A 68-year-old male has a significant past medical history of severe aortic stenosis, peripheral arterial disease, chronic kidney disease, and an abdominal aortic aneurysm treated with a bifurcated interposition aortobiiliac graft. He was admitted to the hospital for an elective one-vessel coronary artery bypass graft and placement of a bioprosthetic aortic valve. Postoperatively, he developed worsening abdominal pain, leukocytosis, and inability to tolerate nutrition by mouth. Computed tomography revealed moderately dilated loops of the small bowel with two transition points in the right lower quadrant. He was taken emergently to the operating room for an exploratory laparotomy, and a 28-cm necrotic jejunal loop was entrapped posterior to the right iliac segment of the graft. In a patient with an intra-abdominal synthetic vascular graft, a closed-loop bowel obstruction caused by entrapment by the vascular graft is exceptionally rare; however, it should be considered in the presence of bowel obstruction. Closed-loop intestinal obstructions are commonly caused by adhesions, internal herniation, or volvulus and can compromise bowel perfusion and progress to intestinal necrosis and perforation . Any intA 68-year-old male has a significant past medical history of severe aortic stenosis, peripheral arterial disease, chronic kidney disease, and an abdominal aortic aneurysm. He lives at home with his spouse and can independently perform routine daily activities of living. Nine years prior, he underwent an endovascular abdominal aortic aneurysm repair, which was complicated by an endoleak and required multiple graft revisions, eventual explantation of the endovascular graft, and placement of a bifurcated interposition aortobiiliac graft. The patient was admitted to the hospital for an elective one-vessel coronary artery bypass graft and placement of a bioprosthetic aortic valve. There were no intraoperative complications and the patient was extubated one day later. Four days after surgery, he developed worsening abdominal pain, leukocytosis, and inability to tolerate nutrition by mouth. Enteral nutrition was stopped, and a nasogastric tube on low intermittent suction was removed with over 1 L of bilious output daily. On postoperative day five, his abdominal pain progressed to focal peritonitis in the right lower quadrant.InvestigationsComputed tomography with intravenous contrast on postoperative day five revealed thickened and moderately dilated loops of the proximal small bowel with a more prominent C-shaped loop of the small bowel and two transition points in the right lower quadrant, medial and posterior to the right iliac segment of the aortobiiliac graft Figure . The conTreatmentThe patient was taken emergently to the operating room for an exploratory laparotomy. Upon entering the abdominal cavity, extensive adhesions were encountered and lysed. While examining the small bowel, a jejunal loop was found fixed deep in the right hemi-pelvis. Upon further dissection, a 28-cm jejunal loop was entrapped behind the right iliac limb of the graft, causing a closed-loop jejunal obstruction and bowel necrosis (Figure He returned to the operating room the next day for a second-look laparotomy, and the remaining bowel appeared viable and well perfused. The jejunum was anastomosed, and the abdomen was closed. Over the next several days, the patient had return of bowel function. His diet was advanced, and he was discharged home on postoperative day eight. At two-week follow-up, the patient was ambulating, eating a regular diet, and performing routine daily activities.This case highlights a rare intraoperative finding of jejunal strangulation posterior to a prosthetic aortobiiliac interposition graft. This closed-loop bowel obstruction occurred several days after elective coronary artery bypass grafting and aortic valve placement. When cardiopulmonary bypass is initiated during a coronary artery bypass graft, various hemodynamic changes take place. A patient’s mean arterial pressure decreases, and systemic vascular resistance decreases . Carrel There is a higher incidence of bowel obstruction and overall reoperation rates with the open technique versus endovascular technique for aortic aneurysm repairs , likely In an unstable patient with peritonitis, emergent operative intervention is required . In hemoAfter an open transabdominal aortic aneurysm repair, extensive adhesive disease can form between the synthetic vascular graft and the surrounding visceral organs. When these patients undergo surgery with cardiopulmonary bypass, hemodynamic changes can occur, causing bowel edema, an increased susceptibility for bowel migration, and an entrapment posterior to the graft. A closed-loop obstruction by direct entrapment by the synthetic vascular graft should be considered.


Despite improvement in the specific treatment, clinical and anatomo-functional central nervous system (CNS) abnormalities of various severities are still observed in cystinosis patients. Patients who develop CNS complications today have a worse compliance to cysteamine treatment. Radiological studies have shown that cortical or central  atrophy is observed in more than two thirds of cystinosis patients’ magnetic resonance imaging (MRI) and correlates with the intelligence quotient score. Half of cystinosis patients have marked aspecific white matter hyperintensities. The development of advanced neuroimaging techniques provides new tools to further investigate CNS complications. A recent neuroimaging study using a voxel-based morphometry approach showed that cystinosis patients present a decreased grey matter volume in the left middle frontal gyrus. Diffusion tensor imaging studies have shown white matter microstructure abnormalities in children and adults with cystinosis, respectively in areas of the dorsal visual pathway and within the corpus callosum’s body. Finally, leucocyte cystine levels are associated with decreased resting cerebral blood flow, measured by arterial spin labelling, in the frontal cortex, which could be associated with the neurocognitive deficits described in these patients. These results reinforce the relevance of neuroimaging studies to further understand the mechanisms that underline CNS impairments. Cystinosis is a rare autosomal recessive disease caused by intracellular cystine accumulation ,2. ThreeNeurological symptoms have been reported in adolescents and young adults with cystinosis in historical cohorts ,6,7. TwoCNS complications are a major concern since the long-term prognosis of adult cystinosis patients appears to be primarily related to neurological complications . Indeed,In historical publications, CNS complications’ occurrence did not correlate with other extra-renal complications of cystinosis, but their frequency was directly correlated with age in the absence of early treatment ,6,7. IndCtns −/− mice, have elevated cystine levels in the hippocampus, cerebellum, forebrain, and brainstem, which increase with age. In addition, cystine crystals have been detected within the choroid plexus and situated adjacent to capillaries. This has been associated with spatial reference and working memory deficits .,35.17,35It has been described that children with cystinosis have a 12-fold higher prevalence of Chiari I malformations than the general pediatric population. Indeed, Chiari malformation is observed in 9.5% to 18% of patients ,43,44. ECazals et al. described a case of an adult patient with perivascular uptake of contrast associated with micronodular T2 hypointensity , which cThe development of advanced neuroimaging techniques has provided new tools to study the underlying neurophysiopathological mechanisms of metabolic diseases. Indeed, different MRI sequences allow noninvasively measuring anatomical and functional brain parameters. In addition, computational statistical software allows comparing these parameters between patients and controls, for instance, as well as to investigate putative correlations with clinical profiles or treatments, providing valuable knowledge in the field. However, so far, very few studies have used this approach to investigate CNS abnormalities in cystinosis patients.In the only study investigating grey matter abnormalities, we compared anatomical images from cystinosis patients to those from controls with nephropathy and from healthy controls . We usedBeyond a radiological description of white matter abnormalities characterized by hyperintensities in MRI scans, which are largely described in cystinosis patients as presented earlier, the white matter microstructure can also be studied using diffusion tensor imaging MRI sequences. Diffusion tensor imaging measures the random motion, or diffusion, of water molecules in neural tissue and allows inferring the structural characteristics of the local tissue environment underlying the movement, i.e., the white matter itself. Two main indices have been investigated: the mean diffusivity (MD), a measurement of the overall magnitude of diffusional motion, and the fractional anisotropy (FA), as an index of the brain white matter microarchitecture . In 2010, Bava et al. studied the cerebral white matter microstructure in 24 young children with cystinosis (age 3–7 years) and examined fractional anisotropy and mean diffusivity . ChildreThe study of resting brain function provides information on the level of activity of the different brain regions as a “baseline”, outside task performance. Using arterial spin labelling MRI (ASL-MRI), cerebral blood flow (CBF) can be measured at the cerebral level using intrinsic physiological contrast by labelling water protons from cervical arteries and measuring them once they are at the cerebral level . In a reFunctional brain activity in cystinosis may be assessed by a high-density electroencephalogram (EEG) . This noHigh-density EEG was used to analyze basic sensory processing in cystinosis, focusing on early auditory sensory processing (N1) and sensory memory (mismatch negativity) . The audNo anomalies have been found in the auditory sensory processing, suggesting that sensory transmission through the auditory system is largely intact in individuals with cystinosis. However, individuals with cystinosis present reduced responses for the longer stimulus onset asynchronies, which could indicate a reduced duration of auditory sensory memory traces, and thus sensory memory impairment, in children and adolescents diagnosed with cystinosis . Adults Despite an improvement in specific treatment and transplantation, CNS abnormalities of various severities are still present in adult cystinosis patients. In particular, neurocognitive impairment has been largely described. Unraveling the brain mechanisms that underline these abnormalities is essential to develop actions to improve long-term patient outcome. By MRI, cortical or central atrophy is observed in more than two-thirds of cystinosis patients and correlates with the total IQ score. The development of advanced neuroimaging techniques provides new tools to investigate CNS complications and the brain correlates of neurocognitive impairments. Therefore, future studies, in particular focusing on brain–behavior correlations, can bring new light to the subject. It is important to note that some findings may not be specific to cystinosis, supporting not only the importance of adding a control group with renal failure when investigating brain abnormalities in cystinosis patients, but also the relevance of neuroimaging investigations in patients with renal failure in general. It was recently shown that leucocyte cystine levels are associated with decreased resting cerebral blood flow in the frontal cortex, which could be related to the neurocognitive deficits described in cystinosis patients. These results further reinforce the importance of compliance to cysteamine treatment, which is a major concern in these adult patients, since it seems to play a major role in cognitive and neurological complications.


