Since the early 1990s, the use of trap-neuter-return (TNR) as a humane alternative to the lethal management of stray and feral cats  has expanded in the United States. Over this time, numerous studies have pointed to the effectiveness of TNR at reducing community cat numbers, although many of these investigations have covered relatively short time periods (3 years or less). A seminal paper by Levy et al. in 2003, documented a significant decline in community cat numbers on the campus of the University of Central Florida (UCF) over a 11-year period. Since 2017, a series of peer-reviewed articles have described other examples of long-term reductions in community cat numbers associated with TNR. The present study adds to this growing body of evidence by revealing the extent to which the results first reported by Levy et al. have been sustained over a subsequent 17-year period. After a total of 28 years, 10 (5%) of 204 total cats enrolled in the UCF TNR program, remain on campus and the campus community cat population has declined by 85% from the completion of an initial census in 1996 to 2019.A growing body of evidence indicates that trap-neuter-return (TNR) is not only effective at reducing community cat numbers, but that such reductions are sustainable over extended periods. Recently, a series of peer-reviewed articles documenting long-term declines in community cat populations associated with TNR have been published. The present study adds to this pool of evidence by updating and reexamining results reported from the campus of the University of Central Florida (UCF) in 2003 by Levy et al. From 1991 to 2019, a total of 204 cats were enrolled in a volunteer-run TNR program on the university grounds; 10 cats (5%) remained on site at the conclusion of the present study. The campus community cat population declined by 85% between 1996, the year an initial census (indicating the presence of 68 cats) was completed, and 2019. In addition, 11 of 16 total colonies were eliminated over a 28-year period. These results occurred despite significant growth in enrollment at UCF over the same time frame, which suggests that with sufficient ongoing management of colony sites, declines in community cat populations associated with TNR are sustainable over long periods and under varying conditions. For decades much controversy has surrounded the management of stray and feral cats (henceforth referred to as community cats). Among the key factors prompting policy makers to take action are concerns over wildlife and public health impacts ,2,3, nuiRecently, a number of TNR studies documenting long-term reductions in community cat populations have been published: nine years by Swarbrick et al. 2018) , 10 year18 , 10 yThe University of Central Florida is located 21 km east of downtown Orlando, Florida, and covers 573 hectares. The campus population of students and staff has risen from an estimated 38,000 in 2002 to over 68,000 students and 13,000 employees in 2018 . IncreasSeveral colonies of cats formed near food service buildings and dormitories soon after the university opened in 1968. Over the ensuing decades, the number of colonies grew and cats were periodically trapped and euthanized in response to nuisance complaints. A concerted management program consisting of trapping, sterilization, vaccination, and return of cats to campus locations or adoption began in 1991.The program protocol called for cats to be trapped in humane box traps, transported by Friends of Campus Cats to a veterinary facility  to be sterilized and vaccinated for rabies and viral rhinotracheitis/calicivirus/panleukopenia (FVRCP). As practicable, follow-up rabies vaccines have been administered to cats who have remained on campus for a number of years. Cats were also ear-tipped and received minor medical care , as warranted and resources allowed. Routine feline immunodeficiency virus/feline leukemia virus (FIV/FeLV) testing of selected cats  was part of the standard program protocol until 2000. Cats testing positive were euthanized. The last cat euthanized for a positive FIV test was in March of 2000, while the last cat euthanized for a positive FeLV test was in September of the same year.A census of the campus cat population, began upon inception of the program in 1991 and completed in 1996, was conducted by four members of Friends of Campus Cats. The number of cats residing on campus has been monitored on an ongoing basis since completion of the initial census. During daily feeding sessions, caretakers noted novel or unusual circumstances at colony sites, including suspected health problems among the cats and changes in attendance . The maximum number of feeding stations  installeCats were recorded and their presence tracked as they were discovered on the UCF campus. Each cat was assigned a name and tracked by colony affiliation on a Microsoft Excel spreadsheet. The enrollment date, description (as well as a photograph), sex, age category (adult or kitten), perceived level of socialization at first appearance , date neutered/neuter status, departure date (if applicable), and final outcome (if applicable) for each cat was documented. Kittens were defined as cats estimated to be six months of age or younger. Level of socialization was determined at enrollment and was not revised to reflect changes over time, even when cats subsequently became more social or friendly to specific individuals .Spreadsheets tracking the data listed above, updated through March of 2019, were analyzed as part of the present study. End-point data were compared to results of the initial census and to published results from the conclusion of the initial observation period in 2002. Semi-structured interviews were conducted with two of the founders of Friends of Campus Cats, who were also co-authors of the 2003 paper, in order to provide context and fill information gaps.Consistent with what was reported by Levy et al. , descripA total of 204 cats were recorded on campus during the combined 28-year reporting period ending in March of 2019; 49 (24%) of the cats arrived on campus after the end of the initial observation period in 2002, at which time 155 (76%) cats had been recorded. At the end of the combined observation period, the total number of cats recorded on campus consisted of 43 (21%) socialized cats and 161 (79%) unsocialized feral cats (at first appearance); 111 (54%) adults and 93 (46%) kittens (age category determined at enrollment); and 114 (56%) males and 90 (44%) females . From thThe median number of cats enrolled in the program during the initial observation period was 11 per year  compared to the median number of 2.5 enrolled per year  during the follow-up observation period. The amount of time between enrollment in the program and sterilization surgery declined from an average of 1 year  for the initial observation period to an average of 0.1 years  for the follow-up observation period .At the end of the combined observation period, 10 (5%) of the 204 total cats remained on campus; 23 (15%) of the original 155 cats remained on campus at the end of the initial observation period, so this change documented between observation periods represents a 57% decline in population over the intervening 17 years. The cats remaining on campus at the end of the combined observation period had resided there for a median of 3 years ; no cats continued to remain on campus from the end of the initial observation period. When the number of cats remaining on campus at the conclusion of the combined observation period was compared to the 68 cats recorded on campus at the end of the initial census, which concluded in 1996, an 85% decline in population size occurred over 23 years. All 10 of the cats who remained on campus in 2019 had been sterilized.In addition to the 10 cats remaining on site at the end of the combined observation period, 92 (45%) cats had been adopted, 49 (24%) disappeared, 23 (11%) were euthanized, 17 (8%) were known to have died, 10 (5%) relocated to one of the wooded areas that surround much of the campus (status unknown), and 3 (1%) were confirmed to have moved to a location on the outskirts of campus outside of the program area .Overall, on an absolute basis, the number of cats adopted from the campus TNR program between 2002  and 2019 (end of the combined observation period) increased from 73 to 92 ; howeverCats not adopted within six months of enrollment (152), regardless of their ultimate disposition, spent an average of 4.2 years  on campus; 91% of such cats were feral, including 34 of 40 who were adopted on an average of 5.1 years  after arriving on campus.Between the end of the initial observation period in 2002 and the combined observation period in 2019, the total number of cats known to have died after being returned increased from 10 (6%) to 17 (8%); eight were killed by automobiles and nine died of unknown causes. Such cats resided on campus for a median of 3.9 years . The number of cats euthanized increased from 17 (11%) to 23 (11%); nine were euthanized due to serious illness, two after a traumatic injury, and 12 after testing positive for FIV (10) or FeLV (2). Cats who were euthanized lived for a median of 4.3 years  on campus.The number of cats who disappeared rose from 23 (15%) to 49 (24%). Cats who disappeared resided on campus for a median of 3.1 years . During the follow-up observation period, one additional cat moved “to woods.” This cat was not observed subsequent to relocation to the wooded research park located adjacent to the southern edge of the UCF campus. Cats last seen in the woods resided on campus for a median of 0.3 years . All but one cat (58/59), who either disappeared or moved to the woods, were feral.Since the end of the initial observation period, three cats were confirmed to have joined a colony on the periphery of the campus grounds, outside of the program area, requiring the creation of a new category of disposition (“to out of area”) as listed in In all, 16 discrete colonies were identified ; in addition, a varying number of cats roamed with an apparent attachment to no particular colony . The maximum number of cats in each of the 16 colonies ranged from 1 to 25; the number of NFC cats ranged from 0 to 11. Overall, 11 of the 16 colonies were eliminated, three had the same population (1) at the end of the combined observation period as at enrollment, and two colonies increased in size (one colony from one to two cats and the other colony from two to three cats); two NFC cats were documented at the inception of the program in 1991; and two (different) NFC cats were documented in 2019.As of 2002, Levy et al. reported that 3 of 11 colonies had been eliminated and that “despite the presence of cats for 7 to 9 years before colonies were disbanded and the ongoing availability of food, these colonies have not been reestablished by new arrivals” [As observed by Levy et al. , not allThe purpose of the present study was to determine the extent to which results of a long-term TNR program, as reported in a seminal work by Levy et al.  in 2003,Levy et al.  reportedSimilarly, the percentage of the total number of cats enrolled in the program who remained on site at the end of the combined study period (5%) was smaller than what was observed at the other three locations . PerhapsIt was suggested by those interviewed for this study that the UCF TNR program’s low profile, including the placement of feeding stations at inconspicuous locations, which allowed cats to wait for food and eat out of public view, was an important factor in reducing abandonment on campus. The university’s ongoing commitment to a “no pets” policy was thought to have discouraged