We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods.Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA  Octopus vulgaris  offers a robust, sensitive, versatile and low-cost method for simultaneous detection of multiple mRNAs in cells or tissues of any organism  imaging of whole embryos or organs, often combined with marker gene labeling techniques. Besides classical confocal microscopy, that allows for high resolution imaging at the cellular level, light sheet fluorescence microscopy (LSFM) revolutionized imaging speed of optically transparent organisms including several aquatic embryonic and larval specimens . In addiO. vulgaris embryos, even in combination with immunohistochemistry. These methods will advance gene expression analysis in non-model species such as cephalopods. Moreover, we present data on developmental stage XV as an experimental stage, which is mid-organogenesis, and the embryos have developed eye pigmentation which is successfully cleared by the proposed protocol in order to visualize the brain by LSFM. We also include immunohistochemistry to prove that sequential detection of mRNA and protein is feasible using our combined method. Combining visualization of mRNA and protein can be useful for a number of reasons. First of all, it provides flexibility to the researcher in the case where not for all genes under investigation antibodies are available. Secondly, expression information gathered at the mRNA level and protein levels might be different. For instance, the respective protein can be secreted, which means that mRNA could be detected in a different location than the protein. Our data confirmed previous findings using HCR v3.0 on transverse sections  imaging. 3D imaging can provide an additional perspective. Here, we add to the existing methodology an automation of probe design, and optimized clearing protocol that retains the signal generated by HCR v3.0 in whole mount sections  and showOctopus vulgaris embryos were received from the Instituto Español de Oceanografía . Embryos were incubated until reaching the developmental stage XV in the closed standalone system located at the Laboratory of Developmental Neurobiology . The size of an octopus egg, from the stalk till micropyle, is 2 mm × 0,7 mm and a stage XV octopus embryo, from the top of the mantle till the end of arms, is approximately 1,25 mm × 0,88 mm. The stage XV embryos were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight, followed by a wash of Diethyl pyrocarbonate-treated phosphate buffered saline (PBS-DEPC). Embryos were manually dechorionated using tweezers  in PBS-Tween (PBST). Embryos were dehydrated into 100% Methanol (MeOH) following a series of graded MeOH/PBST washes, each for 10 min: 25% MeOH/75% PBST, 50% MeOH/50% PBST, 75% MeOH/25% PBST, 100% MeOH, 100% MeOH. Dehydrated embryos were kept at −20°C overnight or until further use.Live in situ mRNA visualization. Ov-apolpp, Ov-ascl1, Ov-elav, and Ov-neuroD were already designed as previously described in Ov-apolpp, Ov-ascl1, Ov-elav, and Ov-neuroD, respectively  (The protocol is based upon the Molecular Instruments’ (MI) HCR v3.0 protocol for whole mount mouse embryos (usculus)  with somWhen HCR was combined with IHC, the incubation in DAPI in the HCR protocol was included in the IHC section of the protocol. The embryos were directly processed for IHC after the last excess hairpin removal wash. The whole protocol of IHC was carried out at 4°C. Embryos were incubated with the primary antibody ) (Millipore 06–570) for the following 2 days after the HCR protocol. Afterwards, the embryos were washed with 5xSSCT three times for 2 h followed by adding the secondary antibody donkey anti-rabbit Alexa 488 (Life Technologies) at a final concentration of 1:300 diluted in antibody diluent (Roche) and incubated overnight. The excess secondary antibody was washed with 5xSSCT twice for 2 h and the embryos were incubated in 1:2000 DAPI in 5xSSCT for 2 h followed by 5xSSCT wash for 5 min. Fructose-glycerol clearing was performed as described above.Imaging was done using Zeiss Z1 Light sheet fluorescence microscopy (LSFM) . The cleared and stained embryos were glued from their mantle on a metal plunger and immersed in low-viscosity immersion oil mix, as described in in situ hybridization chain reaction (HCR) combined with immunohistochemistry, clearing and imaging to visualize Octopus vulgaris embryonic neurogenesis. An overview of the methodology pipeline is depicted in In this study, we report for the first time on optimization of whole mount RNA multiplexed Ov-elav . Easy_HCR is a set of jupyter notebooks made to automate the creation of probe pairs for hybridization chain reaction (HCR). It is based on insitu_probe_generator  with a glial marker (Ov-apolpp). As controls, we measured autofluorescence on each channel as well as performed HCR using only-hairpins without any probe conditions  and neuronal precursor (Ov-neuroD) markers was followed by an immunostaining for the mitotic marker phosphorylated-histone H3 (PH3) . By combining Ov-neuroD HCR with 3D imaging, we confirmed the bow-shaped expression pattern but also revealed that it is actually shaped as a double bow, both sides being connected to each other posteriorly  . Ov-asclal brain . The expteriorly . Also Ovtructure . By combsections . The z-sormation .Four different  water-based clearing methods were compared for their clearing performance as well as to evaluate whether the signal intensity of HCR was retained after clearing on octopus embryos see also  Costant. CUBIC .Arc mRNA (Cre mRNA in a Thy1-Cre mouse brain (HCR with different clearing methods has been published over the last few years . AlthougArc mRNA . PACT (pArc mRNA . HCR in Arc mRNA . RecentlArc mRNA . Apart fArc mRNA . The comArc mRNA . For insse brain .Our aim was to report an optimized protocol for whole mount HCR v3.0 and its compatibility with IHC as well as a water-based tissue clearing method on octopus embryos for understanding neural anatomy and neurogenesis in 3D. We believe that the proposed experimental pipeline can be adapted to other model and non-model organisms. Also, HCR has a wide range of applications in various fields, such as in biomedical purposes. For instance, it can be used for studying development and creating developmental atlases for specific systems or for understanding diseases such as pathogen detection and behavior in chronic infections . Whole m


Background: Patients older than 70 years are the fastest-growing age group of patients requiring renal replacement therapy. This has resulted in a corresponding increase in the number of elderly transplant recipients. We hypothesized that graft survival in this population would be comparable to that seen in the literature on kidney transplant recipients under 70 years of age.Methods: This was a retrospective, single-center review of outcomes of kidney transplant recipients aged ≥70 years. Patients were dichotomized based on whether their allograft originated from a living or deceased donor.Results: A total of 59 recipients aged ≥70 years underwent kidney transplantation. Of these, five (8.5%) were lost to follow-up within the first year post transplant and excluded from the analysis. History of cerebrovascular accident (p = 0.003), coronary artery disease (p = 0.03), postoperative return to the operating room (p = 0.03), and readmission within one year of transplant were predictive of graft loss (p = 0.003). Overall graft survival in our cohort declined from 92.6% at one year to 53.8% at five years. Death-censored graft survival was 100% at one year and decreased to 80.8% at five years. There were no differences seen in patient, graft, or death-censored graft survival based on donor type.Conclusions: Kidney transplant patients over 70 years, as seen in our cohort, had good short-term outcomes. Graft survival is similar to rates seen in younger cohorts but the decline in this rate over time is steeper in the older age group, possibly due to decreased patient survival. These findings could be validated further in larger multi-center studies. The scientific and technological advances over the last century have supplemented increasing public health awareness to increase longevity. This has slowly led to a rising population of older adults that are living significantly longer than their predecessors . It is ePrevious studies have shown improved quality of life and a 41% reduction in mortality risk in elderly patients who received a kidney transplant when compared to dialysis-dependent wait-listed patients -6. Most Study populationThis was a single-center, retrospective cohort study approved by the institutional review board (IRB) of Tulane University, New Orleans, Louisiana, United States  and a waiver of informed consent was granted by the IRB. All kidney transplant recipients who were aged 70 years or more at the time of their transplant at Tulane Medical Center, New Orleans, Louisiana between January 1, 2006, and December 31, 2019, were identified using data submitted to the United Network for Organ Sharing (UNOS) . PatientData collection and study elementsDonor and recipient characteristics were identified using the site-specific UNOS database . RecipieDemographic and clinical outcome descriptors of transplant recipients, such as age, sex, race, BMI, blood type, education level, and waiting list time , comorbidities, polypharmacy (defined as the use of five or more drugs before transplant), functional status at listing (defined as the performance of activities of daily living), anticoagulation/antiplatelet therapy use before transplant, and need for midodrine pre-transplant for hypotension  were surveyed.Operative and perioperative variables collected included warm ischemic time (WIT), hospital length of stay (LOS), 30-day