As you will be aware I only write Editorials infrequently. For example, around the turn of the year I like to thank all our colleagues, Authors, Reviewers, members of the Editorial Board and the staff at SAGE for all their hard work during the year. I especially like to thank our readers for their continuing support. However the 30th June 2021 is a very significant day for me personally as I shall cease to be your Editor in Chief at the end of that day after twenty eight and a half years in the role.It has been an incredible privilege for me to have spent so long with you and I want to thank you all most sincerely for allowing me to share this time with you.Journal of Engineering in Medicine on the 1st January 1993 and since that time we have increased our issues from 4 to 12/year and have expanded our international authorship magnificently. I took over the Editor’s role from my friend and Mentor, Professor Duncan Dowson who, to my delight, remained as a Board member until his passing in January 2020. Duncan and I exchanged telephone calls regularly and he always asked about the Journal which remained so very dear to him.I became Editor-in-Chief of However it is now time for the Journal to move onward and upward and I am delighted to welcome my successor, Professor Liz Tanner who has been a very active Associate Editor with Part H for many years and I trust that you will give her the loyal support that you kindly gave to me. Publishing is changing dramatically and as I hand over to Professor Tanner I am delighted to wish her and all of you, every success for the future.Anthony UnsworthDurham University, Durham, UK Journal of Engineering in Medicine from Tony Unsworth. To serve for nearly 30 years as Editor of a journal was unusual even in the first part of the 20th Century, to do so in the late 20th and early 21st Century must be nearly unique. My first, and very pleasant duty, is to announce that Tony is not really leaving the Journal, merely becoming the Emeritus Editor. The Journal of Engineering in Medicine has been special to me since my first paper was published with my status given as ‘Undergraduate’ when Bernard Lucas the founding editor and Duncan Dowson were the Editors. I am really honoured and delighted to take on the mantle of Bernard Lucas, Tony and Duncan. After 50 years of the Journal to be the fourth Editor is an honour.It is a privilege to take on the Editorship of We have to thank Tony for his efforts bringing the journal from the days of paper and physical photograph submissions to the Institution of Mechanical Engineers to today’s electronic submission and review with our publisher SAGE. I would like to thank the Editorial Board and Associate Editors for the support they have given the Journal over the years since I joined the Editorial Board. I know I will require their expertise and support as I take on this job.Elizabeth TannerQueen Mary University of London, London, UK


Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1–2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In  Together with light and nutrient availability, temperature is one of the major abiotic factors affecting growth of microalgae ,2,3,4. TChlorella sp. (Chlorophyta) [In green algae dividing by multiple fission, a gradual increase in temperature results in a physiological response in which cells at first increase their growth rate and shorten their cell cycle. Upon a further increase in temperature, an optimal point is reached at which growth rate is at its maximum and cell cycle duration is at its minimum. However, after exceeding this optimal temperature, the duration of the cell cycle is gradually prolonged while growth rates remain unaffected . Eventuarophyta) ,7,8,9. Irophyta) .An inherent property of cell division is that it is an energy-demanding process, consuming the majority of the cell’s energy reserves . A simplChlamydomonas reinhardtii has served as a well-established model for a number of years [C. reinhardtii as a biotechnology platform has been limited. Only recently, attempts were made to increase the starch content of C. reinhardtii by utilizing techniques such as nutrient deprivation and temperature stress [C. reinhardtii to almost 49% (w/w), the build-up is rather slow and it can take weeks until the maximum concentration is reached, which reduces overall productivity [C. reinhardtii, the cells at 30 °C initially accumulated starch as they grew in size, but this starch was spent for cell division. The cells at 39 °C grew in size similar to those at 30 °C, but they did not divide. Instead, they continued to increase their cell size and after 24 h, their total starch content was more than two-fold higher than the maximum at 30 °C [of years ,16. Thisof years ,18. Howee stress ,20,21,22C. reinhardtii by supraoptimal temperature, a method that has already been proven to cause a rapid 2-fold increase in starch yields under laboratory conditions [In the present study, we examine the potential for pilot-scale starch production in nditions . In doinChlamydomonas reinhardtii wild type 21gr (CC-1690), obtained from the Chlamydomonas Resource Center at the University of Minnesota . For routine subculturing, the strains were streaked onto culture plates containing standard high salt (HS) medium [The algal strain used in these experiments was the unicellular alga ) medium  solidifi−2 s−1 of photosynthetically active radiation (PAR) provided by light-emitting diode lamps . The cultures were aerated with a mixture of air and CO2  at a flow rate of 0.63 VVM in order to provide a carbon source and mixing of the cell suspension.For the purpose of the experiments, a starting culture was cultivated in a bench-top flat-panel airlift photobioreactor  in the following manner: 400 mL of liquid HS medium was inoculated directly from the culture plates and was cultivated at 30 °C and under constant incident light intensity of 500 µmol photons m4Cl concentration being increased 5-fold. This resulted in a growth medium with the following final composition: 250 g L−1 NH4Cl, 2 CaCl2·2H2O, 20 g L−1 MgSO4·7H2O, 1.84 g L−1 C10H12FeN2NaO8, 0.05 g L−1 Na2MoO4·2H2O, 3.09 g L−1 H3BO3, 1.18 g L−1 MnSO4·7H2O, 1.40 g L−1 CoSO4·7H2O, 1.24 g L−1 CuSO4·5H2O, 1.43 g L−1 ZnSO4·7H2O, 72 g L−1 KH2PO4, 134 g L−1 K2HPO4. For the preparation of the medium, 100× concentrated stock solutions of macro elements and microelements were used. All components, excluding phosphates, were diluted in distilled H2O and autoclaved for 20 min at 121 °C. After cooling, the sterile autoclaved phosphates were added. The medium used for cultivation in the pilot-scale flat-panel photobioreactor was not sterilized and tap water was used to dilute the stock solutions. In the course of the experiments, pH was monitored daily and was maintained at 7.0 ± 0.1 by the addition of 2 M NaOH. Foam formation in the reactor vessels was controlled with the help of 10× diluted antifoam silicone Snapsil RE 20 containing 30% active compound .All experiments were performed under photoautotrophic conditions. The culture medium used for the pilot-scale experiments was based on a HS medium but was modified in order to facilitate high biomass yields with the NH2  and ensured mixing of the suspension culture. Both cultivation vessels were constantly illuminated by a panel of luminescent lamps  delivering 50 µmol photons m−2 s−1 of PAR (measured at the vessel surface). However, the majority of PAR delivered to the cultures was through exposure to natural sunlight.A flat-panel Algae-Germ photobioreactor with two cultivation vessels, each of a total volume of 25 L (20 L of culture volume) , was use7 cells mL−1. The resulting culture, with an initial cell concentration of approximately 1.4 × 106 cells mL−1, was then incubated at 30 °C for 4 to 6 days. The supraoptimal temperature phase started upon reaching a biomass concentration exceeding 1.0 g L−1. At this point the culture was diluted with HS medium and was separated into two cultivation vessels, which were then transferred to 30 °C or 39 °C.All the experiments were performed in the time-span between July 17th and September 17th, 2018 . Each ex−2 s −1) was measured with a LI-COR 190-SA 2π PAR (400–700 nm) quantum sensor . Continuous light data logging was made with a sensor from the same model and manufacturer, mounted outdoors and facing the sky, parallel to the ground.The photon flux density  was calculated by dividing the mean light intensity during 24 h (obtained by continuous light data logging) by the number of viable cells for that period. To obtain a measure of light energy absorbed by the cell suspension grown at different concentrations of cells, the mean light intensity (I) was calculated according to the Lambert-Beer law: 2O), and stored at 4 °C. Cell diameter was measured on microphotographs taken with an Olympus Camedia C-5050 Zoom digital camera. The microphotographs were then analyzed using ImageJ image processing and analysis software . The cell diameter was recalculated to volume by a basic formula assuming a spherical cell shape. Cell number was determined by means of a Bürker counting chamber .One milliliter aliquots of culture suspension were taken, fixed with 10 µL of iodine solution  were taken and centrifuged  for 10 min at 4000 rpm. The supernatant was then removed, and the remaining pellet was transferred to a 2 mL preweighed test tube and dried for 24 h at 105 °C. After cooling for 2 h, the test tube with the pellet was weighed on an analytical balance  and the weight of the pellet was determined by subtracting the weight of the empty test tube.v/v) ethanol to the pellet and incubating in a water bath for 15 min at 68 °C after which the samples were centrifuged  for 2 min at 14,000 rpm and the supernatant was removed. The depigmentation procedure was repeated 3 to 4 times (or until the pellet was completely discolored). After that, 1 mL of α‑amylase  solution (0.5 g·L−1w/v in 0.1 M sodium phosphate buffer (pH 6.9)) was added to the samples and incubated for 1 h at 37 °C. The samples were centrifuged  for 2 min at 14,000 rpm, after which the supernatant was used for the quantification of reducing sugars through the dinitrosalicylic acid (DNSA) color reaction, as described by Miller [w/v) DNSA, 30% (w/v) potassium sodium tartrate tetrahydrate, 20% (v/v) 2 M sodium hydroxide) and incubated for 5 min at 105 °C on a heat block. Following a cooling period of 10 min at room temperature the mixture was diluted five-fold with distilled water, after which the absorbance of the samples was measured at 570 nm. The concentration of starch was estimated through a calibration curve of potato starch  digested with α-amylase.A starch-specific enzymatic method was used to accurately estimate the biomass starch content. Aliquots of culture suspension (10 mL) were harvested and centrifuged  for 10 min at 4000 rpm. The supernatant was then discarded, and the resulting pellets were stored at −20 °C. After thawing, the cells in the pellets were disintegrated by adding 300 µL of glass beads (0.7 mm in diameter) and vortexing vigorously  for 15 min. Depigmentation of the samples was carried out by adding 1 mL of 80%  and 39 °C was compared. At first a ectively .