °C for 2 h. The suspension was subjected to the standard dilution-to-extinction method to 10https://www.ncbi.nlm.nih.gov/) search was carried out to determine the most closely related species.Positive strains were cultured in 5 mL standard synthetic sea salt medium (Sigma-Aldrich) and incubated at 37 °C, 180 rpm for 12 h. Genomic DNA of the positive strains was extracted according to the manufacturer’s instructions of the kit  and was used as the template. Amplification of the 16S rRNA gene was performed using eubacteria primers of Bact-27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and Bact-1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR products were cloned into the pGEM-T vector  and sequenced . Then, the Blastn  and AmyR  were designed according to the α-amylase (WP_036770168) from P. halophilus JSM 076056. After digested with NdeI and XhoI, the PCR product was ligated into pET22b(+)  to generate pET22b(+)-AmyZ1.To clone Escherichia coli BL21 (DE3) containing pET22b (+)-AmyZ1 was cultivated in 1 L Luria Broth containing 100 mg/L ampicillin at 37 °C until OD600 reached 0.6. Protein expression was induced by the addition of 0.2 mM IPTG and the culture was further incubated at 150 rpm and 37 °C for 4 h. Cells were collected at 4 °C by centrifugation at 8000×g for 10 min and resuspended in cold Tris–HCl buffer . Then the cells were disrupted by sonication, followed by centrifugation at 10,000×g and 4 °C for 30 min to isolate AmyZ1 inclusion bodies.2O supplemented with 10 mM CaCl2 was added until the final concentration of urea reached 1 M. After kept at 4 °C for 10 h, the supernatant was pooled and dialyzed against Na2HPO4–KH2PO4 buffer  containing 1 mM CaCl2 for overnight. All experiments were performed at 4 °C.To refold the protein, AmyZ1 inclusion bodies from 1 L cell cultures were dissolved in 75 mL Tris–HCl buffer  containing 8 M urea. Then dHThe refolded protein was evaluated by SDS-PAGE with 15% polyacrylamide gel. Protein concentration was determined by BCA method according to the protocol . To determine the molecular mass of native protein, the purified protein was analyzed by Native-PAGE using the precast polyacrylamide gel , and the unstained protein marker as the standard .http://www.cbs.dtu.dk/services/SignalP/) and simple modular architecture research tool . To classify AmyZ1 into a subfamily, multiple sequence alignment of AmyZ1 with other GH13 α-amylase sequences was performed using ClustalX 2.0. The phylogenetic tree was constructed by MEGA 7 using the Maximum Likelihood method. The conserved regions of enzymes were displayed using GENEDOC (http://www.psc.edu/biomed/genedoc).The presence of a putative signal peptide was predicted using SignalP 4.0 program (http://swissmodel.expasy.org) with α-amylase from B. licheniformis (PDB code: 1BLI) as the template. The structures were visualized using Pymol (http://www.pymol.org/).The three-dimensional structure of AmyZ1 was generated using the automated Swiss-Model protein modeling server  supplemented with 1% raw rice starch and 1 mM CaCl2. After incubating the mixture at 35 °C for 10 min, the reaction was stopped by adding 300 μL of DNS. The reaction mixture was then heated in boiling water for 15 min. The amount of reducing sugars released was monitored at 540 nm. One unit of amylase activity was defined as the amount of enzyme needed to release 1 μmol of reducing sugars as maltose per minute under standard assay conditions described above. Assays with heat treated AmyZ1 was used as control.The enzyme activity of AmyZ1 was determined by measuring the reducing sugars released from the hydrolysis of raw starches base on the dinitrosalicylic acid (DNS) assay. The reaction mixture contained 30 μL enzyme solution, and 570 μL Na2HPO4 buffer  and Na2HPO4–KH2PO4 buffer . In the absence of Ca2+, the thermostability was determined by incubating the enzyme in Na2HPO4–KH2PO4 buffer  at 20–35 °C. At appropriate intervals, the residual activity was measured using the DNS method as mentioned above. The pH stability of AmyZ1 without Ca2+ was determined by dispersing the enzyme in Na2HPO4–KH2PO4 buffer of pH 6.0, 6.5, and 7.0, and the residual activities were measured at appropriate intervals.The effect of temperature on AmyZ1 activity was determined at temperatures ranging from 20 to 70 °C using raw rice starch as the substrate. The optimum pH of AmyZ1 was examined in the pH range of 4.0 to 8.5 at 35 °C in citrate–Na2+ and Na+ on the activity of AmyZ1, additional supplement of CaCl2 and NaCl was included in the reaction mixture that consisted of raw rice starch, appropriate volume of enzyme, and Na2HPO4–KH2PO4 buffer . To determine the thermostability in the presence of Ca2+ or Na+, the enzyme was mixed with different concentrations of CaCl2 or NaCl and incubated at 30 or 35 °C. The residual activities were measured at appropriate intervals.To evaluate the influence of Ca2+, Cu2+, K+, Mg2+, Zn2+, and EDTA. In control, the reaction mixture contained appropriate volume of enzyme and Na2HPO4–KH2PO4 buffer  supplemented with rice raw starch as the substrate. The enzyme activity determined in the control were defined as 100%. In the reaction mixture containing additional metal ions, the enzyme activity was determined and the relative activities were calculated. The relative activities were defined as the activity in the presence of additional metal ions relative to that of control.The effects of other metal ions and chemicals on the activity of AmyZ1 were evaluated under the standard assay conditions with additional supplement of 1, 5 or 10 mM MnThe substrate specificity was determined under the standard assay condition using 1% (w/v) raw starch from various origins including rice, corn, wheat, barley, potato, and bean. Soluble starch, amylose, amylopectin, pullulan, α-cyclodextrin, and β-cyclodextrin were also used as the substrates.Km and Vmax, were measured using raw rice starch as the substrate based on the DNS assay. The reaction was performed by incubating the enzyme in Na2HPO4–KH2PO4  supplemented with varying concentrations of raw rice starch (1.0–20 mg/mL). The reaction was performed at 35 °C for 10 min. The kinetic parameters were calculated by fitting the experimental data to Lineweaver–Burt equation of the Michaelis–Menten model using Origin 8.0.The kinetic constants of AmyZ1, including C. pyrenoidosa. With raw rice, corn, and wheat starch as the substrates, factors including enzyme unit (0.5–10 U/mg starch), substrate concentration , reaction temperature (20–35 °C), and reaction time (1–24 h) were optimized using the single factor analysis. Reactions were carried out in a mixture containing appropriate volume of enzyme, raw starches, and Na2HPO4–KH2PO4 buffer  supplemented with 1 mM CaCl2.Hydrolysis activity of AmyZ1 toward high concentration raw starch was evaluated using raw starches from terrestrial plants including rice, corn, and wheat, as well as aquatic algae C. pyrenoidosa as the substrate, the C. pyrenoidosa powder was suspended in Na2HPO4–KH2PO4 buffer  to a final concentration of 5% (w/v). The mixture was disrupted by sonication for 30 min at a frequency of 40 kHz and an acoustic power up to 450 W [Specially, when using raw starch from to 450 W . Five peThe extent of raw starch hydrolysis was calculated using the following formula:reaction .A. niger. The purified AmyZ1 (1 U/mg raw starch) was added to 30% raw corn starch slurries. The reaction mixture was incubated at 30 °C for 4 h with shaking at 150 rpm. Then, the mixture was supplemented with the commercial glucoamylase (5 U/mg raw starch) and sequentially incubated at 55 °C for another 24 h. Furthermore, the commercial α-amylase from B. licheniformis (BLA) was used as control to hydrolyze raw corn starch performed as mentioned above. At appropriate intervals, the samples were withdrawn and the reducing sugars were measured by the DNS method using glucose as the standard. The rates of hydrolysis were calculated according to the formula described above.Thirty percent (w/v) raw corn starch slurries were employed to evaluate the combined hydrolysis effect of α-amylase AmyZ1 and the commercial glucoamylase from g to recover the pellets. Then the pellets were washed with pure ethanol for three times, followed by drying at 35 °C to a constant weight. The samples were fixed on a specimen holder using a silver plate and coated with Pt using Ion Sputter E-1010 at 5.0 kV and 20 mA for 40 s. The specimens were then viewed with SEM.The raw starch shape before and after hydrolysis was visualized using a Scanning Electron Microscope . Briefly, the reaction was conducted using AmyZ1 (5 U/mg starch) and 5% raw starches from various sources including rice, corn, and wheat. After incubation at 30 °C for 30 min, the mixture was centrifuged at 8000×The hydrolytic products of AmyZ1 from raw starches were determined using high-performance liquid chromatography  equipped with a Carbohydrate ES column  and an evaporative light scattering detector. The mobile phase was acetonitrile and water  with a flow rate of 1.0 mL/min at 25 °C. Glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), and maltopentaose (G5) were used as standards.Pontibacillus sp. ZY and AmyZ1 amino acid sequence have been deposited in the GenBank database with the accession number MH279661 and AXV43605, respectively.The partial 16S rRNA gene sequence of Additional file 1: Figure S1. Alignment of the amino acid sequences of AmyZ1 and other known α-amylases. The four conserved regions (region I-region IV) were boxed. The key catalytic residues were indicated below the sequences by a black triangle. 1BLI: α-amylase from Bacillus licheniformis; ADE44086: α-amylase from Bacillus amyloliquefaciens; WP_058836133: α-amylase from Luteimonas abyssi.Additional file 2: Figure S2. Phylogenic analysis of AmyZ1 and other reported α-amylases of GH13 family. The protein sequences of different subfamilies were retrieved from CAZy database. The tree was built using Maximum Likelihood method of the program MEGA 7. The bootstrap values were calculated based on 1000 replicates.Additional file 3: Figure S3. Homology model structure of AmyZ1. The structure of AmyZ1 was constructed based on its closest structural relative Bacillus licheniformis α-amylase (PDB code: 1BLI). The domains were colored as follows: A, red; B, green; C, blue.Additional file 4: Table S1. Denaturation and renaturation of α-amylase AmyZ1.Additional file 5: Figure S4. Effects of NaCl and CaCl2 on AmyZ1 stability. The assays were performed by incubating the enzyme at 30 °C in Na2HPO4–KH2PO4 buffer  containing additional CaCl2 or NaCl. The residual activities were determined at various time intervals.Additional file 6: Table S2. Effects of metal ions on enzyme activity.Additional file 7: Figure S5. Scanning electron microscopy of raw starch granules hydrolyzed by AmyZ1. a, c and e: raw rice, corn, and wheat starch before hydrolysis; b, d and f: raw rice, corn, and wheat starch treated with AmyZ1.Additional file 8: Table S3. Hydrolysis products of raw starches catalyzed by AmyZ1.