hospital readmission, delayed graft function , and postoperative complications including atrial fibrillation, hypotension, myocardial infarction, and return to the operating room. Immunologic characteristics included ABO incompatibility, human leukocyte antigen (HLA) mismatches, donor-recipient (DR) mismatch, and induction and maintenance immunosuppression regimen used.The primary outcomes of interest were patient survival, graft survival, and death-censored graft survival at one, three, and five years following the kidney transplant. Graft failure was defined as a return to dialysis, repeat kidney transplant, or death with a functioning graft. Death-censored graft survival was used to estimate the probability of graft loss only. In the event of death with a functioning graft, the date of death is taken as the date of the last follow-up. The fatal event is thereby handled as a case lost to follow-up, not as a graft failure. Secondary outcomes of interest included perioperative complications, hospital LOS, re-admissions, and incidence of rejection, infection, or cancer in the follow-up period.Statistical analysisPatients were dichotomized by donor type (living vs deceased). Descriptive statistics were used to examine the cohorts. An examination of factors affecting one-year and three-year graft survival was also performed with patients categorized by graft survival or loss at the specified time frame. Categorical values were reported as frequencies, represented as n (%), and examined by Chi-square analysis. Continuous variables were reported as median (interquartile range (IQR)25 - IQR75)) and examined by the Mann-Whitney U test. The level of statistical significance was set at p < 0.05. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 28.0 .A total of 54 kidney transplant recipients were included in the analysis, of which 17 (31.5%) had living donors and 37 (68.5%) deceased donors. The overall cohort had a mean age of 72 years and a BMI of 27.1, was predominantly of white race (64.8%), and had a high school education or lower (64.8%) . The majority of the recipients had blood group A or O, with the remainder being either AB or B type, but these proportions did not differ between cohorts. The most common comorbidity present at the time of listing was HTN, followed by DM, coronary artery disease (CAD), and a history of cerebrovascular accident (CVA); these proportions did not differ between the cohorts. Most (87%) recipients were taking five or more pharmacological agents at the time of transplant, and half of the total cohort was on anticoagulation/antiplatelet therapy. Most recipients (46.3%) were documented as having a functional status at the listing of 90%, while only 3.7% had a functional status below 70%. The distribution of the degree of functional ability did not differ between cohorts. Living donor recipients had a significantly shorter waiting list time compared to deceased donor recipients (p <0.001). Donor characteristics are detailed in Table The donor cohort was of median age of 45.5 years, median BMI of 26.8, majority male (53.7%), and majority white race (63.0%). The KDPI for the deceased donor group was 59%. The cold ischemia time was higher for the deceased donor group at 16.8 hours vs 0.6 hours in the living donor group (p < 0.001). Kidneys were pumped prior to transplantation in 21.6% of the deceased donor cases.Table Of the postoperative complications examined, atrial fibrillation was seen in 8% of the cohort, hypotension in 4%, and myocardial infarction in 2%. A total of seven patients (14% of cases) required a return to the operating room within that hospital stay, 26.9% were readmitted to a hospital within 30 days of the transplant, and 57.7% had at least one hospital readmission after 30 days but before one year after transplant. There were no differences in readmission rates based on donor organ type. DGF was observed at a higher rate in the deceased donor group (24.3%) when compared to the living donor group (11.8%), but this difference was not significant (p = 0.29). The median LOS postoperatively was six days and was comparable between both living donor and deceased donor groups.The incidence of infection in the first year post transplant was 51.9%, and this decreased to 39.1% after one year. There were no differences in the incidence of infectious complications in the first year post transplant between deceased and living donor groups. The most frequently occurring pathogen types were bacterial, seen in 59.3% of patients (through common UTIs), and viral, seen in 48.2% of patients , BK virus (BKV)). Cancer was seen in 3.8% of the total cohort within one year of transplant, and in 33.3% after one year. The incidence of rejection in the first year post transplant was 9.6%. Acute cellular rejection (ACR) was the most common type of rejection (three patients), followed by combined ACR and antibody-mediated rejection (AMR) in one patient.Rates of one, three, and five-year graft survival, death-censored graft survival, and patient survival are presented in Figures Factors impacting graft survival were then examined. Specifically, recipient characteristics, comorbidities, peri-operative complications, incidence of rejection, cancer, and infections were correlated with one and three-year graft survival Table .Recipient demographics including age, sex, race, education level, blood type, functional status, BMI, and polypharmacy at the listing time did affect one or three-year graft survival in our study population.Patients with a history of cerebrovascular disease were observed to have a higher (50% vs 6%) incidence of graft loss at one year (p = 0.003) and at three years post transplant (p = 0.04). History of CAD also affected graft function, with 46.7% of those with graft loss at three years having a history of CAD (p = 0.03). Out of those with graft failure at one year, 75% had at least one episode of infection and 33.3% had a diagnosis of cancer within the first year post transplant. Cancer within one year after transplant adversely affected 1 and 3-year graft survival with an incidence of 33.3% (p = 0.006) and 15.4% (p = 0.02) in patients who incurred graft loss.Post-operative incidence of atrial fibrillation, hypotension, myocardial infarction, and length of initial hospital stay did not adversely affect one-year or three-year graft survival. Patients that required a return to the operating room after their transplant faired poorer than those who did not- 50% of patients who had graft failure at one year were taken back to the operating room postoperatively, compared with 10.9% with a functioning graft (p = 0.03). Readmission after 30 days but within the first year post transplant was seen in 86.7% of patients with a failed graft at three years whereas only 40.6% in those with surviving grafts (p= 0.003). DGF was seen in 75% of those with a failed graft within the first year (p = 0.005).Kidney transplantation is the preferred modality for the treatment of end-stage renal disease (ESRD) owing to improved survival benefits and quality of life ,14. HoweElderly waitlisted dialysis-dependent candidates have a 50% mortality rate while waiting for a kidney transplant, and this has been on the rise since 2019 ,16. TherThere is limited data regarding transplant outcomes in this subpopulation of elderly patients compared to their younger counterparts. In 2004, Fabrizii et al. reported a higher five-year patient survival amongst kidney transplant recipients in the age group of 50-59 years when compared to patients older than 60 . Huang aAs of 2021, there are over 90,000 patients in the United States waiting for a kidney transplant, with 13 patients dying every day on the waitlist . The oneStudies have reported higher perioperative surgical complications in the elderly . From ouPost-transplant incidence of rejection in our study was 9.6%, with no differences based on donor status. Huang et al., in 2010, compared outcomes in three cohorts of ages 60-69, 70-79, and above 80 years and reported no difference in rates of rejection  between the three groups . SimilarThere are several limitations to the study, which must be addressed. First, the retrospective study design has well-known limitations. Second, this was a single-center study, which allowed consistency in the way patients were evaluated, waitlisted, transplanted, and managed; however, generalizability is limited and the present findings may not be applicable to other institutions or patient populations. The small sample size introduces the possibility of a Type II error. There is a need for other similar studies that look at outcomes in kidney transplant patients solely above 70 years of age in order to confirm our results are not limited to our institution. Owing to the retrospective nature of the study, post-transplant changes in functional independence were not able to be known. Lastly, there are other potential factors such as frailty syndrome, dementia, delirium, and malnutrition that may impact graft survival, which were not accounted for in this analysis.Our study reports acceptable short-term outcomes in a sample of kidney transplant recipients over the age of 70 years. Graft survival is similar to rates seen in studies that looked at younger cohorts but the decline in this rate over time is steeper in the older age group and may possibly be due to decreased patient survival. Thus, the expected transplant advantage in terms of long-term patient survival is not realized in elderly patients included in our study. Considering the shortage of kidney donors, anticipated life expectancy has to be equated with expected outcomes. More studies on quality-of-life benefits and post-transplant changes in functional independence are needed to validate appropriate resource utilization among older recipients.