−1) (−1. The biomass starch content in terms of percentage of starch within the dry matter (DM) was much greater in the culture cultivated at 39 °C, reaching 18% of DM as opposed to 8% of DM at 30 °C  A. In conat 30 °C C. Moreovat 30 °C D.3 and cell division occurring, as expected, during the course of the experiment with mother cells dividing predominantly into eight daughter cells  and 0.8 g L−1 (dense culture); these were then cultivated at 39 °C for 8 more days  in the less dense culture was about 20% higher than in the dense culture. However, the maximum volumetric starch concentration reached at 39 °C was similar (in the case of the less dense culture) or lower (in the case of the dense culture) than that reached during the biomass accumulation phase at 30 °C   in both cultures was nearly identical due to the difference in biomass concentrations within the two cultures  C. Althoucultures D.C. reinhardtii cells were dividing during the biomass accumulation phase at 30 °C . However, this type of treatment required 7 days of nitrogen starvation until the maximum starch content was reached [−1 versus 16 pg cell−1. Notwithstanding, the limitation of pilot-scale cultivation, the starch accumulation in experiments presented here was significantly faster (only 1 to 2 days) compared to the nutrient limitation conditions [Starch synthesis in response to nitrogen depletion in laboratory-scale experiments with  reached . Similar reached . Thus, tnditions ,26. SuchNannochloropsis sp. showed a strong link between photosynthetic efficiency and the accumulation rate of lipids which are the primary energy storage compound in this microalga [A modern biotechnological process requires high productivity and cost efficiency . This caicroalga . As a reC. reinhardtii cultures after a certain period of time. In both cultivations at a starting biomass density of 0.8 mg mL−1, biomass decline was observed after 3 days of exposure to supraoptimal temperature  and supraoptimal temperature seemed to have a negative effect on perature A, and thperature A. In addperature .When synthesis of a substance such as starch is linked to the photosynthetic capacity of the cell, and consequently to light availability, production in large-scale requires efficient utilization of light. In this regard, an effective reactor design that ensures proper light distribution within a high biomass culture is essential ,29,32. TThe main advantage of the experiments presented here, compared to the other treatments, is the speed of starch accumulation . In the Based on the results described here, a tentative industrial-scale starch production strategy based on the use of supraoptimal temperature can employ both batch and repeated batch modes of operation. This is made possible by the fact that the culture can be recovered and reused as an inoculum after the temperature treatment . The proC. reinhardtii at low biomass densities. Moreover, starch synthesis was faster, with the maximum being reached within only 1–2 days, compared to five days at the optimal temperature. Thus, the supraoptimal temperature treatment provides a viable alternative to other starch stimulating methods, such as nutrient depletion. This is especially true when times required for starch accumulation are taken into account. However, technical challenges, such as bioreactor design and improved light availability per cell, still need to be dealt with.The method of supraoptimal temperature treatment was successfully applied in pilot-scale and resulted in a considerable, nearly 3-fold, enhancement of starch content in


Procyanidins are contained in various foods, and their effects on starch hydrolysis have been reported. In Japan, black soybeans, which contain a trimeric procyanidin, procyanidin C1 (proC1), are cooked with rice and used to prepare dumplings. In this study, the effects of proC1 on the pancreatin-induced formation of reducing sugars and starch hydrolysis were studied using potato starch and corn starch. ProC1 inhibited both reactions; the inhibition was greater in potato starch than corn starch when added to heated potato starch and corn starch. When heated with proC1, its inhibitory effects decreased, especially in potato starch, suggesting the important role of proC1 itself for the inhibition of potato starch hydrolysis. ProC1 also inhibited the hydrolysis when added to heated, longer amylose , and the inhibition decreased when heated with the amylose. On the other hand, proC1 could not inhibit the hydrolysis when added to heated, shorter amylose (average molecular weight: 4500), but could when heated with the amylose, suggesting the important role of the degradation products of proC1 for the inhibition. We discuss the mechanism of the proC1-dependent inhibition of amylose hydrolysis, taking the molecular weight into account. Proanthocyanidins are oligomeric and polymeric flavan-3-ols. The flavan-3-ol units linked through the C4→C8 bond or the C4→C6 bond are called B-type proanthocyanidins. An ether bond between C2 and C7 is added to the B-type, and the proanthocyanidins are called A-type. The proanthocyanidins can be classified into three types depending on the hydroxylation patterns of the B-ring of the flavan-3-ol units, namely, propelargonidins, procyanidins, and prodelphinidins. Of the proanthocyanidins, procyanidins are commonly found in various foods ,2.Vigna angularis), apples, black soybeans (Glycine max L.), sorghum, and so on are known as proanthocyanidin-rich foods. The benefits of the foods for human health have been recently reviewed . T. Tg for Hydrolysis of starch in the suspensions of control ppt, ppt-1, and ppt-2 was initiated by adding pancreatin (10 μg/mL) at 37 °C. During the incubation, aliquots (10 and 25 μL) of the reaction mixture were withdrawn at 10 min intervals for 50 and 90 min, respectively. The former aliquot was used to estimate the reducing sugar formation and the latter to estimate the starch hydrolysis. In addition, the control ppt of potato starch, which was suspended in the above buffer solution with 0.15 M of NaCl, was incubated with 7, 8, 9, or 10 μg of pancreatin per mL to understand the effects of the decrease in α-amylase activity on the kinetics of starch hydrolysis.Two types of amylose reagents (Mw4500 and Mw32100) (10 mg) were also treated as in g. The rate of the reducing sugar formation of each sample was estimated from the absorbance increase at 410 nm as a function of incubation time, and the slope was determined by the least squares method. The effects of proC1 were expressed relative to their controls.Reducing sugar formation was estimated by the method reported by Lever . An aliqTo estimate the starch hydrolysis, an aliquot (25 μL) of each reaction mixture, which was withdrawn as described above, and 0.1 mL of 100 mM of iodine solution were added successively to 1 mL of 0.1 M of sodium phosphate (pH 7.0) with 0.15 M of NaCl. After leaving it for 10 min at room temperature (about 25 °C), the absorption spectrum of the supernatant was measured. The starch hydrolysis was a first-order-like reaction. Then, the effect of proC1 could be estimated from the half-life.g for 5 min after the incubation for 10 min at room temperature. The amounts of proC1 in the precipitates  and the respective supernatants were estimated from the cyanidin formation from proC1 as follows [2O to remove the yellowish ammonium iron(III), and then the concentrations of cyanidin in the 1-butanol fraction were estimated from the absorbance at 545 nm using standard cyanidin. Potato starch or corn starch (5 mg each) suspended in 0.5 mL of 0.1 M of sodium phosphate (pH 7.0) with 0.15 M of NaCl was heated with and without 0.4 mM of proC1 in boiling water for 30 min, and 0.4 mM of proC1 was added to the starch heated without proC1 as described in  follows : 2 mL ofThe binding of 0.2 mM of proC1 to 5 mg of reagents amylose (Mw4500 and Mw31200) was also studied. Their control ppt, ppt-1, and ppt-2 were prepared and the amounts of proC1 bound and not bound to the ppt-1 and ppt-2 were estimated as described above.g for 5 min (g for 5 min to prepare the supernatants (SUP-1s) and the precipitates (PPT-1s). The PPT-1s and the SUP-1s would contain proC1 not extracted and extracted with 80% methanol, respectively. The PPTs and PPT-1s suspended in the above buffer with 0.15 M of NaCl (0.5 mL) and an aliquot (1 mL) of the SUP-1s were used to estimate the amounts of proC1, as described in The ppt-1 or ppt-2 prepared using 5 mg of potato starch and 0.2 mM of proC1 were suspended in 0.5 mL of 0.1 M of sodium phosphate (pH 7.0) with 0.15 M of NaCl. The suspensions were incubated with and without pancreatin (20 μg/mL) for 20 min at 37 °C, and then centrifuged at 7700× or 5 min B. The prAll spectrophotometric measurements were performed using UV-2450 combined with a sphere integrating apparatus (ISR-240A) . The path length of the measuring beam was 2 mm if not otherwise specified.t-test with the significance threshold set to 0.05.Each experiment was repeated 3–4 times. The data were presented as averages with standard deviations. Significant differences between the samples were determined using the Student’s


