Aspergillus niger O1. The results showed the transglycosylation product panose was decreased by more than 88.0% in agdA/agdB double knock-out strains than strain O1. Additionally, the B-P1 domain of agdB was found accountable as starch hydrolysis activity only, and B-P1 overexpression in ΔAΔB-21 significantly increased glucoamylase activity whereas keeping the glucoamylase cocktail low transglycosylation activity. The total amounts of the transglycosylation products isomaltose and panose were significantly decreased in final strain B-P1-3 by 40.7% and 44.5%, respectively. The application of engineered strains will decrease the cost and add the value of product for starch biorefinery.Dextrose equivalent of glucose from starch hydrolysis is a critical index for starch-hydrolysis industry. Improving glucose yield and decreasing the non]-fermentable sugars which caused by transglycosylation activity of the enzymes during the starch saccharification is an important direction. In this study, we identified two key α-glucosidases responsible for producing non-fermentable sugars in an industrial glucoamylase-producing strain  Bacillus subtilis, Aspergillus niger and A. oryzae . Strain O1 and the transformants were cultured at 34°C on Czapek-Dox solid medium. The composition of Czapek-Dox solid medium (per 100 ml) was as follows: sucrose 3 g, NaNO3 0.2 g, MgSO4⋅7H2O 0.102 g, KCl 0.05 g, FeSO4⋅7H2O 0.00183 g, K2HPO4 0.1 g, agar 1.5 g, pH 6.0. The semisolid medium, which was used to prepare mycelia for fermentation, had the same composition with agar concentration 0.04% (w/v). The components of the shake-flask fermentation medium were 100 g/L glucose, 30 g/L soybean flour and 30 ml/L corn steep liquor, pH 5.6. Equal weights of mycelia were each inoculated into 50 ml fermentation medium in 250-ml Erlenmeyer flasks. Then, the shake flasks were cultured at 34°C with shaking at 240 rpm for 6 days. Samples of the fermentation broth were collected at 2, 4, and 6 days of cultures for extracellular protein, secretome, and glucoamylase activity analyses. For plasmid proliferation, Escherichia coli Tran1-T1 was cultured at 37°C in Luria-Bertani (LB) medium supplemented with kanamycin (50 mg/L) or ampicillin (100 mg/L). The components of LB medium were as follows: tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L.The agdA (An04g06920) and agdB (An01g10930), gene-deletion cassettes were constructed  to complete the construction of the OE plasmids.According to the protein domain information of agdB, the DNA sequences of B-P1, and B-P2 were amplified from the genome of A. niger was carried out as described previously with some modifications . Total RNA was synthesized to first strand cDNA using ReverTra Ace qPCR RT Kit . qRT-PCR was performed using SYBR Green Real-time PCR Master Mix . The primers used for detection of expression of α-glucosidases are listed . The actT method .Because extracellular protein level and total glucoamylase activity are highest in 6-day supernatant, the enzyme activity and secretome of 6-day supernatants were determined and analyzed. Glucoamylase activity was determined by a modified 3,5-dinitrosalicylic acid method using soluble starch as the substrate. Firstly, the fermentation supernatants were appropriately diluted using 50 mM sodium acetate buffer, pH 4.6. Then, 250 μL 1% (w/v) soluble starch preheated in a water bath at 62°C for 5 min was added to 250 μL diluted supernatant and mixed well. The mixture was incubated at 62°C for a further 10 min and then the reaction was terminated by adding 500 μL 3,5-dinitrosalicylic acid. The samples were boiled for 10 min and cooled on ice for 5 min before adding 1.5 ml distilled water. The glucoamylase activity was evaluated by calculating the amount of reducing sugars released from starch hydrolysis from the absorbance at 540 nm.2SO4 before filtering through 0.22 μm PES membrane . The filtered supernatant was analyzed by high-performance liquid chromatography (HPLC) with an e2695 instrument  equipped with a Waters 2414 refractive index detector and an Aminex HPX-87H column . The mobile phase was 10 mM H2SO4 with a constant flow rate of 0.5 ml/min.To decrease glucose production when maltose was used as the substrate in the transglycosylation activity assay, 0.75 g/L acarbose was used to inhibit the activity of amylolytic enzymes as described in previous work . Firstly2 and the mixture was incubated at 85°C for 2 h. The end point of starch liquefaction was identified using 0.05 M I2-KI standard solution. The reaction mixture was boiled for 10 min to inactivate the enzyme and then cooled to room temperature. Subsequently, the pH was adjusted to pH 4.4 to provide a favorable environment for glucoamylase activity. A 4.5-ml aliquot of the starch liquefaction product mixture was mixed with 300 μL fermentation supernatant (containing excessive amounts of enzymes) from O1 or each of the transformants. The mixture was then incubated at 62°C for 48 h. Ethyl alcohol was used to verify the end point of starch saccharification (when no precipitate was generated). Finally, the products of starch saccharification were analyzed by HPLC and the reducing sugars content was measured using an SGD-IV automatic reducing sugar detector  with Fehling’s solution.For starch liquefaction and saccharification, liquid α-amylase (kindly provided by Ms. Xiaoling Mu) was used for starch liquefaction and the supernatants produced by O1 and variety of transformants were used for starch saccharification. Before the experiment was conducted, corn starch was dried to constant weight at 60°C. First, 100 μL liquid α-amylase was added to a 30 ml slurry of 30% w/v corn starch  with 0.2% CaClAspergillus niger O1 and transformants were cultured in fermentation medium and samples were collected at 2, 4, and 6 days of cultures. The samples were centrifuged and the supernatants were filtered through 0.22 μm PES membrane (Millipore). The protein concentration in the culture supernatant was determined using a Bio-Rad protein assay kit according to the manufacturer’s instructions . Bovine serum albumin (BSA) was used as the standard, and the absorbance was measured at 595 nm. The supernatant of 6-day cultures was assayed by SDS-PAGE. The subsequent LC-MS/MS analyses and identification of proteins in the secretome were performed as described previously  and An01g10930 (agdB) were high in the transcription level analyses of 2-day, 4-day, 6-day cultures (agdB (encoded by An01g10930), the most abundant α-glucosidase in the secretome, and agdA (An04g06920), the highest transcript level of all the seven α-glucosidase genes, were selected as the target genes for decreasing transglycosylation activity.According to the results of previous studies, α-glucosidases generate transglycosylated products in bacteria and fungi . To idencultures     . The gluctively) .agdB single KO or agdA/agdB double KO were almost the same as that of O1 except for A2QAC1, for which no corresponding peptides were detected , B-P2 , and B-P3 . ΔAΔB-21 was selected as the original strain for domain-specific overexpression because it had the highest specific activity of glucoamylase. After protoplast transformation, transformants were acquired and identified by diagnostic PCR  were found to be the structural basis of transglycosylation activity, corresponding to the middle part of Glyco_hydro_31 domain in agdB  responsible for starch hydrolysis. Because glucoamylase activity and panose content were unaffected in the B-P3 OE strains, and on the basis of results and information in the literatures, we concluded that the Glyco_hydro_31 domain is an incomplete transglycosylation domain. The whole transglycosylation domain may contain all or part of the GH31_N domain, whole sequence of Glyco_hydro_31, all or some of the domain with unknown function at the C-terminal of agdB.Aspergillus niger strain O1 is an industrial glucoamylase-producing strain, but the α-glucosidases are also present in fermentation cultures affecting the purity of glucose, and the product of starch degradation. Therefore, two steps of starch liquefaction and saccharification were conducted under laboratory conditions using commercial α-amylase and the supernatants of strains O1, ΔAΔB-21, and transformants to assess the effect of genetic manipulation. After starch liquefaction and saccharification, the products were diluted and assayed by HPLC. As shown in AΔB-21 and B-P1-3 produced 0.4% and 0.9% more glucose, respectively  was the substrate with the inhibitor acarbose, the product was simple (panose is the sole transglycosylation product). However, when the mixture of starch liquefaction was used as the substrate, its composition was more complex, the concentration of maltose was lower than 20%, and no inhibitor was present in the reaction system. These differences may have led to difference products. In summary, overexpression of the B-P1 domain containing the NtCtMGAM_N domain increased the reducing sugars content, which enhanced the purity of glucose, thereby adding to its value.agdA and agdB have been identified responsible for transglycosylation in the industrial glucoamylase-producing A. niger strain O1. The transglycosylation product panose was decreased more than 88% in the agdA/agdB double KO strains than in O1. Additionally, agdB-P1 was identified as the specific domain responsible for starch hydrolysis activity in agdB. Overexpression of B-P1 domain of agdB in strain ΔAΔB-21 significantly increased glucoamylase activity. In products detection of starch liquefaction and saccharification, the transglycosylation products isomaltose and panose decreased significantly as compared with strain O1. The reducing sugars content of the B-P1-3 product increased accordingly. Together, the glucoamylase activity and the glucose content of B-P1 OE strain were increased, as well as the transglycosylated products were decreased. The application of the engineered strains will simplify the production process and decrease the cost of product, increase production of amylose and organic acids, and add the value of product for starch biorefinery.In this study, two α-glucosidase genes PXD036777 (http://www.ebi.ac.uk/pride/archive/projects/PXD 036777).The data presented in this study are deposited in the PRIDE repository , accessiWG contributed to the methodology, data curation, investigation, writing – original draft, and writing – review and editing. DL contributed to the methodology and investigation. JL contributed to the methodology. WS contributed to the project administration. TS contributed to the investigation. XW and KW contributed to the resources and methodology. QL contributed to the methodology, writing – review and editing, supervision, and funding acquisition. CT contributed to the methodology, funding acquisition, writing – review and editing, and supervision. All authors contributed to the article and approved the submitted version.