Animal Models and Experimental Medicine (AMEM) has received 396 manuscripts from 46 countries and regions, including Chinese mainland, Iran, the United States, India, Israel, Nigeria, Brazil, Bangladesh, Sri Lanka, the Netherlands, Denmark, Australia, Japan, and Spain. It has become an important international exchange and display platform for innovative research results in the field of laboratory animal science and basic medicine. From 2018 to 2021, AMEM was indexed by CSCD, DOAJ, PubMed Central, WJCI, MEDLINE, ESCI, and other domestic and foreign scientific databases. In 2022, it was included in Elsevier's Scopus database, indicating the further improvement of AMEM's international influence. In the beginning of 2023, good news kept pouring in for AMEM. First, after the first year of inclusion in the Scopus database, the first CiteScore will be obtained this year. Second, in 2023, it will receive the first Impact Factor (JIF) moving another big step forward for it to become a world‐class journal.After 5 years of accumulation, AMEM has accelerated the construction of international frontier hotspots column. So far, seven hot topics that focus on themed issues of international basic medical research have been published, such as the study of intestinal microflora, cardiovascular and cerebrovascular disease, and neurodegenerative disease. In October 2022, the themed section on the interaction between virus and host was launched. Research papers and comments by domestic scholars on infectious diseases, such as monkeypox virus and COVID‐19 related diseases, were published on that issue. AMEM editorial department vigorously promoted the research results and insights of scientists from all over the world in this field online and offline. In 2023, AMEM will continue to make efforts to make hot columns regular ones to improve its core competitiveness and influence.After changing to a bimonthly journal, AMEM's editorial team was unprecedentedly united and active in reviewing, organizing, and contributing after the convening of the editorial board meeting, meeting the challenges of vigorous development and competition in Chinese journals in the post‐epidemic era. In the face of new opportunities and challenges, our goal is to invite manuscripts from international peers, increase the number of manuscripts of AMEM, maintain the quality of AMEM's manuscripts, forge ahead, stand out among similar journals, and move toward becoming the world's first‐class journal!In 2021, under the leadership of Editor‐in‐chief Chuan Qin, AMEM. We would like to express our gratitude for the support and trust of experts in laboratory animal industry and thank experts from editorial team for their hard work. AMEM will stay true to its original intention of its inception, continue to work hard in this field, forge ahead bravely, and go on creating brilliance. We wish everyone a happy new year!At the beginning of the New Year, we warmly welcome more domestic and foreign experts to contribute articles to


The enhancement of the PLA thermomechanical properties is significant due to its suitability as a replacement for primary synthetic polymer use in diverse industrial production. The amphiphilic chitin was used as a compatibilizer in PLA/starch biocomposite. The properties of plasticised polylactic acid blended with starch, and amphiphilic chitin was studied for enhanced thermomechanical and viscoelastic properties. Chitin was modified using acetylated substitution reaction and blended with plasticised PLA/starch biocomposite. The biocomposite was prepared with combined compression and melt extrusion techniques. The biocomposite’s thermomechanical, thermal, mechanical, and morphological properties were studied using dynamic mechanical analysis, TGA-DSC, tensile test, and scanning electron microscopy. The storage and loss modulus were significantly enhanced with increased amphiphilic chitin content. Similarly, the single peak of tan delta showed good miscibility of the polymeric blend. Additionally, the modulus increases with frequency change from 1 Hz to 10 Hz. The thermal stability of the biocomposite was observed to be lower than the neat PLA. The tensile properties of the biocomposite increased significantly more than the neat PLA, with P4S4C having the highest tensile strength and modulus of 87 MPa and 7600 MPa. The SEM images show good miscibility with no significant void in the fractured surface. The viscoelastic properties of PLA were enhanced considerably with plasticizer and amphiphilic chitin with improved biodegradability. The properties of the biocomposite can be adapted for various industrial applications. Biopolymers based on lactic acid (PLA) are the most promising renewable polymers. Biopolymers are compostable, biocompatible, and easily processed with similar techniques used in conventional plastic manufacturing ,2. PLA tPLA has been reinforced with other biopolymers, of which starch, chitin, and cellulose are the most abundant. Starch is a naturally occurring plant and animal product. Biopolymer is abundant and inexpensive ,9. ChitiThus, starch has been used as reinforcement in PLA without impairing its biodegradability. Nevertheless, as the starch concentration of the biocomposite increases, the biocomposite’s tensile and elastic modulus properties have been found to decrease ,14,15,16Additionally, Aranda-Garcia  noted thOn the other hand, chitin has been employed to reinforce PLA with excellent dispersion but slightly improve tensile strength . As a rePLA/Starch biocomposite has been reported with agglomeration and low tensile strength due to differences in the nature of the polymer. Several researchers have enhanced the miscibility between PLA and Starch due to their suitability for multiple applications. PLA is hydrophobic, and starch is hydrophilic. Therefore, in this study, amphiphilic chitin has both hydrophobic and hydrophilic ends that act as a nonreactive compatibilizer to enhance their miscibility and strength. This research explored a novel green approach to using amphiphilic chitin as a compatibilizer between PLA and Starch against complex chemicals. Amphiphilic chitin has been reported to be a non-toxic and biodegradable derivative of chitin. Chitin was modified to amphiphilic chitin using two-step acylation to incorporate both hydrophilic and hydrophobic functional groups. The use of two-step acylation in this study has not been reported before. Additionally, amphiphilic chitin as a compatibiliser in plasticised PLA/starch has not been researched. The amphiphilic chitin was filled in plasticised PLA/starch to form a biocomposite to improve its miscibility and enhance its strength. The use of amphiphilic chitin in a PLA-starch biocomposite has not been reported. Furthermore, this study develops a novel green approach to using amphiphilic chitin as a compatibilizer between PLA and starch to enhance its properties for industrial application.2. PLA (4032D) has a molecular mass of 100 kg/mole on average, a specific gravity of 1.24, and an MFR (melt flow rate) of g/10 min . Chitin was commercially obtained in Sigma Aldrich, South Africa.Polylactic acid, starch, and amphiphilic chitin are the biocomposite components. Nature Works  supplied pelletized poly(lactic acid) (4032D) for this study. PLA (4032D) was used because of its outstanding resistance to oil, grease, twist, machinability, and dead fold properties. PLA melts between 150 and 170 degrees Celsius and has a glass transition temperature of 65 °C. The PLA has a tensile strength of 53.5 MPa, a modulus of 3500 MPa, and an impact strength of 2.99 kJ/m3 and filtered off. The gel was freeze-dried after washing in distilled water and grounded to powder.Amphiphilic chitin was produced using a two-step acetylation reaction, as shown in PLA in pellets form was reduced to a smaller size of 0.045–0.71 mm with a granulator AA-150 power  to enhance miscibility. The ground particles were dried at 60 °C in a dryer Luxor 50  for 4 h and then dissolved in chloroform (plasticiser). The amphiphilic chitin and starch of varying compositions were mixed with PLA solution in a Thermo Fisher Scientific Rheomixer 03 thermoelectric . The polymer blend was extruded to filament with a Thermo Fisher Scientific twin-screw extruder Process 11  at a temperature profile of 120–170 °C and quenched in water. The filament was pelletized with a Thermo Fisher Scientific pelletizer 11 , and Carver pressed into characterisation shapes for 15 min using a compression moulding machine  at 10 MPa, 170 °C. The percentage of PLA in the polymer blend composite is highest in the matrix. The test samples , tensile testing, thermogravimetry analysis (TGA and DTG), differential scanning calorimetry (DSC), and scanning electron microscopy were used to characterise the thermomechanical, mechanical, thermal, and morphological properties, respectively. The thermomechanical characteristics of materials were determined using dynamic mechanical analysis over a temperature and frequency range. This was accomplished with the assistance of a DMA 8000 PerkinElmer Inc. . The shape and dimensions of the samples were determined using the ASTM D4065 for polymer composites at −50 to 150 °C for 1 to 50 Hz. The change in modulus storage (E′), loss (E″), and loss factor (tan δ) were obtained.The thermal characterisation of the biocomposite was determined using a TG-IR-GCMS thermogravimetric analyser . The thermogravimetric test was conducted in accordance with ASTM E1131, 15–20 mg of sample, at 10 °C/min from ambient temperature to 600 °C in air. Origin software was used to analyse and plot the data (Pro 8.1). Additionally, differential scanning calorimetry was used to analyse the material’s behaviour as a function of temperature change using a DSC model 6 . Temperatures of the glass transition, crystallisation, and melting were measured. The samples used had a mass of between 5 and 7 mg. Tensile testing was performed on a 20 kN load using an Intron Universal Testing Machine (model 5966) . The crosshead speed was kept constant at 2 mm/s. Biocomposite samples with a cross-sectional size of 5 mm by 10 mm and a gauge length of 30 mm were created and submitted to the American standard (ASTM) for materials testing. The tensile test is performed in accordance with ASTM D3039. The microstructure of the tensile fractured surface of the biocomposite was examined with a scanning electron microscope (SEM). SEM was utilised to analyse the microstructure after the gold-coated material using secondary electron (S.E.) and backscattered electron (BSE) signals. The SEM was operated at a 2.00 kV SEI accelerating voltage.A soil burial test was also used to determine the material’s biodegradability qualities. The samples were cut into 2 cm × 2 cm squares and buried 10 cm into organic soil for 150 days. The pre-weighed PLA and biocomposite samples were weighed before burial and again after one month to determine the deterioration rate. The samples are removed and rinsed with distilled water before being dried in an oven for 24 h at 40 degrees Celsius before being weighed. Previous research and ISO 846  were used to conduct the soil burial test. The relative humidity of the compost soil was kept between 40 and 50 percent at room temperature, and the soil was injected with distilled water on a regular basis to keep it moist enough for microbial activity. The weight loss was calculated from the percentage difference between the final and the initial weight of the material at 0, 100 and 150 days.Water absorption was conducted to measure the wettability of the composite based on ASTM D570. The samples were pre-weighed and immersed in water for 24 h, and the initial and final weight differences were recorded. A dimension was 1 cm by 1 cm by 3 cm was used for all samples.