G3: Genes|Genomes|Genetics to the Genetics Society of America’s publishing portfolio a decade ago, we discussed two compelling reasons for doing so . Our expert board of editors thoughtfully synthesize reviews and communicate clear decisions, so you’re not left wondering what to do next.We believe that no journal should make the process of publishing a paper any harder than it needs to be. That’s why In our first 10 years, you’ve come to trust us as a place that treats you, your work, and your time with respect and care. A place that innovates and grows alongside the science and the scientists in our sphere. We hope to keep serving you with that same ethos for the next 10 years—and the 10 after that.The author declare that there is no conflict of interest.


Sixty implants with an internal connection system were used. Two-piece zirconia abutments were used with the titanium inserts. Combinations of different titanium insert axial heights (mm) and zirconia abutment labial heights (mm) constituted five groups: Gr1 (1–3), Gr2 (3–3), Gr3 (3–5), Gr4 (5–3), and Gr5 (5–5). After thermocycling, a fracture load test was performed with a universal testing machine. The initial deformation load and the fracture load were measured and analyzed. The fractured surface and cross-section of the specimens were examined by scanning electron microscopy (SEM). The groups of titanium inserts with axial heights of 3 mm and 5 mm showed significantly greater initial deformation load and fracture load than the group with an axial height of 1 mm (p < 0.05), but there was no significant difference between the two groups with axial heights of 3 mm and 5 mm. The labial height of the zirconia abutment had no significant influence on the initial deformation load and fracture load. In some specimens in Gr4 and Gr5, cracking or bending of the titanium insert and abutment screw was observed on SEM. The axial height of the titanium insert should be designed to not be less than 3 mm to increase the fracture strength and promote the long-term stability of implants.This study aimed to analyze fracture strength  Titanium insert-reinforced two-piece zirconia abutments have been developed and used to complement the mechanical stability of one-piece zirconia abutments .A zirconia abutment with a tooth-like color represents aesthetic advantages in preventing unnatural metal shadows appearing through thin gingival tissue and the exposure of titanium abutments after gingival recession , 3. In aNevertheless, mechanical limitations of one-piece zirconia abutments have also been reported. One thing to consider is the high brittleness of zirconia. Zirconia is often considered a metallic ceramic because of its high strength. However, it is more vulnerable to fracture than conventional titanium abutments when a thin piece of zirconia is used in a prosthesis. Particularly for internally connected types of one-piece zirconia abutments, the thickness of the zirconia abutment is thin at the site of connection inside the fixture. Regarding preloading, the joint stability could be established by applying a preload to obtain the clamping force using the tension between a screw and a component in the titanium abutment; however, zirconia abutments do not allow the plastic deformation of components to determine a safety value for the joint screw and may fracture due to stress concentration , 7.In addition, zirconia is known to undergo low-temperature degradation in a moist environment, which weakens its mechanical properties , 9. ZircThe marginal gap at the point of contact between a zirconia abutment and titanium implant may be defective due to an error in the abutment manufacturing process or long-term clinical use. A poor fit between the abutment and implant can lead to mechanical complications, such as fracture, loosening of the abutment screw, and deformation of the components, as well as biological complications, such as bone resorption because of stress concentration in the marginal area and peri-implantitis due to bacterial retention . Since pA defect in the fit of the zirconia abutment–implant interface resulting from long-term clinical use may be induced by both a difference in hardness between the zirconia and titanium and the micromobility generated during mastication or deglutition . The harA titanium insert, as a complementary component, was designed and attached to the bottom of the zirconia abutment and allowed the zirconia abutment to be connected to the implant with titanium-to-titanium contact, ensuring force distribution along the internal connection and overcoming mechanical limitations related to inherent problems of zirconia material and processing , 11, 15.The mechanical stability, including the flexural strength, fatigue load and fracture resistance, of titanium insert-reinforced zirconia abutments was proven to be significantly higher than that of one-piece zirconia abutments , 5. The The factors that influence the mechanical strength of titanium insert-reinforced zirconia abutments are the height, diameter and thickness of the titanium insert and the method of retention. Concerning the efficacy of the retention method, one investigation compared three groups of titanium inserts and zirconia abutments joined with bonded retention, friction fit retention, and ring friction and treated with cyclic loading; they found that the bonded group showed the highest fracture strength, followed by the friction fit group and ring friction group in order . In realin vitro study was designed to measure the fracture strength and observe the fracture patterns of titanium insert-reinforced zirconia abutments fabricated by varying the axial height of the titanium insert and the labial height of the zirconia abutment to suggest an ideal titanium insert axial height in terms of the integrity of the abutment–implant complex.This Sixty screw-shaped implants 4.0 mm in diameter and 10.0 mm in length  were used, and a total of sixty titanium-reinforced zirconia abutments were divided into five groups based on the axial height of the titanium insert and the labial height of the zirconia abutment in this study. The implants tested in this study were composed of SLA surface-treated pure titanium and were consistent with those used clinically.The bottom of the titanium insert was 4.0 mm in diameter, and the axial height from the bottom to the top  varied fA total of 60 specimens were divided into 5 groups, with 12 specimens in each group, based on the axial height of the titanium insert and the labial height of the zirconia abutment: Gr1 (1–3), Gr2 (3–3), Gr3 (3–5), Gr4 (5–3), and Gr5 (5–5).To assemble the titanium insert and zirconia abutment, the inner surface of the zirconia abutment was treated with silica-modified aluminum oxide sandblasting  to enhance the bonding strength with the titanium insert, and then a ceramic primer  was applied. The zirconia abutment was bonded with the titanium insert using dual-cure self-adhesive resin cement  according to the manufacturer’s instructions. Remaining adhesive was removed under a microscope at 10X magnification.The abutment was connected to the implant through a titanium abutment screw using a torque wrench  with 30 Ncm of tightening torque, as recommended by the manufacturer. Implant–abutment joint stability was ensured by retightening the screw to establish sufficient preloading , 17.Then, the abutment was stored in distilled water at 37°C for 24 hours to eliminate the effect of volume change due to adhesive hydration. Thermocycling  was performed at 5–55°C in a water bath at intervals of 15 seconds and 10 seconds to simulate five years of exposure to moist oral conditions .To investigate the fracture strength in this test set up, the abutment–implant specimen was fixed to the universal testing machine  using a metal holder that was positioned with a vertical distance of 3 mm downward from the platform of the implant to simulate 3 mm of crestal bone resorption . The loaAlthough the load–deformation curve shows progressive proportionality, the sudden reduction of the load in some segments indicates cracking, bending or loosening of the component, which means a potential failure in the function of the prosthesis –22. In tThe fracture load was automatically recorded at the point where the fracture occurred through Instron Series IX Software .The fractured surface of three specimens from each group was observed under a scanning electron microscope . The superior aspect of the implant (20X magnification) was observed to determine the damage in the inner hexagonal surface of the titanium insert. The lateral aspect of the connecting part of the fixture and abutment (18X and 30X magnification) was observed to verify deformation in the titanium insert, abutment, and fixture. The surface of fractured zirconia (40X magnification) was observed to examine the fracture patterns of the zirconia abutment.Blocks were fabricated by burying two specimens from each group in acrylic resin  to determine the deformation pattern of the abutment–implant interface after the load test. Specimens were prepared by cutting along the longitudinal axis of the implant and were observed under a microscope ; 12.5X, 40X, 100X.U test were used for data analysis. Bonferroni correction was used to adjust the confidence interval. The level of statistical significance was set at less than 1.7% .The initial deformation