We describe the rapid diagnosis with point of care ultrasound (POCUS) of two acute pseudoaneurysms of a bovine arteriovenous dialysis graft with superimposed cellulitis in a 44-year old male patient who presented with pain over his upper arm graft site. POCUS evaluation decreased the time to diagnosis and vascular surgery consultation.  Point of care ultrasound (POCUS) using a linear transducer revealed two pseudoaneurysms  of the graft that were not initially identified on physical exam due to severe tenderness on exam and tissue edema. After informing the patient of this finding, he revealed that he had in fact noticed two painful bumps develop immediately after his last dialyses session. The graft was otherwise patent without thrombus in the lumen. Each pseudoaneurysm appeared as a 4mm and 5mm defect within the graft wall, which is referred to as the neck of the pseudoaneurysm. The neck led to a sac where blood could be seen swirling within the two similarly sized pseudoaneurysms on B-mode. Each measured approximately 1.5 x 3 x 3 cm. Color Doppler showed a pulsating jet entering the pseudoaneurysm from the neck . Additionally, cobblestoning of the subcutaneous tissue was observed directly adjacent to the graft at the proximal aspect, supporting the suspected underlying diagnosis of cellulitis . The patient was treated with vancomycin and cefazolin. POCUS prompted consultation of vascular surgery who removed the graft on day four of admission with a post-operative diagnosis of graft infection. Angiography with computed tomography was performed for preoperative planning . Surgical pathology showed myxoid degeneration of part of the graft wall. Tissue cultures from the graft site taken on the fourth day of antibiotic therapy were negative, including acid-fast bacilli, anerobic bacteria, and fungus. Gram stain was negative. Blood cultures drawn prior to antibiotic administration were negative. Transthoracic echocardiogram showed no valvular vegetations. The patient was discharged after eight days of hospital admission on cefazolin with a tunneled dialysis catheter. A pseudoaneurysm is sometimes called a false aneurysm because it lacks all three normal elements of the arterial wall. The intima and media are disrupted and the adventitia acts as the outer layer of the pseudoaneurysm, which is why it is sometimes referred to as a contained rupture. The predominate risk is complete rupture, leading to life threatening bleeding or secondary infection. They are often caused by needle puncture and frequently associated with infection The patient consented for use of ultrasound images and clinical information for academic purposes. The authors have no conflicts of interest to declare. Video S1 Video S2


The present study tested and compared the biomechanical properties of four different triplicate graft tendon techniques.32 tripled tendons from the common extensor muscle of bovine fingers were tested on a material testing machine, passing the end loop over a metal rod of a clevis connected to the load cell on the upper side, and fixing the lower end to a clamp. The samples were divided into four groups: (A) tripled with a free end sutured only to one of the two fixed bundles (B) tripled with a free end positioned between the two fixed strands and sutured to both (C) tripled with an S-shape and all the three strands sutured together at the upper and lower extremities of the graft (D) partially quadrupled with the free end sutured together with the other three bundles at the upper extremity. Each sample was pretensioned at 50 N for 10 min and then subjected to 1000 load control cycles between 50 and 250 N. Finally, each sample was subjected to a load to failure test.Authors also present some preliminary results on the feasibility of a non-contact and full-field Thermoelastic Stress Analysis technique, based on Infrared Thermography, to evaluate the level of stress on the whole graft, and hence on each strand, during fatigue loading.Eighty five percent of the samples failed at the level of the clamp. The cyclical elongation progressively decreased in all the samples and there was a simultaneous increase in stiffness. An increased stiffness was noted between Group 2 vs Group 3 and Group 2 vs Group 4 at the 500th and 1000th cycle. The failure loads were as follows: (a) 569.10 N, (b) 632.28 N, (c) 571.68 N, (d) 616.95 N. None of the parameters showed a statistically significant difference between the four groups.This study reported similar biomechanical behavior of four different models of tripled grafts suitable for ACL reconstruction. In addition, the biomechanics of overall tripled tendon grafts seems more affected by the viscoelastic property of the tendon itself rather than the preparation method. Anterior Cruciate Ligament (ACL) Reconstruction is the gold standard for patients who develop knee instability after ACL rupture. Over the years, surgical reconstruction techniques have changed with technological progress and a deeper knowledge of graft biomechanics . FurtherThe assumed of iso-stress condition among the strands has yet to be proved and little work is available about the influence of a number of external factors that can influence the ability of strands to sustain loads. These may include the ability of sutures to transfer and distribute loads uniformly among the strands, the initial pretension of each strand, and the role of the fixing and anchoring sites on the loading transfer to the strands .The purpose of our study was to compare the biomechanical properties of four different methods of suture fixation to prepare tripled tendon grafts when a rigid suspensory fixation device is used. We hypothesized that the method of preparation of tripled tendon grafts does not affect the load to failure and displacement properties.The work also presents some preliminary results of the use of Thermoelastic Stress Analysis  (TSA) toThe present study and the experimental protocol were approved and performed in accordance with the author’s Institution guidelines and regulation for the use of experimental animal tissue. Furthermore, the study was authorized by the local ethical committee (Department of Orthopaedic Surgery—DiChirOnS). Fresh-frozen bovine common digital extensor tendons were harvested from front legs of 8 mature bovine aging from 18 to 24 months . TendonsContinuous saline graft irrigation using syringes was performed throughout the preparation and mechanical testing to prevent drying. For the tests, an electro-mechanic two-columns universal testing machine (Instron 3367), equipped with a 30 KN load cell , was used. Since the experiments carried out in this work required load comprised between 50 and 700 N, the accuracy of the load cell was verified to be within ± 0.5% of the reading in the above range, which is considered sufficient for the evaluations made in this work.The final tendon configuration see Fig.  was obtaAll couplings between the various parts where axially tightened by threaded flanges, so as to eliminate any axial clearance. The lower end of the samples was fixed by a Zwick Roell wedge-screw grip, clamping the samples for a length of 2 cm. The lower grip was firmly connected to the test machine rig, again eliminating any possible axial clearance. In this way, the crosshead displacement during the test could account only for the grafts stretching and/or slippage on the lower clamp.Grafts were fixed with a distance of 70 mm from the clamp to the rod, to simulate the femoral tunnel length (40 mm) and intra-articular space of the ACL (30 mm) that could be obtained with more recent femoral fixation devices Fig. .Each tendon was tripled following four different methods of folding and suturing the strands, creating four different groups of eight grafts. These techniques of graft tripling were chosen after a literature review of all tripling graft techniques described so far.The tendon was looped around the rod with the free end positioned between the two fixed strands. The free end was sutured only to one of the two fixed bundles Fig. a.Fig. 2IThe tendon was looped around the rod with the free end positioned between the two fixed strands and sutured to both Fig. b.The tendon was looped around the rod with the free end positioned externally to the two fixed strands to form an inverted “S” configuration. All the three strands were sutured together at the upper and lower extremities of the graft Fig. c.Tendon was looped twice around the rod creating a partially quadruplicated graft with the free end sutured together with the other three bundles at the upper extremity Fig. d.Non-absorbable sutures  were used for graft preparation. Each suture was passed under a slight tensile load of the graft, to avoid permanent graft elongation, due to the slippage of the wire over the tendon tissue during loads , 28. TheStatic pre-conditioning by holding the tendon at a stable tensile load of 50 N for 10 minCyclic loading for 1000 cycles, between 50 and 250 N, with a triangle wave applied at 1 Hz;Final monotonic tensile loading up to failure, performed in displacement control at a machine crosshead speed of 1 mm/s.A specific loading protocol was set up, by exploiting the Instron Blue Hill v 2.0 remote control software of the testing machine. The loading protocol set in this study comprised three successive stages Fig. :Static pThe pre-conditioning stage was performed to stabilize the graft’s mechanical properties , 29, 30.The amplitude of graft elongation during a peak-to-peak fatigue cycle was determined, given by the difference in crosshead displacement between the load peak and the valley. For comparison purposes among the different lots of samples, the amplitude was measured at three stages of the cyclic loading window, and specifically: at the first applied fatigue cycle [L1], at the 500th cycle [L500] and at the last cycle [L1000].The graft slippage [L4] .The cyclic elongation [D0-500], defined as the difference in crosshead displacement between the condition at the end of the 50-N static pre-conditioning hold and the condition at the max applied load in the 500th fatigue cycle.The final elongation [D0-1000] .The initial stiffness [K10], i.e. the slope of the secant line joining minimum and maximum points of the loading wave in the load–displacement curve, measured at the 10th cycle).Cyclic stiffness at 500th cycle [K500] (as described previously but at the 500th cycle).Pull-out stiffness [KL], i.e. the initial slope of the load–displacement curve at the final monotonic loading. The initial slope corresponds to the steepest straight-line tangent to the curve.Ultimate failure load [Fr], i.e. the peak force of the final load–elongation curve.For each specimen, load–displacement curves were recorded and analyzed to determine specific parameters. Specifically:The mechanism of final static failure for each specimen was also observed and recorded.Thermoelastic Stress Analysis (TSA) is a non-contact and full-field technique, based on the measurement of temperature by means of an Infrared Thermal camera, able to evaluate the level of elastic straining on solid matter when stressed under adiabatic conditions , 21. In Thermoelastic Stress Analysis (TSA) exploits the thermoelastic effect observed in solid matter, according to which a solid structure, subject to elastic deformation, undergoes a volume change that produces a temperature change proportional to the stress metric that, for linear isotropic materials, coincides with the first stress invariant, i.e., the sum of the normal stresses. One peculiar feature of the thermoelastic effect is that it gives rise to a reversible temperature change. When a load is applied via a cyclic wave, this