abandonment as well. Limiting the abandonment of pet cats and vigilance in monitoring of the program area for new arrivals are credited with helping to sustain the reductions in free-roaming cat numbers in the long term ; this coThe various outcomes experienced by cats in the program , when expressed as a percentage of the total number of cats enrolled, changed little from 2002 to 2019 . Two notWhen the outcomes over the combined observation period are compared to the disposition of cats enrolled in two other long-term TNR programs, Chicago and Sydney, Australia, a smaller percentage of cats remained on site  and disappeared , a greater percentage of cats were adopted , and the percentage of cats euthanized  or known to have died  fell in between what occurred at the other two locations ,20. Of nLevy et al. reported that at the end of the initial observation period, “in general, the cats were in adequate physical condition, and only 4% were euthanized for humane reasons” . ThroughAs noted by Levy et al. , adoptioA long-term commitment to the sterilization of the UCF community cat population resulted in a sharp decline in the number of kittens on campus. The percentage of cats categorized as kittens at inclusion in the program decreased from 56% during the 11-year initial observation period to 12% during the 17-year follow-up observation period. All six of the kittens enrolled between 2002 and 2019 were feral immigrants. The last kitten known to have been born on campus was in 1995, four years after program initiation. This is consistent with what has been observed at sites of other long-term TNR programs, where kitten births were eliminated after two to eight years . In all,Levy et al.  concludeFrom 2002 to 2019, the total number of colonies discovered on the UCF campus rose from 11 to 16; however, over the same period, the number of colonies eliminated increased from 3 to 11. The proportion of colonies eliminated exceeds what was observed in an urban Chicago neighborhood, where 8 of 20 colonies were eliminated over a 10-year period ; howeverOf the 24 cats who moved from one colony to another, 88% (21/24) were sterilized at the time of their first colony change; however, two male cats, who changed colonies once, went unsterilized (one for 1.8 years and the other for 7.5 years) before disappearing, and another male cat changed colonies three times (including once to the NFC group for a period of 2.9 years) before being sterilized after 7.7 years on campus (and disappearing 2.3 years later). Gunther et al. observed significantly higher rates of emigration among unneutered cats ; the afoThe limitations of the present study include those commonly encountered when conducting a retrospective investigation. The absence of certain data  constrained the types of analyses that could be performed. The inability to examine all of the same documents used to calculate results at the end of the initial observation period caused a nominal degree of uncertainty in the reconciliation of some details. Moreover, UCF’s TNR program was set up without control group colonies to allow for the comparison of results. Colony control groups, as established by Nutter , offer oIn their 2003 paper, Levy et al.  concludeThe present study reveals that free-roaming cat numbers on the UCF campus have declined significantly for nearly three decades since the inception of the TNR program. During the follow-up observation period, on average, only three new arrivals were enrolled in the program per year, even as the UCF campus grew into the largest public university (by enrollment for the 2018–2019 academic year) in the U.S. . The durAs suggested by Levy et al. , the pai


Cephalometric analysis is used to evaluate facial growth, to study the anatomical relationships within the face. Cephalometric assessment is based on 2D radiographic images, either the sagittal or coronal planes and is an inherently inaccurate methodology. The wide availability of 3D imaging techniques, such as computed tomography and magnetic resonance imaging make routine 3D analysis of facial morphology feasible. 3D cephalometry may not only provide a more accurate quantification of the craniofacial morphology and longitudinal growth, but also the differentiation of subtle changes in occlusion. However, a reliable protocol for the computation of craniofacial symmetry and quantification of craniofacial morphology is still a topic of extensive research. Here, a protocol for 3D cephalometric analysis for both the identification of the natural head position (NHP) and the accurate quantification of facial growth and facial asymmetry is proposed and evaluated. A phantom study was conducted to assess the performance of the protocol and to quantify the ability to repeatedly and reliably align skulls with the NHP and quantify the degree of accuracy with which facial growth and facial asymmetry can be measured.The results obtained show that the protocol allows consistent alignment with the NHP, with an overall average error (and standard deviation) of just 0.17 (9.10e-6) mm, with variations of 0.21 (2.77e-17) mm in the frontonasal suture and 0.30 (5.55e-17) mm in the most prominent point in the chin. The average errors associated with simulated facial growth ranged from 1.83 to 3.75% for 2 years’ growth and from − 9.57 to 14.69% for 4 years, while the error in the quantification of facial asymmetry ranged from − 11.38 to 9.31%.The protocol for 3D skull alignment produces accurate and landmark free estimation of the true symmetry of the head. It allows a reliable alignment of the skull in the NHP independently of user-defined landmarks, as well as an accurate quantification of facial growth and asymmetry. Cephalometric analysis is used to evaluate facial growth, to study the anatomical relationships within the face and as a routinely used tool for treatment planning in orthodontics and craniomaxillofacial deformity surgery. A standard cephalometric assessment is based on 2D radiographic images taken in either the sagittal  or coronal planes , where multiple landmarks, lines and angles are identified to quantify vertical and horizontal relationships in the face. The analysis is primarily dependent on the accurate and repeatable definition of a standardized head position, therefore patients are imaged in customized image acquisition systems . Regardl2D cephalometrics is an inherently inaccurate methodology, regarding the quantification of small increments of facial growth and the analysis of the transverse dimensions, in particular the analysis of facial asymmetry. The accuracy of contemporary cephalometric analysis is further complicated by the variation, inconsistency and errors in image capture, image quality and landmark identification, as well as limited intra and inter observer reliability and reproducibility , with stThe wide availability of 3D imaging techniques, such as computed tomography (CT), cone-beam CT (CBCT) and magnetic resonance imaging (MRI), makes routine 3D analysis of facial morphology feasible. 3D reconstructed cephalometry possesses several advantages when compared with traditional cephalometry. It avoids the need for standardized fixation of the head and allows an a posteriori definition of the anatomical head position. It also prevents structure magnification and distortion inherent to imaging process, allowing at the same time the full assessment of the 3D craniofacial morphology, which is important for pre-operative evaluation, surgical planning and post-operative evaluation . 3D imagThe most critical aspect of 3D cephalometric assessment is the accurate positioning of the skull in space, requiring accurate definition of both the craniofacial symmetry plane and the appropriate anatomical frame for the head. In 2D analysis of the lateral cephalogram, the skull is positioned through the identification of the true horizontal or the true vertical direction, and the identification of the S, as the reference point for image superimposition and comparison. The Frankfurt horizontal (FH) plane is used to orient the lateral cephalometric image in some specific analyses, requiring the identification of two bilateral landmarks , which do not lie in the same plane as assessed using 3D imaging due to the natural asymmetry of the human face. In addition, FH related landmarks are amongst the most subjective landmarks and therefore can be prone to erroneous identification , 10. TheThe development of a consistent and reliable 3D cephalometric workflow will provide clinicians with a valuable tool for the 3D characterization and quantification of facial growth, for the evaluation of dentofacial relationships and subsequent anatomically accurate orthodontic and surgical planning, and for the evaluation of treatment outcomes. We present a protocol for the accurate estimation of craniofacial symmetry, evaluating this using random skull orientations, facial growth vectors and different types of facial asymmetry. The null hypothesis tested was that the 3D method for analysing facial growth and asymmetry was not reliably quantifiable. The alternative hypothesis was that both craniofacial symmetry plane, facial growth and facial asymmetry could be accurately quantified with the proposed 3D algorithm.https://grabcad.com/), and skull landmarking  and landmarked by a clinical expert  of the Euclidean distances between the nearest points in the original and reflected models, along the Apply ICP to register the original and reflected mesh created in step 3;Perform the PCA over the combined models from step 4 and reorient the original model according to the newly obtained eigenvectors To determine the correct symmetry plane of the skull Principal Component Analysis (PCA)  and Iterpoint-to-plane alignment provides very accurate alignment between equal meshes  mm). ICP registration with point-to-plane alignment was therefore set as the default in step 4 .Evaluation of craniofacial morphology and growth is quantified considering a standardized anatomical position and therefore recovery of the NHP of the raw model may provide a consistent framework for 3D cephalometric assessment in clinical practice. The NHP can be defined by two landmarks along the symmetry plane, for example N and Pog, with the subsequent definition of the N-Pog line as the true vertical direction. However, it is important to note that use of the N-Pog line is not mandatory and two other landmarks along the symmetry plane could also be used to define the true vertical direction .gold standard model  assigned to the basilar surface , where:MSD)d stands for the 3D Euclidean norm and m and n are the number of points in A and B, respectively [Evaluation of the cephalometric alignment for NHP and craniofacial growth and asymmetry was performed considering several measures, namely the alignment and distances between cephalometric landmarks against their respective landmarks on the ectively .gold standard model, the ideal alignment between gold standard and warped mesh was considered to be the initial alignment after warping and before any rigid transformation . In the evaluation of alignment of morphologically different skulls (section 2.3), the Hd, MSD and the deviation between the N and Pog were computed. To understand how much information about facial growth and asymmetry is preserved after transformation and realignment, the distances between gold standard landmarks and morphed landmarks were therefore also calculated.In facial growth and asymmetry tests, there is no longer an exact correspondence between cephalometric landmarks. Since mesh warping was applied to the ion Fig. d. In botMSD and Hd (with standard deviations) between the gold