−1, indicating O.H. and N.H. stretches, respectively. On the unaltered chitin, a minor peak was found between 2800 and 2930 cm−1, indicating aldehyde -C-H stretches. Additionally, from the amino group’s amide functional group and acetylation, a brief peak at 1600–1680 cm−1 is referred to as C=O and -C=C-. The alkene sp2 C-H bends are 1550 cm−1 (CO2−), 1400 cm−1 band sp3 -C-H bend, 1154 cm−1 C-O-C bridge, 800 cm−1, and 650 cm−1. The existence of 800 cm−1 bands in amphiphilic chitin indicated the presence of an alkene C-H bend, while an increase in 650 cm−1 bands indicated the presence of a C-H bond, indicating the presence of an alkyl group. Changes in the bands 1500 cm−1 and 800 cm−1 and the formation of new peaks are typical of alkyl group attachment (substitution) to the chitin chain. The alkyl group attachment occurred in the amine functional group present in the chitin in previous research on this modification method [2 were replaced [n method . During replaced . Figure replaced . Accordireplaced ,26.This DMA is an adaptable thermomechanical analysis technique that is frequently used to analyse polymeric composites’ viscoelastic and structural behaviour to identify their relevant damping and stiffness properties for various applications. Periodic stress or strain deforms the materials . DMA wasThe storage modulus of the biocomposite (E′) lowers progressively with an increase in temperature and more significantly as the biocomposite enters the glass transition area owing to the entire polymer chain’s free mobility . The E′ gE′ before glassy region) and 130 °C (rE′ before rubbery region) of the composite. Additionally, gE′ before glassy region) and 130 °C (rE′ before rubbery region) of the matrix. The rate of reduction of E′ value for both biocomposites and neat PLA across the glass transition area is identical. This is probably due to the hydrodynamic effects of the filler material introduced into the PLA matrix and mechanical constraints on the PLA matrix’s mobility and deformability . SimilarC is inversely proportional to the filler’s effectiveness [The value of tiveness . Table 2The lowest was obtained for biocomposite with P4S4C, and the highest was P8C. Therefore, this shows that the combined starch–amphiphilic chitin addition has more effectiveness than amphiphilic chitin or starch used separately. These coefficient values justify the increased storage modulus results and the combined use of starch and amphiphilic chitin fillers. This can be scientifically explained as a bridging effect of amphiphilic chitin having a compatibiliser effect due to its double functional group , allowing its bonding with starch and PLA.The loss modulus gives the energy lost per deformation cycle of a system at constant strain or stress amplitude . The losTan δ is a damping term that refers to the impact resistance of a material . As the Temperature, time, and frequency influence a material’s viscoelastic properties . This isThe graph of storage modulus, loss modulus, and dissipation factor are displayed in The effect of frequency on the loss modulus (E″) of the tidy PLA and biocomposite is shown in g values of all the samples with varying frequencies. Frequency, temperature, and time all affect a material’s viscoelastic characteristics. If material is subjected to constant stress, its elastic modulus will decrease. This is due to the material’s molecular rearrangement, which reduces localised stress. Modulus measurements made over a short period (high frequency) yield higher results, whereas measurements taken over a long period (low frequency) yield lower results [g of all samples significantly increases with increasing frequency. The increase in the Tg values means that the relaxation movement of PLA chains is delayed at high frequency. High frequencies can induce more elastic-like behaviour. If the frequency is chosen to be high enough, a material will behave stiffer than it can be. results ,42. The g of biocomposites is somewhat reduced for P8S, P6S2C, and P2S6C, but increased above that of PLA for P4S4C, and P8C, as indicated in g values are probably due to disruption in the matrix due to the addition of the starch filler, which is not compatible with the PLA. However, optimum Tg was obtained for P4S4C, with an equal percentage of amphiphilic chitin and starch due to the compatibility effect of amphiphilic chitin enhancing miscibility between PLA and starch. Previous studies show reduced Tg when degradable fillers are incorporated in PLA [g values reduce.Increased amphiphilic chitin (reduced starch) in the PLA matrix lowers the tan peak height, which can be related to the restriction of polymer molecule mobility. That is, the chain segments in the neat PLA are unrestricted. On the other hand, Td in PLA ,43. ThisThe single relaxation peaks of the DMA analysis can further be double-checked using the Cole–Cole analysis of viscoelastic response ,45. ColeThe biocomposite shows a well-separated Cole–Cole plot, which means the change in composition variation significantly affects their relaxation time and exhibits distinct dispersions . The devg) of PLA is 62.5 °C. The addition of filler to the PLA matrix reduces the Tg value slightly, hence increasing the material’s thermal degradability.The influence of filler on the melting behaviour of PLA was determined using DSC. c) drops from 107.5 °C to 106.2 °C as filler is added. This suggests that including a filler with lower crystallinity reduces the matrix’s nucleating ability, which results in a more amorphous biocomposite. The semicrystalline nature of chitin and starch resulted in a sharper crystalline temperature peak, as observed in samples P8S and P8C [The use of natural filler has been reported to enhance the thermal degradability of PLA ,28. Thes and P8C . Howeverm) is at 162.3 °C and reduced by adding the two fillers. Biocomposite with both fillers was observed to have two melting peaks. However, this melting peak disappears with either amphiphilic chitin or starch alone as fillers. This could be because when a single filler is employed instead of a double filler, less-perfect crystals have more time to melt and reorganise into crystals with better structural perfection before melting at a higher temperature [m value of PLA.It was also observed that PLA’s melting point  was used in this study to assess the thermal stability of polymeric materials. The result of the ultimate tensile strength (UTS) test reveals the load-carrying ability of the material. The tensile strength of the polymer biocomposites is shown in The biocomposite’s tensile strength was significantly greater than that of the neat PLA (53.5 MPa). However, the graph indicates that PC8 and PC6S2 have lower values due to decreased tensile strength values. The sample PC4S4, with a tensile strength of 87.5 MPa, had the highest tensile strength of the set of samples, followed by PC8 and PC2S6 with only amphiphilic chitin and starch filler. The results indicate that amphiphilic chitin and starch improve the tensile strength of the biocomposite. The higher tensile values of PC4S4 and PC8 justify the modification of amphiphilic chitin. The increase in tensile strength is most likely caused by physical or chemical interaction between the three polymeric components . TensileThe elongation properties of the biocomposite are presented in The tensile modulus value in Furthermore, the modulus value with an increased percentage of amphiphilic chitin has higher values than those of increased starch. Based on the result, the percentage increase is significant with amphiphilic chitin addition because of the amphiphilic properties of the modified chitin. Additionally, compared with the neat PLA, the combined effect of equal percentage amphiphilic chitin and starch on the tensile modulus of neat PLA is well above those of individual starch or amphiphilic chitin reinforcement. Based on this result, incorporating reinforcement in the PLA matrix enhanced the material’s toughness and reduced its brittleness. Furthermore, the addition of amphiphilic chitin increased the interfacial interaction of the polymeric material, which increased the modulus .The SEM images demonstrate a smooth surface, indicating the three-polymer blend’s compatibility or adherence. The neat PLA has a plane surface while other biocomposites showed patched filler. The PLA-starch (PS8) sample reveals distinct starch patches in the biocomposite, while PLA-amphiphilic chitin (PC8) almost did not reveal a different coloration from the PLA matrix. This is most likely owing to the chitin modification utilised. The tensile fracture surface morphology of the PLA–amphiphilic chitin–starch composite a–e exhibThe SEM images reveal an even distribution in the P4C4S sample d, which The networks’ bridging effect prevents voids and cracking and enables efficient stress transfer . PC8 Fif demonstThe percentage weight loss for the neat PLA and the biocomposites are shown in The neat PLA showed 1.1 ± 0.1%, 2.0 ± 0.11%, and 6.0 ± 0.2% weight loss at 50, 100 and 150 days, respectively. The reduction in PLA degradation rate is not spontaneous, probably due to its hydrophobic nature. PLA/starch (P8S) is seen to show the highest degradation percentage of 5.1 ± 0.13%, 10.0 ± 0.13%, and 19.1 ± 0.09%, at 50, 100 and 150 days, respectively. This high degradation could be due to the hydrophilic nature of starch, which absorbed the moisture content in the soil. Furthermore, PLA/amphiphilic chitin (P8S) was also seen to show a higher degradation percentage of 4.6 ± 0.12%, 9.5 ± 0.11%, and 18.5 ± 0.09%, at 50, 100 and 150 days, respectively, compared to the biocomposite with both fillers. The P6S2C, P4S4C, and P2S6C biocomposites showed an enhanced degradation rate than the neat PLA but lower than those with a single filler. Overall, neat PLA has the lowest percentage of weight loss while P8S has the highest.The weight loss shows that the biodegradability of the biocomposite was significantly improved