Tg) and gelatinization temperature significantly increase (p < 0.05) after microwave heating. According to correlation analysis, the power intensity and heating time were correlated negatively with the lifetime of o-Ps. In addition, the changes of amorphous region and Tg of rice starch were strongly related to FV changes. These results provided a theoretical basis for further research on the directional regulation of FV and improvement the quality of starch-based food by using microwave treatment.The aim of this work was to investigate the role of microwave parameters and moisture content on the free volume (FV) changes of rice starch by positron annihilation lifetime spectroscopy analysis (PALS) and to explore the potential relationship between the changes of FV and physicochemical properties of rice starch. Microwave heating and water molecules lead to the increasing of FV of starch. However, this result is largely influenced by the plasticization of water molecule. The anti-plasticization caused by water evaporation resulting in a decrease in the size and concentration of FV during microwave heating. Significant decrease (p < 0.05) in the thickness of amorphous region of microwave-heated rice starch was found by small angle X-ray scattering (SAXS), and the glass transition temperature ( It has been confirmed that FV is temperature-dependent, and the temperature at which the FV is insufficient for changing molecular conformation, is termed as glass transition temperature (Tg)3. Therefore, the changes of FV hole will interfere the dynamic and physical properties of polymer. For example, the viscosity of polymer system reduces upon increasing in FV, which is attributed to the enhancement on diffusion ability of the molecular chains4.Free volume (FV), the concept of which was first proposed by Fox and Flory with the motivation for illustrating the glass transition process, was defined as the unoccupied volume composed by interspace of molecular chain or imperfections in random arrangement of molecular5. The starches with diverse amylose/amylopectin ratios possess the different FV, which will determine the structure, phase transitions and physicochemical properties of starches, and ultimately affect the functional characteristics of starchy food6. An earlier study in 1999, Benczedi reported that FV of starch became larger with a higher water concentration7, while in other blended starch systems, the role of water was found to be significantly more complex. Hughes indicated that the holes in starch/sucrose blend decreased firstly and then increased with rising moisture content8. Therefore, it was speculated that water could influence the starch FV in two ways through the nanoholes occupation and inter-molecular hydrogen bonding formation9.Starch is a complexly semi-crystalline biopolymer consisting of amorphous amylose and crystalline amylopectin lamellae10. The main reason for temperature dependence is that segments of the molecular chains are activated to initiates the thermal motion when the thermal energy is sufficient, which determines the size and distribution of the FV in the polymer. Therefore, various heating approaches, heating rate and heating time are bound to change the FV holes. Microwave heating has an extensive application in food processing because of its rapid heating rate and safe handling11. In our previous works, we have demonstrated that microwave can change the lamellar architecture of rice starch12, which suggested that it might play an important role in regulating the FV of starch. However, there are no available publications regarding the effects of microwave heating on the FV holes of starch although it can manipulate FV of other polymer materials13. In addition, it is noteworthy that water is a vital dipole molecule, which had a high response to the applied microwave electromagnetic field14. Moisture content, impacting the hydration process of starch, is an important factor in the process of microwave heating starch as we previously reported15. However, it is still not clear, microwave heating and water molecule, which plays a more important roles in the FV changes of starch.Actually, temperature significantly changes the FV of polymer materials16, which involves the detection of the lifetimes of positron injected into a polymer. The first objective of this study was to evaluate the effect of moisture content and microwave parameters on the FV changes of rice starch during microwave heating. The second objective was to provide insights into the relationship between the changes of FV and physicochemical properties of rice starch upon the microwave processing.Currently, positron annihilation lifetime spectroscopy (PALS) is a highly suitable technique for characterization of FV holes in starchet al.17. The moisture content of rice starch was adjusted to 10%, 15%, 20%, 25%, 30% and 35% and equilibrated for 12 h at ambient temperature.Native rice starch (up to 95% pure) was purchased from BENEO Asia Pacific Pte. Ltd. The amylose and protein contents (dry mass) of the starch were detected according to method reported by Lin 18. After cooling at ambient temperature, parts of samples were screened directly through an 80 mesh sieve for PALS and Tg analysis. The others were dried at 30 °C until the moisture content stabilized, and then screening for small angle X-ray scattering (SAXS) and differential scanning calorimeter (DSC) analysis.Batches, one gram, of rice starch were processed at a varieties of power intensity  by using a Milestone single-mode/multimode microwave synthesizer chamber , which was equipped with an infrared temperature probe to real-time monitor the temperature of samples22Na was deposited in a BaF2 probe9. Eight grams of starch samples were sealed with Mylar film (7 mm thick) to prevent water evaporation during measurement; the source was placed in the center of the sample to form a sandwich structure. The time resolution [full width at half maximum (FWHM)] of prompt spectra was 0.23 ns and the time spectrum was measured until ~106 total counts were accumulated in the peak channel. The lifetime τ3 and the intensity I3 of o-Ps represent the FV hole size and the number of FV cavities per unit volume of starch, respectively, which were analyzed by using the spectral software PATFIT (discrete spectrum) and LT (continuous spectrum).The lifetime measurements were made with a standard fast-fast coincidence system, and the positron source 3 and the FV of the hole radius is calculated20.According to the Tao-Eldrup formula, the relationship between the value τΣ = R + ΔR and ΔR is 0.166 nm, which is an empirical parameter; that is, the thickness of the uniform electron layer in the spherical potential well is assumed, and o-Ps is trapped solely in the electron layer.The FV radius R is calculated with formula , and thev, which can be calculated by formula  monitored by a photomultiplier. The rotating anode device was operated at 50 kV and 0.6 mA, and the exposure time was 15 min. The scattering vector in the range of 0.07 to 2.3 nm−1 was detected . The formula of the one-dimensional correlation function is as follows, where x represents the actual distance, q is the scattering vector, and I is the scattering intensity.For SAXS analysis, the moisture content of rice starch was adjusted to 50% in order to remove the water in the upper layer of starch. The lamellar structure of rice starch was performed on a SAXS nanostructure analyzer , with an incident X-ray beam  horizontal line and the oblique line on the Z coordinate. da is the average thickness of the amorphous region (da = L − dc). L is the average period of the layered structure (long period); the value corresponds to the first maximum (peak) of γ(x) on the Z coordinate. Φ represents the volume fraction of the crystalline zone in the long period, that is, the ratio of dc to L. The graphical representation of the mentioned parameters was shown in Figure The information related to the Small angle X-ray scattering (SAXS) spectra of the starch samples was analyzed with a one-dimensional function, and the related parameters were calculated by referring to the methods of Zhu Tg of the native starch-water system was carried by using DSC . 5 ± 0.2 mg of starch samples was sealed in an aluminum pan and heated at a rate of 10 °C/min from 10 °C to 130 °C, then samples were cooled at 20 °C/min to 10 °C, and heated at 5 °C/min to 130 °C according to two heating scan method24. The Tg values were estimated from the midpoint of the heat capacity change observed during the second heating scan.17. The parameters of To (onset temperature), Tp (peak temperature), Tc (conclusion temperature), and ΔH  were obtained based on the DSC analysis.The gelatinization properties of starch were performed as described to evaluate molecular activity by using a DSC . The moisture content of sample was measured before DSC analysis, and appropriate amount of rice starch was mixed with deionized water to ensure a weight ratio of starch and water was 1:2. 8–10 mg samples were placed in an aluminum pan and heated at 30 °C for 5 min, followed by heating from 30 °C to 90 °C at a scanning rate of 10 °C/minOriginPro 8 SR1  and Microsoft Office Excel® 2013 was used to conduct the graphical analysis and statistical calculations. Values were expressed as mean values ± standard deviations. Significant differences among values were calculated based on Tukey’s procedure at p < 0.05, using the software SPSS statistics (version 17). All the measurements were repeated at least three times for each sample.3 and I3 values of starch samples, which indicated that the FV in the starch was expanded by water adsorption. Water molecules regulate the FV of starch through occupying the free spaces and changing the hydrogen bond interactions in the biopolymer9. The I3 value of pure water is 26.9%, indicating the generation probability of positronium in the pure water is higher than that in the plasticized starch25. The I3 values of samples were less than those in the pure water and increased with increasing moisture content, which indicated that the FV of starch was not filled with water molecules. The I3 value may be the result of the average of o-Ps intensity between the small molecules and starch polymer. To further evaluate the effect of moisture content on FV in the rice starch, the size distribution of the FV in starch systems with various moisture contents was analyzed from the continuous distribution spectrum  were selected for the following studies.The FV of rice starch with different moisture content was evaluated by PLAS analysis Fig. . The inc3 and I3) decreased in the microwave-irradiated samples26. The FV of rice starch at varieties of power intensity were calculated by the Tao-Eldrup formula   and in a range of heating time (0 to 10 min) by PLAS analysis. The lifetime and intensity values of o-Ps appeared to a downward trend as the power intensity increased Fig. , suggestla Table 19,20. Wein) Fig. . The FV ly Table . Therefo28. In addition, the effective of microwave absorption resulted in the temperature of samples raised rapidly, which had a positive effect on the FV9. However, our previous study observed that the changes of FV size and concentration were different by comparing microwave and conventional heating methods29. Therefore, it could be concluded that the plasticization of water molecules play a synergetic effect in heating process and the microwave heating resulted in the FV changes of rice starch, but the anti-plasticization caused by the evaporation of water is the main reason for the reduction of FV in the samples during microwave heating.To further verify whether the FV changes of rice starch were caused by the evaporation