standard and target models are 0.17 (9.10e-6) mm and 0.52 (4.65e-5) mm for the full data, respectively. The true vertical was recovered with an average error of just 0.21 mm for the N and 0.30 mm for the Pog landmarks. For partial data, the MSD and Hd were 0.27 (2.23e-5) mm and 0.52 (4.26e-5), whereas the N and Pog landmark error were 0.32 (2.63e-7) and 0.23 (1.76e-7), respectively. Figure The reliability in the recovery of the NHP was tested by applying a random rigid transformation Fig. c to the and 0.52 .65e-5 mmand 0.52 .65e-5 mmand 0.52 .65e-5 mmThe ability to quantify facial growth and asymmetry were tested by considering theoretical growths of 2 and 4-years Fig. a-c. The Table The alignment of the asymmetric models showed good agreement with the theoretical models Fig. a-c, and gold standard and warped models is one to two orders of magnitude smaller than the initial perturbation, the facial growth and asymmetry assessments were assumed to be accurate and therefore the null hypothesis was rejected.3D medical imaging techniques offer the opportunity for more accurate evaluation of the craniofacial anatomy, however most assessment protocols are still based on the concepts of 2D cephalometry. Here, a protocol for general 3D cephalometric analysis is proposed, which aims firstly to accurately estimate facial symmetry and the NHP and secondly to use the basicranium to superimpose other morphologically different skulls. A phantom study was conducted to quantify the level of accuracy and reliability with which small morphological differences within or between subjects can be evaluated. Since the measured differences between the true vertical direction, and (2) a method for consistent alignment between different skulls, which is independent of the standard cephalometric landmarks. The true vertical direction is given by two user-defined landmarks (here the N and Pog), however these are internally corrected once the true symmetry plane is computed. To consistently align different skulls , the basilar surface is used as the reference structure, instead of the geometrical centre of the sella turcica (Sella point (S)). In previously reported studies, the estimation of the symmetry plane was accomplished through the identification of multiple anatomical landmarks. Landmarks that were then mirrored about an arbitrary plane and matched using Procrustes superimposition [According to the results obtained, the protocol provides: (1) consistent definition of the craniofacial symmetry plane and the position –25. Hereλi that minimizes the root-mean-squared error (RMSE) of the Euclidean distances between the nearest points in the original and reflected models. The algorithm performed consistently for both full and partial skull data  and re-applying the PCA method to the matched original and mirrored skulls (section 2.1 step 5). It is important to stress that the definition of the symmetry plane is only as accurate as the ability to match the original and the mirrored models . To obta.1 step 2In the definition of the NHP and with the newly defined symmetry plane, new estimates of the N and Pog landmarks and N-Pog line/plane are computed by estimating the nearest node to the landmark projection along the symmetry plane. However, it is important to stress that in the presence of a mandibular asymmetry Fig. c the ideIn facial growth and asymmetry quantification, the final skull alignment is defined by the facial symmetry procedure and the ICP with weights, which minimizes distances across the basilar surface. Since in adolescence and adulthood there is a stabilization of the craniofacial base and foramen magnum , this anThe alignment protocol allowed an accurate quantification of facial growth, with an error that ranged from − 11.38 to 9.31% for 2-years and from − 11.38 to 2.77% for 4-years facial growth  determination of the orientation of dental occlusion plane relative to the craniofacial morphology; (3) quantification of mandibular asymmetry and biomechanical investigations in masticatory asymmetry [gold standard model. It can be combined with other mid-plane cephalometric landmarks (such as N and Pog) to position the skull in a standard anatomical position. The alignment error was quantified for both NHP, facial growth and facial asymmetry and was evaluated against the ideal alignment defined by a clinical expert. The results obtained demonstrate that symmetry alignment is accurate and independent of errors associated with the user-defined landmarks. In addition, the correct alignment of morphologically disparate skulls can be accomplished using the basilar surface instead of the traditional sella turcina (S) landmark. This is an important advance alongside the contemporary trend towards 3D imaging and the need to be able to reliably quantify longitudinal changes in 3D, with landmark free superimposition producing greater precision and reliability than previously used methods. With the craniofacial images being derivable from MRI, it is also non-invasive, clinically useful in orthodontics and maxillofacial surgery, versatile and applicable to other anatomical areas and even other fields of interest.The new protocol to determine the true craniofacial symmetry plane has numerous clinical applications, namely (1) as a part of a symmetry , (4) it symmetry , and mansymmetry . The proThe protocol proposed for 3D skull alignment is fully automated and produces an accurate and landmark free estimation of the true symmetry plane of the human skull. It also allows the accurate alignment of the skull in the NHP, taking into account the true symmetry plane of the skull and two used-defined landmarks along the facial profile that specify the true vertical direction. After the definition of the true symmetry plane, the user-defined landmarks are internally corrected and the final alignment is completely independent of any user-defined data. The algorithm produces accurate quantifications of both facial growth and asymmetry, which renders it useful for clinical applications.


This study aimed to investigate the coverage of hepatitis B vaccine (Hep B) and its completeness and timeliness for birth cohorts from 1999 to 2017 in Zhejiang province, East China. Demographic characteristics and vaccination records of Hep B of children born from 1 January 1999 to 31 June 2017 were extracted from the Zhejiang provincial immunization information system. The timeliness of the first dose of Hep B (Hep B1) was defined as the proportion of children who received the Hep B1 within 24 h after birth among the target population. The completeness of Hep B was defined as the proportion of children who completed the three-dose series of Hep B before 7 years of age. The demographic characteristics of the target population were described. The coverage of each dose of Hep B, the timeliness of Hep B1, and the completeness of Hep B for each birth cohort were described. A logistic regression model was applied to detect the determinants of the timeliness of Hep B1 and the completeness of Hep B vaccination. The coverage of Hep B1 increased from 90.3% to 98.3%, the coverage of Hep B2 increased from 88.8% to 96.1%, and the coverage of Hep B3 increased from 86.4% to 94.2%. The timeliness of Hep B1 increased from 80.3% to 91.3%. The completeness of Hep B increased from 81.3% to 91.5%. The determinants of timeliness of Hep B1 included children’s delivery place, immigration status, maternal education level, and economic development level of resident area. The determinants of completeness of Hep B included children’s delivery place, immigration status, maternal education level, economic development level of resident area, maternal occupation, and frequency of vaccination service. Zhejiang province had achieved significant improvements in the timely administration of Hep B1 and the completeness of Hep B. To accelerate progress toward additional reductions in the transmission of hepatitis B virus, further efforts need to be focused on improving the timeliness of Hep B1 vaccination and reducing the drop-outs among disadvantaged children with the risk factors identified in this study. Hepatitis B is a viral infection that spreads through the blood or bodily fluids. Globally, approximately 2 billion people are currently infected by hepatitis B virus (HBV), and an estimated 240 million people are chronic HBV carriers. More than 780,000 people die every year due to the consequences of HBV infection, such as hepatic failure, cirrhosis, and hepatocellular carcinoma . The proHBV infection is a serious health problem in China, causing a substantial disease burden. The national serum epidemiological survey of HBV in 1992 indicated that the prevalence of hepatitis B surface antigen (HBsAg) was 9.8% in the general population, and 9–12% in children under 5 years of age . The recThe Chinese policy on Hep B vaccination has undergone some changes in the last two decades. In 1992, the Chinese Ministry of Health (MoH) recommended that children’s Hep B be classified as a category two vaccine, which required the parents to cover the expense. User fees charged to parents by local health departments for the vaccine’s purchase and its administration were two serious barriers to uptake at that time. Hep B has been included in the Chinese expanded program on immunization (CEPI) since 2002 and provided to all children for free, while parents were still required to pay the administration fee. Hep B1 should be administrated in the first 24 h at the delivery hospital, according to the “who delivers the infant gives the birth dose” policy. It meant that the hospital who delivered the infant had the responsibility to ensure that Hep B1 was given in a timely manner. Meanwhile, the Chinese MoH recommended the screening of all pregnant women for HBsAg, and demanded that all infants born to HBsAg-positive mothers should have administered the Hep B1 and hepatitis B immunoglobulin simultaneously within 24 h of birth. Since 2007, all vaccinations for children under 15 years of age, including Hep B, have been offered without any charge, and the amount of HBsAg of Hep B increased from 5 ug to 10 ug. Since 2016, the latest Chinese vaccination schedule updated the recommendations of Hep B for low-birth-weight infants and premature infants. It stipulated that Hep B1 should be administered to all newborns even if they were low-weight or premature. Hep B1 should not be counted in the routine schedule among infants with a low birth weight or premature infants, and these infants should restart the three-dose schedule at one month of age. Alongside the improvements in Hep B vaccination policy, extensive efforts on maternal health and other aspects of the public health service were made in the same period, including an increase in the hospital delivery rate and staff training, the development of a local vaccination strategy, and improvements on the awareness of HBV infection. As a result, both the coverage for Hep B vaccination and the timeliness of Hep B1 increased dramatically from 1992 to 2012 [Zhejiang province is located in East China, with 70 million inhabitants living in 11 cities and an annual birth cohort of approximately 700,000 newborns. Just like the whole nation, the carrier rate of HBsAg in Zhejiang has decreased remarkably in the general population and in children under 5 years of age since the introduction of Hep B into CEPI in 2002. However, previous studies had always evaluated the coverage of the individual Hep B dose at a cross-sectional level and did not focus on the completeness and the timely administration of Hep B1 . FurtherWe obtained data from the Zhejiang provincial immunization information system (ZJIIS), which consists of two parts: client software and central server. ZJIIS is deployed in the Zhejiang provincial health network, which is a local area network that is isolated from the Internet to protect the data’s privacy while covering the center for disease control and prevention (CDC) at different administrative levels and immunization clinics. Any data extracted from ZJIIS should be de-identified.The client software is deployed in immunization clinics and delivery rooms in the maternity hospitals. The client software is used to collect demographic information , and vaccination records. All of this information will be uploaded to the central server in real time or updated if there is any change, and can be downloaded or shared among different vaccination clinics in Zhejiang Province. It is worth noting that the client software deployed in delivery rooms can create a record of every newborn after his/her birth, which includes the demographic information of the child and his/her mother and the vaccination record of Hep B1. When a child is brought to the vaccination clinic at one month old to receive the next vaccinations, the staff of immunization clinics can download his/her vaccination record from the center server before making new records on it. For migrant children from other provinces, their demographic information and the complete historic vaccination records are required to be entered into the client software when they first visit any immunization clinic in Zhejiang province. Additionally, the information on the frequency of sessions and working time of immunization clinics are also uploaded to the central server. The frequency of sessions means the number of times an immunization clinic has been open during a specific time period .The central server is deployed in Zhejiang Provincial CDC. It receives, stores, and processes all of the data collected from the client software, and makes them exchangeable among different immunization clinics. To date, over 10 million records of children born from 1 January 1999 are stored in the central server of ZJIIS. The “Zhejiang Provincial Immunization Information Management Platform (ZJIISMP)” is a website that can help staff of CDCs at different administrative levels to monitor the process and outcome of the EPI work. ZJIISMP can generate plenty of tables and figures, including vaccination coverage rates by vaccine or by dose, completeness and timeliness of vaccination for a specific birth cohort, drop-out rates, and lists of unvaccinated children.Children born from 1 January 1999 to 31 June 2017 and registered in ZJIIS were enrolled in this study. Appropriately anonymized individual records of children, including Hep B vaccination records, were extracted from ZJIIS on 1 July 2018. The 12-month-wide cohort method was adopted to calculate the vaccination coverage of each dose of Hep B, the timeliness of Hep B1, and the completeness of the three-dose series of Hep B. For example, the 1999 cohort consisted of children born between 1 January 1999 and 31 December 1999. However, the 2017 cohort consisted of children born between 1 January 2017 and 31 June 2017. The reason for only including half the birth cohort of 2017 in our analyses was that we needed to ensure that every child had the opportunity to finish the three-dose series of Hep B.Zhejiang province consists of 11 cities in total. Since Huzhou city did not participate in ZJIIS, our analyses were performed in the remaining 10 cities. In 2016, the number of newborns of Zhejiang province was 739,650, and the number of newborns of Huzhou was 23,311, with a proportion of 3.2%. The 10 cities were classified into three strata according to the Gross Domestic Product (GDP) per capita, by using the census data of 2017. Hangzhou (HZ), Ningbo (NB), and Zhoushan (ZS) were classified as the high level for GDP per capita >16,000 USD; Shaoxing (SX), Jinhua (JH), and Jiaxing (JX) were classified as the middle level for GDP per capita between 10,000 and 16,000 USD; and Taizhou (TZ), Quzhou (QZ), Lishui (LS), and Wenzhou (WZ) were classified as the low level for GDP per capita <10,000 USD.The coverage was defined as the proportion of vaccinated individuals among the target population. The timeliness of Hep B1 was defined as the proportion of individuals who received Hep B1 within 24 h after birth among the target population. The completeness of Hep B was defined as the proportion of individuals who completed the three-dose series of Hep B before 7 years of age. The second dose of Hep B (Hep B2) was considered to be invalid if it was administrated <28 days from Hep B1. The third dose of Hep B (Hep B3) was considered to be invalid if it was administrated <60 days from Hep B2 . Invalidij: probability of the ith child in the jth community to be immunized in a timely manner with Hep B1 or be complete with the Hep B series, 0β: intercept, nβ: regression coefficient, xij: independent variables, uj: community level errors, and eij: individual-level errors. All variables on demographics, the economic status of resident areas, and vaccination service provision were included in the final models. The adjusted odds ratio (AOR) with 95% CI for each variable was calculated. The statistical significance was set at 0.05.All analyses were performed with STATA MP version 14.0 . The demographic characteristics were described by different birth cohorts. The coverage of each dose of Hep B, the timeliness of Hep B1, and the completeness of Hep B were estimated by city and birth cohort, along with the 95% confidence interval (CI). In order to detect the determinants of the timeliness of Hep B1 and the completeness of Hep B vaccination, the generalized mixed linear model was adopted to account for the clustered nature of the data from ZJIIS (nested within cities) and the binary response of the outcome variable. The city was treated as a random effect, and other demographic characteristics were treated as fixed in our analyses. The Final model was: This study was exempt from ethical review since it involved the assessment of de-identified data.In total, 10,911,530 children were enrolled in this analysis for the birth cohorts dating from 1999 to 2017. The male-to-female ratio was 1.03. Of the enrolled children, 92.9% were delivered in hospital, and 42.3% were migrant. The proportion of mothers with fixed jobs and with an education background of senior middle school or above was 87.1% and 90.4%, respectively. Almost 40% of the enrolled children were from areas with the high economic development level, and almost 16% of the enrolled children received the vaccination service from clinics with a service frequency of 5 times per week or more .p < 0.01) after controlling for other factors. The values of intra-class correlation (ICC) were 17.2% for the model of timeliness of Hep B1 and 15.7% for the model of completeness of Hep B, showing the relevant proportions of the variation due to the clustered nature of the data.The results from the two generalized mixed linear models are presented in There are numerous studies on the development of the Chinese Hep B vaccination program . These sGenerally, the coverage of each dose of Hep B improved from the birth cohort 1999 to the birth cohort 2017 and had maintained the goal of 90% since the birth cohort 2004. This trend was similar to that at the national level . The covIn this study, we found the timeliness of Hep B1 had increased from 81.2% to 91.3%. It was in line with the trend of this indicator at the national level, which increased from 22.2% for the 1992 birth cohort to 90% for the 2012 birth cohort ,24. Due An occupied mother was an inverse factor of the completeness of Hep B vaccination. It was consistent with our previous study, and we assumed that they did not have enough time to spare for primary healthcare and were less aware of the information on vaccination ,33. A hiOur analyses supplemented the previous studies based on the nationwide serum surveys, the administrative coverage aggregated at the county level, or a combination of different data sources. This approach also had a wider scope than household surveys from limited geographical regions. This study has some limitations. First, our findings were based on 10 cities, and could not be generalized to the entire province. Second, our analyses included the children registered in ZJIIS, and the children who were not registered might have a lower coverage for all vaccines . Based oZhejiang province has made a great achievement in integrating Hep B into routine immunization programs, achieving significant improvements in the timely administration of Hep B1 and the completeness of Hep B. However, there were still some children with a delayed Hep B1 or an incomplete Hep B vaccination. To reduce the prevalence of HBV infection, and to accelerate progress toward additional reductions in HBV transmission, further efforts need to be focused on improving the timely administration of Hep B1 and reducing the number of drop-outs among disadvantaged children with the risk factors identified in this study.


The EC50 for 2,4-D was 55.7–59.8 mg/L. The addition of the test substances resulted in changes of the population structure of the microbiota which formed the fermentation pulp. The research was based on 16S rDNA analysis with the use of the Next Generation Sequencing method and the MiSeq platform . There was a significant decrease in bacteria belonging to Firmicutes and Archaea belonging to Euryarchaeota. A significant decrease of the biodiversity of the methane fermentation microbiota was also established, which was expressed by the decrease of the operational taxonomic units (OTUs) and the value of Shannon’s entropy. In order to determine the functional potential of bacterial metapopulations based on the 16SrDNAprofile, the PICRUSt(Phylogenetic Investigation of Communities by Reconstruction of Unobserved States)tool was used, which allowed to determine the gene potency of microorganisms and their ability to biodegrade the herbicides. In the framework of the conducted analysis, no key genes related to the biodegradation of MCPA or 2,4-D were found, and the observed decrease of their content in the supernatant liquid was caused by their sorption on bacterial biomass.The aim of the study was to evaluate the effect of herbicidal ionic liquids on the population changes of microorganisms used in a batch anaerobic digester. The influence of the following ionic liquids: benzalkonium acetate (BA), benzalkonium (4-chloro-2-methylphenoxy)acetate (BA)(MCPA), didecyldimethylammonium acetate (DDA), didecyldimethylammonium (4-chloro-2-methylphenoxy)acetate (DDA)(MCPA), as well as reference herbicides (4-chloro-2-methylphenoxy)acetic acid (MCPA) and acetic acid  in the form of sodium salts on biogas production efficiency was investigated. The effective concentration (EC50) values were determined for all tested compounds. (MCPA) Anaerobic digestion is a process that uses anaerobic microorganisms, which is widely utilized for processing vegetable biomass into biogas . MethaneThe correct balance of carbon, nitrogen, phosphorus and sulphur sources, as well as micronutrients such as iron, nickel, molybdenum, tungsten, selenium or cobalt, are extremely important for the proper development and functioning of microorganisms involved in anaerobic digestion ,4,5. In Herbicides are another important group of compounds used in high quantities in agricultural production. However, due to their numerous undesirable environmental effects, particularly their possible accumulation, there is an ongoing search for substances with herbicidal properties characterized by low toxicity towards a wide range