compared with the PLA. Furthermore, the biocomposite with single filler (P8S and P8C) has a higher weight loss compared with those with double fillers. This is probably due to improved interaction in the microstructure of the biocomposite with two fillers due to the compatibility effect of amphiphilic chitin. The interaction possibly resulted in a stronger bonding which reduced the biodegradable rate. The reduction in biodegradability when a compatibiliser or plasticiser is used has been reported by Phua et al. . The autThe water absorption percentage of the neat PLA and the biocomposite is shown in g of all samples increase with increasing frequency due to the delayed relaxing of PLA chains when the high frequency is applied. If the frequency is set high enough, biocomposites would be stiffer. The DSC research reveals that PLA’s Tc peak declines with filler incorporation. This shows that the inclusion of both fillers with low crystallinity results in more amorphous biocomposite. All of the biocomposites had a somewhat lower Tg than the pure PLA. The TGA data indicate that the To of biocomposites decreases as the filler content increases. This implies that biocomposites have lower thermal stability than neat PLA, which is attributable to the filler’s lower thermal degradation. According to the findings of this study, a biocomposite composed of 4 wt% percent equal parts amphiphilic chitin and starch is deemed to be the optimal condition. The biocomposite developed in this study has potential application in packaging and biomedical implants due to its green compatibilizer and enhanced mechanical strength.Amphiphilic chitin was prepared using acetylation, and the biocomposite was successfully prepared with combined melt extrusion, pelletisation, and compression moulding technique. The storage modulus (E′) is between that of pure PLA and that of biocomposites. The E′ value increases as the amphiphilic chitin content grows and decreases as the starch content increases due to the matrix’s compatibility with the fillers, limiting the PLA matrix’s mobility and deformability. E″ is the modulus of loss that increases as the amphiphilic chitin content increases and decreases as the starch content increases. The damping (tan δ) value reduces as the amphiphilic chitin content increases and increases as the starch content increases. The effect of frequency on the DMA vales of biocomposites demonstrates a significant change in its properties. It is established that the E″ and the T


Heat-moisture treatment (HMT) is a safe, environmentally friendly starch modification method that reduces the digestibility of starch and changes its physicochemical properties while maintaining its granular state. Normal potato starch (NPS) and waxy potato starch (WPS) were subjected to HMT at different temperatures. Due to erosion by high-temperature water vapor, both starches developed indentations and cracks after HMT. Changes were not evident in the amylose content since the interaction between the starch molecules affected the complexation of amylose and iodine. HMT increased pasting temperature of NPS from 64.37 °C to 91.25 °C and WPS from 68.06 °C to 74.44 °C. The peak viscosity of NPS decreased from 504 BU to 105 BU and WPS decreased from 384 BU to 334 BU. The crystallinity of NPS decreased from 33.0% to 24.6% and WPS decreased from 35.4% to 29.5%. While the enthalpy values of the NPS declined from 15.74 (J/g) to 6.75 (J/g) and WPS declined from 14.68 (J/g) to 8.31 (J/g) at 120 °C. The solubility and swelling power of NPS decreased while that of WPS increased at 95 °C. Due to the lack of amylose in WPS, at the same HMT processing temperature, the reduction in peak viscosity of treated WPS compared to that of native starch was smaller than that of NPS. The resistant starch (RS) content of NPS after HMT at 120 °C was 73.0%. The slowly digestible starch (SDS) content of WPS after HMT at 110 °C was 37.6%. According to the International Diabetic Federation (IDF), 537 million people are currently living with diabetes, and this number is projected to increase to 783 million by 2045 . Type 2 The HMT is a common physical method to improve the RS content of starch. It involves exposing starch granules to high temperatures 80–120 °C) at low moisture levels 10–30%) for a specific period of time between 15 min and 16 h . Compare0–120 °C –30% for The NPS was donated by Huaou Starch Co., Ltd., Huhehaote, China, while the WPS was purchased from AVEBE, Veendam, The Netherlands. The porcine trypsin (P7545) and amyloglucosidase (A7095) were purchased from Sigma-Aldrich Chemical Co. , while the glucose oxidase-peroxidase (GOPOD) assay kit was obtained from Megazyme International Ireland Ltd. . All other chemical reagents were of analytical grade.Starch (20 g) was weighed, and water was added until the starch moisture content reached 25%. The sample was sealed in a stainless-steel reactor, equilibrated at room temperature for 24 h, and allowed to react for 2.5 h at 100 °C, 110 °C, and 120 °C, respectively. The samples were dried in an oven at 45 °C and sieved using a 100-mesh sieve. The NPS samples treated with HMT at 100 °C, 110 °C, and 120 °C were labeled NPS100, NPS110, and NPS120, while the corresponding WPS samples were labeled WPS100, WPS110, and WPS120, respectively. Native normal potato starch (NNPS) and native waxy potato starch (NWPS) represented the two native starches. A scanning electron microscope , operated at an accelerating voltage of 10–20 kV, was used to observe the morphology of the starch granules. The starch was evenly dispersed on conductive adhesive and sprayed with gold for 3 min , after which its morphology was observed at 500× magnification, and a suitable viewing angle was selected [A small amount of 1% starch suspension was collected and deposited onto glass slides. The focal length of the polarizing microscope was adjusted, and a suitable viewing angle was selected at 200× natural light, which was then adjusted to polarized light conditions to observe the Maltese crosses of different starches .g/g) were calculated as follows:The swelling degree was measured using a method similar to the one described by Yassaroh . Starch 2) were added, and then diluted with distilled water to 100 mL. The solution was allowed to stand for 10 min at ambient temperature prior to absorbance measurements at 620 nm. The amylose content was calculated from a standard curve plotted for mixtures of amylose and amylopectin from potatoes (0–100% amylose).The iodine absorbance method was used to determine the amylose content according to the national standard GB/T15683-2008 determination of amylose content in rice. The 100 mg starch sample (dry basis) was mixed with 95% ethyl alcohol (1 mL) and 1 M sodium hydroxide (9 mL). To induce gelatinization, the mixture was heated and cooled to room temperature. Next, the starch solution was transferred to a 100 mL volumetric flask and diluted with distilled water to 100 mL. From that, 5 mL were taken, and volume was adjusted to 50 mL with diluent. Subsequently, 1 mL acetic acid and 2 mL of iodide (KI) and iodine (Io), peak temperature (Tp), and conclusion temperature (Tc) were recorded. The enthalpy of gelatinization (ΔH) was estimated by integrating the area between the thermogram and the subpeak baseline, which was expressed in joules per gram (J/g) of dry starch [Water was added to 3 mg dry basis starch until reaching a total weight of 10 mg. The gold plate was sealed and equilibrated at room temperature for more than 24 h. The temperature was maintained at 30 °C for 1 min, after which it was increased to 120°C at a rate of 10 °C/min and then cooled from 120 °C to 30 °C at the same rate. All samples were measured at least in duplicate. The onset temperature . A 6% starch suspension was heated from 30 °C to 95 °C, which was maintained for 5 min, and cooled to 50 °C, which was maintained for 5 min. The heating and cooling rates were 7.5 °C/min.The prepared samples were measured using an X-ray diffractometer  at 40 kV and 40 mA with Cu Kα radiation (λ = 0.154 nm). Sample powder was packed tightly in rectangular glass cells and scanned at an angle range of 5° to 40° .The starch samples were digested refer to a method described by Englyst et al. . The enzp < 0.05.The Origin Program 8.0  was used to analyze and report the mean values and standard deviations. An analysis of variance (ANOVA) was conducted, followed by Duncan’s multiple-range test using SPSS 19.0. The significance level was set at SEM allows for a convenient and intuitional characterization of starch granules. Various studies have shown that no obvious grain starch deformation is evident after HMT modification ,22. The The swelling power can reflect the bonding degree of the internal bonds in the particles to some extent. The starch expanded from a relatively loose amorphous region, followed by expansion in an amorphous region near the crystalline region, and finally, the expansion of the crystalline region . The sweThe solubility of the NPS and WPS starch was reduced after HMT at 65 °C and 80 °C. The solubility of WPS increased significantly after HMT at 95 °C compared to NPS . The redAlthough HMT may increase the amylose fragments , our stuo, Tp, and Tc increased  absorbed during the gelatinization was monitored with DSC. The enthalpy characteristics of the two types of starches varied significantly after HMT, while the Tncreased , which w HMT. Li  believeds region . The ents region . Lan [40The viscograph was used to determine the pasting properties of starches. Temperature significantly affects the paste properties. The pasting temperature of the two processed starches increased compared to the native samples . As a reXRD confirmed that the starch crystallinity could be divided into A, B, and C types, and was affected by amylopectin content, average chain length, the orientation of the double helices to the X-ray beam, and crystallite size . The X-rStarch digestibility is influenced by two factors. One is the ability of amylolytic enzymes to bind to starch granules, and the other factor concerns how many binding sites are present and how they are surrounded . As showWPS and NPS are compared to better understand how HMT affects their physico-chemical properties. Under the same treatment conditions, WPS were more sensitive than NPS to HMT. Increasing the value of amylopectin to amylose makes starch granules active by absorbing more water during treatment, which promotes their expansion and favors morphological changes driven by thermal force. Compared with the original starch, the swelling degree and solubility of the two treated starches decreased, while the solubility and swelling of WPS increased at 95 °C because of completely gelatinization. After HMT, the gelatinization temperature and thermal stability of the two starches increased while the enthalpy decreased. This means HMT is able to offer a more stable and resistant architecture, allowing it to be used in products that are easy to age. Hot paste stability and swell power of WPS after HMT were superior. Hence, it is used as a thickener to increase the smoothness and stability of caramel and gummy. The crystalline type of the starch is converted from type B to type A, while the crystallinity is decreased after HMT. Furthermore, the higher the amylose content, the more RS formed during HMT and, in contrast, the higher SDS content. The physical and chemical properties of the two starches change after HMT at the same temperatures, indicating that the presence of amylose affects the amylopectin reorganization. The fine structure of amylopectin and amylose is a key factor forming resistant starch. More attention can be paid to the nano-level changes in HMT starch granules and the specific locations of degraded molecular chains. In the future, starch raw materials and processing conditions can be selected according to the demand for starch digestibility to achieve the intelligent control of starch digestibility. Due to the high RS content, HMT-treated potato starch can be used to develop low GI foods for glycemic control in patients with diabetes mellitus. Considering the compositional complexity of food, the changes in the properties of the complexes composed of starch and other components, such as proteins, fats, and polyphenols, after HMT require further investigation. Furthermore, the influence of the HMT heating source, working pressure, and cooling procedure on the properties of starch needs exploration.


Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are widely used in the treatment of coronary heart disease, but the best revascularization method for multivessel coronary artery disease (MVD) patients is still controversial. Hybrid coronary revascularization (HCR), together with CABG and PCI, have been proved to be feasible methods, but the long-term effect of HCR is not as clear as CABG.By October 2020, we retrieved articles from PubMed, Web of science, EMBASE and Cochrane library databases. The main results are based on major adverse cardiovascular and cerebral events (MACCE).A total of 18 articles  and 15 observational studies) were included in this meta-analysis. The outcomes of MACCE in the HCR group at perioperative, short-term (30 days to 1 year), medium-term (1 year to 5 years) and long-term (5 years and above) follow-up period were similar to those in the CABG group. The mortality rates of patients in perioperative, short-term and medium-term follow-up were similar to those in the CABG group, but lower than that in the CABG group at long-term follow-up . The revascularization rate was higher in the HCR group during the perioperative period , short-term  and mid-term follow-up .Our results reveal that HCR is a safe and therapeutically effective alternative in treatments for MVD patients. It has not only less short-term adverse effect, but also better long-term effect, especially in death.The online version contains supplementary material available at 10.1186/s13019-022-01903-w. According to the World Health Organization report, in the 2016, Cardiovascular diseases (CVDs) are the number one cause of mortality globally, killing approximately 17.9 million each year . CoronarHybrid coronary revascularization (HCR), grafting LIMA to LAD along with PCI in the non-LAD vessels, is a less invasive alternative for MVD patients while maintaining durability. HCR may decrease repeat revascularization rate and enhance long term outcomes through benefiting from LIMA-LAD revascularization. As a minimum invasive intervention, HCR reduces risk of bleeding and infection, time of mechanical ventilation, and length of stay , 14.In the recent years, HCR has received considerable attention as being the most suitable revascularization strategy for patients with MVD. In the current meta-analysis of HCR and CABG, no significant change in terms of in-hospital death, MI and stroke was observed between the two groups. Moreover, the need for red blood transfusion, length of hospitalization, length of intensive care unit (ICU) stay, and ventilation time were all better in HCR group –17. HYBRSeveral RCTs and observational studies have compared the long-term outcomes of HCR and CABG. Herein, a meta-analysis was conducted to compare the long-term difference between HCR and CABG in patients with MVD.Systematic literatures were retrieved using these databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) : PubMed,Studies that have met the following criteria were included: (1) original research that compared HCR and CABG for MVD patients; (2) RCTs or observational studies (cohort or case–control studies); (3) relevant data reported. Exclusion criteria were as followed: (1) ongoing studies or with unavailable data; (2) duplicate reports. The latest article will be included / selected if there were duplicate publications. (3) patient with previous experience of coronary revascularization. (4) The language was not English.Two independent authors evaluated the included RCTs and observational studies via the Cochrane Collaboration’s tool  and the 2 statistic. The fixed effects model was applied when p > 0.1 otherwise the random effects model would be used. p < 0.05 was considered statistically significant.We used Review Manager 5.3 (The Cochrane Collaboration) to analyze the extracted data. The odds ratio (OR) and 95% confidence interval (CI) were calculated to establish dichotomous variables while the standardized mean difference (SMD) and the weighted mean difference (WMD) was were calculated for continuous variables. Heterogeneity among studies was quantified using IThe search strategy originally generated 6,277 relevant clinical records from the four mentioned databases. There were eventually 18 studies  MACCE between HCR and CABG . MACCE were followed up as time went on. 11.3% patients treated with HCR and 8.9% patients with CABG had suffered from MACCE during one-year follow-up (30 days to 1 year) . A total of 13.7% patients treated with HCR and 8.7% patients who had undergone CABG have suffered from at least one kind of the MACCE during mid-term follow-up (1 year to 5 years) . MACCE occurred in 28.6% patients after HCR and 30.2% patients after CABG during the long-term follow-up (5 years and above) . The outcomes of MACCE at short-term, mid-term and long-term follow-up were not statistically dissimilar. Detailed data are shown in Table 0 days MATwelve studies referred to the MI. No significant difference was observed to the incidence of MI between HCR group and CABG group during perioperative period ≤ 30 days) ; short-term (30 days to 1 year) , mid-term (1 year to 5 years)  and long-term follow-up (5 years and above) . Death was reported in eight studies. The mortality in perioperative period , short-term  and mid-term follow-up  between HCR group and CABG group were not significant. Three articles have mentioned the death during long-term follow-up. Patients who had undergone HCR had a lower risk of mortality rate during long-term follow-up compared with those who had undergone CABG  , short-term and mid-term follow-up . But the prevalence of long-term follow-up repeat revascularization was not significantly different between the two groups . Patients treated with HCR did not display a significant reduction in risk of developing a stroke as compared to those who underwent CABG in perioperative period , short-term  and long-term follow-up   Stents cannot (but bypass grafting can) prevent disease progression near the injured site . (2) LonOur data seems to have revealed a more pronounced ascent trend in mortality rate in CABG group compared with HCR group as follow-up duration increased. There was no significant difference in perioperative period; short-term, mid-term follow-up between patients underwent HCR and CABG respectively. However, in the long-term follow-up, patients who had undergone HCR had a drastic advantage over those who had undergone CABG. The speculative mortality trends conclusion raised by another study is in accordance with ours . This maBesides, there was no difference in stroke and MI in each period between two groups. This conclusion is consistent among all existing RCTs , 26. SarIn a large number of randomized trials, the combination of clopidogrel and aspirin has been proved to drastically reduce the incidence of stent thrombosis in PCI patients . TogetheWe found that the rate of AF was relatively low in the perioperative period of HCR. A series of reports investigated that the incidence of AF after minimally invasive CABG (MIDCAB) is between 4 and 23% . These dIn general, HCR has better perioperative results with a good trend of long-term survival rate, but its repeat revascularization rate is higher in the short- and medium-term period. Although repeat revascularization is often evaluated, it is far less important for patients than other endpoints, such as death, MI. It is a complicated to define whether repeating revascularization for patients with stable condition is essential or not. Among all the influential factors, the most vital ones are instructions and preferences, while other endpoints are more qualitative rather than quantitative assessed . TherefoThough in theory, HCR combines the advantages of both procedures, it is still suggested that HCR actually combines the unfavorable disadvantages. For example, performing two types of invasive surgery in a short period of time requires a high degree of cooperation between the two surgical teams, not mentioning the higher cost. Therefore, HCR needs to be generalized with greater advantages in follow-up.Our study also has some limitations. Firstly, there are only three RCTs compareing the safety of HCR and CABG while more observational studies have been included. Therefore, chaos between the trial population and selection bias may affect the results. Secondly, the definition of MACCE varies from study to study. Most studies define MACCE as a composite endpoint of MI, stroke, mortality, and repeat revascularization. Others replace repeat revascularization with target vessel revascularization (TVR), or delete stroke. After all, there are few studies with different definitions, hence the general speculation is not significant.In summary, HCR is relatively less invasive with better perioperative results, and hold a higher trend in long-term survival rate for MVD patients. HCR has the same therapeutic effect but lower long-term mortality incidence compared with CABG. Therefore, we believe that HCR is a promising alternative methods for MVD patients though more long-term RCTs with large-scale are needed in the future.Additional file 1: Table S1. Characteristics of all the studies included in the meta-analysis.Additional file 2: Table S2. Quality assessment of studies included.