produces a temperature change that is modulated at the same frequency of the applied load. Therefore, the temperature changes filtered form the harmonic frequency content of temperature provides the thermoelastic signal, which carries the stress information needed.The experimental implementation of TSA was performed in a couples of graft samples, using a high thermal resolution FLIR X6540sc IR camera, set with an integration time of 3000 ms, and acquiring thermograms during cyclic loading at a sampling framerate of 50 Hz and for a sampling time windows of 10 s. The thermoelastic acquisitions were randomly taken during the cycling stage of the tendons testing, and therefore the applied load consisted of a triangular wave at a frequency of 1 Hz. The temperature changes at the same frequency of the applied load (1 Hz) were measured from the acquired temperature by means a Discrete Fourier Transform-based signal post-processing. This allowed to filter all the temperature harmonics that had frequencies different from the load frequency. The central portions of all three strands of the graft should develop a uniform uniaxial stress change during load cycling, which is considered proportional to the measured thermoelastic signal. Therefore, TSA has the potential to reveal if and how the stress is redistributed among the three strands, by comparing the thermoelastic signal maps obtained. Furthermore, it is specified that no hydration was applied immediately before and during the few seconds of temperature measurement by the IR camera, since water behaves as a black body making the infrared signal coming from the tendons. Even so, a small degree of humidity, as left by initial hydration, at the start of the cyclic testing, was found to have no disturbing effects on the ability to perform TSA. Hydration may also cause the sample to undergo some slow temperature changes, such us cooling due to evaporation. These temperature changes, irreversible in nature, would not influence the thermoelastic signal since they are slow and not modulated at the load frequency, and therefore filtered out during the TSA signal processing.α = 0.05. Further, each parameter  was compared between groups using an independent t test, with a significant level placed at p < 0.05.Data were analyzed using SPSS statistical software, version 18.0 . Means and standard deviations were calculated for all parameters of each group. Grafts tensile properties between the four graft groups were analyzed using the ANOVA method with a level of significance at Each tripled graft was 9 cm long while the mean length of samples for each group from the rod to the clamp was 7.06 cm in Group I, 7 cm in Group II 6.87 cm in Group III, and 7 cm in Group IV. No differences in terms of graft diameter were observed among the 4 groups (> 0.05). All specimens completed phase 1 and phase 2 of the loading protocol  and all graft failed during the final monotonic tensile load stage. In Group I, all samples failed at the level of the clamp; in Group II, rupture occurred at the level of the clamp in 75% of grafts and in the middle of the bundles in 25% of them.; in Group III and IV, 87.5% of the samples failed at the level of the clamp and in 12.5% failed in the middle of the grafts.p = 0.013) and Group II vs Group IV . Further, a difference was observed at the 1000th cycle between Group II vs Group III  and Group II vs Group IV . No differences were noted between the four groups concerning all other parameters evaluated during cyclic loading (p > 0.05). Furthermore, no differences were noted between the four groups concerning pull-out stiffness and ultimate failure load (p > 0.05). The amplitude of graft elongation has gradually decreased simultaneously and consistently with the increase of the stiffness. All data and their related p values concerning statistical significance are represented in Table Concerning stiffness, a difference was observed at the 500th cycle between Group II vs Group III , the analyzed area comprised of the whole strand and part of the upper and lower connections to the testing machine. Figure In particular, area Ar1 is taken across a horizontal border of the upper clevis, area Ar2 is taken on the central gage portion of one of the three strands, and area Ar3 is taken near the clamped lower end of the same strand. The area Ar1 is in part covered by the background and in part by the sidewall of the clevis. Since the clevis is warmer than the background, the signal acquired from Ar1 is modulated due to the stretching of the tendon. In particular, while the load is applied, a different amount of background and clevis wall is found to fill Ar1, and therefore, the average temperature versus time is an exact reproduction of the loading wave. When the wave has a peak temperature, this corresponds to the lower position of the clevis, i.e., lower load, while a trough temperature is achieved when the clevis is in its upper position, at higher load. The plots from areas Ar2 and Ar3 show that the temperature of the tendon is modulated according to the load, which is an unmistakable indication that a thermoelastic reversible temperature change component is present in the temperature signal. It is interesting to observe that the temperature change is in the same phase as the load in Ar2 and in the opposite phase  in Ar3.Figure The number of processed frames was modified to find the conditions for minimum spectral leakage at the expected frequency of the thermoelastic signal . After tFigures The main finding of this study is that the biomechanical behavior of 4 different models of tripled grafts suitable for ACL reconstruction have been shown to be similar. Furthermore, we proposed an original implementation of an experimental stress analysis technique to evaluate the mechanical stress undergone by each strand of the grafts.In our study, eight samples were tested for each group and this number is in line with current literature , 17, 30.Regarding the failure mechanism of the graft, in the study of Yoo et al.  and FabbIn agreement with Fabbri et al. , stiffneT) at the load frequency on a fatigue-loaded graft. The linear relationship describing the correlation between the temperature change at the load frequency and the stress change, for linear elastic and isotropic solid matter, can be written as follows:The implementation of TSA reported in this work is an attempt to evaluate the contribution of each tendon strand to resist to external loads. To our knowledge, no previous studies have tried to measure the thermoelastic effect induced temperature changes in tendons. For this reason, a Thermoelastic Stress Analysis technique was implemented, evaluating strands temperature change  “To” is the initial body temperature, “k” a material constant and the stress term is the range of variation of the sum of in-plane normal stress components, i.e., the first stress invariant [where Δnvariant , 20.In the present work, it was found that at the mid-length of the graft the left strand (sutured free-strand) has a significantly higher thermoelastic signal than the other two strands Fig. a. This iEven if we tried to reproduce a scenario as similar as possible to the human one, this is only a laboratory biomechanical study and the comparison to the clinical practice could not be so immediate. First of all, we used bovine tendons for the mechanical simulations, however, some studies have shown a behavior similar to human tissue . We usedThis study reported similar biomechanical behavior of four different models of tripled grafts suitable for ACL reconstruction. The biomechanics of overall tripled tendon grafts seems more affected by the viscoelastic property of the tendon itself rather than the preparation method. Further, Thermoelastic Stress Analysis could represent an alternative technique to evaluate the mechanical stress experienced by each strand of the grafts during loads.


This retrospective study describes the reconstruction of 18 nasal defects with chondrocutaneous (composite) grafts. Composite grafts are versatile one-stage options for defects ≤2.5 cm at the lower third of the nose and are particularly useful in reconstruction of small full-thickness defects and superficial defects bordering or involving the alar rim. Reconstruction of nasal defects involving the nasal alar or soft triangle can be challenging due to the complex three-dimensional anatomy of the nose with high functional and esthetical demands. Soft-tissue reconstruction without a structural component can lead to collapse of the nostril, causing stenosis. This lack of structural support can also cause a cephalad retraction of the alar rim resulting in an aesthetically unpleasing outcome, and uncomfortable exposure of nasal mucosa. These challenges can be managed with a composite graft, consisting of cartilage, subcutaneous fat, and the overlying skin, providing stability from the cartilage and the skin needed to cover the defect, for both superficial and full-thickness nasal defects. Using a composite graft from the ear provides tissue with a 3 D-structure, color, texture, and thickness matching the nose to manage both the aesthetic and functional demands. This cartilage provides stability to not only avoid alar retraction but also ensure a patent airway. The auricular donor site allows for the graft to be harvested with skin on both sides if inner lining is needed for full thickness defects. Furthermore, it provides a well-tolerated donor site away from the midface.Composite grafts were first described more than a century ago . Their uAt our unit, the use of composite grafts has increased since 2017 and this case series will present the lessons we have learnt. We describe our series of patients with nasal defects bordering or involving the alar rim managed either primary after surgical resection or secondary to correct alar retraction or stenosis with composite grafts harvested from the auricle.This study is a retrospective study of eighteen consecutive patients who had nasal reconstruction performed with composite grafts at the Department of Plastic Surgery at Herlev and Gentofte Hospital, Copenhagen University Denmark, from July 2017 to February 2022. Data were extracted from the electronical recorded medical notes (Epic Systems Corporation) and clinical photographs. This study was undertaken as a quality control, with the patients’ written informed content, and approved by the hospital administration and no ethical approvement was therefore necessary.The surgical procedure was generally performed as described: The recipient site was cleared of cancer or prepared for the corrective procedure. Careful hemostasis was achieved, limiting the use of cautery to avoid closing the dermal and subcutaneous vessels necessary for graft take. The cartilage graft was then marked on the donor site with the width of the defect at the alar rim 1:1 adding another approx. three to five mm of cartilage at each end as shown with the solid yellow lines in Postoperative restrictions were advised until the dressing was removed (after 7 days) to avoid haematoma. If there was any concern about graft take, or epidermolysis, the patient was seen regularly until the graft had taken or re-epithelialized. Depending on the preferences of the surgeon, prophylactic antibiotics were prescribed. Some surgeons advocated for postoperative cooling of the graft (such as 10–15 min/hour for the first 24 h). All patients were followed closely, and if the patient and surgeon were satisfied with the outcome at 3 months follow-up, most were transferred for follow-up at their general practitioner or dermatologist.All results are descriptive and shown as number (%) and mean (range).Eighteen Caucasian patients, who had composite grafts performed by either of the authors or five additional colleagues, were included . Most caAll cases were reviewed by first and last authors and categorized as whether a composite graft would have been chosen with our current knowledge, which was the case for all patients (100%). The outcomes were categorized as poor, acceptable, or good in four (22%), five (28%), and nine (50%) cases, respectively.A composite graft for nasal reconstruction is a one-stage procedure which can be performed under local anesthesia as an out-patient procedure. Most cases in our series were secondary to cancer. As the surrounding tissue is left untouched, it leads to safe and easy cancer surveillance. If local recurrence is diagnosed; local tissue has been left uncompromised and is available for further reconstruction. In other parts of the world, the use of composite grafts has been described more often for reconstructions of nasal deformities due to burn injury and congenital defects ,5 These An alternative to excision of carcinomas of the nose and composite graft reconstruction is radiotherapy. Despite an often excellent functional and cosmetic outcome after radiation therapy, the treatment is time consuming, lengthy, and may cause long-term nasal crusting or dryness, postinflammatory hypo- or hyperpigmentation, atrophy, fibrotic tissue, alar retraction, and telangiectasia covering an area larger than the tumor site . In a raComposite grafts are dependent on revascularization and bridging phenomenon over the cartilage for survival. It is therefore paramount for graft take that a vascularized wound bed is available for capillary ingrowth and that the graft is immobilized to avoid detrimental shearing forces causing graft failure. The former can be ensured by enlarging the surrounding superficial defect and to avoid excessive cautery to the wound bed. The maximum graft width in our series were just over two cm. As illustrated by the largest two defects in our series, a maximum distance of approx. 1 cm from a vascular source can be applied. This is in accordance with one of the most quoted measurements for the maximum size of a graft by Ruch stating that ‘no part of the graft should be much more than 1 cm from a free edge’ ,11. OtheIn smaller defects, where most of the skin graft overlies the cartilage, which acts as a barrier for imbibition, inosculation, and revascularization of the graft, the graft take, which is mostly at the margins of the defect, can therefore be more challenging than with larger grafts. In these cases, harvesting or leaving as little cartilage under the skin as possible, without losing its stabilizing properties, might enhance graft take as this would provide more surface area for revascularization. The thicker the graft, the higher the metabolic demand. As the subcutaneous tissue between the skin and cartilage can only be trimmed minimally not to affect the blood supply of the graft, choosing a donor site with the least subcutaneous tissue might improve graft take. In our series, most grafts were harvested from the root of helix where the subcutaneous tissue usually is only a few mm thick. If the defect is superficial and no inner lining is needed, the postauricular donor site is also an option. This site was used in one of our cases. A potential drawback of a composite graft is the long-term shrinkage or atrophy of the graft. In our series, the longest follow-up was 22 months. While no significant shrinkage or atrophy of the graft was noted during this period, our follow-up period does not allow for long-term assessment of the technique.The literature on risk factors associated with composite grafts is scarce. In a study by Wang et al. assessing the outcome of reconstruction using flaps or grafts, former and current smokers, increased defect size, and free cartilage grafts were found to be associated with an increased rate of acute complications defined as any postsurgical infection, dehiscence, hematoma, uncontrolled bleeding, and tissue necrosis that required medical counselling or intervention . In thatNone of our patients had received radiotherapy to the operating field. Using a composite graft in a radiated field may be associated with a higher risk of graft failure. It does, however, still have a place as the surrounding tissue is left untouched and if failure occurs, secondary procedures such as a forehead flap can be performed at no worse state. Others authors have, however, used composite grafts successfully following irradiation. . The onlMost of the grafts in this series were harvested from the root of the helix. Other parts of the ear, such as the antitragus or the posterior aspect of the ear, can also be used ,19. The The grafts were made to fit the defect, which, was the right choice in most cases. In some cases, however, alar retraction was noted. This could be due to contracture of the graft, loss of tissue or insufficient stability of the cartilage. To address the former, the grafts could be designed slightly larger to accommodate the contracture. This might in some cases lead to bulkiness and thus a need for revision surgery. The instability of the cartilage caused by a mechanical pull or warping of the cartilage might also be alleviated by using a larger and more resistant cartilage graft . RevisioWith the increase of the older population, and the increase in non-melanoma skin cancer (NMSC) worldwide, particularly in the Caucasian populations, NMSC predilection for the nose and the decrease in available resources, composite grafts should be in the reconstructive surgeon’s toolbox as it offers an out-patient single-stage technique with excellent outcomes ,21,22.The technique can be performed by even less experienced surgeons, as illustrated by our series, where 2 (of 9) of the surgeons performed the reconstructions while in-training. We did, however, notice potential improvable outcomes in cases performed by surgeons with low number of cases, which was particularly evident for less experienced surgeons, which may be explained by the complexity of the procedure requiring more experienced surgeons, or simply a matter of a learning curve. Our eighteen cases were performed by nine different surgeons. This likely reflects the outcomes. With increase case numbers and experience the outcomes are anticipated to improve. All cases were reviewed by first and last authors and categorized as whether a composite graft would have been chosen, with our current knowledge, which was the case for all patients (100%). With our current knowledge, however, we would have made a few minor adjustments to our technique in some of the cases as described above.The overall outcomes were categorized as poor, acceptable, or good in four (22%), five 28%), and nine (50%) cases, respectively. The four poor results were caused by partial or complete tissue loss, of which three patients declined revision surgery  and 9, a%, and niIn conclusion, many defects near or involving the alar rim, both superficial and full thickness, can be managed successfully with composite grafts, harvested from the auricle. In our experience, composite grafts are versatile and can be used for reconstruction of the lower third of the nose after oncological resection, and to correct collapse of the nostril and alar retraction. Thorough analysis of the location and the dimensions of the defect is crucial. Composite grafts are an excellent method of reconstruction to master for reconstructive surgeons; they are one-stage procedures with no additional midface scars, and the procedure can be carried out under local anaesthesia and is well tolerated by patients. This especially applies to defects at the alar rim or close to the alar rim. With through and through defects there are other good alternatives for reconstruction. These, however, often require multiple procedures, often under sedation or general anaesthesia. Many older patients as in our case series, need and/or prefer a simple immediate reconstruction, which can be performed in the outpatient clinic under local anaesthesia. Composite graft for alar reconstruction is thus a method that should be considered in selected cases. As with all reconstructions the choice of patient/defect is crucial when choosing the method of reconstruction.


Metastasis of breast cancer have caused the majority of cancer-related death worldwide. The circRNAs are associated with tumorigenesis and metastasis in breast cancer according to recent research. However, the biological mechanism of circRNAs in liver metastatic breast cancer remains ambiguous yet.Microarray analysis of three pairs of primary BC tissues and matched hepatic metastatic specimens identified circEZH2. We used RT-qPCR and FISH assays to confirm circEZH2 existence, characteristics, and expression. Both in vivo and in vitro, circEZH2 played an oncogenic role which promoted metastasis as well. A range of bioinformatic analysis, Western blot, RNA pull-down, RIP, ChIP, and animal experiments were used to define the feedback loop involving FUS, circEZH2, miR-217-5p, KLF5, FUS, CXCR4 as well as epithelial and mesenchymal transition.In our research, circEZH2 was proved to be upregulated in liver metastases in BC and predicted the worse prognosis in breast cancer patients. Overexpression of circEZH2 notably accentuated the vitality and invasion of BC cells, whereas knockdown of circEZH2 elicited the literally opposite effects. Besides, overexpressed circEZH2 promoted tumorigenesis and liver metastasis in vivo. Moreover, circEZH2 could adsorb miR-217-5p to upregulate KLF5 thus leading to activate FUS transcription which would facilitate the back-splicing program of circEZH2. Meanwhile, KLF5 could upregulated CXCR4 transcriptionally to accelerate epithelial and mesenchymal transition of breast cancer.Consequently, a novel feedback loop FUS/circEZH2/KLF5/CXCR4 was established while circEZH2 could be novel biomarker and potential target for BC patients’ therapy.The online version contains supplementary material available at 10.1186/s12943-022-01653-2. Breast cancer (BC) has been the most prevalent cancer in the population of women which has been the capital reason for cancer-relative female mortality globally. According to the recent research, the overall survival rate for primary BC in 5 years has been nearly 90%. Nevertheless, 33% of BC patients appear non-nodal distant metastases that shorten overall survival rate of 5 years down to 23% . ClinicaOnly 2% of the cellular transcriptome is transcribed into mRNAs, while the majority is noncoding RNAs . CircRNAIn our article, a new circRNA hsa_circ_0008324 (circEZH2) was verified via microarray which was upregulated significantly in BCLM and predicted poor survival of BC patients. CircEZH2 enhanced tumorigenesis and metastasis in vitro and vivo by upregulating KLF5 protein expression through sponging with miR-217-5p, while the transcriptional activation of FUS by KLF5 promoted the process of back-splicing of circEZH2. Eventually, KLF5 activated CXCR4 transcription to induce EMT in BC.P value < 0.005 (2) fold-change ≥2 (3) recorded in circBase, deepbase and Rybak-Wolf 2015 dataset (4) Gene symbol was metastatic related according to recent research (5) detected and upregulated in clinical BCLM specimens compared to BC primary samples, circEZH2 (hsa_circ_0008324) was identified among all the upregulated circRNAs   P value In order to investigate circEZH2’s potential functions in BC, we constructed a circEZH2-overexpression plasmid and two small interfering RNAs (siRNAs) Fig. : Fig. 1aIn order to identify the effects of circEZH2 on tumorigenesis of BC tumors in vivo, human-derived BC xenograft