of organisms and new methods of their application . The novIonic liquids belong to a relatively new class of compounds which occur as liquids at the temperature below 100 °C. They consist of a cation and an anion, which determine the traits and properties of a given ionic liquid. These substances are characterized by very low vapor pressure and unique properties due to interactions between ions based on Coulomb and Van der Waals forces, as well as hydrogen bonds. An important feature of these compounds is their ability to dissolve both organic and inorganic materials. Due to this reason they are applied in the chemical, electrochemical, pharmaceutical and biotechnological industries, mainly in the extraction and separation processes of various compounds . HerbiciHowever, in addition to their anti-weed effectiveness, HILs are often characterized by high toxicity and may be introduced into groundwater when used at high doses, which creates a potential hazard to humans. The risk of accumulation of herbicides in various elements of the environment may adversely impact the bioconversion processes, in cases where plant biomass which has had contact with HILs is used as the raw material.The aim of this study was to evaluate the influence of herbicidal ionic liquids on the efficiency of methane production. The potential of anaerobic microorganisms which biodegrade the herbicides, the genetic potential of the environment and changes in microbial communities which form the fermentation pulp were established by determining the indicators of α- and β-biodiversity.The following ionic liquids were used for the tests: benzalkonium acetate (BA), benzalkonium (4-chloro-2-methylphenoxy)acetate (BA)(MCPA), didecyldimethylammonium acetate (DDA), didecyldimethylammonium (4-chloro-2-methylphenoxy)acetate (DDA)(MCPA).The compounds were obtained by chemical synthesis at the Faculty of Technology Chemical University of Technology . Commercially available herbicides in the form of acides were obtained from Sigma-Aldrich . Structural formulas of the studied herbicides and herbicidal ionic liquids are presented in 2, O2, CH4, NH3 and H2S measurements. The analyser was connected to the valve located on the storage tube with a hose. The volume of produced biogas was measured every 24 h and biogas analysis was carried out with a minimum gas volume of 500 cm3. All fermentation experiments were carried out in triplicate.The fermentation was carried out in glass bioreactors with a capacity of 2 L which were inoculated with 1000 g of inoculum originating from an active agricultural biogas plant. Then, an appropriate amount of HIL ranging from 0 to 125 mg/L was introduced into each bioreactor, respectively, based on the herbicidal component. In the next stage, a carbon source in the form of whey permeate was introduced in the amount of 100 g/L. The initial dry matter content was equal to 76 g/L. After sealing, the bioreactors were placed in a water bath with a constant temperature of 37 °C and mixed several times a day in order to obtain a uniform fermentation pulp. In the upper part of the bioreactor the biogas was discharged by a hose into a 4.5 Lpolycarbonate tube with a scale to measure the amount of gas produced. The analysis of the chemical composition of the gas was carried out using a GA5000 gas analyser from Geotech  equipped with a head for COThe samples were filtered through a pre-weighed 3h grade filter paper from Munktell&Filtrak . The obtained filtrate was diluted with a methanol: water mixture (80:20 v/v) and further filtered through a 0.2 µm polytetrafluoroethylene (PTFE) syringe filter  before the high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, whereas the biomass sludge was dried on the filter paper and weighed. The amount of biomass was calculated by subtracting the filter mass. Next, the filter paper containing the biomass was cut and HILs were extracted using the ultrasound bath with 25 mL methanol for 15 min. The obtained extract was diluted with a methanol: water mixture (80:20 v/v) and filtered through a 0.2 µm PTFE syringe filter before the HPLC-MS analysis.−3 mol/L of ammonium formate in water (phase A) and methanol (phase B) at a flow rate of 0.2 mL/ min. Gradient elution was performed by linearly increasing the percentage of phase B from 85 to 100% during 2 min and maintained at 100% for 3 min. The LC column effluent was directed to the API 4000 QTRAP triple quadrupole mass spectrometer from AB Sciex  through the electrospray ionization source (Turbo Ion Spray) which operated in positive ion mode for analyses of cations and in negative ion mode for analyses of anions. The dwell time for each mass transition detected in the MS/MS multiple reaction monitoring mode was set to 100 ms. Nitrogen was used as the curtain gas (10 psi), nebulizer gas (40 psi), auxiliary gas (40 psi) and collision gas (medium). The source temperature was equal to 400 °C and the ion spray voltage was at 4500 V for cations and −4500 V for anions. The declustering potential was equal to 50 V for cations and −50 V for anions. The detected mass transitions and specific parameters of each analyte are summarized in The HPLC-MS analyses were performed using the UltiMate 3000 RSLC chromatograph from Dionex . Two µL samples were injected into a Hypersil GOLD column (100 mm × 2.1 mm L.D. (Length and Diameter); 1.9 µm) with a 2.1 mm L.D. pre-filter cartridge (0.2 µm) from Thermo Scientific . The mobile phase consisted of 5 × 10In order to conduct a metabiome analysis, samples of 5 mL of each replicate were taken, mixed together, averaged and used as a starting material for DNA isolation.DNA extraction: Total DNA was extracted from 1 mL of sample using a Genomic Mini AX Soil kit  according to manufacturer’s instruction. The extracted DNA was quantified using Quant-iT HS ds.-DNA assay kit  on Qubit 3 fluorometer; 2 µL of extracts were examined on a 0.8% agarose gel .® DNA Library Prep Master Mix Set for Illumina . Then the libraries were pooled at equimolar concentration. Pooled libraries were quantified using a Qubit Fluorimeter and dsDNA HS assay kit . The library was denatured with 0.2 N NaOH and diluted with HT1 buffer  to a final concentration of 8 pM. To balance the overall lack of sequence diversity, a spike-in of denatured Phix was added to the concentration of 40%. Sequencing was conducted on an Illumina MiSeq  using paired-end (2 × 250) MiSeq Reagent Kits v2 . Sequencing primers were based on Caporasoet al. [Region IV of bacterial 16S rDNA gene was amplified using universal primers 515F and 806R: containing reverse complement of 3’ Illumina adapter, golay barcode, reverse primer pad, reverse primer linker and reverse primer . Genomicsoet al.  . Total number of reads ranged from 182,354 to 223,487. After sequencing, the reads were demultiplexed to the probes, the overlapping paired-end reads were merged  and trimmed to yield fragments of 289 nt. Only fragments which passed the merging were retained for downstream processing. Chimeric reads  were filtered and remaining sequences were assigned to operational taxonomic units (OTUs).The number of reads which passed merging and trimming ranged from 84,128 to 1,116,529. Reads were clustered against the SILVA v119 99% 16S rDNA gene database ,23,24. RTo determine the predicted gene content from each OTU table constructed against GreenGenes 13.5, the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) tool was used . The outDuring the anaerobic digestion in the system without the addition of herbicidal ionic liquids (HILs), the process yield reached 260 mL of methane/kg dry mass of sediment. Addition of HILs in an amount up to 125 mg/L significantly reduced the methane production of the system .− or − anions indicated that ionic liquids with the (MCPA)− were more toxic in low concentration, than those with −. Similarly, an inhibition of biogas production occurred when MCPA and 2,4-D were used. In this case, the MCPA herbicide was also more toxic than 2,4-D. The addition of 125 mg/L of the herbicide  completely inhibited biogas production. The EC50 values determined for the anaerobic digestion process are shown in The comparison of the effect of HILs comprising (MCPA)− compared to − towards aerobic bacteria from different environments. However, the determined EC50 values for MCPA and 2,4-D were higher than those obtained in this work. It can therefore be concluded that anaerobic microorganisms are more sensitive to the presence of herbicides compared to aerobic bacteria. Similar results were obtained by Sanchis et al. [The obtained data confirm the results of studies conducted by Ławniczak et al. , which as et al.  in the fs et al. .During the course of anaerobic digestion, the residues of herbicides were determined and their concentration was analysed both in the sludge and in the supernatant. The initial concentration of HILs added to the fermentation pulp was 50.0 mg/L . In the case of herbicides, their presence was found both in the liquid and in the sediment. However, their concentration did not differ statistically. A higher concentration of herbicides was observed in the supernatant. Their amount absorbed on the sediment biomass was much lower and did not exceed 10 mg/kg. The determined concentrations of herbicides in the fermentation pulp lead to the conclusion that these compounds were not biodegraded and the decrease of their concentration in the supernatant was related to their adsorption on bacterial biomass . Other aTaxonomic identification based on the hypervariable region of 16s rDNA using the SILVA v119 database allowed for the detection of microorganisms comprising the fermentation pulp. In all tests, both Bacteria and Archaea were found, which belonged to 82 classes .Methanobacteria was the dominant class among Archaea (25% of Archaea). Clostridia was the predominant Bacteria domain-related class (37% of Bacteria). Such composition is characteristic for microbial communities which carry out the anaerobic digestion process [Firmicutes and Euryarchaeota were particularly sensitive phyla to the presence of herbicides. The decrease in the ratio of Archaea belonging to the Methanobacteria class in all variants of the experiment in comparison to the control sample was particularly noticeable. Their content decreased to 3% after addition of (DDA)(MCPA).In the fermentation pulp without the addition of herbicides (control), the ratio of Archaea was highest and amounted to 32%.  