Sophora flavescens, has great potential to alleviate pulmonary emphysema via its anti-inflammatory and antioxidative activities. Pulmonary administration is the most preferable way for the treatment of lung diseases. To improve the in vivo stability and pulmonary retention of OMT, OMT-loaded liposome with carboxymethyl chitosan (CMCS) modification was developed. The CMCS was modified on the surface of OMT liposomes via electrostatic attraction and covalent conjugation to obtain Lipo/OMT@CMCS and CMCS-Lipo/OMT, respectively. A porcine pancreatic elastase (PPE)-induced emphysema mice model was established to evaluate the alleviation effects of OMT on alveolar expansion and destruction. CMCS-modified liposomal OMT exhibited superior ameliorative effects on emphysema regardless of the preparation methods, and higher sedimentation and longer retention in the lung were observed in the CMCS-Lipo group. The mechanisms of OMT on emphysema were related to the downregulation of inflammatory cytokines and the rebalancing of antioxidant/oxidation via the Nrf2/HO-1 and NF-κB/IκB-α signaling pathways, leading to reduced cell apoptosis. Moreover, the OMT liposomal preparations further enhanced its anti-inflammatory and antioxidative effects. In conclusion, pulmonary administration of OMT is a potential strategy for the treatment of emphysema and the therapeutic effects can be further improved by CMCS-modified liposomes.Pulmonary emphysema is a fatal lung disease caused by the progressive thinning, enlargement and destruction of alveoli that is closely related to inflammation and oxidative stress. Oxymatrine (OMT), as a bioactive constituent of traditional Chinese herbal  Pulmonary emphysema is caused by damage to the air sacs in the lungs and is characterized by the gradual enlargement and destruction of alveoli in the lung, which is one of the lung conditions that causes breathing difficulties . AlthougOxymatrine (OMT) is a quinazoline alkaloid extracted from Sophora flavescens. Previous studies have revealed that OMT exerts anti-inflammatory, antioxidative stress and anti-apoptotic effects on various diseases, such as liver failure , diabetiFor the treatment of respiratory diseases, pulmonary administration is preferred due to its advantages in escaping from first-pass metabolism and directly targeting delivery to lung tissue, leading to high bioavailability, rapid absorption, and fewer adverse effects ,18. NeveSurface modification is a promising strategy for preventing liposomes from fast elimination by fast blood flow and immune recognition, resulting in an improved half-life in vivo . CarboxyIn this study, we developed two CMCS-modified OMT liposomes via physical adsorption and covalent conjugation, respectively, for pulmonary administration. The ameliorative effects of OMT liposomal preparations on an emphysema mice model were primarily evaluated, and the mechanisms were explored in terms of anti-inflammation and antioxidation . This stOxymatrine (OMT), polyoxyethylene stearate (SPEG), rhodamine B (RB), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and carboxymethyl chitosan  were offered by Macklin Biochemical Co., Ltd. , respectively. Octadecylamine (ODA) and N-Hydroxysuccinimide (NHS) were supplied by Adamas-beta . Soybean phospholipid (SPC) was supplied by A.V.T Pharmaceutical Co., Ltd. . Cholesterol (Chol) was purchased from VetecTM reagent grade . Porcine pancreatic elastase (PPE) was purchased from Macklin Biochemical Co., Ltd. .Six-week-old C57BL/6 mice of either sex (18–22 g) were supplied by the Experimental Animal Center of Guizhou Medical University. All animal care and experiments have been approved by the Animal Welfare and Ethics Committee of Guizhou Medical University (No: 1804102). Mice were raised at room temperature (23 ± 2 °C) and constant humidity (45 ± 10%) in an SPF condition. One week before starting the experiments, animals were placed for adaptation.The OMT liposomal preparations were prepared using the pH-gradient method. In brief, SPC, Chol, ODA and SPEG were dissolved in dichloromethane at a mass of 10:2:1:1 and evaporated from lipid film under reduced pressure for 30 min using a rotary evaporator . The lipid film was resuspended with 0.1 M critic acid buffer (pH 4.0) and then incubated in a water bath for 30 min at 60 °C. Next, the film was hydrated using a water bath sonicator (200 W) for 3 min. The obtained Lipo were extruded through a 0.45 μm filter. After the formation of the empty Lipo, the external pH was adjusted to pH 7.0 with a 2 M NaOH solution. Then, OMT was added to the Lipo solution. The Lipo/OMT were then incubated for 30 min at 40 °C with occasional shaking. For the preparation of Lipo/OMT@CMCS, CMCS was coated on a liposomal surface through electrostatic adsorption. CMCS solution, dissolved in phosphate-buffered saline (PBS) at a concentration of 20 mg/mL, was added dropwise to the freshly prepared Lipo/OMT solution at a volume ratio of 1:60 and stirred at 500 rpm for 30 min. Furthermore, CMCS-Lipo/OMT was obtained via an amide reaction. The carboxyl groups in CMCS were activated by EDCI and NHS, and then the activated carboxyl groups were bonded to primary amines in ODA . In brieThe RB-encapsulated liposomes were prepared via a film dispersion method. Briefly, 4 mL of RB solution at 0.5 mg/mL was incubated with the thin lipid film in a water bath at 60 °C for 30 min. The Lipo/RB was obtained after passing through a 0.45 μm filter. After the modification of CMCS via the same methods described above, Lipo/RB@CMCS and CMCS-Lipo/RB were obtained.v/v) at a flow rate of 1 mL/min and was detected at a wavelength of 210 nm. Equations (1) and (2) were used to calculate the drug loading efficiency (LE) and loading capacity (LC). The in vitro release profile was measured using the dialysis method. A dialysis bag  holding 2 mL of OMT-encapsulated liposomal preparations was immersed in 15 mL of PBS in a capped bottle and incubated at 37 °C with stirring at a speed of 50 rpm. At a predetermined time point, 500 μL of the medium was withdrawn and replenished with fresh PBS at the same volume. The amount of OMT released into the medium was determined using the HPLC method described above. The release medium satisfied the sink conditions, and the experiments were performed in triplicate. To determine the stability of the formulations, Lipo/OMT, Lipo/OMT@CMCS and CMCS-Lipo/OMT were kept in an incubator at 4 °C for one week. The particle size, PDI and zeta potential were measured over the storage periods.The morphology of Lipo/OMT, Lipo/OMT@CMCS and CMCS-Lipo/OMT were observed by transmission electron microscope . The particle diameter, polydispersity (PDI), and zeta potential of Lipo/OMT, Lipo/OMT@CMCS and CMCS-Lipo/OMT were measured by a NanoBrook 90Plus PALS . The concentration of the OMT in liposomal preparations was assayed by high-performance liquid chromatography (HPLC), which used an Agilent 1260 HPLC system with a reversed-phase column Hanbon C98200305228  at 30 °C. The mobile phase consisted of acetonitrile and 0.2% triethylamine  was used for the intratracheal spraying of free RB and RB-labeled liposomes as previously described [2 inhalation and put in a supine position. Once the sprayer needle was inserted into the trachea to reach the carina, it was retraced 1–2 mm to avoid the splash of the suspension. Then, 50 μL of suspension was administered in one push. Above all, the standard curves of plasma and various organs  were established. Then, the mice were grouped randomly and quantitively pulmonary administered with free RB and RB-labeled liposomes at an RB dose of 750 μg/kg. Different organs and blood samples were collected at the fixed intervals of 0.5, 1, 2, 4 and 8 h. The lung tissue was homogenized in PBS and the plasma was separated by centrifugation at 10,000× g for 10 min. Ethanol was added to