nude mice models were constructed via subcutaneously injection by MCF-7 cells and BT-549 cells overexpressing circEZH2 stably, and corresponding control cells in the BALB/C nude mice, while the circEZH2-overexpressed tumors were significantly larger and weighed higher than their relevant control tumors Fig. a-c. To sAccording to circInteractome , there wTo further identify biological mechanism of circEZH2, bioinformatic analysis based on miRanda  and circIn order to uncover the deep interplay mechanism by which circEZH2/miR-217-5p axis worked, we performed bioinformatics analysis among miRMap , miRDB , TargetSAccording to the RT-qPCR analysis of clinical BC samples from SYSUCC, FUS expression had notably positive correlation with KLF5 in mRNA level Fig. l. When wAccording to the STARBASE, we found that KLF5 was positive correlated with CXCR4 in RNA expression  in Guangzhou, China which were conducted by RNA later instantly after gathered. We obtained all samples from patients with informed consent under institutional review board consented protocols.There are six human BC cell lines, MDA-MB-231, BT-549, T-47D, MCF-7, and SKBR-3 and the breast normal epithelial cell line, MCF-10A with HEK293T used in this study . A selection of cell lines, including MDA-MB-231, SKBR-3, BT-549, T-47D, MCF-7, HEK293T, and SKBR-3, were all grown in DMEM medium , while MCF-10A cells were grown in DMEM/F-12 medium . The media mentioned above were replenished with 10% fetal bovine serum  and 50 U/ml streptomycin and penicillin . This study used a humidified incubator  to culture all the cells at 37 °C in 5% CO2.6 in PBS) with stably overexpressed circEZH2 or control cells into the mice that were sacrificed 4 weeks after injection. The volume of tumors was measured weekly and analyzed in line with the standard formula: volume = length x width2 / 2, while the weight of tumors was measured after the sacrifice of the mice. In the mice liver metastasis experiments, 1 × 106 MCF-7 or BT-549 cells overexpressing circEZH2 steadily, as well as luciferase, were given injection into the spleen of four- to five-week-old BALB/c female mice, and control cells were given same injection meanwhile. After 60 days, we performed bioluminescence by injecting 100 μl 15 μg/μl D-luciferin intraperitoneally into the mice and captured images by Xenogen IVIS Lumina Series II and processed the results via Living Image 2.11 software (Xenogen Corp.). After that, livers of the mice were removed as soon as they were sacrificed for further pathological research. For xenograft-induced metastatic mice model, firstly, BT-549 cells (2 × 106 in PBS) were injectied into mammary pad of BALB/C nude mice. Secondly, when the short diameter approach 2 cm, tumorectomy was performed to remove the xenograft tumor. After 8 weeks, bioluminescence was performed to observe the status of metastasis. Once metastases were noticed, the mice were sacrificed to separate the multi-organ metastases to extract RNA and protein for further analysis. Sun Yat-Sen University’s Animal Care and Use Committee has approved all the procedures above.Female BALB/C nude mice were obtained from Vital River Laboratory  and fostered in the Center for Experimental Animals of Sun Yat-Sen University under standard conditions. For xenograft proliferation mice models, we subcutaneously injected MCF-7 and BT-549 cells  and GraphPad Prism 9.1  were used for statistical analysis. For detecting the significances between groups, we used the student’s t test, while the Spearman correlation coefficient was used to determine the correlation between groups. The survival analysis was conducted by Kaplan-Meier method, while the statistical significance was determined by the log-rank test.Metastasis is the major cause of BC-related death globally . And theResearch has shown that a variety of regulatory factors are involved in the biogenesis of circRNAs from their precursor mRNA, like transcription factor Twist1 , variablThe function of circRNAs is highly related with its location within the cell. In our article, circEZH2 was proved to be distributed mostly in the cytoplasm and bioinformatic analysis, RNA pull-down assay showed that circEZH2 could bind to miRNA hsa-miR-217-5p. Various types of cancer have been validated to contain miR-217-5p as an anti-tumor miRNA –45. In oKnown transcription factor KLF5, it has been found to be overexpressed in basal-subtype BC . In BC, While BC primary tumors are cured via surgical resection and systematic chemotherapy, cancer with metastasis is mostly incurable or untreatable because currently available therapeutic drugs are ineffective against disseminated BC tumor cells . HoweverA novel circRNA named circEZH2 was identified to promote BC oncogenesis and liver metastasis in BC while BCLM exhibited an upregulation of circEZH2 which correlated with worse prognosis. Furthermore, we found that circEZH2 could reverse KLF5 post-transcriptional inhibition by sponging miR-217-5p which could accelerate CXCR4-induced EMT of BC. Moreover, FUS which was able to help back-splicing process of circEZH2 could be upregulated transcriptionally via KLF5 thus leading to FUS/circEZH2/KLF5/CXCR4 positive feedback loop, which potentially serving as a biomarker for BC metastasis and a target for biological BC therapy.Additional file 1. Supplemental Figure 1Additional file 2: Table S1. Sequences of siRNAs used in this studyAdditional file 3: Table S2. Primer sequences used in RT-qPCR and PCR analysisAdditional file 4. Supplemental Method


Philosophical Transactions B following the end of the Editorship term of Professor John Pickett CBE, FRS, with the last issue of 2022. I have known John for many years, and his infectious engagement with all things scientific very much personifies the interdisciplinary focus of the journal. He will be a hard act to follow, although I suspect we will be in touch a lot!I am excited to assume the role of Editor of Phil Trans B made me realize that working with this journal would be a very different experience from handling manuscripts for most specialist journals, or even a general interest journal such as PNAS, on whose board I currently serve. There is much to be said for learning new things as you get older, and the broad, interdisciplinary scope of Phil Trans B will certainly make sure that happens.After taking partial retirement from the University of North Texas in October 2021, I felt that I had the time to be involved in more editorial work. But for what journal? My three years as Editorial Board member for https://bdi.unt.edu/) in 2015. I am now Professor Emeritus at UNT, maintaining a much smaller laboratory mainly remotely from my home in Tulsa, Oklahoma.For those who do not know me, just a little about my background. I am a plant biochemist who graduated from Oxford, did two years postdoctoral work in Cambridge, and then spent 9 years on the faculty in Biochemistry at Royal Holloway College, University of London. In 1988 I threw caution to the wind and assumed the position of Director of Plant Biology at the Noble Foundation, a private research institute in Ardmore, Oklahoma, dedicated to promoting good agricultural practices. This turned out to be a very good decision, and for 25 years the Foundation was a wonderful place for both basic and applied plant research. It was there that I saw first-hand the power of interdisciplinary research to translate basic science to benefits for the ultimate end users, the farmers and ranchers who were supported by the Foundation's Agricultural Division. In 2013 I left the Foundation to become Distinguished Research Professor in the Department of Biological Sciences at the University of North Texas, joining a group of excellent plant scientists and founding the BioDiscovery Institute (Phil. Trans. B.My research interests centre on the biochemistry, molecular biology and metabolic engineering of plant natural product pathways and their implications for agriculture and human health, and the engineering of lignocellulosic biomass for the improvement of forage and bioenergy feedstocks. Over the years, I have worked in several large interdisciplinary research teams addressing questions from overcoming biomass recalcitrance for processing to fuels and chemicals to understanding metabolic conversions of dietary plant flavonoids in relation to amelioration of cognitive dysfunction. I have enjoyed and appreciated how collaborations beyond one's own area of expertise or even interest can result in gains in knowledge and understanding that would not otherwise occur, and such synergy is clearly a major feature of Phil. Trans. B. was an article I co-authored in 1986 in the proceedings of a discussion meeting on differential gene expression in plants. Just being there at the Royal Society with many of the leaders of the field was an amazing experience for a new faculty member. Re-reading the article now makes apparent the incredible advances in technology in the biological sciences over the past 30–40 years. Back in 1986 we were asking the right questions but had limited tools to help us answer them. Now we seem to have a different problem—the age of ‘big data' has made it possible to generate, both rapidly and cheaply, far more information than one could have dreamed of back in the day. In some cases this has led to spectacular advances, but it can also lead some into blind alleys in which the wood is not seen for the trees. I very much like the focus of Phil. Trans. B on questions of deep biological significance.My first and only experience in publishing in Phil. Trans. B is clearly seen as the premier place for review articles and themed issues by a number of communities and has an enthusiastic user/reader base in these areas that should be maintained. I do not have a specific agenda for changes in direction, other to than to ensure that we actively encourage issues across the whole spectrum of interest areas covered by the journal, including the climate : agriculture interface mentioned by Professor Pickett in his concluding editorial.My philosophy for the journal is simple—to maintain the high standards of quality, thematic diversity, author/editor diversity, and international collaboration that the journal currently enjoys. Phil. Trans. B. International collaborations are suffering in some cases from heightened suspicion of scientists' intentions from their representative governments. More isolationist policies in many countries have reduced funding for international collaborations. These issues highlight the need for the journal to maintain, or increase, the diversity of nationalities in its thematic issues; even if transfer of materials and personnel across international boundaries is undergoing a reduction, transfer of ideas and synthesis of new concepts among the broader scientific community should be free and receive continued encouragement. Furthermore, as the climate crisis and changing political and social norms affect, in different ways and extents, countries and societies across the world, the journal should continue to encourage scientists from under-represented countries to participate in the themed issues.The present uncertain political climate further highlights the value of a journal such as I am looking forward to working with the excellent Editorial Team at the Royal Society and also to taking the opportunity of getting back to visit my home country after the restrictions of the Covid pandemic. I urge readers to come forward with suggestions for theme issues across the whole subject area of the journal—we are always looking for new topics and all scientists are welcome to submit a proposal. Please visit the journal's website for more information.