of water after microwave heating, the FV of samples with various of moisture contents were analyzed. As the results shown in Fig. 30. As shown in Tables 31, which implied that the structure of starch was matching with the three-phase model. This model consists of crystalline region, moving amorphous region, and amorphous zone with a semi-crystalline lamellar structure, which is consistent with previous reports32. However, compared to the native starch, the average thickness of amorphous region of microwave-heated rice starch had a tendency to decrease, which was possible caused by the compression of the amorphous region after microwave heating. Therefore, it is speculated that there is a correlation between FV and amorphous region of starch lamellar structure.The effect of microwave heating on the lamellae structure of rice starch was investigated by SAXS. There was no significant difference in the scattering peak position of the sample with the increasing of power intensity and heating time, which indicated that the thickness of lamellae structure of rice starch was little changed by microwave heating. One dimensional function Fig.  was usedTg, as an important characteristic of amorphous polymers, contributes to the physical properties of food33. As shown in Fig. Tg increased with the microwave power intensity. This result is consistant with previously reported by Zhao et al., which the power intensity affected the water distribution and Tg was negatively correlated with the areas of cytoplasmic bulk water34. The fluctuation was observed during microwave heating in a range of time (Fig. Tg was increased. As the previously reported that the changes of the Tg had a strong association with FV35, and the variation of Tg was corresponding to FV changes of rice starch. In addition, configurational rearrangements of starch chain backbones will occur extremely slowly at this critical temperature36. Therefore, the changes in Tg may be attributed to the decrease of FV and molecular mobility of rice starch.The ime Fig. , but the37. Based on the microwave heating, more polar groups of rice starch will be available for microwave absorption leading to maximum interaction to rearrange the crystallization and amorphous area of starch granules to form a more orderly crystal matrix38. The rearrangement resulted in the increasing of crystalline area and the amorphous region reduction of rice starch, and the changes of structure possibly resulted in the decrease of the FV and molecular mobility5. These results seem that the increased in Tg and gelatinization enthalpy might be related to the changes of the FV in rice starch. In addition, excessive power intensity may induce partial structural degradation of starch, which resulted in the reduction of gelatinization enthalpy39.After microwave heating, the gelatinization properties of rice starch were investigated by DSC analysis. As the results shown in Tables 40. For the correlation between FV and specific physical properties of rice starch based on the results of samples with different heating time, da had a significant positive correlation with τ3 and I3, which suggested that amorphous region had a strong correlation with the size and concentration of FV. In addition, the shrink of FV in polymers will have an impact on the chain segment movement, resulting rise in viscosity and Tg33, thus significant negative correlation was observed between Tg and the FV concentration of rice starch.The results of the correlation analysis Tables  and 6 shThis study provides novel and deeper insights on the FV changes of rice starch during microwave heating. More importantly, we reveal that the microwave heating and the plasticization of water molecules play a synergetic effect on the FV changes of rice starch, and the physical properties of rice starch were also strongly related to FV changes in the samples. These results may facilitate optimization of starch-based microwave food process to achieve better quality features. The detailed regulation between the FV changes involved in microwave parameters and the quality of products requires further investigation with improved strategies.Supporting Information


Application areas of robotic grasping extend to delicate objects like groceries. The intrinsic elasticity offered by variable-stiffness actuators (VSA) appears to be promising in terms of being able to adapt to the object shape, to withstand collisions with the environment during the grasp acquisition, and to resist the weight applied to the fingers by a lifted object during the actual grasp. It is hypothesized that these properties are particularly useful in the absence of high-quality sensory feedback, which would otherwise be able to guide the shape adaptation and collision avoidance, and that in this case, VSA hands perform better than hands with fixed stiffness. This hypothesis is tested in an experiment where small-fruit containers are picked and placed using a newly developed variable-stiffness robotic hand. The grasp performance is measured under different sensory feedback conditions: full or impaired visual feedback, full or impaired force feedback. The hand is switched between a variable-stiffness mode and two fixed-stiffness modes. Strategies for modulating the stiffness and exploiting environmental constraints are observed from human operators that control the robotic hand. The results show consistently successful grasps under all stiffness and feedback conditions. However, the performance is affected by the amount of available visual feedback. Different stiffness modes turn out to be beneficial in different feedback conditions and with respect to different performance criteria, but a general advantage of VSA over fixed stiffness cannot be shown for the present task. Guidance of the fingers along cracks and gaps is observed, which may inspire the programming of autonomously grasping robots. Online grocery sales are an intensively growing field with growth rates of more than 25% year-over-year in the USA . It focuses on the development of manipulation systems with simple, robust, and efficient robotic hands with embodied compliance, which both endure and require the exploitation of environmental constraints like surfaces and edges for guiding their motion.Solutions to these problems are investigated within the European project Concepts for embodied compliance include series-elastic actuators —is presented and its performance in a particular grocery manipulation task—picking small-fruit containers from storage boxes—is examined.In this paper, a VSA robotic hand prototype—the The WHISG bases its VSA technology on the one of the DLR Hand Arm System  of intrinsic hand muscles according to Höppner et al. , a methoTeleimpedance using sEMG has also been studied for VSA hands  and two underactuated fingers with two DoF each, which oppose the thumb. The two thumb DoF closest to the base constitute the metacarpophalangeal (MCP) joint. It allows sideways (adduction/abduction) and bending (flexion/extension) movement. The MCP is followed by the proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints with one flexion/extension DoF each. The opposing fingers contain only one PIP and one DIP joint each.For adjusting the stiffness of the joints, the Flexible Antagonistic Spring  allows setting the stiffness of each joint separately from the other joints. This concept is followed, e.g., in the DLR Hand Arm System. To reduce weight and cost, the WHISG hand uses less than 2N motors.Having two motors per DoF  Bluebird BMS 390 DMH servo motors together  by Force Dimensions Figure  top leftIn the variable-stiffness mode, the pretension of the WHISG thumb and fingers, and thereby their stiffness behavior, was controlled via the operator's grip stiffness, which was acquired via sEMG signals from intrinsic hand muscles according to Höppner et al. . To measA block diagram depicting the connections between the subsystems is shown in Figure Six healthy subjects, all male, five right-handed and one left-handed, age 24–30, all naive to the experiment, took part as operators and performed the experimental protocol described below. The whole procedure lasted between 60 and 75 min per participant. Oral and written descriptions of the experiment were provided to the subjects. After all questions were answered a written consent form was signed by all participants. These experiments are compliant with the World Medical Association's Declaration of Helsinki, regarding the ethical principles for medical research involving human subjects, last version, as approved at the 59th WMA General Assembly, Seoul, October 2008. Necessary approvals for the subject studies were received from the organization-wide works council of the German Aerospace Center as well as its institutional board for data privacy. A physician is part of the works council. The collection and processing of experimental data were approved by both committees.Operators were asked to grip one out of eight tightly packed small-fruit containers (LxWxH: 140 mm x 90 mm x 75 mm) out of a cardboard box (LxWxH: 400 mm x 300 mm x 90 mm) using the WHISG hand , low fixed stiffness (LS), and high fixed stiffness (HS). VS corresponded to the range of 0 to 55% pretension of the FAS mechanisms, LS to 5% pretension and HS to 55% pretension. Each subject was assigned a different permutation of stiffness modes to reduce effects of learning or fatigue on the results.visual feedback (VF) only: the operator had an unimpaired view of the experiment area and the force generation of the GFM was switched off;no feedback (NF): the visual feedback was impaired by altered welding goggles and the force generation of the GFM was switched off; the welding visor glasses were replaced with plastic disks which were processed with sandpaper and an added adhering plastic sheet; objects and their outlines seen through these glasses were blurred;full feedback (VF+FF): the operator had an unimpaired view of the experiment area and the force generation of the GFM was switched on;force feedback (FF) only: the visual feedback was impaired and the force generation of the GFM was switched on.Within each stiffness mode, the following feedback modes were tested:A questionnaire  was filled about the usefulness of the different stiffness modes, about exploiting the environment to get a secure grasp on the object and about the helpfulness of obtaining force feedback.To answer the research questions and evaluate the hypotheses of the study, several types of data were collected and analyzed during the trials.the grasp success rate; a grasp was counted as successful whenever the small-fruit container was grasped, lifted, and placed on the table next to the cardboard box within the time limit of 2 min; the grasp success rate was calculated for each experimental condition  as the number of trials with grasp success divided by the total number of trials within this experimental condition over all operators and repetitions;the task completion time of each trial, i.e., in case of successful grasps, the time it took to complete the task, and otherwise, the 2 min after which the trial was aborted;the number of grasping attempts for each trial, i.e., the number of times that the commanded gripper opening angle crosses a certain threshold and thereby starts a gripping phase;the mean thumb gripping torque for each trial, i.e., the mean torque of the thumb joints during the last gripping phase before placing the container on the table.For investigating the effects of stiffness and feedback modes on the grasp performance and for evaluating the hypothesis that variable stiffness helps to compensate lack of sensory feedback better than fixed stiffness, four performance measures