process . The addMethanomicrobia class, where the ratio was decreased to 0.45–2.3%.A particularly toxic effect was observed after the addition of MCPA in case of the Methanosaeta genus (which was predominant in this class of microorganisms) to the presence of herbicides. The addition of MCPA and HILs with (MCPA)− to the fermentation pulp in an amount of 50 mg/L resulted in its complete elimination from the community of microorganisms conducting the methanogenesis process. In contrast, the addition of 2,4-D or HILs with − caused a 52–76% decrease in its ratio with respect to the anaerobic digestion process without the addition of herbicides.This was caused by the extremely high sensitivity of Archaea belonging to the − and − also increased the toxicity and caused a significant decrease of both Methanobacteria and Clostridia classes. The Methanobacterium genus (which was predominant in the Methanobacteria class) was characterized by higher resistance to the presence of herbicides compared to the Methanosarcina genus (which was predominant in the Methanomicrobia class). In this case, a decrease of its ratio in the population was observed and the presence of herbicides did not eliminate it from the environment. Based on the comparison of the taxonomic composition of the pulp, the most notable changes caused by the addition of HILs concerned bacteria belonging to the Bacilli and Clostridia classes. However, in all cases, the increase of the percentage ratio of the Proteobacteria class in relation to the metapopulation of the fermentation pulp can be noticed. The decrease of the ratio of Bacilli and Clostridia classes in the microbial community caused an increase of the ratio of Bacteroidetes phylum (the abundance of which in the control sample amounted to 0.3%). These bacteria were characterized by very high resistance to the presence of herbicides and HILs. As a result, their ratio in the population increased after addition of 2,4-D and MCPA to 6.5% and 9.1%, respectively. On the other hand, the addition of herbicides in the form of HILs resulted in the increase of their abundance in the microbial community from 32.0% to 41%. The Porphyromonadaceae and Prevotellaceae families as well as numerous uncultured bacteria showed particularly high resistance to herbicides. The fermentation pulp is a dynamic multi-phase system, which contributes to the diversity of the local metapopulation structure of indigenous microorganisms, and thus their metabolic profiles. Alpha-diversity analysis expressed as number of OTUs showed significant differences between all variants of experiments (The introduction of the herbicide in the form of HILs containing (MCPA)eriments  compared− resulted in a greater reduction of biodiversity than the addition of HILs with −. This phenomenon is often described in the literature and is related to the influence of toxic substances which change the composition of the microflora. This is particularly true for environments characterized by high anthropopressure, especially in heavily polluted areas, where a smaller number of OTUs is found than in pollution-free environments. This may be due to the fact that only microorganisms with enzymatic profiles specialized for biodegradation of xenobiotics and characterized by high resistance to the existing stress factors are able to grow as a result of long-term exposure to pollutants. Often, the decline in biodiversity may also be related to the formation of dead-end products during the biodegradation of various xenobiotics, which have a toxic effect on some of the metapopulations in the environment [This indicates a significant decrease of biodiversity in the fermentation pulp caused by the addition of herbicidal substances. The addition of HILs with (MCPA)ironment .The Principal coordinates analysis (PCoA) with Bray-Curtis dissimilarity  also shoThe most notable changes in comparison to the composition of the control population occurred after the addition of (BA)(MCPA) and (BA) into the pulp. The distances between the samples presented in Evaluation of distances in the Bray-Curtis dissimilarity analysis for the addition of herbicidal ionic liquids indicated that the changes in the population of microorganisms resulting from the influence of HILs were targeted and non-random.Metapopulations of microorganisms are characterized by the dynamics of qualitative and quantitative changes under the influence of varying environmental parameters and exposure to toxic compounds . The PICPrediction of the presence of the sum of genes responsible for the biological degradation of 2,4-D and MCPA indicated a decrease in their number relative to the control . In all This was confirmed by the biodegradation results, in which no decrease of herbicide concentration was established during the anaerobic digestion process. In order to explain this phenomenon, the PICRUSt program algorithm should be taken into consideration. During categorization to the metabolic pathway level, all genes included in it are counted (based on the KEGG database). However, taking into account the selected orthologs units, a clear tendency of decline in the abundance of the majority of crucial orthologs in the first stages of biodegradation of herbicides  can be observed. It is considered that the moment of ring splitting is the key stage of biodegradation of numerous xenobiotics, which determines the efficiency of the process. In associated with the above, it can be assumed that the lack of or low number of one of the first enzymes may result in the inhibition of the biodegradation processes, despite the generally high indications at the level of the metabolic pathway.The presence of some genes responsible for the biodegradation of herbicides is related both to the fact of coexistence of various metabolic pathways of the enzymatic degradation of xenobiotics as well as the limitations associated with insufficient data in the databases which are the basis for the operation of bioinformatic algorithms. In order to use the estimation method based on 16SrDNA data, it is worth conducting an analysis of all available orthologs. This also refers to the construction of other molecular tools for the genetic evaluation of biodegradation potential, e.g., microarrays. Comprehensive multivariate analysis may contribute to the increased effectiveness of such tools. The dynamics of microbial metapopulation changes under the influence of herbicide contamination is undoubtedly a targeted process, however, due to the complexity of the microbial community, more research is needed in this field.Proteobacteria, Actinobacteria and Bacteroidetes in the microbial community with a simultaneous decrease of the Firmicutes ratio. Additionally, it was reported that phosphonic-based HILs can be a stress factor for soil microorganisms and affect their biodiversity changes, particularly by increasing the ratio of microorganisms well known for biodegradation of hydrocarbons, such as the Sphingomonas and Pseudomonas genera [Biodegradation of HILs is possible under aerobic conditions both in soil  and in ts genera . Other as genera . On the s genera  and an aThe dynamics of metapopulation changes caused by the presence of HILs in the fermentation pulp is a targeted process, which results in a decrease of methane production efficiency mainly due to the reduction of the ratio of Archaea responsible for the production of methane. Due to the lack of key genes related to the biodegradation of MCPA and 2,4-D, the microorganisms which carry out the methane fermentation process were exposed to the phenomenon of accumulation of herbicides in the biomass. This causes changes in the microbial population structure and, consequently, inhibits methane production. However, due to the complexity of the fermentation environment, further research into the toxicity of herbicides in relation to the anaerobic microbiota is necessary.


Ubiquitination plays a pivotal role in several cellular processes and is critical for protein degradation and signaling. E3 ubiquitin ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition. In order to achieve selectivity and specificity on their substrates, HECT E3 enzymes are tightly regulated and exert their function in a spatially and temporally controlled fashion in the cells. These characteristics made HECT E3s intriguing targets in drug discovery in the context of cancer biology. Post-translational modification of proteins by the addition of a ubiquitin (Ub) moiety can induce alteration in protein stability, function, activity, localization and interaction . This tiIn this review, we focus on the HECT-containing E3 ligases. Invariably at their C-terminus, all HECT E3s present the catalytic HECT domain, composed of a bulkier N-terminal lobe (N-lobe) that contains the E2 binding domain, and a C-terminal lobe (C-lobe) carrying the catalytic cysteine. The two lobes are connected by a flexible hinge region that allows the C-lobe to move around in order to facilitate the Ub transfer from the E2 to the E3 .According to the domain organization present in the N-terminal part of the proteins, the HECT E3s can be subdivided into three main families . The besThe activity of HECT E3s is tightly regulated in terms of chain specificity (mono- or poly-ubiquitination and Ub chain linkage), interaction with the E2 and recognition of the substrate.The ability to build linkage-specific poly-Ub chains appears to be an intrinsic feature of the HECT enzymes, as they are able to generate distinct Ub chains regardless of the paired E2 enzymes. NEDD4 family members primarily synthesize K63 chains , while EPrecise control of E3 ligase activity is needed to ensure that their functions are restricted until required. Several HECT E3s are kept in a catalytically inactive state by intramolecular interactions between the N-terminal region (either the C2 or the linker region between the WW domains) and the C-terminal HECT domain . For othA third level of regulation is represented by adaptor proteins that can modulate both the E2–E3 interaction and the interaction with the substrate. An example of the former is represented by SMAD7. SMURF1 and SMURF2 have low binding affinities for the E2-conjugating enzyme UbcH7, providing a point of control for regulating the Ub ligase activity through the action of auxiliary proteins. Indeed, SMAD7, functioning as a bridge between the E2 and E3, stabilizes an active complex and promotes, thus, the ligase activity . In otheNotably, HECTs themselves can function as adaptors for other conjugating enzymes as in the case of HERC2 whose binding to the N-terminal domain of E6AP increases the catalytic activity of E6AP .Finally, the catalytic activity of the HECT enzymes is often spatially and temporally regulated by post-translational modifications. Activating modifications can contribute to the release of auto-inhibiting conformational states of the E3s. For example, the phosphorylation of ITCH on the three residues of the proline-rich region releases the auto-inhibitory state generated by the binding of the C2 and the first WW domain to the HECT domain . LikewisUbiquitin ligases regulate a wide range of cellular processes and are involved in many human pathologies. Abnormal expression or dysfunction of HECT E3s have been shown in many different cancers. The current knowledge often suggests a dual role for these ligases in tumorigenesis, which might depend on the tissue context and/or additional events that affect their activity. Here, we will review the recent literature on E6AP, NEDD4, and HUWE1, and highlight excellent reviews for additional reading .The classical example of an HECT associated with cancer is E6AP. Since its discovery in 1993, it was evident that E6AP drives human papilloma virus (HPV)-induced cervical carcinogenesis, exerting its activity toward the tumor suppressor p53 through its association with the viral protein E6. E6 is an adaptor protein of the HPV and it is capable of binding to the N-terminal of EA6P and the DNA-binding domain of p53 , acting vs  as a more potent inhibitor. However, the binding mode and the specificity of this compound has not been experimentally validated.With the idea of identifying covalent modifiers of the catalytic cysteine of NEDD4, trations . ThroughA recent approach suggests the use of specific Ub mutants identified by phage display  to modulate HECT catalysis . The scrin silico screening of the hydrophobic pocket of the WW domains of SMURF1 led to the identification of compounds that possess features similar to the PPXY motif. These compounds bind the ligase and block SMAD1 ubiquitination, possibly disrupting the WW domain:SMAD1 interaction (A few molecules have been found to inhibit the HECTs, impairing substrate binding. An eraction . However50 values in the low micromolar range, leaving NEDD4 family members or the E1 and E2 enzymes unaffected. The compounds reduced the growth of