The effectiveness of liver transplantation to cure numerous diseases, alleviate suffering, and improve patient survival has led to an ever increasing demand. Improvements in preoperative management, surgical technique, and postoperative care have allowed increasingly complicated and high-risk patients to be safely transplanted. As a result, many patients are safely transplanted in the modern era that would have been considered untransplantable in times gone by. Despite this, more gains are possible as the science behind transplantation is increasingly understood. Normothermic machine perfusion of liver grafts builds on these gains further by increasing the safe use of grafts with suboptimal features, through objective assessment of both hepatocyte and cholangiocyte function. This technology can minimize cold ischemia, but prolong total preservation time, with particular benefits for suboptimal grafts and surgically challenging recipients. In addition to more physiological and favorable preservation conditions for grafts with risk factors for poor outcome, the extended preservation time benefits operative logistics by allowing a careful explant and complicated vascular reconstruction when presented with challenging surgical scenarios. This technology represents a significant advancement in graft preservation techniques and the transplant community must continue to incorporate this technology to ensure the benefits of liver transplant are maximized. Since the introduction of liver transplant as a treatment option for end stage liver disease and liver cancer in the 1960s, its role and potential benefits have expanded substantially . In the The assessment that a patient, with an accepted indication, can be transplanted with a good outcome is reliant on social, psychological, medical, and surgical factors. Further to this, the risk - benefit assessment must be individualized as not all perspective recipients present with the same risks, nor should they all be expected to glean the same benefits. Despite research into overall risk , 11, theSelecting donors and organs that can be safely used in the transplantation process is a challenging task. Organs chosen for transplantation should pose a minimal risk of donor-derived infection or malignancy, and ideally function in both the short and longer term. Graft-recipient matching goes beyond the size and ABO blood type compatibility, it relies on clinical judgement of the donor organ and its expected graft function, with the recipient stability to withstand the physiological disturbance caused by ischemia reperfusion injury . The majvia SCS. Endovascular stents and thrombi in major vascular structures or even extending into the right portion of the heart may require extensive logistics for use of a veno-venous bypass, and involvement of cardiothoracic teams. The ability of NMP to abbreviate the cold ischemic period but prolong the graft preservation in a safe manner provides a mechanism to expand graft options for these patients, accommodate the complex logistics, and mitigate the risk of unanticipated surgical factors.A liver transplant is a challenging operation, with numerous risks due to both the technical aspects of the procedure and the recipients’ physiology. Despite advances in technique, such as preservation of the recipient inferior vena cava , 19 and It must be emphasized that with the combination of NMP, a high risk recipient, and a suboptimal graft, the implantation time must be kept short as the graft is already warm and therefore ATP depletion will proceed at a faster rate than following SCS. Scientific evidence for this is yet to be published, however more prolonged implantation times with NMP-preserved grafts are likely unfavorable. Alternative surgical strategies can be used in complex scenarios with NMP as opposed to cold storage. Rather than striving for a short cold ischemic period with the standard preservation method and early reperfusion of the graft, NMP allows time for a careful dissection to gain proper vascular control prior to the implantation phase and this is particularly useful in the setting of portal vein or late hepatic artery thrombosis when the implantation can be complicated . The lenAppropriate graft-recipient matching has been shown to be an important factor in ensuring optimal liver transplant outcomes, with marginal grafts generally preferred for recipients with lower model for end stage liver (MELD) scores . The ratvia ex situ machine perfusion remains an imperfect science, with many unanswered questions  due to the high concentrations of this substance within preserved bags of human erythrocytes . FollowiOptimizing the safe utilization of deceased donor livers should be an ambition of all transplant programs. Despite recent improvements, the organ discard rate for retrieved organs from deceased donors in the US and UK is 8.4% and 18.2%, respectively , 44. PreThe only absolute contraindications to transplanting a deceased donor’s liver based on their medical history is an established diagnosis of cirrhosis, primary central nervous system lymphoma, hematological or metastatic malignancy, active JC viral infection, or a transmissible spongiform encephalopathy , 48. TheThe physiological effects of age have less of an impact on the liver compared with the kidney and heart. However, both structural and functional differences exist in livers of older donors. The metabolic function of the hepatocyte has been shown to be decreased in the elderly. Peterson et al. demonstrated that the phosphorylation and oxidative capacity of mitochondria within the hepatocytes of the elderly (61–84 years) was reduced by 40% compared with young controls (18–39 years) . This waThe threshold for considering a donor to be of advanced age varies considerably, ranging from ≥40 to ≥80 years of age –56. ReceAlcohol-induced liver damage progresses in severity from hepatic steatosis to alcoholic steatohepatitis and then cirrhosis . Approxip = 0.75)  . These a = 0.75) . However = 0.75) . This maThe commonly described “liver function tests”, actually provide minimal insight into the liver’s synthetic function, if at all. Despite this, even minimal elevations of liver enzymes are considered as criteria for defining marginal donors as per Eurotransplant criteria , 62. AlaThe viability assessment provided by NMP is the ideal preservation platform for these grafts in the three aforementioned clinical scenarios that lead to organ discard, and has previously been shown to be effective even when the transaminase elevation is accompanied by significant hepatocyte necrosis . Our insIn the context of deceased organ donation, consideration of the events and pathological process that resulted in brain death (or the consideration for DCD donation) is required. Consensus guidelines are available for the scenario of donor malignancy or infection and are beyond the scope of this review , 48. In A donor ICU length of stay >7 days is considered an extended criteria donor by Eurotransplant, whereas previous consensus meetings have stipulated an ICU length of stay <5 days is ideal for DCD donors , 62. It A blunt traumatic injury may result in serious intracranial injuries and brain death as a result. There may also be an associated traumatic liver injury, which is most commonly lacerations or contusions of the liver parenchyma . These tA frequent reason for a graft to be assessed as suboptimal and discarded is the presence of steatosis . AlthougMultiple studies have demonstrated that even severe microsteatosis does not have a deleterious effect on transplant outcome –75. It iThe extent of macrosteatosis that represents a graft that is totally unusable remains unclear . StudiesNMP has previously demonstrated benefits in the setting of steatotic livers, and probably represents one of the most frequent indications for its use , 13, 82.Reducing the cellular temperature to approximately 4°C slows cellular metabolism and therefore slows ATP consumption to approximately 10% of activity at normal body temperature . NeverthFeng et al. demonstrated that in reference to a graft with an 8 h CIT, every additional hour resulted in a 1% decrease in 1 year graft survival . This suIn this section we discuss how we tie up the previously discussed issues together—the novel technology of NMP and the utility of this in the context of high-risk recipients who are significantly disadvantaged due to the scarcity of good-quality organs and timely transplantation of these candidates with marginal grafts. At our institution, Queen Elizabeth Hospital Birmingham, we have incorporated NMP technology into our service on a selected basis for the high-risk graft-recipient combination. The overall benefit of NMP technology over and above cold storage for standard graft-recipient combinations remains debatable. Although NMP is known to mitigate ischemia reperfusion injury and somewhat mitigate short-term beneficial outcomes, it is our belief that NMP should be utilized with a greater aim. Therefore, we utilize NMP in a manner that will allow high-risk recipients to benefit through improved graft access. The results of this approach have been previously published and presented at international conferences , 20. To The ability to accept and safely transplant marginal and ‘orphan’ livers into high-risk recipients has been made possible with NMP due to a number of reasons. Firstly, the NMP viability assessment of a graft provides additional confidence that the graft will function adequately in the early post operative period. This is supported by the fact that PNF has not occurred in any of the 92 recipients of a liver preserved with NMP. Secondly, the ability to inspect and connect the liver to NMP expediently on arrival at our center allows the CIT to be kept to an acceptable minimum. This means that grafts that are declined at other transplant centers, in other parts of the UK, can still be reperfused within an adequate time period. The additional insult of a prolonged CIT on a graft with marginal attributes should not be underestimated, as undoubtedly there is an interaction between these variables as described previously. Finally, with the current national organ retrieval system in the UK being relatively inflexible about donor retrieval timing, it allows for a difficult operation to be performed during daylight hours with adequate and appropriate staffing.P=<0.01). Furthermore, in this expanded cohort, the peak alanine transaminase in the initial post operative 7 days was significantly lower  in the NMP group, and the EAD rate was not significantly different (47% vs. 37%). The rate of early acute rejection was higher  in the NMP cohort and has been discussed in a previous publication. We acknowledge that the incidence EAD rate is higher than previous reports, however this did not translate to early graft loss. Furthermore, a large majority were “biochemical” EAD due to raised bilirubin only (>177 mmol/L) in the presence of severe immune-mediated rejection, without any clinically relevant organ failure.In regards to graft physiology following NMP, we have experienced a lower than expected incidence of reperfusion syndrome and clinically significant early allograft dysfunction than would be expected given the graft and recipient characteristics . In the The transplantability of a liver graft has long been a subjective assessment. It is understandable that surgeons may therefore err on the side of caution, in an effort to do no harm. This is of course particularly important when the predicted surgical risks are already high. The ability of NMP technology to extend liver graft preservation time can allow more complex recipients to be transplanted in a controlled and safe manner. Furthermore, it can safely facilitate the usage of liver grafts for these recipients that would otherwise not have been considered. Evidence supporting the use of NMP for marginal organs and high-risk recipients is starting to emerge with promising data in the retransplantation setting. The problem inherent with researching this topic is the lack of appropriate controls for comparison. Retrospective control cohorts, or even propensity matching, have their limitations as under different preservation conditions these grafts would not have been transplanted previously . The benData collected for this study, including individual participant data and the data dictionary, will be made available to others at publication. The data will be in an anonymized form to protect participants’ privacy. The authorship agrees to provide access to all additional study documents.