were recorded:For the grasp success rate, 12 values for twelve the experimental conditions were recorded, while the other three recorded performance measures contained 288 values for 288 trials.The three latter performance criteria were analyzed with a linear mixed regression model,yijkmn is the response variable, i.e., any of the three above-mentioned performance measures—task completion time, number of grasping attempts or mean thumb gripping torque—, i is the stiffness mode, j denotes the presence of unimpaired visual feedback, k denotes the availability of gripping force feedback, m is the within-operator trial number, n is the operator number, β0 is the intercept, which is a constant term, βistiffness_mode, is the fixed effect of the stiffness mode, βjvisual_feedback, is the fixed effect of visual feedback, βkforce_feedback, is the fixed effect of force feedback, βijstiffness_mode×visual_feedback,, βikstiffness_mode×force_feedback,, βjkvisual_feedback×force_feedback,, and βijkstiffness_mode×visual_feedback×force_feedback, are the fixed effects of their interactions, mβtrial_number is the trial-number-dependent fixed effect of learning or fatigue, ϵnoperator, is the operator-specific random effect and ϵmn is the residual random error. The random effects are assumed to follow normal distributions as follows:where How operators experienced the helpfulness of the different stiffness modes and the availability of force feedback was measured using a questionnaire  with three questions for the stiffness modes and one question for force feedback. The answers were grouped into positive, neutral, and negative groups and reported summarily over all operators.For the analysis of the stiffness modulation strategies in the variable-stiffness mode, the mean normalized stiffness values in the searching phase and the grasping phase were recorded.They were analyzed using the following mixed regression model:Kjklmnmean, is the response variable, i.e., the mean normalized stiffness, l denotes the phase (searching or gripping phase), βlphase, its fixed effect on the response variable, βjlvisual_feedback×phase, and βklforce_feedback×phase, its interactions with the feedback modalities and the other variables as in Equation (1).where lmer function of the lme4 library  the effects of stiffness and feedback modes on the grasp performance and user experience, (b) in the case of the variable-stiffness mode, observations of stiffness modulation strategies, and (c) observations of strategies for exploiting environmental constraints.The effect of the trial number, which can account for learning or fatigue, is two to three orders of magnitude smaller than the effects of stiffness and feedback.The grasp performance is measured by four criteria: grasp success rate, task completion time, number of grasp attempts, and mean gripping torque.The grasp success rate is 100% for all stiffness and feedback modes.T, number of grasp attempts Nga, and mean gripping torque τmean—, the absolute measurement values at the different combinations of stiffness and feedback modes are shown as samples and box plots in Figure For the other three grasp performance measures—task completion time tendencies. Since the significance level is not corrected for multiple comparisons, the confidence intervals are interpreted in conjunction with each other as aggregate results, rather than independently as separate results.How these three measures are affected by the presence or lack of sensory feedback and by the variable or fixed grip stiffness modes is shown in Figures The distributions of the residuals of the mixed models of task completion time and number of grasping attempts were somewhat positively skewed compared to a normal distribution, but still unimodal and smooth. While the method is robust to deviations from the normal distribution, this adds some uncertainty to the results.T ≈ +11 s…+15 s, ΔNga ≈ +2…+3, Δτmean ≈ +0.1 Nm… +0.2 Nm; Figures T ≈ +4 s…+5 s, ΔNga ≈ +0.2…+0.8, Δτmean ≈ +0.04 Nm… +0.1 Nm; Figures mean ≈ +0.08 Nm… +0.15 Nm; Figure T ≈ −3 s, ΔNga ≈ −1, Δτmean ≈ −0.05 Nm; Figures The effect of the sensory feedback is shown in Figure Figure In the questionnaire, operators gave one neutral and five negative responses for the low stiffness, five positive and one neutral for the high stiffness and four positive and two neutral for the variable stiffness setting. Four out of six operators found the presence force feedback helpful.To answer the question how the operators adjust the stiffness in the variable-stiffness mode during the course of the trial, it is interesting how the stiffness changes between the searching phase and the grasping phase and how it is affected by the sensory feedback mode. Figure The effect of sensory feedback itself on the mean normalized stiffness is shown in Figure In the questionnaire, five out of six operators reported using the environment to get a secure grasp on the object.Figure In this study, picking and placing of small-fruit containers from a tightly packed set by the variable-stiffness WHISG robotic hand was investigated under different stiffness modes and different levels of sensory feedback. The hand was positioned and controlled by human operators as proxies for a robotic arm and a control computer. It was shown that the WHISG hand is suitable for consistently performing the task well within the time constraint of 2 min even when the controller received only a limited amount of sensory feedback. However, it was also shown that the task could even be completed by a mechanically simpler hand with a fixed stiffness behavior.Regarding the influence of sensory feedback on the grasping performance, it was shown that high-quality visual feedback tends to decrease task completion time, number of grasping attempts, and gripping torques. The influence of force feedback was less decisive, with a tendency of increasing task completion time, number of grasping attempts, and gripping torques when using a low-stiffness hand in the absence of visual feedback, but slightly decreasing gripping torques otherwise. This matches the result of Laghi et al.  that visDespite the lack of clear benefit on the measured grasping performance, the majority of operators reported in the questionnaire that they found force feedback helpful. This is similar to the findings of Godfrey et al. , where sThe hypothesis that variable stiffness can better compensate lack of sensory feedback than fixed stiffness could not be confirmed comprehensively. Under conditions of impaired visual feedback, high fixed stiffness tended to provide the lowest task completion times and least number of grasping attempts. When high-quality visual feedback was given, low fixed stiffness tended to perform at similar speed and number of attempts as the high-stiffness and variable-stiffness hands, but tended to apply somewhat lower gripping forces, which may be beneficial for the handling of delicate goods. Variable stiffness tended to provide a compromise between high and low stiffness when both visual and force feedback were lacking. While under these conditions it performed similarly fast and with a similar number of grasping attempts as high fixed stiffness and faster and with less attempts than low fixed stiffness, it tended to apply somewhat lower gripping torques than high fixed stiffness but higher gripping torques than low fixed stiffness.The grasp success rate was 100% under all stiffness and feedback modes, which we found surprising. Apart from proving the suitability of the WHISG hand for the given task, this also shows that a more difficult task might have been needed for better discovering possible benefits of variable stiffness in situations of sensory deprivation. In future studies, the task could be made more difficult by tightening the time constraint, by grasping more delicate objects without hurting them and by positioning the robotic hand by a robotic arm to eliminate the inadvertent force feedback on the operator's forearm via the splint.The investigation of stiffness variation strategies showed that operators apply higher stiffness in the grasping phase than in the searching phase and somewhat higher stiffness in the presence of force feedback than in its absence. The higher stiffness in the grasping phase corresponds to the presumption that the fingers need to withstand the weight of the object during lifting. The higher stiffness in the presence of force feedback could be explained by the fact that the stiffness of the robotic hand is not only a function of the robotic grasping force, but also of the sEMG-controlled FAS pretension level, which is in turn influenced by the contraction and cocontraction of the operator's muscles. In the absence of force feedback, no forces are applied to the operator's fingers, hence the operator can only modulate the stiffness by cocontraction. When force feedback is turned on, the operator additionally contracts to counteract the forces on the finger, which further increases his grip stiffness and the sEMG signals. This is actually more an artifact of the teleimpedance setup than an insight into an optimal stiffness modulation strategy.The observation of environmental constraint strategies yielded four distinct grasping patterns, one of which enjoyed a striking preference by being used in 94% of the trials. Interestingly, this pattern, which exploits gaps between the containers near their corners, places only two fingers in grasping positions, as opposed to the other patterns, which place three fingers and exploit the cracks between the containers in addition to the gaps. One possible explanation lies in the fact that subjects had difficulties to see the second finger that often ended up unused. A further possible explanation is that forcing the fingers in the crack between the containers required more force and was therefore harder to realize than the placement in the corner gaps.In conclusion, the study showed that the VSA WHISG hand is suitable for the task of picking and placing small fruit containers from a tightly packed set but that the task may also be done with a simpler SEA hand with fixed stiffness. Furthermore, it showed operators' strategies of exploiting the cracks and gaps between containers to guide compliant fingers to relevant grasp contact points, which may also be useful for autonomously operated robots.MH contributed to literature review, implementation of electronics, calibration methods, experiments, data analysis and paper writing. WF developed the robotic hand, supervised the project overall, and contributed in proof reading. GS contributed to the interpretation of data, writing, and proof reading the manuscript. HH developed the idea, supervised the project overall and contributed in data analysis, writing, and proof reading.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Journal continues its policy of presenting to its readership the latest progress in the fields of science and practice of cardiology in an easily readable and timely manner.The concept of devoting an issue of the Journal to one topical subject, was recently started to present virtually all aspects of a chosen topic by experts in the field, presenting the latest progress of knowledge in their area.In the current issue, HCM was chosen because of the recent sustained increase in data relating to both management of, and research into, this relatively common inherited disease which could be of interest to a wide range of our readership.The current issue represents the proceedings of a recent meeting organised by the Guest Editors in Barcelona. We are grateful to all of them for making this project a reality, and hope that the issue of the Journal will achieve its declared objectives.