colorectal cancer cells, but not that of HUWE1-depleted or normal epithelial cells of the colon (For many small molecules and inhibitors, the binding sites, the mechanism or the specificity have not been determined. By a high-throughput screening of small molecules, Eilers and co-workers identified two compounds that selectively inhibit the enzyme activity of HUWE1, as seen by a reduced substrate ubiquitination. Both compounds were found to inhibit the ligase activity with IChe colon . Howeverhe colon . With a he colon . The authe colon .Although tackling the ubiquitination system rather than the proteasome seems to be a promising avenue for therapeutic drug discovery, targeting HECT E3s to manipulate their activity is challenging for several reasons. First of all, we still lack the complete picture of their ubiquitome and their mechanism of action. Which substrates do HECT E3s ubiquitinate? What impact does ubiquitination have on their function proteolytic or non-proteolytic? How are these substrates recognized and how is their ubiquitination regulated in time and space and in different cellular conditions? What are the mechanisms the different HECT E3s apply to ubiquitinate their targets? So far, we only have a few answers for a small number of ligases and substrates due to the fact that ubiquitination is a dynamic and highly regulated process, and that the interaction with substrates is often transient with a low binding affinity. Besides the PPxY motif that is recognized by the WW domains of NEDD4 family members, no other substrate binding motif is known. An additional challenge is represented by redundancy. While E3s target multiple substrates, a specific substrate may be modulated by several E3s, depending also on the cell context. The high conservation of the HECT domain within the HECT family makes it a difficult target for which to develop specific inhibitory compounds. Finally, most of the HECTs act as both tumor suppressors and oncogenes, and more information is needed in order to find specific and effective compounds. Thus, acquiring more insights into the structural composition and the ubiquitination mechanism used by the different HECT E3s is of paramount importance in order to open new avenues for therapeutic interventions.JW, EM, and SP conceptualized and wrote this review. JW prepared the figure. All authors approved the final version of the manuscript and agreed to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast, the temperatures of fingertips increased and then returned to the baseline levels during the second minute of MS period. In Group III, the temperature of stimulation point increased every time with the operation of AC, accompanied by the temperature decrease of middle fingertip. The findings suggest that moxibustion may trigger the sympathetic nervous system and induce the reduction of microcirculation, accompanied by a reduction of fingertip temperature. In addition, the operation of AC caused repeated cycles of thermal stimulation on the stimulation point, which may repetitively activate cutaneous sympathetic nerve fibres and evoke the temperature reduction of fingertips.The aim of this study is to investigate the response of local and distal skin temperature to moxibustion stimulation (MS) and explore the effects of MS on sympathetic nerve activity. The distal skin temperatures of fingertips, as an indicator for sympathetic reflex response, were recorded using infrared camera during resting period (10 min), MS period (10 min), and natural cooling period (15 min), respectively. The MS without ash cleaning (AC) was applied to acupoints Quze (PC3) (Group I) and Lao Gong (PC8) (Group II), respectively. In Group III, the MS with the operation of AC was performed on PC8. The temperature responses of the local stimulation points and corresponding control points were also investigated. At the beginning of MS period, a significant increase of temperature on the stimulation point accompanied by a simultaneous reduction of temperature on fingertips was observed. A marked negative correlation was also obtained between temperature changes in the stimulation point and in the fingertips. At the end of natural cooling period ( Artemisia vulgaris, Mugwort) to stimulate the meridians of human body [Moxibustion plays an important role in Traditional Chinese Medicine treatments, which treats and prevents diseases by burning a herb preparation containing Moxa , MS without AC on PC8 , MS with AC on PC8 , and a Control group (n = 8).In this study, suspended moxibustion was used to carry out experiments, which was performed by holding an ignited moxa stick at a certain distance above the patient's skin surface, keeping the spot warm and making it reddened without burns. Based on the theory of acupuncture and moxibustion in Traditional Chinese Medicine, the Pericardium Meridian is the main vessel of the heart that governs blood circulation in the whole body . The eleThe schematic of the experiment and the positions of monitoring points are shown in  t = 10 min. The distance between the bottom surface of moxa sticks and the skin surface was 3 cm, which is the most comfortable distance in the clinical moxibustion treatment [ t = 20 min, the burning moxa stick was moved away from skin surface. For Group II, the burning moxa stick was applied 3 cm above the skin surface of PC8. For Group III, during MS period, the burning moxa stick was removed slightly away from above PC8 to clean the ash content on its bottom surface every 3 min  and then moved back with the same distance of 3 cm between the bottom of moxa sticks and skin surface after every operation of AC. For Control group, participants sat on the chair and the infrared images of left forearms were recorded continuously throughout 35 min including 10 min of baseline, 10 min of MS period with unlit moxa sticks above the skin surface, and 15 min of natural cooling period.As shown in reatment . At t =  t-test was employed for within-group temperature comparison. The Friedman test was used for temperature comparison among four temperature changes in Group III. Correlations between temperature of the stimulation point and the middle fingertip during moxibustion were analyzed using simple regression and Spearman's rank correlation. Significant differences were indicated by ∗ p < 0.05 or ∗∗ p < 0.01.The results are presented as mean values ± standard deviation. Statistical analysis was performed using SPSS 21.0. One-way ANOVA was performed for temperature comparison among groups, and post hoc tests were used for further analysis with significant effects. The pairedi), middle fingertip (Pm), and ring fingertip (Pr) were monitored to study the response of distal skin temperature to MS on PC3. T) between transient temperatures and the mean values of baselines. As shown in m during the first minute of MS period was analyzed in r = -0.981, p < 0.05). As shown in m at different times with baseline values using 2-tailed t-test for paired data, and the temperature drop was obtained by subtracting the mean value of baseline from the transient temperature of middle fingertip. It can be found that significant temperature drops occurred at the 21 s after moxibustion started and lasted for 82 s. In addition, the temperature of control point PC8 has no obvious change throughout the MS period.The MS was applied on PC3, and the temperature of PC8 was measured as a control point. The temperatures of index fingertip (PT) between transient temperatures and the mean values of baselines. As shown in m during the first minute of MS period was analyzed in r = -0.957, p < 0.05). The temperatures of Pm at different times were compared with baseline values statistically using 2-tailed t-test for paired data, and the results were depicted in r was lower than that of Pi at the beginning of MS period, although they showed similar variation tendency. Meanwhile, the temperature of control point PC3 had no obvious change throughout the MS period.The MS was applied on PC8, and the temperature of PC3 was measured as a control point. The temperatures of three fingertips were monitored to study the response of distal skin temperature to MS on PC8. m decreased significantly from 16 s of MS period and this obvious temperature reduction lasted to 137 s. The detailed temperature-time history of PC8 and Pm in the first operation of AC is shown in m temperature occurred at 212 s after the temperature rise of PC8 began and lasted to 271 s. The other two operations of AC were performed at 330 s and 510 s of MS period. After each operation of AC, the temperature of PC8 began to increase due to the heat effect of MS, and the obvious temperature reductions of Pm occurred with the temperature rise of PC8. These results indicated that the temperature of Pm experienced three additional temperature reductions with three repeated increases of PC8 temperature induced by the operation of AC.m was analyzed during the first minute of MS period, and the significant negative correlation  was observed in m were compared with their baseline values statistically using paired t-test, respectively. Also, Friedman test was employed to compare four temperature rises and four temperature drops in Group III, respectively. The results demonstrated that there is no obvious difference among four maximum temperatures on PC8 and among four minimum temperatures on Pm.The correlation between temperature changes of PC8 and P et al. [ et al. . ConsideIn  et al. [In the present study, the temperature evolution on skin surface was recorded during MS. The hand and finger temperature fluctuated significantly when the body was near a neutral thermal state, and the possible explanation is that the body is actively using hand vasodilation and constriction to regulate heat loss to around the neutral conditions . Thus, t et al.  measured et al. [ et al. [Some research has been reported to study the relation between fingertip temperature decrease and sympathetic nerve activity , 21–23.  et al.  proposed et al. [As mentioned in , 24, bot et al.  also fouGroup I and Group II were compared to study the effects of stimulation position on fingertips' temperature. Both the temperature rises of stimulation points (PC3 and PC8) and the temperature drop of fingertips were observed in Group I and Group II . The maxT of stimulation point at the end of MS period (t = 20 min) in Group II was smaller than that of Group I (p < 0.05) , the temperature of PC3 in The temperature on stimulation points decreased after reaching the maximum values in both groups due to ash formation on the surface of burning moxa sticks Figures . However Figures . The resBecause the ash deposition on the surface of burning moxa stick has a negative effect on stimulation temperature , the ope et al. [Repeated stimulation may cause fatigue or sensitization of nervous system , 29. Fat et al.  indicate et al. , heat-se et al. . AlthougIn summary, the temperature responses of stimulation points and three fingertips were monitored during MS by means of infrared camera. At the beginning of MS, increased temperature of the stimulation point was accompanied by reduced temperatures of fingertips. The heat effect of MS on the stimulation point lasted until natural cooling period, but the temperatures of fingertips returned to baseline levels during the second minute of MS period. The main findings of the present study imply that MS induces transient activation of skin sympathetic nerves and the repeated cycles of thermal stimulation on the stimulation point caused by operation of AC may repetitively activate cutaneous sympathetic nerve fibers and evoke the temperature reduction of fingertips.