load and fracture load were processed statistically using SPSS . The power of the experiment was sufficiently high to show that all differences were statistically significant. A power analysis was therefore not required. The nonparametric Kruskal-Wallis test and Mann-Whitney A total of nine zirconia abutment–titanium insert specimens were excluded from the analysis due to not only abutment dislodgement from the titanium insert (one in Gr3 and one in Gr5 during screwing into the implants and two in Gr1 during the thermocycling procedure) but also zirconia fracture at an early stage of the load test . Therefore, the test results of fifty-one specimens were subjected to analysis: Gr1 (n = 8), Gr2 (n = 12), Gr3 (n = 9), Gr4 (n = 11), and Gr5 (n = 11).The mean initial deformation load in each group was 89.19 N, 190.46 N, 179.86 N, 180.63 N, and 177.88 N in Gr1, Gr2, Gr3, Gr4 and Gr5, respectively . AccordiThe mean fracture load in each group was 361.7 N, 509.07 N, 558.5 N, 814.88 N, and 608.54 N in Gr1, Gr2, Gr3, Gr4 and Gr5, respectively . AccordiThe zirconia abutment completely dislodged from the titanium insert or partially fractured . In Gr1,The fractured surface of three specimens from each group was observed under a SEM. A superior and lateral view of the implant and the fractured surface of the zirconia abutment were examined . In the Two specimens from each group were cut along the long axis of the specimen, and the sectioned surface was observed under a microscope . MeanwhiThis study shows that a zirconia abutment reinforced by a titanium insert with an axial height of at least 3 mm could resist the biomechanical stress exerted during oral functions, such as mastication and chewing. Physiological masticatory force developed during mastication and deglutition is known to be 90–370 N for anterior teeth and, 200~900 N for posterior teeth, while 300 N has been suggested as an appropriate load for a single premolar and 700 N as a maximum load for the second molar region, with a monotonic increase in the load along the dental arch; however, there are still conflicting findings –28. Due Considering the low mean initial deformation load (89.19 N) and fracture load (361.7 N) of zirconia abutments with a titanium insert with an axial height of 1 mm in this study, a titanium insert with an axial height of 1 mm seems inappropriate for clinical application due to the low strength regardless of the location of the prosthesis.Since the mean initial deformation load in the 3 mm and 5 mm titanium insert groups was 185.92 N and 179.26 N and the fracture load in the 3 mm and 5 mm titanium insert groups was 530.26 N and 711.70 N, respectively, a titanium insert with an axial height of 3 mm or 5 mm was proven to be suitable for anterior teeth replacement. A study of the effect of various cements on the retentiveness of zirconia abutments with respect to the titanium insert axial height reported that there was a significant increase regardless of the cement used and that the axial height of titanium inserts should be at least 3 mm to maintain zirconia abutment retention .However, in the case of posterior restorations, it is necessary to design prostheses to reduce stress by reducing the size of the occlusal table or lowering the cusp inclination of crowns. One previous study concluded that a zirconia abutment with a titanium insert appeared to be a suitable treatment option in the anterior and premolar regions after an observation period of 6 years . HoweverThe vertical crown height or interocclusal space is also an important factor to consider when designing titanium insert reinforced zirconia abutments. The interocclusal space refers to the distance between the ridge crest and the occlusal plane comprising the depth of the soft tissue above the fixture, the height of the abutment, and the thickness of the implant prosthesis . Based oThe initial deformation load was measured in addition to the fracture load. In the early stage of the load–deformation curve, a small amount of fluctuation could be seen, the first point of which was the initial deformation load. This point could be considered to indicate the potential loss of functional stability because it signifies a change in the original structural integrity, such as initial cracking or bending, began to occur due to external compressive loading. Even if the fracture load is high, a restoration with a low initial deformation load is prone to fracture under cyclic fatigue loading. The conventional load–deformation curve shows a sharp drop in load at the end of the curve, indicating the fracture or plastic deformation of components , but in The values of the load test did not significantly change with changes in the labial height of the zirconia abutment in this study. It can be concluded that the labial height of zirconia is unrelated to the fracture load and initial deformation load of titanium insert-reinforced zirconia abutments.Previously published studies have investigated the effect of the zirconia abutment thickness on the fracture strength and fatigue load, which was not considered in this study, and reported that the fracture strength was highest in the 0.5 mm group; additionally, there was a significant difference between this group and other groups with preparation depths of 0.7 and 0.9 mm, suggesting that a zirconia abutment preparation depth of 0.5 mm is advisable for titanium insert-reinforced zirconia abutments , 32. AltIt was inferred that a higher torque and stress is expected on the area of contact between the thin zirconia wall and the axial titanium wall than on the interface between the screw head and abutment or the abutment and implant; therefore, the volume of the labial side of the zirconia abutment could affect the fracture strength comparison, based on reports in the literature indicating that the labial side of an anterior zirconia abutment is 1.7 times stronger under compression than the palatal surface under tensile stress . An initIn Gr1, with the lowest titanium insert axial height, all zirconia abutments completely detached from the titanium inserts during the fracture load test without deformation of the titanium insert, abutment screw, or fixture on SEM. It was assumed that this result was caused by low retention force due to the short axial height on the basis of an investigation of changes in the retention force with varying abutment height . With inThe fractured zirconia abutments showed a vertical fracture pattern in the longitudinal direction of the implant, and in all fractured specimens, the cervical portion of the abutment was involved. It could be inferred that cracks started from the cervical portion of the abutment due to tensile stress transformed from compressive force on the top of the titanium insert and propagated along the abutment. At this point, the axial height of the titanium insert is presumed to play an important role in the fracture strength of the abutment.The present study was not designed to determine whether fatigue loading affects the fracture strength and integrity of the abutment–implant complex. Considering the initial deformation load, under cyclic loading, specimens with a low initial deformation load are more prone to be vulnerable to deformation of the abutment screw or the abutment itself. As factors related to the retentiveness of the abutment and the initial deformation load, the height, surface area, slope, surface treatment, and adhesive could be considered , 36, butThermocycling treatment in this study was performed to evaluate clinical stability by simulating five years of exposure to moist oral conditions , 37. ManThe uneven and small numbers of specimens per group (Gr 1 (8), Gr 2 (12), Gr 3 (9), Gr 4 (11), and Gr 5 (11)) is considered one of the limitations of this study. To avoid bias in the data analysis, nine failed specimens in the preparation stages for the screwing, thermocycling, and loading test were excluded. In addition, non-anatomical, internal connection type abutments were used in this study. Further experimental studies using anatomically designed zirconia prostheses and large-size specimens are required to achieve more clinically meaningful results. An experimental study is needed to investigate the fatigue load and labial thickness of titanium insert-reinforced zirconia abutments to maintain the integrity of the abutment complex under dynamic functional loading using a greater number of specimens and a load applicator to mimic the conditions of occlusion between anterior teeth, followed by supporting clinical research.in vitro study, the conclusion regarding clinical use is that the axial height of the titanium insert should be designed to not be less than 3 mm to increase the fracture strength and promote the long-term stability of implant-supported fixed prostheses with titanium insert-reinforced zirconia abutments. In addition, considering the initial deformation load, the occlusion scheme and prosthetic structure should be designed to reduce stress and distribute the functional load on implant-supported prostheses for anterior teeth.Within the limitations of this S1 Data(XLSX)Click here for additional data file. 22 Feb 2021PONE-D-21-03699Fracture strength analysis of titanium insert reinforced zirconia abutment by axial height of titanium insert with internal connectionPLOS ONEDear Dr. Jung,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Apr 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Antonio Riveiro Rodríguez, PhDAcademic EditorPLOS ONEJournal requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: There is no specification of implant type regarding sterile and clinical usage type of implants, while those implants on the figures attached have smooth surface and they should not be used for clinical purposes, the surface should be rough and specific related to the manufacturer! This in vitro study should be performed on the original, sterile packed dental implants prepared for clinical use.English should be carefully checked by the native speaker and the manuscript need serious proof editing before final consideration for publication.Reviewer #2: I have reviewed the manuscript Fracture strength analysis of titanium insert reinforced zirconia abutment by axial height of titanium insert with internal connection” submitted to “PLOS ONE” for publication. In this study, authors have analyzed the fracture strength in vitro by varying the axial height of the titanium insert and the labial height of the zirconia abutment in an internal connection implant to identify the titanium insert axial height with optimal mechanical stability. The manuscript fits well within the scope of the journal; it needs some major improvements; there are a few suggestions that authors may consider improving it further:The use of English language is reasonable, however, there are a number of minor punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader.Abstract: covered all key informationIntroduction; is very detailed and covering the background information and the rationale of the study effectively. Some of the statements have missing citations; such as for this statementA number of studies have proven the relevance…..: author may cite the following referencesCustomized therapeutic surface coatings for dental implants. Coatings. 