The flow behavior of the solution-treated Mg-3.2Bi-0.8Ca  alloy was systematically investigated during hot compression at different deformation conditions. In the present study, the strain-related Arrhenius constitutive model and dynamic recrystallization (DRX) kinetic model were established, and the results showed that both two models had high predictability for the flow curves and the DRX behavior during hot compression. In addition, the hot processing maps were also made to confirm a suitable hot working range. Under the assistance of a hot processing map, the extrusion parameters were selected as 573 K and 0.5 mm/s. After extrusion, the as-extruded alloy exhibited a smooth surface, a fine DRX structure with weak off-basal texture and good strength–ductility synergy. The newly developed strong and ductile BX31 alloy will be helpful for enriching low-cost, high-performance wrought Mg alloy series for extensive applications in industries. Magnesium and its alloys, as one of the green materials, have shown wide application prospects in transportation, electronics and military industry due to its excellent characteristics, i.e., low density, high specific strength/stiffness, good vibration-reducing performance, etc. . In receUp to now, some new Mg alloys, such as Mg-Sm- ,8, Mg-CaHowever, until now, the flow behavior, constitutive model, dynamic recrystallization (DRX) kinetic model and hot processing map of Mg-Bi-Ca series alloys during hot deformation are rarely reported systematically. It is important to understand the hot flow behavior of Mg-Bi-Ca series alloys to obtain the desired microstructure and to achieve excellent mechanical properties. The Arrhenius-type constitutive model is commonly used to describe the hot flow behavior, predicting the hot-deformed microstructure and optimizing the hot processing parameters of Mg alloys ,25. In MTherefore, in order to investigate the hot deformation behavior of Mg-Bi-Ca series alloys, in this work, taking a new RE-free Mg-Bi-Ca alloy as an example, hot compressive tests were performed at different conditions. The Arrhenius constitutive model and the DRX kinetics of this alloy were established. Based on the data of hot compressive flow curves, the suitable hot processing range was measured. Finally, the extrusion parameter was selected to gain the fine complete DRX structure with weak off-texture and obtain the strength–ductility balance in an as-extruded Mg-Bi-Ca alloy.6:CO2 = 1:99). After a series of sitting and slagging, the melt was poured into a steel mold with Φ 80 (in diameter) × 200 (in height) mm preheated to 350 °C. The chemical composition of the as-cast alloy was detected by an X-ray fluorescence spectrometer  , and the real chemical composition is 96.0 wt.% Mg, 3.2 wt.% Bi, and 0.8 wt.% Ca. It can be labeled as BX31. Before hot compression, the solid solution of the as-cast BX31 alloy was treated at 773 K for 24 h. Subsequently, hot compression tests for samples with a size of Φ 8 × 12 mm (diameter × height) were operated on a Gleeble-3500 thermo-mechanical simulator  at different conditions . In addition, the solution-treated BX31 alloy was also extruded into the bar with Φ 22 mm in diameter at 573 K using an extrusion ratio of 21:1 and a die-exit speed of 0.5 mm/s by XJ-500 horizontal extruder . The room-temperature (RT) tensile properties of the as-extruded sample with 25 mm in gauge length and Φ 5 mm in gauge diameter were machined from the as-extruded bar along extrusion direction (ED) and measured using CMT6305-300 kN universal tensile testing machine  at a strain rate of 1 × 10−3 s−1.Commercial pure Mg ingot (≥99.99%), Mg-10Bi (wt.%) and Mg-25Ca (wt.%) master alloys were melted at 720 °C in an electric resistance furnace  under a protective atmosphere   equipped with an energy dispersive spectrometer (EDS)  and electron backscattered diffraction  device . The phase constitutions were identified by X-ray diffraction  . The preparation of EBSD samples was composed of grinding, washing, blow-drying and electro-polishing at 20 V and 0.03 A for 90 s at 298 K using AC2. The scanning step size was set as 0.5 μm. All EBSD data were analyzed using ATEX software v2.01.3 .2Bi2Ca ones, as displayed in 3Bi2 phases, even Mg2Ca ones, also could be found in wrought Mg-Bi-Ca series alloys [3Bi2 and Mg2Ca particles are the products of a combined effect from deformation force and temperature. On the other hand, the thermal stability of Mg2Bi2Ca phases is superior to Mg3Bi2 and Mg2Ca ones, based on the thermodynamics of phase formation [2Bi2Ca. In order to further verify this statement, an XRD test was carried out, and the result is shown in 2Bi2Ca, which is in accordance with the SEM observation.s alloys ,18,19,20ormation . TherefoT is the temperature; β, A, α and n are material constants; Q is the activation energy; and R is the mole gas constant (8.314 J/mol K). The β and α can be described as follows [The Arrhenius constitutive models are given :(1)ε˙=A1 follows :(4)α=βn1Z) parameter is introduced to construct the relationship among the Z, T. It can be shown as follows [Zener–Hollomon ( follows :(5)Z=ε˙eσ as a function of the Z is expressed as follows [For Equation (3) with Equation (5) together, the  follows :(6)σ=1αlQ, n and A are calculated by Equations (7)–(11).Under the peak stress condition, these material constants of Both sides of Equations (1) and (2) are taken natural logarithms as follows:Both sides of Equation (3) are taken natural logarithms as follows:n value (~5.390).Both sides of Equation (9) are taken partial derivatives as follows:S value is the slope of this linear relationship, ~3.390. Therefore, the Q value at the peak stress condition is about 151.9 kJ/mol, slightly larger than that of pure Mg . Fine p5 kJ/mol ,18,19,20i et al.  pointed g et al. . This kiCombining Equation (3) with Equation (5), the following relationship is shown:Z can be calculated at different deformation conditions by taking into the Q value. lnA is ~26.430 as the intercept of this function. As mentioned above, the Arrhenius constitutive model at the peak stress condition can be established as follows:The values of Q, n and A are closely associated with the strain. By using 6-order polynomial equations expresses their relations as follows:R) and the average absolute relative error (AARE), and the relative error (δ) as follows [n is the size of flow stress set. R is ~0.9919 and the AARE is ~4.28%, as shown in Under the case of the strain-related Arrhenius constitutive model, the material constants of  follows ,42:(17)RZ parameter given as follows [θ-σ relationship (θ represents the work hardening rate), the −1) as an example in In order to describe the DRX kinetics model, in general, the following equation can be used and expressed ,44:(20)Xθ-σ curve and Equation (23), According to the calculation o ε at different deformation conditions. As for each curve, as it increased the ε, the −1, the ε is up to 0.7. As the strain rate enlarges, low temperature obviously declines the DRX degree, especially for the deformation condition of 573 K and 1 s−1 (only ~40% at the 0.7 strain).−1 are selected to observe the DRX area fractions at different strains of 0.3 and 0.5. The result is demonstrated in In order to verify the accuracy of the DRX kinetics model, the hot compressive samples at 623 K and 0.01 sFurthermore, we also observed that the DRX nucleation preferentially appears at the original grain boundaries. At a high temperature, the CRSSs of non-basal slips may decline, likely activating the non-basal slips within grains that easily induce the obvious orientation gradient. It is also a type of important DRX mechanism (known as continuous DRX) to promote the formation of the DRXed grains, together with the discontinuous DRX mechanisms . As the strain increases, the DRXed grains grow, and its average grain size gradually increases from ~10.6 to 13.9 μm, as seen in P, G and J represent the power dissipated by the workpiece, plastic deformation and microstructural evolution, respectively. The K and m are a material constant and the strain rate sensitivity coefficient, respectively. Together Equation (27) with Equation (28), J is expressed as follows:A dynamic material model is valid to describe the hot processing map. In this model, an equation is given :(27)P=G+is given :(28)σ=KεThe efficiency of power dissipation ( follows :(30)η=JJBased on the extreme principle, the instability criterion is proposed as follows :(31)ξε˙¯=In this work, The solution-treated BX31 alloy is perfectly extruded into a bar at a given deformation condition. 2Bi2Ca ternary phases. It indicates that they belong to the broken remains from the second phase in the solution-treated BX31 alloy after extrusion. These fine second phases accelerate the DRX nucleation, known as the PSN effect.Additionally, the SEM analysis of the as-extruded BX31 alloy is shown in In this work, the hot deformation behavior of the solution-treated BX31 alloy was systematically studied. The strain-related Arrhenius constitutive model presented a high predictability for the flow curves during hot compression. The flow curves followed the DRX-dominated softening mechanism. The DRX nucleation preferentially appears at the original grain boundaries, and subsequently, non-basal slips might be activated to promote the continuous DRX at a high deformation temperature. For a given deformation condition, the DRX benefited from the texture weakening, and interestingly, with increasing the strain, the DRX growth further reduced the basal texture intensity. This result might be attributed to the preferred growth of non-basal-oriented grains due to the solute segregation. According to the hot processing map, a suitable extrusion parameter could be selected, and the as-extruded BX31 alloy showed a balance of strength and ductility. Such good comprehensive tensile properties were mainly attributed to the following three aspects: (i) fine grain size, (ii) homogeneous complete DRX structure and (iii) the formation of off-basal texture. The newly developed strong and ductile BX31 alloy will be helpful for enriching low-cost, high-performance wrought Mg alloy series for extensive applications in industries.