The evaluation of drug-induced cytotoxicity is of great importance for the clinical application of pharmaceutical products, and human-induced pluripotent stem cells (hiPSCs) have received considerable scrutiny as a cell source for in vitro cytotoxicity testing. The aim of this study is to validate the concept of cytotoxicity testing using hiPSC-derived retinal pigment epithelium (hiPSC-RPE) by comparing the responsiveness of human fetal RPE (hfRPE) and human RPE cell line (ARPE19) to recombinant tissue plasminogen activator (rtPA).  HfRPE, two types of hiPSC-RPE, and ARPE19 were cultured in media with or without rtPA. A lactate dehydrogenase release assay was performed to investigate the dose- and time-dependent effects of rtPA on cell death. RPE function was evaluated by measuring the secretion of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) and RPE-specific gene expression. μg/ml) for 1 hour, whereas ARPE19 cell damage was increased by supplementation with rtPA at concentrations higher than 50 μg/ml. Although 100 μg/ml rtPA for 24 hours did not affect RPE cell function, sustained rtPA exposure induced prolonged cytotoxic effects in hfRPE and two hiPSC-RPE, but not ARPE19.  Rates of cell damage in hfRPE and both hiPS-RPE were increased by rtPA supplementation (2000 and 4000  The responsiveness of hiPSC-RPE to rtPA is similar to that of hfRPE in terms of cell death and cell function. Thus, hiPSC-RPE is a valuable cell source for in vitro cytotoxicity testing. The evaluation of drug-induced cytotoxicity is of great importance for the clinical application of pharmaceutical products. Although human cell lines or primary rodent cells have been used for cytotoxicity testing in vitro, these cells might possess different characteristics from native human cells due to immortalization or different race. In recent years, human-induced pluripotent stem cells (hiPSCs)  have recμg/ml appears to be toxic to the neural retina and RPE. In vitro, rtPA damages nerve cells in the ganglion cell layer or inner nuclear layer by enhancing N-methyl-D-aspartate signalling and apoptosis [Subretinal hemorrhage is the presence of a blood clot between the photoreceptors and RPE and typically results in poor visual outcomes . To addrpoptosis , 11. HowIn the present study, we compared the responsiveness of hiPSC-RPE, human fetal RPE (hfRPE) as native RPE, and a human RPE cell line (ARPE19) to rtPA by evaluating cell morphology, cell death, and cell function to validate the concept of cytotoxicity testing using hiPSC-RPE. In addition, the potential effects of rtPA administration on cell function were elucidated by evaluating rtPA cytotoxicity in vitro at clinically used and toxic concentrations.μM, Sigma-Aldrich Corp.)) after reaching confluence. The medium was changed every 2 to 3 days. Cultured RPE were recorded using a laser scanning confocal microscope .The hiPS cell lines 454E2  and 253G5 cells/cm2 on 96-well plates, and preconfluent medium was switched to postconfluent medium for 2 weeks after confluence was reached. RPE were cultured in postconfluent medium with eight different dilutions of rtPA  for 1 hour or 24 hours, and culture supernatants were evaluated at 1, 7, and 28 days after rtPA administration (each n=5). The absorbance of each supernatant treated with a chemiluminescent reagent was recorded using a multimode microplate reader , and the cell damage rate was calculated with the following equation:The cell damage rate was measured by performing a lactate dehydrogenase (LDH) cytotoxicity test (Wako) following the manufacturer's instructions. rtPA (monteplase) was purchased from Eisai . RPE in preconfluent medium were seeded at a density of 1.0 × 10The positive control was RPE supernatant treated with 0.2% Tween 20 for 45 min at 37°C.RPE grown in Transwell® inserts were fixed in 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, dehydrated with increasing concentrations of ethanol, and visualized with a scanning electron microscope .μg/ml; Molecular Probes, Thermo Fisher Scientific). Samples were imaged using a laser scanning confocal microscope .The methods used for RPE immunocytochemistry have been previously described . ZO-1 wan=3). PEDF [The culture medium from confluent RPE treated with and without rtPA was collected 24 hours after the medium was changed (each 3). PEDF  and VEGF3). PEDF  secretion=3). RNA concentration and quality were assessed with a NanoDrop 1000 spectrophotometer (Thermo Scientific). RNA was reverse transcribed in a 20 μl reaction containing 11 μl of RNA (1000 ng) in DNase- and RNase-free water (QIAGEN), 1 μl of 50 μM Oligo (dT) 20 (Invitrogen), 1 μl of 10 mM dNTP Mix (Invitrogen), 1 μl of RNaseOUT , and SuperScript™ III Reverse Transcriptase . cDNA synthesis was performed using the following reaction conditions: 5 min at 25°C, 60 min at 50°C, and 15 min at 70°C. Reverse transcription PCR reactions were performed using Ex Taq® DNA polymerase , and real-time PCR was performed using SYBRTM Green master mix . The following thermal cycling conditions were applied: one cycle at 50°C for 120 sec and 95°C for 120 sec, followed by 40 cycles at 95°C for 15 sec and 58°C for 60 sec. Relative gene expression of RPE65, VMD2, RLBP1, and MERTK was determined using the 2−(ΔΔCt) method, with TFRC as a housekeeping gene which was unaffected by rtPA administration. The following forward and reverse primer (FP and RP) sequences  were used to amplify four RPE-specific genes  and two housekeeping genes .Total RNA was extracted from RPE treated with and without rtPA using an RNeasy Plus Mini Kit®  according to the manufacturer's instructions . LDH release assay, PEDF and VEGF secretion, and relative RPE-specific gene expression were analyzed by performing one-way analysis of variance (ANOVA) followed by Scheffe's test.Values are expressed as the mean ± SEM, and μg/ml) for 1 hour, and each RPE and corresponding culture supernatants were evaluated 1 day after rtPA administration. HfRPE treated with 0 to 1000 μg/ml rtPA showed no obvious morphological changes although floating cells were observed in the medium containing 2000 and 4000 μg/ml rtPA , and ARPE19. All RPE were treated with eight different dilutions of rtPA  for 24 hours. Each culture condition and supernatant was evaluated at 1, 7, and 28 days after rtPA administration. All RPE exhibited no morphological changes at all time points examined  induced evidence of prolonged cytotoxic effects in hfRPE and the two hiPSC-RPE but not ARPE19.Next, we investigated whether sustained rtPA exposure affects cell death. Previous reports showed that rtPA at a dose greater than 100  Figures . As demo Figures , and 50 on day 7 . The twoon day 7 . The celon day 7 . The cliμg/ml) for 24 hours. All RPE treated with 20  and 100  μg/ml rtPA expressed the tight junction marker ZO-1 in an immunocytochemistry assay  and showed no observable morphological changes under scanning electron microscopy . PEDF and VEGF concentrations in the apical and basal media of RPE cells grown in these inserts were measured by ELISA at 1, 7, and 28 days after tPA administration, and no significant differences were observed at all time points , and RPE-specific gene expression was evaluated by performing real-time qRT-PCR at 1, 7, and 28 days after rtPA administration. Expression levels of all RPE hallmark genes in 20 and 100 μg/ml rtPA-treated hiPSC-RPE were comparable to those in control  but not in eyes that received 50 μg of rtPA (0/7). Additionally, Chen et al. [μg of rtPA. In this study, rtPA exhibited time-dependent RPE toxicity; hiPSC-RPE and hfRPE treated with 50 and 100 μg/ml rtPA showed no cytotoxicity after rtPA exposure for 1 hour, whereas 24-hour rtPA exposure caused RPE toxicity at the clinical concentration of 20 μg/ml as well as 50 and 100 μg/ml rtPA. These results suggested that clinical rtPA-induced RPE toxicity occurs between 1 hr and 24 hr rtPA (100 μg/ml) exposures in vitro. The exposure of 100 μg/ml rtPA for 24 hours induced no cytotoxicity in terms of RPE cell morphology, VEGF and PEDF secretion, and RPE-specific gene expression. Thus, 20 μg/ml rtPA used in clinical is the safe concentration, and it is reasonable to assess drug-induced cytotoxicity using in vitro concentrations estimated by comparing in vitro and in vivo results.rtPA converts plasminogen to plasmin, resulting in clot lysis; thus, rtPA needs to touch subretinal hemorrhage directly. The neural retina contains membrane tissues formed by Müller cells, termed the inner limiting membrane (ILM) and external limiting membrane (ELM), which restrict the diffusion of external substances in the retina. According to previous reports, ILM prevents the diffusion of rtPA 70 kDa) but not 20 kDa dextran , whereas0 kDa butn et al.  reportedOne limitation of our study is the use of human fetal RPE as control, which may influence the outcomes due to cell immaturity. We showed that the responsiveness of hiPSC-RPE to rtPA, but not ARPE19 (adult RPE), were similar to that of hfRPE, suggesting that the RPE in patients could show different responsiveness. However, in the previous report , the genWe report here that the responsiveness of hiPSC-RPE to rtPA is remarkably similar to that of hfRPE in terms of cell morphology, cell death, and cell function. These findings suggest that hiPSC-RPE is a valuable cell source for further in vitro cytotoxicity testing. In addition, the concentration of rtPA estimated by comparing in vitro and in vivo results induced no cytotoxicity in terms of RPE cell morphology, VEGF and PEDF secretion, and RPE-specific gene expression. The clinically used rtPA-assisted displacement of subretinal hemorrhage is a safe treatment method. These results provide additional information supporting the use of hiPSCs for drug development.