2020 Jun;10(6):568.Dental Implants: Materials, Coatings, Surface Modifications and Interfaces with Oral Tissues. Woodhead Publishing; 2020 Jul 23.https://doi.org/10.1016/B978-0-12-819586-4.00002-0Similarly, the text in the second paragraph of the introduction require citations.A titanium insert, as a complementary component….. require citations as given above can be used.Methods: why not used same number of samples after exclusion (n=?) for each groupGr1 (n=8), Gr2 (n=12), Gr3 (n=9), Gr4 (n=11), Gr5 (n=11).?Results and data are well explained and beautifully presented.In discussion: please include discussion in terms of reduced vertical height; or reduced inter occlusal space (deep bite) case. Is there any relation?What are the limitations of the study?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  3 Mar 20211. Title:REVISION: PONE-D-21-03699Fracture strength analysis of titanium insert reinforced zirconia abutment according to the axial height of titanium insert with internal connection2. Corresponding author’s name, mailing address, phone number and fax number:Prof. Bock -Young Jungjby1004@yuhs.acE-mail address: Phone: (+82)2-2228-8980, Fax: (+82)2-2227-89063. Names of authors:Seung-rye Song, Kyeong-Mee Park, Bock-Young Jung This response letter accompanies our online resubmission of the manuscript titled “ Fracture strength analysis of titanium insert reinforced zirconia abutment according to the axial height of titanium insert with internal connection ” for the publication in “ PLOS ONE ”.We really thank the reviewer’s valuable comments which made our manuscript more sound and reasonable scientifically. We are re-submitting after revisions as requested by the reviewers. We tried to address all the comments carefully in the revised manuscript. We hope that our revised manuscript meets the high standards of your journal.We hope that this paper would be published in ‘PLOS ONE’. We thank you in advance for your time and attention.Sincerely yours,27th February, 2021Bock Young, Jung Response letterManuscript ID: PONE-D-21-03699 MS-Type: Original ArticleTitle: " Fracture strength analysis of titanium insert reinforced zirconia abutment according to the axial height of titanium insert with internal connection." Correspondence Author: Bock- Young, JungReviewer #11. There is no specification of implant type regarding sterile and clinical usage type of implants, while those implants on the figures attached have smooth surface and they should not be used for clinical purposes, the surface should be rough and specific related to the manufacturer! This in vitro study should be performed on the original, sterile packed dental implants prepared for clinical use.Response: We respect and appreciate the comment of the reviewer. We have modified ‘material and methods’ section and specified the information related to the implants tested.Text Change: See “Material and methods”.“The implants tested in this study were composed of SLA surface-treated pure titanium and were consistent with those used clinically.”2. English should be carefully checked by the native speaker and the manuscript need serious proof editing before final consideration for publication Response: Thank you for the comment. We carefully tried to correct any errors in spelling, grammar, and terminology of this manuscript.Text Change: The corrected words and phrases were highlighted. Reviewer #2I have reviewed the manuscript Fracture strength analysis of titanium insert reinforced zirconia abutment by axial height of titanium insert with internal connection” submitted to “PLOS ONE” for publication. In this study, authors have analyzed the fracture strength in vitro by varying the axial height of the titanium insert and the labial height of the zirconia abutment in an internal connection implant to identify the titanium insert axial height with optimal mechanical stability. The manuscript fits well within the scope of the journal; it needs some major improvements; there are a few suggestions that authors may consider improving it further:1. The use of English language is reasonable, however, there are a number of minor punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader. Response:We appreciated this comments and this manuscript was reedited by one of native experts whose specialty was dentistry according the advice of the reviewer. Text Change: See the marked-up manuscript. 2. Introduction; is very detailed and covering the background information and the rationale of the study effectively. Some of the statements have missing citations; such as for this statement. A number of studies have proven the relevance…..: author may cite the following references. Similarly, the text in the second paragraph of the introduction require citations.A titanium insert, as a complementary component….. require citations as given above can be used. Response:We appreciate this important comment of the reviewer. We have specified the references we cited.Text Change: See “Introduction” section.3. Methods: why not used same number of samples after exclusion (n=?) for each groupGr1 (n=8), Gr2 (n=12), Gr3 (n=9), Gr4 (n=11), Gr5 (n=11).?Response:We appreciated this valuable comment of the reviewer. This study was originally prepared by dividing 60 specimens into 5 groups, 12 pieces for each group. However, we excluded nine specimens. Four specimens during the preparation for screwing and thermocycling procedures and 5 specimens during the preparation for loading were failed, which could affect the results of the experiment. Text Change: We have modified the end section of discussion and added statement on the reason for the uneven distribution of specimens.4. In discussion: please include discussion in terms of reduced vertical height; or reduced inter occlusal space (deep bite) case. Is there any relation?ResponseThank you for the valuable comment. According to the advice of reviewer, we have introduced and added more description regarding the clinical implications that the reduced interocclusal distance has.Text Change: See “Discussion” in page.“Vertical crown height or interocclusal space is also an important factor to consider when designing titanium insert reinforced zirconia abutments. The inter occlusal space means the distance between the ridge crest and the occlusal plane comprising the depth of the soft tissue above the fixture, the height of the abutment, and the thickness of the implant prosthesis [17]. Based on the results of this study that the axial height of the titanium insert should be 3mm or more to ensure structural integrity, the limited interocclusal space can be a contra-indication to titanium insert-reinforced zirconia prosthesis unless the margin of zirconia prosthesis is located subgingivally.” 5. What are the limitations of the study?Response: Thank you for the valid comment of reviewer. We have modified “Discussion” adding several limitations of this study and presented the next research for the further investigation.Text Change: See “ Discussion” in page.“ The uneven and small numbers of specimens per group (Gr 1 (8), Gr 2 (12), Gr 3 (9), Gr 4 (11), and Gr 5 (11)) is considered one of the limitations of this study. To avoid bias in the data analysis, nine failed specimens in the preparation stages for the screwing, thermocycling, and loading test were excluded. In addition, non-anatomical, internal connection type abutments were used in this study. Further experimental studies using anatomically designed zirconia prostheses and large-size specimens are required to achieve more clinically meaningful results. An experimental study is needed to investigate the fatigue load and labial thickness of titanium insert-reinforced zirconia abutments to maintain the integrity of the abutment complex under dynamic functional loading using a greater number of specimens and a load applicator to mimic the conditions of occlusion between anterior teeth, followed by supporting clinical research.“Thank you very much again.We really thank the reviewer’s comments which really solidify our manuscript more reasonably. Sincerely,Bock Young Jung(Corresponding author)AttachmentReponse letter for plos-one.docxSubmitted filename: Click here for additional data file. 15 Mar 2021Fracture strength analysis of titanium insert-reinforced zirconia abutments according to the axial height of titanium insert with an internal connectionPONE-D-21-03699R1Dear Dr. Jung,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Antonio Riveiro Rodríguez, PhDAcademic EditorPLOS ONEReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: (No Response)**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: (No Response)**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I have no comments or suggestions, authors have made all changes suggested earlier and explain them in the cover letter.Reviewer #2: Many thanks for the revision and incorporating all suggested changes to the manuscript and the quality of the manuscript has been improved.