To explore the feasibility of utilizing recycled aggregate concrete (RAC) in revetment construction of the lower Yellow River, a series of mix proportions with local recycled aggregates (RA) were designed to evaluate its mechanical properties and durability. The morphology and micro-hardness of the interface transition zone (ITZ) were also characterized to explain the performance of RAC. Based on the compressive strength data of 13 groups of mixtures, which is larger than 30 MPa, and with the RA substitution rate not less than 50%, the RAC containing 50% recycled fine aggregate (RFA) (HDX50), 70% RFA (HDX70), and 50% recycled coarse aggregate (RCA) (HDC50) were selected. The experiment results suggest that the mechanical performance, frost resistance, and carbonation resistance of the selected RAC is generally poorer than that of natural aggregate concrete (NAC), but can meet the performance requirement of concrete for the revetment construction of the lower Yellow River. The comprehensive performance of these three mixtures ranks as: HDX50 > HDX70 > HDC50. When considering the RA substitution ratio as a priority, HDX70 would be the best choice and can be applied in the revetment engineering. A number of defects are observed on the surface of RA with old pastes attached. Furthermore, the ITZs formed around RA are loose and with low micro-hardness, which is deemed to be the dominating reasons leading to the weaker performance of RAC than that of NAC. The Yellow River, the second largest river in China, has nurtured generations of Chinese people, but its river flood has also caused great losses to human’s lives and properties for thousands of years. In recent decades, the Yellow River conducted integrated management, and the construction of revetment was one of the most important steps ,2 becausConcrete is believed to be a promising replacement, considering that it is a cheap construction material with excellent properties, and it has been successfully used in some river management projects. The biggest problem is that the aggregates needed for concrete, such as gravels and river sands, are also in shortage, and their exploitation will cause damage to the environment like natural stone masonry. Replacing natural aggregates (NAs) with recycled aggregates (RAs) to produce recycled aggregate concrete (RAC) blocks provides an eco-friendly and cost-effective alternative, especially given that tons of waste concrete are produced every year in cities along the Yellow River due to the demolition of old buildings ,4.RAC has been studied for several decades, and many researches ,6,7,8,9 The frost resistance, carbonation resistance, chloride penetration resistance, and impermeability are often used to evaluate the durability of concrete. Generally, RAC is not recommended to use in harsh conditions  because Similar to the mechanical properties and frost resistance, the carbonation resistance of RAC usually decreases with the increase of RA dosage ,26,27. SFurthermore, according to the previous investigations ,29,30, tBased on the above, it can be found that there have been a number of studies about RAC, and almost all of them indicate that the properties of RAC are generally poorer than that of NAC. A few contradictory findings are mainly caused by the different material properties of RA, which can be further traced back to the sources and locations, mix proportions, impurities, such as red block residues and soil, and the service history. All the differences mentioned above make the microstructure of the interface transition zones (ITZ) between the RA and the new cement paste and the RA and the old mortar differ a lot and are very complicated ,38, whicRAC has been applied to many recent projects ,40, suchThe raw materials used in this study consisted of cement, NA, RA, water, and water reducer. The cement was PO 42.5 (Portland Cement), produced by Shanshui Cement Co., Ltd. , and its chemical composition is shown in RA was from the derelict buildings in Jinan city, Shandong province, and had been processed by Shandong Mingran Renewable Resources Utilization Co., Ltd. The processing was mainly about removing the steel bars and most of the red bricks. The appearance of RA is shown in Three groups of RAC were designed: single mixed with RCA, single mixed with RFA, and double mixed with RCA and RFA. Each series includes 4 mix proportions: The substitution ratio of RA being 30%, 50%, 70%, and 100% of NA by mass, respectively. The label of all mixes and the detailed information are shown in After stirring, concrete paste was poured into molds of different sizes. Specimens for compressive strength and carbonation resistance tests were cast in molds with a size of 100 mm × 100 mm × 100 mm, while specimens for flexural strength and frost resistance tests were cast in molds with a size of 100 mm × 100 mm × 400 mm. After casting, the mold surfaces were sealed with thin film to prevent moisture evaporation. Then specimens were demolded after 24 h and cured at 23 ± 1 °C, at relative humidity of 95%. Specimens for compressive strength were cured for 3 days, 7 days, 28 days, 56 days, and 90 days separately, and all other specimens were cured for 28 days.Note that the slump of all mixtures in casting falls in the range of 150~180 mm. The slump of RAC is slightly lower than that of NAC with the same dosage of water reducer. Additionally, the slump of RFA concrete approximates that of RCA concrete with different RA admixtures, attributed to the fact that the water absorptivity of the former is higher, while the actual dosage of the latter is greater. The slump of concrete with mixed RFA and RFC is a little lower. Therefore, when their dosage is higher than 50%, more water reducer is added to reach similar workability to that of concrete with only RFA or RFC, which aims to alleviate the impact of workability on the performance of different mixtures as much as possible.The compressive strength and flexural strength were tested abiding by the GB/T 50081-2002 standard for test method of mechanical properties on ordinary concrete . As intrAccording to the DG/TJ 08-2018-2007 technical code on the application of recycled aggregate concrete , the froiP is the relative dynamic modulus of specimens after n freezing and thawing cycles; nif is the dynamic modulus of specimens after n freezing and thawing cycles, Hz; oif is the original dynamic modulus of specimens before the test, Hz.niΔW is the mass loss rates of specimens after n freezing and thawing cycles, %; niW is the mass of concrete after n freezing and thawing cycles, g; oiW is the original mass of concrete before the test, g.The frost resistance test was conducted abiding by the GB/T 50082-2009 standard for test methods of long-term performance and durability of ordinary concrete . Specime2 concentration at (20 ± 1)%, relative humidity at (70 ± 5)%, and the temperature at (23 ± 2) °C. It should be noted that the distance between specimens should be larger than 50 mm to guarantee all the specimens have the same boundary conditions. The lab photo of the carbonation resistance test is presented in The carbonation resistance test was conducted abiding by the GB/T 50082-2009 standard for test methods of long-term performance and durability of ordinary concrete . Cubic sk can be calculated.x is the carbonation depth, mm; k is the carbonation rate; t is the carbonation time, d.The carbonation depth was tested with the method below. We sprayed phenolphthalein of 1% concentration on the split surface. Due to the alkaline solution of concrete before carbonation, the uncarbonated area turned to purple, whereas the carbonated area stayed unchanged. We marked 12 equally spaced test points along the boundary between the purple area and unchanged color area and measured the distance between each test point and the end surface of the unchanged color area. The carbonation depth was obtained by averaging the 12 measured distances. The carbonation time and depth was reported to follow the empirical relation described in Equation (3), by which the carbonation rate Cubic samples with side lengths smaller than 10 mm were cut out from specimens exposed to different conditions with caution to make sure that at least one of the cutting faces contained both aggregates and slurry. These cubic samples were dried at 45 °C in a vacuum drying oven for 7 days. The macro-appearance of aggregates and interface were observed with an industrial electronic microscope at magnification of 50~100 times. The microscopic morphology of ITZ was observed by a scanning electronic microscope (Quanta 250) at an accelerating voltage of 20 kV and current of 20 mA.The samples for micro-hardness measurement were prepared in the same way as mentioned in (1)Compressive StrengthMoreover, it can be seen from (2)Flexural StrengthITZ is the weak region in either NAC or RAC, which is especially worse for RAC because the interface is usually attached with old slurry see . Figure As for the inherent defects of RA, (1)Process and Characterization of DamageThe appearance changes of RAC and NAC are basically the same with the increase of freezing and thawing cycles. After 25 cycles, the appearance of specimens generally remains intact, and only a small part of RAC specimens have a few edge damages and a small number of voids. After 50 cycles, numerous voids started to appear on the concrete surface, presenting the onset of a pitted surface. The appearance of H0 and HDX50 remained roughly intact, while only a few voids appeared, whereas the surface of HDC50 and HDX70 had a number of voids, and some of them were even connected, leading to a partial pitted surface. After 75 cycles, a pitted surface appeared on the surface of all concrete specimens, and the appearance damage of HDC50 and HDX70 was more severe with large-scale paste spalling. The detailed damage information of the four mix-proportion concrete specimens is presented in (2)Mass Loss(3)Relative Elasticity ModulusCombining the experiment results of appearance, mass loss, and relative elasticity modulus of specimens, it can be found out that the frost resistance of RAC with RFA (HDX50/HDX70) was not necessarily worse than that of NAC, and its elasticity modulus was even higher. However, the frost resistance of RAC with RCA was significantly poorer than that of NAC. RA itself and ITZ were probably the major factors affecting the frost resistance of RAC.It can be observed from Based on the above, it can be drawn that the frost resistance of RAC with RFA (HDX50/HDX70) was slightly weaker than that of NAC, while that of RAC with RCA was significantly worse than that of NAC. This is consistent with the findings of Salih et al. ,22,23, w(1)Carbonation Depth2 infiltration provided by the micro-cracks and large porosity of RA. The carbonation depth of HDC50 with RCA was the highest, reaching 12.4 mm at 28 days, and that of H0 at 28 days was 7.2 mm, the former of which was 1.7 times higher than the latter, and lower than that the result (2.5 times) of Silva et al. [2 infiltration, leading to a more severe carbonation degree. Moreover, since specimens mixed with RFA possess certain inner curing capacities, the hydration degree was more adequate compared with that of specimens mixed with RCA, and the matrix of RFA was denser, which made CO2 infiltration harder. In addition, RFA contained more pastes, which can consume more CO2 compared with RCA. These two factors lead to the result that the carbonation depth of RFA concrete was lower than that of RCA concrete.The development of carbonation depth of specimens is shown in a et al. . This de(2)Carbonation Rate2 infiltration, mitigating the following carbonation reaction, and thus slowed down the carbonation rate. As is shown in 2. Moreover, the Vickers hardness of the ITZ and matrix zone around RA in HDC50 after carbonation is shown in 2 infiltration, and the weaker the carbonation degree. In addition, the carbonation rate of RAC was significantly higher than that of NAC, with HDC50 being the fastest, followed by HDX70 and HDX50, successively.According to the above analysis, it can be summarized that the carbonation resistance of RAC was weaker than that of NAC. However, the carbonation depth of RAC specimens at 28 was smaller than 20 mm, which fully met the carbonation-resistance requirement for the revetment construction of the lower Yellow River . AdditioAbove all, the chosen mix proportions  of RAC successfully met the performance requirement of concrete for the revetment construction of the lower Yellow River, in terms of compressive strength, flexural strength, frost resistance, and carbonation resistance. Therefore, the chosen RAC can be applied to the actual revetment engineering of the Yellow river. After analyzing and combining all the experiment results, the engineering application performance of the three mix proportions is ranked as: HDX50 > HDX70 > HDC50. If the engineering application performance and economic performance of material are considered, RAC HDX70 is the best and is recommended to be used in the actual revetment engineering of the lower Yellow River. Moreover, the RAC designed in this study with local raw material possesses superior mechanical properties and durability, which are as good as those in other studies. Therefore, it can be wildly applied, except for the bank revetment engineering of the Yellow River.(1)Based on the compressive strength data at 28 days, the RAC of the mix proportions HDX50, HDX70, and HDC50 were selected for compressive strength over 30 Mpa and a RA substitution rate no less than 50%.(2)Considering the combination of the appearance, mass loss, and relative elasticity modulus situations of specimens after freeze-thawing cycles, HDX50 possessed the best frost resistance performance, followed by HDX70 and HDC50, successively. Improvement on the relative elasticity modulus of HDX50/HDX70 was observed, compared to NAC.(3)In view of the carbonation depth and rate of RAC, the carbonation resistance of HDX50 was the best, followed by HDX70 and HDC50. Moreover, carbonation could densify the ITZ of RAC and increase its micro-hardness.(4)RA attached with old pastes had lots of micro-cracks and voids, and the ITZ formed around RA were loose and with low micro-hardness, which were deemed to be the dominating reasons leading to the poorer performance of RAC than that of NAC. In addition, RFA concrete had better performance than RFC concrete, since RFA possessed certain inner curing capacities.(5)The selected HDX50, HDX70, and HDC50 meet the performance requirement of concrete for the revetment construction of the lower Yellow River in terms of mechanical and durability performance. Combing performance and RA utilization rate, HDX70 ranks first and is recommended to be used in actual revetment engineering.