This cross-sectional study evaluated women’s attitudes toward the certification logos, labels, and advertisements for organic disposable sanitary pads (OSPs) and investigated what could be the main reason for them. Additionally, the present study examined whether a relationship could be found between these attitudes and OSPs purchasing behavior.This study was conducted using a self-reported online survey of Korean adult women who have purchased OSPs. The study questionnaire had four sections, covering (1) characteristics of OSP purchasing behavior, (2) attitudes toward OSP certification logos, labels, and advertisements, (3) demand on government and companies for proper management, and (4) respondent’s sociodemographic information. The Cronbach’s alpha value of the questionnaire was 0.857.P < 0.05). The most significant demand from the respondents for OSP companies and the government was to clearly indicate hazardous ingredients on the OSP packaging (42.0%) and to strengthen the sanctions for false advertising (37.8%), respectively.A total of 500 respondents completed the questionnaire. Overall, high reliability was found for the certification logos (3.73 ± 0.61), labels on the product packaging (3.71 ± 0.63), and advertisements of OSPs (3.41 ± 0.62). Respondents indicated that these had fairly positive effects on their decision-making regarding product reliability, product image, and their own purchasing behavior. The aspects most frequently affected from the informants were safety to human health. All attitudes toward OSP certification logos, labels, and advertisements that were evaluated in this study became more positive in the direction from non-buyers to occasional buyers and to habitual buyers (all The results of this study clearly indicate the importance of using certification logos, labels, and advertisements in the OSP market. These results can be utilized by OSP companies to improve the effectiveness of their marketing strategies or by policy makers and certifying bodies to manage the informants properly in the OSP market.The online version contains supplementary material available at 10.1186/s12905-022-01723-z. Disposable sanitary pads are considered a necessity for women. Monthly menstruation occurs for about 40 years of the average woman’s life, and most women regularly use disposable sanitary pads during this period. It is estimated that a woman uses approximately 11,400 pads over her lifetime , 3.As awareness of health issues grows, there are efforts to ensure the safety of disposable sanitary pads for women’s health. In South Korea, the Pharmaceutical Affairs Act of Korea, which addresses medicine and quasi-drugs, has been amended to require that all ingredients used in all such products be indicated on the label of the packaging . New YorDespite the growing use of certification logos, detailed labels on product packaging, and advertisements for OSPs around the world in response to concerns about conventional disposable sanitary pads, few studies have focused on women’s attitudes toward this feature and its impact. Although several studies have evaluated consumers’ perceptions and attitudes toward organic logos or labels for foods –12, it iThis is a cross-sectional self-reported online survey of adult women  contains organic ingredients?”; “When you buy an OSP, what do you expect most?” Respondents who answered that they had purchased OSPs one time were considered “non-buyers,” those who bought them from 2 to 6 times were “occasional OSP buyers,” and those who had bought them 7 to 10 times were “habitual OSP buyers.” These values were drawn from previous studies that dealt with targeted and tailored communication strategies regarding different buying-frequency consumer groups , 18.In this section, respondents were asked about their attitudes toward OSP certification logos, labels, and advertisements. Questions included “Do you trust certification logos/labels/advertisements of OSP products? ,” “Do the certification logos/labels/advertisements of OSP products create a positive image for the product? ,” “Do the certification logos/labels/advertisements of OSP products make you build trust in the product? ,” and “Do the certification logos/labels/advertisements of OSP products affect your decision to purchase the product?” In addition to these, respondents were asked other questions in separate parts: for certification logos, “Do you know the meaning of the certification logos of OSP products?” for labels, “Do you read what the label says?” “Do you understand what the label says?” and “Do you perceive the label’s content to be important?” for advertisements, “Have you ever seen or heard advertisements for OSPs?” “How do you usually see or hear advertisements for OSPs?” and “What interests you the most when you see or hear advertisements for OSP products?” For these questions, a 5-point interval scale was used; “very unlikely” (1), “unlikely” (2), “neutral” (3), “likely” (4), and “very likely” (5). A higher score indicates a more positive attitude, and in this study, 3-point was considered as a cut-off value.In this section, the respondents were asked about their demand of the government and companies regarding the appropriate management of certification logos, labels, and advertisements of OSP products.In this section, respondents were asked about their age, residence, and educational background.P-values less than 0.05 were considered to represent statistical significance. All analyses were performed using SAS 9.4 .After the survey results were collected, errors and missing data were reviewed, and only the completed questionnaires were coded into a file for statistical analysis. Categorical variables were presented as values and percentages and compared using Pearson’s chi-square tests. Continuous variables were summarized as mean and standard deviation (SD). Analysis of Variance (ANOVA) was used to compare the differences between the three purchase-frequency groups consisting of continuous variables, and the Bonferroni correction was applied. Two-sided A total of 500 adult women completed the questionnaire and were included in the analysis. Table The average number of OSP purchases of the last 10 times was 5.06 ± 2.95). Approximately 10% of respondents were identified as non-buyers, while 57.2% and 32.4% of respondents were classified as occasional and habitual buyers, respectively. As shown in Table .95. ApprP < 0.001). Habitual consumers had the highest awareness (3.28 ± 0.73), followed by occasional buyers (3.07 ± 0.69) and non-buyers (2.62 ± 0.72). All the items asking about the attitude toward the certification logo were rated above 3, which can be interpreted as a positive attitude of consumers toward the OSP certification logo (Table P < 0.001).For the OSP certification logo, the average score for the respondents’ awareness was 3.09 (± 0.73), and there was a significant difference among the three purchase frequency groups (P < 0.001). Regarding the labels, the respondents’ attitudes toward the detailed contents of the labels were also identified .Only those respondents who had ever seen or heard an OSP advertisement n = 435, 87%) were included in the advertisement analysis. About 90% of the respondents had encountered OSP advertisements, either on the air or online . The respondents had a positive attitude toward the reliability of OSP advertisements (3.41 ± 0.62), and the effects of advertisements on product image (3.64 ± 0.63), product reliability (3.54 ± 0.63), and purchase decision (3.58 ± 0.72) . The second-ranked demand was to clearly indicate the specific parts  of OSPs made from organic ingredients (33.0%) in their advertisements or on the packaging. This was followed by providing detailed information on hazardous ingredients and proof of certification logo acquisition on the product website . For governments, the respondents strongly requested greater sanctions for unfair advertising (37.8%) and the establishment of regulations of the use of certification logos (32.7%). Other demands included consumer education for more intelligent consumption (15.2%) and corporate education to prevent unfair advertising (14.4%).Women’s demands for OSPs are growing with the expectations for their safety for body. However, it is hard for consumers to verify whether a given product was produced according to the promised characteristics , 19 becaThis study showed that the sociodemographic characteristics, including age, education background, and residential area, were not significantly different among the three purchase frequency groups. These results are similar to those of previous studies on organic foods , 22. BecThe respondents in this study showed generally positive attitudes toward the OSP certification logos, labels on the product packaging, and advertisements. The results indicate that women trust those informants, and the informants had positive effects on women’s decisions about the product’s reliability and image, and even on their purchasing behavior. In the credence market, third-party certification or a provision of information via labels or advertisements could be an instrument to gain consumer trust . By thisIn this study, the respondents indicated that the most expected aspect when purchasing an OSP and the aspect that the informants contribute the most to the belief in and impression of the product is safety for human health. However, it is interesting that OSP certification logos, labels, and the word “organic,” used in advertisements, do not guarantee that the product is healthier or that it is free from hazardous ingredients –31. It hFurthermore, this study found that the degree of women’s positive attitude was closely linked to their purchasing behavior. The attitude toward the OSP informants has become more positive going from non-buyers to occasional buyers to habitual buyers. These results suggest that in the OSP market, not only whether women have a positive attitude toward an informant but also the degree of positivity is important. This finding is in line with several previous studies that have found that negative attitudes attributed to uncertainty and lack of trust in organic food logos can act as barriers to purchasing organic food –36.There are some limitations in our study. First, the respondent group was slightly biased toward better educated respondents (about 80% have a degree above college education). This may be attributed to the use of an electronic survey method. Second, this study only focused on OSPs because we observed that, as with previous studies, the most used menstrual hygiene products were disposable menstrual pads, due to their convenience and easiness of discarding , 37, 38.Despite these limitations, the findings of this study are valuable because, to the best of our knowledge, there has been no previous study that has evaluated women’s attitudes toward OSP certified logos, labels, or advertisements in combination with purchasing behavior. Therefore, although this study was conducted in Korea, it can be used as basic data to understand women’s attitudes toward OSP informants in other countries where the demand for OSP is also increasing.This study indicates that certified logos, labels, and advertisements are important tools for ensuring women maintain a positive attitude toward OSP products and key drivers for OSP consumption. The results of this study can contribute to improvements to the effectiveness of the marketing strategies of OSP companies, and it can also provide insight for policy makers or certifying bodies on the importance of the informants and their management in the OSP market.Additional file 1. Checklist for reporting internet E-Survey results. A file entitled “Checklist for Reporting Results of Internet E-Surveys (CHERRIES)".Additional file 2. Checklist for reporting cross-sectional studies. A file named “STROBE statement checklist”.Additional file 3. Contains all the questions of the survey asked for the study participants. A file named "Survey questionnaire"