American Journal of Tropical Medicine and Hygiene. This milestone offers an opportunity to reflect on our Journal. The American Society of Tropical Medicine founded The American Journal of Tropical Medicine in 1921. Nearly 100 years later, the subject matter of that journal (available at http://www.ajtmh.org) is familiar, although it was influenced by the international events of the day. Among articles in the first issue were the following: “An attempt to explain the greater pathogenicity of Plasmodium falciparum as compared with other species,” “On the prevalence of carriers of Endamoebadysenteriae among soldiers returned from overseas service,” and “Study of a case of yaws, contracted by an American soldier in France.” The American Journal of Tropical Medicine and Hygiene was formed in 1952, as a merger of the American Journal of Tropical Medicine and the Journal of the National Malaria Society, coincident with the dissolution of the National Malaria Society after the elimination of autochthonous malaria in the United States. With a name change, changes in physical appearance, recent emphasis on electronic publication, and geopolitical changes in tropical and nontropical regions of the world over the past 100 years, the focus of our Journal on improving global health remains steadfast.This issue marks Volume 100 of the American Journal of Tropical Medicine and Hygiene, published in January 1952, included articles on dengue, yellow fever, scrub typhus, malaria, and amebiasis . Authors included the tropical medicine giants Albert Sabin, Paul Russell, and Robert Coatney. The Journal has remained focused since that time on the great tropical disease problems, most of which are still with us, and has also addressed the new ones, from AIDS to Zika.The first issue of the Journal is broad, with articles on common parasitic, viral, and bacterial infections, and attention to many other tropical infectious diseases. Over the years the number of articles published has increased steadily, but the focus on different topics has changed, including decreased attention to schistosomiasis since about 1980, fewer articles on malaria since early in this century, and increased attention to viral infections and tuberculosis in recent years. Articles on diseases that are not infectious in etiology remain a minority, but important topics include snakebite, diabetes, and various types of poisoning. Some years ago consideration was given to removing “Hygiene” from our title, as it was suggested that this term was outdated, but in fact attention to hygiene has increased in recent years, including 19 articles published in 2017. Notably, the Journal is truly international. In 2017 corresponding authors were from 90 countries, and only 29% of published articles had a corresponding author from the United States.The scope of our Journal has been available online since 2003; only 5% of our membership now elects to receive a print copy. Innovations in the past two decades have included the Images in Clinical Tropical Medicine section, introduced in 2008, and the Stories from the Field section, introduced in 2018. The Journal has published numerous supplements focused on specific tropical medicine topics. However, our main focus has not changed: our primary purpose remains to publish primary research articles pertaining to all aspects of tropical medicine and public health. Our definition of tropical medicine is broad, so articles on arthropod-borne infections and parasites transmitted outside the tropics are within our scope. Increasing numbers of submissions and an enlarging geographic scope attest to continued relevance of our Journal as we reach Volume 100.The Journal is not only a leading international tropical medicine journal, but it also serves as the journal of the American Society of Tropical Medicine and Hygiene. As such, we encourage our members to submit important reports to the Journal. In addition to standard research reports, we encourage perspectives, reviews, Images in Clinical Tropical Medicine, and Stories from the Field. Our Journal has high impact in our fields only because our members and others offer timely and expert peer review of submitted research articles. We encourage Society members and others with interest in tropical medicine to continue to contribute their valuable insights by reviewing submitted manuscripts. These insights are essential to guide the editors in publishing the most impactful work in the Journal.The American Journal of Tropical Medicine and Hygiene will serve the membership of the American Society of Tropical Medicine and Hygiene by continuing to report on the most important advances in tropical medicine and public health.With Volume 100 we are proud to look back at a journal that has contributed importantly to progress in tropical medicine research and to the control of tropical disease. With continued progress, we might put ourselves out of business, as global control and elimination efforts come to fruition for one infectious disease after another. However, as the fight against tropical diseases is not easy , it seems likely that this will not occur any time soon. Also, we know that noninfectious diseases will fill any voids that arise as we conquer infectious tropical diseases, but suffer increasingly from maladies associated with an improved standard of living. So, we anticipate volumes 150 and 200, hopefully with eager future editors celebrating these milestones along with continued progress against tropical diseases. Through this changing landscape, the


SBEIIb and one allele of SSIIa co-segregated with the ASV phenotype and contributed to distinct starch quality traits important for food-processing applications.Seven novel alleles of Sobic.004G163700 and Sobic.010G093400. Comparative genomics identified Sobic.010G093400 as a starch synthase IIa and Sobic.004G163700 as a starch branching enzyme IIb. Segregation analyses showed that mutations in Sobic.010G093400 or Sobic.004G163700 co-segregated with the ASV phenotype.Mutants in SSIIa exhibited no change in amylose content but expressed lower final viscosity and lower starch gelatinization temperature (GT) than starches from non-mutant plants. The sbeIIb mutants exhibited significantly higher amylose levels and starch GT and lower viscosity compared to non-mutant starches and ssIIa mutants. Mutations in SBEIIb had a dosage-dependent effect on amylose content. Double mutants of sbeIIb and ssIIa resembled their sbeIIb parent in amylose content, starch thermal properties and viscosity profiles. These variants will provide opportunities to produce sorghum varieties with modified starch end-use qualities important for the beer brewing and baking industries and specialty foods for humans with diabetes.Sorghum is an important food crop for millions of people in Africa and Asia. Whole-genome re-sequencing of sorghum EMS mutants exhibiting an alkali spreading value (ASV) phenotype revealed candidate SNPs in The online version of this article (10.1007/s00122-019-03430-0) contains supplementary material, which is available to authorized users. Sorghum bicolor (L.) Moench] is important in drought-prone environments  in sorghum  is a mutant starch type resulting in high amylose starches in maize  and negatively with breakdown  is commonly used in rice breeding to select for genotypes with varying starch GT  exhibiting an ASV+ phenotype were used to identify the genes controlling the alkali spreading phenotype of sorghum based on whole-genome re-sequencing and genetic segregation analyses. Genotypes with single or double mutations were created to assess starch properties including amylose content, starch GT and paste viscosity profiles. Grain samples produced from these mutants were also characterized for variation in starch quality and related food-processing parameters. Genes, alleles and SNP markers for use in breeding sorghum varieties with varying starch GT and composition are reported.Given reports in rice, the initial hypothesis was that a Sorghum bicolor EMS (SbEMS) mutants with altered starch phenotypes .To conduct an allele dosage test, A-lines for each mutant were developed by crossing each mutant line as a pollen donor to ATx623 followed by backcrossing to the F1 seeds were grown in the greenhouse and genotyped for both parental alleles. The verified F1 plants were self-pollinated, and the F2 populations were grown in a greenhouse and genotyped to identify homozygous double mutant F2 plants. The homozygous double mutants were transplanted in the field in 2018, self-pollinated and harvested as single panicles. The parental genotypes were also grown in the field in 2018. The seeds of double mutant lines and two biological replicates for each of the parent lines were subjected to further phenotypic analyses.Double mutants were created by hand emasculating and crossing selected EMS mutants in 2017 . The harvested . The aqueous layer was transferred to a new Eppendorf tube and diluted with 1070µL dilution buffer  followed by precipitation with 530µL wash buffer . The samples were incubated for 15 min at room temperature, centrifuged at 12,000 (3220 × g) for 10 min, and the pellet was re-suspended in 53 µL high-salt TE . The samples were incubated at 60 °C for 15 min and then diluted with 212 µL H2O. DNA was precipitated with 530 µL pure EtOH at − 80 °C for 30 min, centrifuged (3220 × g) for 10 min, and the pellet was washed with 70% ethanol. The pellets were