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: No 17 Mar 2021PONE-D-21-03699R1 Fracture strength analysis of titanium insert-reinforced zirconia abutments according to the axial height of the titanium insert with an internal connection Dear Dr. Jung:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. 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In recent decades, the prevalence of diabetic kidney disease has remained stable and appears to be a wide heterogeneity. Normoalbuminuric diabetes with renal insufficiency, which is characterized by a decline in the glomerular filtration rate in the absence of albuminuria, has been identified as an albuminuria-independent phenotype of diabetic kidney disease. Epidemiological data demonstrate that normoalbuminuric phenotype is prevalent. Compared to albuminuric phenotype, normoalbuminuric phenotype has distinct clinical characteristics and a wide heterogeneity of pathological features. Currently, the pathogenesis of normoalbuminuric phenotype remains unclear. Additionally, the flow of diagnosing normoalbuminuric phenotype is not perfect. In this article, we review the latest studies addressing the epidemiology, clinical characteristics, and pathology of normoalbuminuric phenotype. Based on the studies of clinical features and renal histopathologic changes, we attempt to propose an underlying pathogenesis model and a flow chart for diagnosing normoalbuminuric phenotype. However, this classical view has been challenged by evidence that a considerable proportion of patients with diabetes have renal insufficiency in the absence of albuminuria, referred to normoalbuminuric diabetic kidney disease , 2 or noe of DKD , 7. The e of DKD .The combination of renal insufficiency and normoalbuminuria in diabetes was first highlighted by Lane et al. in 1992 . Even thCurrently, the natural course and potential pathogenesis of NADRI are still elusive. In this article, we review the latest studies addressing the epidemiology, clinical characteristics, and pathology of NADRI. Based on the studies of clinical features and renal histopathologic changes, we attempt to propose an underlying pathogenesis model and a flow chart for diagnosing NADRI.Two early studies reported that renal insufficiency may occur in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) with normoalbuminuria , 21, andIn T2DM, the prevalence of NADRI ranges from 14 to 57% in cross-sectional studies, with differences among surveys depending on the geographic area and the calculation formula of eGFR –36. A hiThese data are in line with the increasing prevalence of declined eGFR and the decreasing incidence of albuminuria observed in diabetes , 51. TheThe reported clinical features of NADRI include a higher proportion of females , 37, 47,Compared to patients with micro-ADRI and/or macro-ADRI, the clinical features of NADRI include a higher proportion of females , 18, 57,2), individuals with NADRI are older [In addition, compared to patients with normoalbuminuric preserved renal function  independent of albuminuria in diabetes, levels of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and retinol-binding protein (RBP), tubular damage biomarkers were increased in NADRI, significantly correlated with eGFR and may serve as noninvasive biomarkers of NADRI , 67, to As compared to those with normoalbuminuria and preserved eGFR, vascular lesions in patients with NADRI were more advanced. Likewise, compared to patients with ADRI, vascular lesions of individuals with NADRI were similar or more advanced in contrast to glomerular lesions .Previous clinical observations found, compared to those with or without albuminuria and preserved eGFR, that patients with NADRI are related to a higher risk of major cardiovascular events or cardiovascular morbidity and death , 68, 69,2 have a higher renal artery resistance index and a greater degree of intrarenal vascular disease  than patients with preserved eGFR, and this difference is independent of UAER[Diabetic patients with an eGFR < 60 mL/min per 1.73 mt of UAER. Furthert of UAER and moret of UAER. Moreovet of UAER. Besidest of UAER.Currently, the pathogenesis of NADRI remains to be fully elucidated. The proposed pathogenic mechanisms that underlie NADRI are multifactorial, complex and may involve age-associated renal senescence, hypertension, dyslipidemia, obesity, renal hypertensive and interstitial fibrosis, vascular disease, arteriosclerosis, cholesterol microemboli, lipid toxicity, inflammation, and masking of albuminuria by RAAS inhibitors , 70–72. The presence of low eGFR in diabetic patients with normoalbuminuria is associated with the presence of metabolic syndrome . Risk fa2) to severe obesity (body mass index > 35 kg/m2) was related to reduced renal function in the absence of albuminuria [In T2DM, obesity from the overweight category  , 61, 95 The markers of renal tubular injury, such as NGAL, RBP, and KIM-1, have been found significantly increased in NADRI and weakly but significantly correlated with eGFR, suggesting that renal tubular interstitial injury may be associated with the development of NADRI , 96.Further studies show that the decline in GFR in T2DM with or without microalbuminuria is associated with an increase in carotid intimal-medial thickness, carotid stiffness, and intrarenal arterial resistance index , which sMoreover, interstitial fibrosis, tubular atrophy, and arteriosclerosis in renal biopsy specimens are the pathological factors to develop kidney events . BesidesSome clinical studies of NADRI show that patients with NADRI are more likely to be older, and age is an independent risk factors for renal insufficiency in T2DM patients with normoalbuminuria , 66. ThiAging can lead to various anatomical and physiological changes of the kidney, including structural changes of aging with age, declining GFR, and decreased tubular function , 106. InMore significant heterogeneity in renal structural changes of NADRI  than in ADRI suggested aging may be one of the key contributors to the atypical nephropathy changes observed in NADRI and contributes to decline in kidney function independently of albuminuria . In partThe prevalence of NADRI varies by ethnic groups, indicating the development of the disease may involve genetic susceptibility. Polymorphism of the protein kinase C-β gene was related to accelerated decline of eGFR in T2DM without overt proteinuria, and the T-G haplotype of protein kinase C-β gene (PRKCB1) may be a valuable predictive factor for the rapid deterioration of kidney function .In addition, the relationship between polymorphism of angiotensin type 1 receptor gene and normoalbuminuric chronic kidney disease among South Indian T2DM patients was described. Compared to ADRI, the relative risk of AC genotype and C allele in NADRI was 4 times higher. Significant correlation of AGTR1 A1166C polymorphism was noted in NADRI as compared to ADRI .The effects of gender on renal injury are controversial. Lotfinejad et al. noted renal damage in diabetes was directly related to male gender, but not to female gender . In vitrEpidemiological studies show that the prevalence of NADRI is higher in female gender. The female predominance may be due to sex differences in CKD risk . Female Subsequent studies have demonstrated women with diabetes are at greater risk than men for accelerated GFR decline in the absence of albuminuria . This efTumor necrosis factor alpha (TNF-α), interferon γ, interleukin (IL)-10, IL-6, KIM-1, and other inflammatory cytokines are involved in the immune inflammatory response of DKD , 118. FuThe concentration of serum Fas-pathways cytokines (soluble Fas and Fas ligand) and TNF-α  are related to reduced eGFR independent of urinary albumin excretion in diabetes , 121–123In addition, serum IL-17A, IL-18, and macrophage inflammatory protein 1 alpha were predictors of NADRI , 126. UrBesides immune inflammation, oxidative stress may contribute to the development of NADRI. 8-hydroxydeoxyguanosine, as a sensitive marker of intracellular oxidative stress, is more highly expressed in high intima media thickness than in normal intima media thickness and is positively related to coronary heart disease risk scores . The cliIn addition, urinary NGAL and RBP are biomarkers for NADRI, which may be caused by inflammation and oxidative stress and lead to tubular damage , 129.For the past decades, the use of RAAS blockers has increased  and receAt present, there are two hypotheses on the pathogenesis. One is that the application of RAAS blockers slows the progression of albuminuria in diabetes, while the decline in eGFR is not significantly improved, even possibly resulting in an acute decline of eGFR , 55, 132The other proposed mechanism is that the use of RAAS blockers in diabetes may result in a decline in the prevalence of glomerular damage and allowed the occurrence of other renal injury, such as interstitium and vascular damage, which may lead to progressive renal injury without triggering albuminuria .In diabetes, due to various factors, such as ischemia, infection, nephrotoxicity, or obstruction, acute kidney injury (AKI) often occurs in a mild and unrecognizable form, resulting in progressive decline in kidney function independent of albuminuria . OnuigboSeveral mechanisms have been proposed for the progression of AKI to CKD, such as nephron loss, inflammation, endothelial injury with vascular rarefaction and hypoxia, epigenetic changes, and cell cycle arrest