the homogenates and plasma under constant stirring at 500 rpm for 30 min, respectively. Then, the mixture was centrifuged at 3000× g for 15 min and the supernatant was determined via a fluorescence spectrophotometer .Organ distributions were obtained after pulmonary administration of free RB, Lipo/RB, Lipo/RB@CMCS and CMCS-Lipo/RB. A handheld aerosolizer  was collected on the right lung of mice in each group by washing three times with 0.8 mL of ice-cold PBS. Then, the right lung was carefully excised and stored at −20 °C for biochemical analyses and Western blotting. The left lung was removed and inflated in 4% paraformaldehyde  for histological analysis.We performed a mice model of emphysema induced by PPE, as previously described . Mice reg for 10 min at 4 °C, and the sedimented cells were resuspended in 0.5 mL of PBS and dropped onto the slide. After drying, Wright Giemsa dye was added for staining. Two hundred leukocytes were counted under the optical microscope, and the proportion of macrophages, neutrophils and lymphocytes was recognized via morphological characteristics and calculated. Briefly, the cytoplasm of the neutrophils was in the form of pink, rod-shaped nuclei with variable leaves and was mainly trilobal with uniform and fine granules in the cytoplasm. In comparison to the neutrophils, the macrophages were larger and possessed a more rounded soma and cytoplasm, with fine and loose chromatin, and were relatively pale in coloration. The nuclei of lymphocytes are large, dark purple, and densely stained with minimal cytoplasm [The BALF from each mouse was centrifuged at 4000× ytoplasm ,35,36.The fixed left lung tissues of mice were dehydrated in a series of graded ethanol and embedded in paraffin. The slides of 5 μm thickness were stained with hematoxylin and eosin (H&E) staining for morphometric and histopathological evaluation. The mean linear intercept (MLI) was assessed by qualifying the airspace enlargement and destruction. The MLI was calculated by counting the number of alveolar walls crossed by the reticulum in ten randomly selected fields of tissue sections per lung sample as previously described . Furtherg for 10 min. Tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) were quantified in plasma samples using a specific enzyme-linked immunosorbent assay (ELISA) with a mouse anti-mouse monoclonal antibody. The procedure complied with the manufacturer’s instructions .The plasma was separated from the blood samples by centrifugation at 10,000× g for 10 min, the supernatants were collected and dissolved in extraction buffer for the analysis of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) content. The total protein in the sample was determined using the BCA Protein Assay Kit . To examine the level of lipid peroxidation in the lung tissue, the MDA concentration was assessed by the lipid peroxidation MDA Assay Kit . For antioxidative enzyme activities in the lung tissue, SOD and CAT levels were detected with a total SOD Assay Kit with WST-8  and a CAT assay kit .The lung tissue was homogenized in PBS and centrifuged at 3500× g for 10 min. The supernatant was collected, and the protein concentrations were evaluated using the BCA Protein Assay Kit  according to the manufacturer’s instructions. The protein sample (45 μg) was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes . Subsequently, primary antibodies against Nrf2, HO-1, B-Cell Leukemia/Lymphoma 2 (Bcl2), BCL2 Associated X (Bax), IκB alpha (IκB-α) and beta-actin (β-actin) were purchased from ProteinTech . Primary antibodies against phosphor-NF-κB p65 antibody were purchased from Cell Signaling Technology . After incubating with the corresponding secondary antibody, the proteins were performed using an enhanced chemiluminescence kit  and Syngene Gel Imaging system . The levels of proteins in the membranes were quantified by Image Lab software .Lung tissues were lysed in radioimmunoprecipitation assay buffer  containing 1% protease inhibitor phenylmethylsulfonyl fluoride . The lung samples were homogenized and centrifuged at 10,000× t-test. Multiple groups were made using one-way ANOVA followed by a post hoc Tukey test among the model and treatment groups. The significance was defined as p-values of less than 0.05.Data are presented as mean ± SD. Comparisons between the experimental groups were assessed by an unpaired, two-tailed Student’s In the preparation of OMT liposomes (Lipo/OMT), the weight ratio of SPC: Chol and SPC: OMT was optimized in terms of LE and LC of OMT . Chol afUpon OMT encapsulation and CMCS modification, the particle size, PDI and zeta potential of the OMT liposomal preparations were evaluated a,b. The To evaluate the stability of the OMT liposomal preparations, particle size, PDI and the zeta potential of the OMT liposomal preparations stored at 4 °C were detected for 7 days . A reasoThe pulmonary sedimentation of preparations is closely related to the diameter of nebulized droplets, which mainly depends on the device for pulmonary administration . RB-encaWe investigated the effects of OMT and its liposomal preparations on PPE-induced emphysema in mice via quantitative pulmonary administration. A scheme of the experimental protocol is shown in Apoptosis is a key process associated with the development of emphysema . We examAccumulating evidence shows that overwhelmed inflammatory responses and excessive oxidative stress play essential roles in the pathogenesis of emphysema ,43. AbovThe accumulation of macrophages and neutrophils in the lung of model mice not only boosts inflammatory cytokines and cell release but also enhances the production of reactive oxygen species (ROS). The PPE-treatment leads to an excessive generation of MDA and reduced activity of antioxidant enzymes SOD and CAT in the lung of mice g–i. AfteTo further investigate the protective mechanisms of OMT and its liposomal preparations on PPE-induced emphysema, the expression of NF-κB and Nrf2 was detected. Researchers found that the expression of NF-κB p65 is significantly increased in emphysema patients and animal models ,48. As aIn our study, Nrf2, HO-1 and IκBα were markedly reduced and phosphorylated NF-κB p65 was significantly elevated in the PPE-stimulated emphysema model in comparison with that of the control group . PulmonaIn summary, pulmonary administration of OMT could exert its ameliorative effects in emphysema mice by regulating NF-κB p65 and Nrf2, which suppressed the inflammatory reactions and oxidative stress. Its anti-inflammatory and antioxidant effects were increased after liposome encapsulation and CMCS modification. Moreover, covalent conjugated CMCS on the periphery of liposomes exhibited longer retention in the lung and superior antioxidant effects. It is hypothesized that covalent conjugation of CMCS might provide a more stable OMT liposomal preparation compared with the CMCS modification via electrostatic adsorption. However, no significance was detected between the CMCS-modified OMT liposomes in terms of MLI, apoptosis, and anti-inflammation.In the present study, we prepared Lipo/OMT by a pH-gradient method and two CMCS-modified Lipo/OMTs via electrostatic attraction (Lipo/OMT@CMCS) and covalent conjugation (CMCS-Lipo/OMT). It was found that pulmonary administration of OMT could exert its anti-inflammatory and antioxidant effects to alleviate PPE-induced emphysema by regulating the Nrf2/HO-1 and NF-κB p65 signaling pathways. CMCS-modified OMT liposomal preparations enhanced the therapeutic effects of OMT on emphysema. Despite the superior sedimentation and retention of CMCS-Lipo/RB in the lung than Lipo/RB@CMCS, no significant difference was detected between Lipo/OMT@CMCS and CMCS-Lipo/OMT in the treatment of emphysema. Hence, CMCS-modified liposome might be a promising drug delivery system for pulmonary administration of OMT to treat emphysema.