re-suspended in TE buffer .DNA was extracted from the SbEMS mutants exhibiting an ASV+ phenotype . The other eight EMS mutants SbEMS 2703, SbEMS 2773, SbEMS 3194, SbEMS 3403, SbEMS3568, SbEMS 3920, SbEMS4308 and SbEMS 5890 were processed as described below. The library construction was conducted using the TruSeq® DNA PCR-Free Sample Preparation Guide from Illumina. The samples SbEMS 2773, SbEMS 3194, SbEMS 3568 and SbEMS 5890 were PCR amplified as the final library concentration was too low after the PCR-free protocol step. The samples were sequenced using Illumina HiSeq 2500 with 100 bp paired-end reads. The filtered NGS read files (R1 and R2) obtained from the Purdue Genomics Core were cleaned of contaminants by aligning the R1 and R2 files to the indexed Phix174 genome using Bowtie 2 short reads aligner  sequence read archive at https://www.ncbi.nlm.nih.gov/sra.Whole-genome re-sequencing data for all the sorghum samples were generated at the Purdue Genomic Core Facility. One sorghum mutant, SbEMS 3218, had undergone next-generation sequencing as described by Addo-Quaye et al. , and SNPSorghum bicolor reference genome BTx623  were downloaded from Phytozome database version 11  and the genome annotation information (gff3 file) obtained from Phytozome database version 11  markers were created for the mutant alleles sbeIIb-3194-3. The sequence around the SNP was obtained from Phytozome. The dCAPS Finder 2.0  were created for the mutant allele ssIIa-4308-2, sbeIIb-2703-1, sbeIIb-3403-5 and sbeIIb-3218-4. The sequence around the SNP was obtained from the reference genome. The software PRIMER1: primer design for tetra-primer ARMS-PCR, freely available at http://primer1.soton.ac.uk/primer1.html, was used to create tetra-primer  was digested with HpyCH4III, sbeIIb-2773-2 (2773_04_SNP_51302688) with ApoI-HF, sbeIIb-5890-7 (5890_04_SNP_51301340) with MboI and sbeIIb-3194-3 (3194_04_SNP_51298115) with CviRI=HpyCH4 V. All samples from enzyme digest or tetra-primers were examined in 2.5–3% TAE high-resolution agarose gels, depending on the fragment size.Each PCR product from CAPS and dCAPS was digested with enzymes from New England Biolabs under recommended conditions. The allele F1 plants were selected and self-pollinated to create bi-parental F2 populations. Panicles of F2 plants were self-pollinated, and F2:3 seeds were screened for ASV with 32 seeds per panicle and in rare cases less if limited by seed number. The number of panicles per population screened varied from 30 for SbEMS3403 to 112 for SbEMS2773. F3 plants of those F2:3 panicles were grown on a bench containing sand, and leaf tissues were sampled and genotyped. Each panicles’ ASV phenotype and the corresponding SNP genotype  was determined. A Chi-square test was conducted to determine goodness of fit for the 3:1 phenotypic ratio as well as the 1:2:1 genotypic ratio.The SbEMS mutants were crossed to BTx623 (except SbEMS3920 was un-successful in crossing). An allele dosage test was conducted to determine whether the mutant alleles influenced amylose content in an allele-dependent manner. The A-lines and B-lines for each mutant were intercrossed to produce the hybrids for testing inheritance in the triploid endosperm. To obtain zero doses of the mutant allele, BTx623 was used as pollen donator and crossed to ATx623. A single dosage of the mutant allele was created by crossing the mutants onto ATx623. Two dosages of the mutant allele were created by crossing BTx623 onto the A-line (or hand emasculated panicle) of each EMS mutant. Three doses were created by self-pollinating the B-line (West Lafayette 2017) or by crossing the B-line to the corresponding A-line (West Lafayette 2018). The hybrid seeds were analyzed for ASV and amylose content. The experiment was set up to obtain two biological replicates per genotype and allele dosage, but depended on crossing success and seed set in the field. In 2017, the A-line for SbEMS2703 was not available so hybrids were made by hand emasculation. The SbEMS3920 mutant was not included as no co-segregation data were available at that point.The alkali spreading test was conducted as described in Griebel et al.  with 1.8The flour preparation was conducted as follows. The seed samples from PR16/17, WL17 and WL18 described above were milled into a fine flour using a ball mill  as described in Griebel et al. .To), peak (Tp), conclusion (Tc) starch GT, range of starch GT (Tc − To) and enthalpy.The starch extraction and thermal properties were conducted as previously described from flour samples of double mutants and parents  was used at 510 nm.Statistical analyses were performed using SAS version 9.4.  The GLIMMIX procedure was used to perform a linear mixed model analysis of variance (ANOVA) with the Tukey–Kramer multiple comparison adjustment to compare starch GT, amylose and viscosity properties across genotypes and environments.Sobic.004G163700, Sobic.010G093400) of 19 genes associated with starch biosynthesis , which was not further evaluated as it is an Intron-SNP and the ASV phenotype co-segregated with an exon-SNP in Sobic.004G163700 for that mutant. Similarly, SbEMS 5890 has a second homozygous SNP in a starch synthase gene (Sobic.010G093400); predicted to be a synonymous variant and was, therefore, excluded from further consideration.Whole-genome re-sequencing studies characterizing the sorghum mutants described by Griebel et al.  revealedSobic.004G163700 and Sobic.010G09340  are predicted for the SNPs in the alleles of sbeIIb-2773-2, sbeIIb-3403-5 and sbeIIb-3194-3. In Sobic.010G093400, the SNP in the allele ssIIa-3920-1 is predicted as a missense variant and the SNP in the allele ssIIa-4308-2 is predicted as a missense and splice region variant.The snpEff tool   for the alleles sbeIIb-2703-1, sbeIIb-3194-3, sbeIIb-3568-6 and sbeIIb-5890-7. Genotyping studies showed that the sbeIIb-2703-1, sbeIIb-2773-2, sbeIIb-3218-4 and ssIIa-4308-2 alleles segregated in a 1:2:1 genotypic ratio but sbeIIb-3194-3, sbeIIb-3403-5, sbeIIb-3568-6 and sbeIIb-5890-7 alleles exhibited distorted ratios  compared to the ssIIa mutants ssIIa-3920-1 and ssIIa-4308-2 and the controls. In all three seasons, SbEMS 3194 had the highest amylose values. The SbEMS 2703, SbEMS 3218 and SbEMS 5890 were ranked 2nd to 4th in two environments, respectively. The SSIIa mutations represented by ssIIa-3920-1 and ssIIa-4308-2 produced similar amylose values and did not show higher amylose values compared to the controls.The sbeIIb-2703-1, sbeIIb-2773-2, sbeIIb-3194-3, sbeIIb-3218-4, sbeIIb-3403-5, sbeIIb-3568-6 and sbeIIb-5890-7 allelic variants . Analyses of samples for ASV in 2018 showed an increase in percentage of seeds with an ASV phenotype depending on whether one, two or three mutant sbeIIb or ssIIa alleles were present in the endosperm. No ASV phenotype was observed in genotypes carrying one dose of the allele.Based on the higher amylose values of ssIIa and sbeIIb mutants in both environments. There was a significant genotype by environment effect for the pasting viscosity profiles (Table S2); therefore, data are presented by individual environment  and two new alleles of a starch synthase (Sobic.010G093400) were identified in this study. Comparative genomics provided evidence that Sobic.010G93400 encodes a SSIIa. Sobic.010G093400 is most similar to SSSII-3 in rice and SSIIa in sorghum and other crop species. Sequence comparisons suggest that Sobic.004G163700 codes for a SBEIIb with high sequence similarity to the one reported previously by Mutisya et al. . The SBEIIb of sorghum reported by Mutisya et al.  encoding a SBEIIb.Whole-genome re-sequencing of ASV mutants in sorghum revealed SNPs in starch biosynthesis genes. Seven new alleles of a starch branching enzyme  and sbeIIb-3218-4 (W548*) might be also in the catalytic domain of SBEIIb, as this is close to a region of importance for catalytic activity in maize SBEIIb  into intermediate size chains (DP 12–24)  gene is the major gene that regulates starch GT (starch GT measured by ASV) in rice   is similar in sequence and function to Ae1 in maize. The new sorghum sbeIIb alleles result in varying amylose content with higher values overall in comparison with the ssIIa mutants, which is similar to ae maize and rice starches  and in a wider starch gelatinization range compared to the wild-type , both traits being negatively correlated  gene, SSIII-2, SSI and PUL  gene  have been reported to exhibit amylose values and starch GT more similar to the amylose extender parent in other crops  gene in rice did not contribute strongly to changes in paste viscosity profiles as observed for the wx locus  than wild-type samples or ssIIa mutants. The ssIIa mutant alleles resulted in a drop of final viscosity, less gel consistency, lower thickening power, same amylose values and lower starch GT compared to wild-type samples from Tx623. These mutations provide opportunities to produce sorghum varieties with modified starch end-use qualities likely important for the beer brewing and baking industries and specialty foods for humans with diabetes. The fast and cheap ASV assay adapted to sorghum can be easily implemented as a pre-selection step in plant breeding programs around the globe. However, the ASV assay will not completely replace the use of DSC and RVA technologies and requires those analyses on pre-selected ASV+ genotypes.Nine novel alleles of a Supplementary material 1 (DOCX 22 kb)Below is the link to the electronic supplementary material.