in epithelial cells, ultimately leading to progressive glomerulosclerosis and tubulointerstitial fibrosis –139.2, at least twice in three measurements, and exclude eGFR reduction caused by AKI and other reasons; and (4) to exclude other secondary nondiabetic kidney diseases  guidelines recommend that the diagnosis of DKD should be based on monitoring UACR or UAER, fundus changes, and evaluation of eGFR. Based on KDOQI guidelines recommendations and clinical studies of NADRI, we attempt to propose the diagnostic criteria of NADRI: (1) to meet the latest diabetes diagnostic standards of World Health Organization or American Diabetes Association (ADA); 2) to test and prove within 6 months, UAER < 30 mg/24 h or UAER < 20 µg/min or UACR < 30 mg/g, at least twice in three measurements, to avoid confounding by transient increases in conditions, such as exercise, fever, hematuria, urinary tract infection, and congestive heart failure; (3) to  to test eGFR estimation formula based on serum creatinine is generally recommended for diagnosis of DKD, including Cockcroft-Gault formula, modification of diet in renal disease formula, and CKD epidemiology collaboration (CKD-EPI) formula , 142. HoEpidemiological data demonstrate that NADRI is a prevalent clinical phenotype of DKD. Moreover, NADRI exhibits distinct clinical characteristics and a wide heterogeneity of histology features. Based on the studies of clinical features and renal histopathologic changes of NADRI, we attempt to propose an underlying pathogenesis model and a flow chart for diagnosing NADRI, which increases the practicality and reliability of the study. The underlying pathogenesis associated with NADRI involves multiple factors and comprises the usual suspects, including obesity, hyperlipidemia, tubulointerstitial lesion and vascular disease, age, gene polymorphism, female gender, immune inflammation and oxidative stress, drugs, and AKI, while appears to be at least partially independent of hyperglycemia and hypertension and does not exclude the possible role of hyperuricemia. Early diagnosis and a deeper understanding of pathogenesis may provide theoretical guidance for the therapy of NADRI and prevent or delay the CKD progression, end-stage renal disease, and diabetes complications as far as possible.Although many of factors are discussed in this review, the list is not exhaustive. Multilevel, multicenter, large-sample clinical studies are needed to further clarify the pathogenesis of the disease, discover specific biomarkers, establish clear and precise laboratory diagnostic standards for the disease, and provide an important theoretical basis for preventing the occurrence, development, early intervention, and treatment of the disease.


Biomimetics (ISSN 2313-7673), published by MDPI, has developed into a robust scientific journal that is appreciated in the field. Biomimetics is an open access journal regarding biomimetics, bioinspiration and bionics, dedicated to research that relates to the most basic aspects of living organisms and the transfer of their properties to human applications. The journal aims to provide a forum and survey for researchers and professionals in the fields of materials science, mechanical engineering, nanotechnology and biomedicine interested in exploiting biologically inspired designs in engineering systems, as well as technology and biomedicine aimed to developing novel solutions that enable sustainable innovation. Biomimetics invites submissions on a wide range of topics, including, but not limited to: biomimetic mechanism and design, biomimetic robotics, biofabrication and characterization, biomimetic and bioinspired chemistry, biosensing, nanotribology, nanomechanics, micro/nanoscale studies, plant biomechanics, synthetic systems, self-organization and cooperative behavior, tissue engineering and bioinspired materials.Since its inaugural issue published in 2016, the scientific journal Biomimetics is based on publishing articles written by specialist scientists as well as experts who work in an inter- or transdisciplinary way in all fields of biomimetics, including professional education, research and development. The journal has contributed immensely to consolidating biomimetics as a field of research and supports it in its growth. I wish to convey my warmest congratulations to the journal Biomimetics on the occasion of receiving its first impact factor in 2022, and keenly look forward to witnessing the continuous achievements of the journal along its mission of being dedicated to the advancement of communication and cooperation among all scholars, and the dissemination of knowledge and education in the field of biomimetics.The success of Biomimetics is a field that is a little bit different from various other scientific fields, because of its inherent inter- and transdisciplinarity. Biomimetics experts understand the basic biology and current challenges. Furthermore, their goal is to abstract and transfer biological principles into engineering, architecture or art—and eventually in a way the resulting products and processes are sustainable. Such a broad and at the same time deep approach is difficult to realize in a single person. Biomimetics as a field is very attractive to students and the general population. The challenge is how to teach and communicate biomimetics in a way that creates a positive effect for science and society, on various levels. Living nature provides us a glimpse into the possible, into what can be done. Additionally, with curiosity, eagerness and dedication, biomimetics experts can learn from this great master . ConventBiomimetics has made valuable contributions to serve as a platform for biomimetics experts around the world—for informing their colleagues and being informed. It has further contributed to combining science with values, resulting in approaches that focus on a sustainable future . On. On28]. The second aspect of biomimetics that is arguably fundamental to its future success is related to the complexity of the physics utilized in organisms. Contrary to potential ordinary belief, the complexity of the physics of organisms does not correlate with their size or location in the phylogenetic tree. Single celled algae or bacteria can be as great an inspiration for new approaches in biomimetics as the largest whale or the most complex ecosystem. This allows knowledge gain in a vast variety of research environments and promises great future developments!


When compared to the neat films , the incorporation of at least 15% SFO/KOH gave rise to transparent, hydrophobic (water contact angles of ca. 90°), stretchable (ca. 20×), elastic (ca. 5×), and water tolerant starch-based films, contrary to the films produced without the catalyst. ATR-FTIR and 1H NMR revealed structural differences among the produced films, suggesting that starch was modified with the SFO-derived fatty acids. Therefore, adding KOH during the potato starch/spent frying oil-based film’s production was determined to be a promising in situ strategy to develop starch-based materials with improved hydrophobicity and flexibility, while valorizing the potato chip industry’s byproducts.Starch is a promising candidate for preparing biodegradable films with useful gas barriers and thermoplastic capabilities. However, these materials are hydrophilic and brittle, thus limiting their application range. To overcome these drawbacks, it has been hypothesized that starch can be hydrophobized and plasticized during the starch-based film production using a single-step approach and following transesterification principles. In this work, KOH powder and spent frying oil (SFO) were used as an alkaline catalyst and a source for triacylglycerides, respectively, to promote the modification of starch. Different ratios of SFO ( In. In10]. ectively . Moreoveectively .1H NMR spectrum of starch-based films with 15% SFO, the characteristic proton resonance bands of glucose units were still present, but new signals appeared at 0.87 ppm associated to the protons of the terminal methyl group (CH3), at 2.22 ppm associated to the methylene group (CH2) that occurs immediately before the carbonyl group of the ester, denoted as CH2(CO), which was linked to a second oxygen atom bonded to the carbon atom in the carbonyl group by a single bond. Other proton peaks at 1.24 ppm, 1.44 ppm, and 1.96 ppm, associated to other protons of different methylene groups of the SFO fatty acid chain [2 groups) of the fatty acid chain , as well as on the region of the terminal methyl groups at 0.76 ppm, denoted as CH3, were possible due to the chemical environment changes promoted by the starch modification in the presence of KOH. Furthermore, changes in the proton resonance of the OH-2 and OH-3 glucose units were observed . However, the presence of KOH minimized the weight loss (ca. 18%) of starch/15% SFO-based films, thus increasing their water tolerance  lost olerance a. This pThe presence of KOH allowed the significant decrease of the WVTR of the starch-based films b. The in1H NMR suggested the possible modification of the starch structure by the presence of chemically bounded SFO-derived fatty acids. Therefore, this work establishes an in situ single-step approach for the simultaneous hydrophobization and plasticization of potato starch-based films, offering a new valorization strategy for the potato chip industry byproducts. The results obtained with the casting process used paves the way for the application of starch/SFO/KOH formulations by the plastic industries in their extrusion processes.The incorporation of KOH during the starch/SFO-based films production revealed to be a promissory strategy for developing water tolerant biobased materials with enhanced stretchability and elasticity without changing their transparency, opening the possibility to extend the use of starch-based bioplastics to fields that include packaging applications. FTIR and