Living donor liver transplantation (LDLT) has emerged as an equally viable option to deceased donor liver transplant for treating end stage liver disease patients. Optimising the recipient outcome without compromising donor safety is the primary goal of LDLT. Achieving the adequate graft to recipient weight ratio (GRWR) is important to prevent small for size syndrome which is an uncommon but potentially lethal complication of LDLT.Here we describe a case of successful dual lobe liver transplant for a 32 years old patient with ethanol related end stage liver disease. A right lobe graft without middle hepatic vein and another left lateral sector graft were transplanted successfully. Recipient and both donors recovered uneventfully.Dual lobe liver transplant is a feasible strategy to achieve adequate GRWR without compromising donor safety. Liver transplantation is a gold standard treatment for a patient with end-stage liver disease.Living donor liver transplantation (LDLT) rate is increasing due to the shortage of deceased liver donations. Small for size syndrome and small donor liver remnant are two uncommon but potentially lethal complications of LDLT .Small for size syndrome (SFSS) generally occurs when the graft recipient weight ratio (GRWR) is less than 0.8. There is disequilibrium between increased metabolic demand and liver regeneration which leads to a severe graft dysfunction . The cliLDLT is the most common type of liver transplant in India. Almost 33% of potential live donors are rejected as liver donors for adult recipients because of calculations suggesting a small for size (SFS) graft, a small future liver remnant (FLR) and steatosis .In recent years the subset of morbidly obese patients undergoing liver transplant is raising due to the increase in the incidence of non-alcoholic steatohepatitis (NASH) related end stage liver disease (ESLD). In order to overcome the problems of SFSS, optimise donor safety and increase the donor pool, dual graft liver transplant is the best feasible option. The first successful dual lobe LDLT using left lobe graft and left lateral section was performed by Lee et al. in year 2000 . In this2) diagnosed with ethanol related ESLD, decompensated with jaundice, ascites, and hydrothorax   and right hepatic artery (RHA). RHA had an extra-hepatic length of 27 mm. Segment 4 artery was arising from LHA and there were no other accessory arteries identified  and VIII (2.8 mm) veins were draining into MHV  was dividing into LHA and RHA. LHA was arising from HAP immediately at its origin and supplied segments 2 and 3. Segment 4 artery was arising from RHA  and Middle hepatic vein (MHV) measuring 5.8 mm in length  were 0.63 and 0.62 respectively. As neither of the two probable donors were suitable as a single donor , right hepatic artery (RHA), right portal vein (RPV), right hepatic vein (RHV), and right inferior hepatic vein (RIHV). Left lateral lobe graft was harvested from Donor II after the division of the left hepatic duct, left hepatic artery (LHA), left portal vein (LPV) and LHV.Actual harvested graft weights were 503 g (right lobe without MHV graft) and 374 g  Fig.  giving GOperative time for Donor I and Donor II hepatectomy was 360 min and 310 min respectively with a blood loss of 450 ml and 370 ml.Grafts were perfused with UW solution. As hepatic vein tributaries of segment V and VIII were less than 3.5 mm diameter, venous drainage of these segments was not established. In both grafts, no other back table reconstruction was needed.The right lobe without MHV graft was orthotopically implanted on the right side of IVC.IVC was clamped using a side biting clamp. Cavotomy was performed at the orifice of RHV.Hepatic venous anastomosisGraft RHV was anastomosed to recipient IVC in an end to side manner with 5–0 polypropylene continuous suture.Graft RIHV was anastomosed to recipient IVC in an end to side manner with 6–0 polypropylene continuous suture.Portal Venous anastomosisGraft RPV was anastomosed to recipient RPV (end to end manner) using a 6–0 polypropylene continuous suture with appropriate growth factor.Graft reperfusion was immediate and uniform.Arterial anastomosis:Graft RHA anastomosed with recipient RHA with 8–0 polypropylene interrupted suture under three time’s magnification.After arterial reperfusion intra-operative doppler ultrasound showed regular portal venous, hepatic venous and hepatic arterial flow.Hepatic venous anastomosisGraft LHV was anastomosed to cavotomy made on recipient IVC at the orifice of LHV and MHV (end to side manner) with 5–0 polypropylene continuous suture.Portal Venous anastomosisGraft LPV was anastomosed to recipient LPV (end to end manner) using a 6–0 polypropylene continuous sutureGraft reperfusion was immediate and uniform.Arterial anastomosis:Graft LHA anastomosed with recipient segment IV hepatic artery with 8–0 polypropylene interrupted suture under three times magnification.After arterial reperfusion intra-operative doppler ultrasound showed regular portal venous, hepatic venous and hepatic arterial flow in the left lateral graft as well.Biliary anastomosisNumber of graft ducts: two in right lobe graft (RASD and RPSD) and 1 in left lateral graftNumber of anastomosis: 3Anastomosis no 1: Graft RASD was anastomosed to the recipient right hepatic duct (RHD) with 6–0 Polydioxanone in an interrupted manner.Anastomosis 2: Graft RPSD to the recipient cystic duct anastomosed with 6–0 Polydioxanone in an interrupted manner.Anastomosis 3: Graft left hepatic duct (LHD) to the recipient LHD with 6–0 Polydioxanone in an interrupted manner. Fig. .Recipient recovered well postoperatively. He received standard triple immunosuppressant. The patient was discharged on the eleventh postoperative day with normal liver graft function and good general condition.Both donors recovered well postoperatively. They were discharged in good general condition and with normal liver function tests.The recipient was followed up weekly for the first 2 months and then every monthly. During follow up the patient was examined clinically and by laboratory tests including liver function tests, renal function tests, and serum tacrolimus level. A radiological assessment of hepatic vasculature was done whenever indicated. The long-term outcome, evaluated after a 48-month follow-up, was optimal with normal graft function, good general condition and superior quality of life.There is an increasing incidence of ESLD and shortage of deceased donor organ pool especially in East Asian countries including India. LDLT has emerged as a crucial solution to tackle the situation of disparity between demand and supply of liver allografts. The evolution of LDLT is a history of search for an optimal graft to minimize donor risk and maximize recipient outcome simultaneously .It has been shown that minimum GRWR of 0.8 is required to reduce the occurrence of SFSS . Also moThe surgical risk to the donor is always an ethical dilemma even with increasing application and success of LDLT. Donor death has an immense impact on the surviving recipient, other family members, and the transplant team. This ethical dilemma is compounded in dual lobe LDLT as two donors are at risk.Left lobe donor hepatectomy is associated with lower morbidity and mortality than right lobe donor hepatectomy. Dual left lobe LDLT with two minor left lobe donor hepatectomies may actually have less combine donor risk than one major right lobe donor hepatectomy . While dIn the present case, it could have been possible to use reversed left lobe graft. However, we chose the Right lobe graft over the left lobe as we have maximum experience in Right Lobe LDLT. We have 100% safety record for all our Right lobe donors (220 Cases). Moreover, reverse left dual graft surgery is technically challenging and associated with more complications.The issue of donor risk can be partially defeated by expert dual lobe transplantation teams, technical advances and deliberately choosing the dual left lobe grafts or accepting the low GRWR single lobe graft with inflow modulation techniques like splenectomy, splenic artery ligation or porto-caval shunt. In our institute we prefer to use a porto-caval shunt if intra-operative actual GRWR is less than 0.7 . Higher In this case, there is a disparity between volumetric estimated graft weight (EGW- 295 g) and actual graft weight (AGW- 374 g) of the left lateral segment of donor II. Right and left lobes EGW on CT Volumetry is overestimated as compare to AGW. For the Left lateral segment (LLS) graft, CT may underestimate the volume because the actual surgical plane of transection is around 1 cm to the right of a falciform ligament whereas radiological plane is at falciform ligament. In addition to that non-hepatic tissues like falciform ligament which are excluded in CT volumetry are a part of LLS allograft included in AGW .Surgical planning and decision-making process are complex and difficult in dual graft LDLT. Both donor and type of graft selection are very crucial for the optimal outcome. In our institute, we investigate donor as per protocol which includes laboratory investigations, estimation of liver attenuation index (LAI) on computed tomography, triphasic CT abdomen with the estimation of liver volumes. We asses biliary anatomy with MRCP preoperatively and with intra-operative cholangiogram.Decision-making process of dual graft LDLT needs to be standardized and to achieve consensus across the transplant community, Yinzhe Xu et al. have proposed a decision making pathway for same  Fig. 1313. The iPost dual graft LDLT biliary complications incidence is around 21%. Biliary reconstruction of both grafts can be performed using duct to duct anastomosis or combination of duct to duct and hepatico-jejunostomy (HJ) to each graft. A biliary reconstruction strategy is therefore critical to the outcome of dual graft LDLT as well. Dual duct-to-duct anatomoses are preferred over HJ for being more physiologic and allowing better access for post-transplant endoscopic and interventional radiological procedures .Furthermore, unnecessary dissection of recipient bile ducts in the hilar plate should be avoided and as much as connective tissue should be preserved to avoid ischemic biliary damage. Dual graft LDLT is an extremely complex procedure, its outcome relies on the appropriate patient and donor selection, detailed donor evaluation, precise surgical planning, and meticulous surgical techniques .In conclusion, the dual lobe LDLT is complex and technically challenging procedure and it should be considered a viable option to increase the donor pool and overcome the problem of the small-for-size syndrome.